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This program is sponsored by Emory University School of Medicine

GAJ

GAJ Global

antiviral Journal

Volume 12, Supplement 1

February 20-21, 2016Boston Marriott Copley Place • Boston, Massachusetts

Final Program and Abstract Book

HIV DRUG RESISTANCEw o r k s h o p

Aims and ScopeGlobal Antiviral Journal publishes peer-reviewed original works related to international efforts to advance antiviral discovery and development, including full-length articles and short papers, as well as solicited review articles, conference reports, letters and book reviews. Occasional supplements contain conference abstracts presentations and/or posters from international meetings in the fields of virology and antiviral research. The scope of the journal encompasses chemistry and biological advances in the fundamental and clinical study of antiviral diseases and their treatment. Areas covered include HIV, hepatitis B, hepatitis C and emerging viruses, co-infections, vaccines, animal models, pharmacology, microbicides, alternative therapies, viral dynamics and resistance issues.

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ISSN (print): 1556-9047ISSN (online): 1556-9055

GAJ Global

antiviral Journal

Final Program and Abstract Book

HIV DRUG RESISTANCEw o r k s h o p

February 20-21, 2016Boston, Massachusetts USA

International HIV Drug Resistance Workshop iiGlobal Antiviral Journal Volume 12 Supplement 1

Table of Contents Page

Corporate Supporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iii

Conference Committees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .v

Abstract Listing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .viii

Keynote Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..1

Poster Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Preclinical Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

New Resistance Technologies and Interpretations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Clinical Implications of Resistance for Treatment and Prevention Strategies . . . . . . . . . . . . 21

Epidemiology of Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

International HIV Drug Resistance Workshop iiiGlobal Antiviral Journal Volume 12 Supplement 1

Table of Contents | Program | Abstract Listing

Support for 2016 International HIV Drug Resistance Workshop has been provided by the following:

Major Supporter

Contributors

Supporters

The mark “CDC” is owned by the US Dept. of Health and Human Services and is used with permission. Use of this logo is not an endorsement by HHS or CDC of any particular product, service, or enterprise.

International HIV Drug Resistance Workshop ivGlobal Antiviral Journal Volume 12 Supplement 1


Organizing Committee John MellorsUniversity of Pittsburgh, USA

Carlo-Federico PernoUniversity of Rome Tor Vergata, Italy

Deenan PillayUniversity College London, UK

Douglas RichmanVA San Diego Healthcare System and University of California San Diego, USA

Scientific CommitteeElaine AbramsICAP, Mailman School of Public Health, Columbia University, USA

Silvia BertagnolioWorld Health Organization, Switzerland

Vincent CalvezHôpital Pitié Salpêtrière, France

Tulio de OliveiraUniversity of KwaZulu-Natal, South Africa

Jim DemarestViiV Healthcare LLC, USA

Richard HarriganBC Centre for Excellence in HIV/AIDS, Canada

Walid HeneineCenters for Disease Control and Prevention, USA

Gillian HuntNational Institute for Communicable Diseases (NICD), South Africa

Ilesh JaniInstituto Nacional de Saúde (INS), Mozambique

Pontiano KaleebuMRC/UVRI Uganda Research Unit on AIDS, Uganda

Daniel KuritzkesBrigham and Women's Hospital, USA

Frank MaldarelliNational Institutes of Health (NIH), USA

Michael MillerGilead Sciences, Inc., USA

Roger ParedesirsiCaixa AIDS Research Institute, Spain

Christos PetropoulosMonogram Biosciences, USA

Elliot RaizesCenters for Disease Control and Prevention, USA

Jonathan SchapiroNational Hemophilia Center, Israel

Raymond SchinaziEmory University/VA Medical Center, USA

Robert ShaferStanford University, USA

Amilcar TanuriUniversidade Federal do Rio de Janeiro (UFRJ), Brazil

Marco VitoriaWorld Health Organization, Switzerland

Mark WainbergMcGill University AIDS Centre, Canada

Carole WallisLancet Laboratories and BARC-SA, South Africa

Annemarie WensingUniversity Medical Center Utrecht, the Netherlands

International HIV Drug Resistance Workshop vGlobal Antiviral Journal Volume 12 Supplement 1


Saturday, February 20, 2016

Opening Session

13:00 Opening Remarks and Introduction of the Keynote Presentation Deenan Pillay University College London, United Kingdom

13:05 Keynote Presentation: Modelling the Spread and Impact of Resistance K1 under the New WHO ART Initiation Guidelines Andrew Phillips University College London, United Kingdom

13:35 Discussion

Preclinical Resistance Chairs: Walid Heneine Centers for Disease Control and Prevention, United States Carlo-Federico Perno Universita Di Tor Vergata, Italy

13:45 Second Generation HIV-1 Maturation Inhibitor BMS-955176: 1 Antiviral Optimization and Cross-Resistance Profiling Ira Dicker Bristol-Myers Squibb, United States

14:00 Resistance Pathways for Potent and Broadly Active HIV-1 Maturation Inhibitors 2 Emiko Urano National Cancer Institute, United States

14:15 In Vitro Sensitivity of HIV-2 Isolates and Integrase Mutants to Cabotegravir 4 Raltegravir + Darunavir/Ritonavir vs. Tenofovir/Emtricitabine + Darunavir/Ritonavir Robert Smith University of Washington, United States

14:30 Break

New Resistance Technologies and InterpretationsChairs: Vincent Calvez Hôpital Pitié Salpêtrière, France Robert Shafer Stanford University, United States 15:00 Spatio-Temporal Dynamics of Drug Resistance Evolution and Persistence of RT-SHIV 6 Alison Feder Stanford University, United States

15:15 Rare Linked Drug Resistance Mutations Detected by New Ultrasensitive SGS Assay 15 Valerie Boltz National Cancer Institute, United States

15:30 Performance Evaluation System for Next Generation Sequencing-Based 10 HIV Drug Resistance Genotyping Assay Dun Liang MOgeneDx LC, United States

Abstract

PROGRAM

International HIV Drug Resistance Workshop viGlobal Antiviral Journal Volume 12 Supplement 1


Abstract

Saturday, February 21, 2016 (cont.)

Children and Adolescents: Clinical Implications of Resistance for Treatment and Prevention StrategiesChairs: Elaine Abrams ICAP, Mailman School of Public Health, Columbia University, United States Frank Maldarelli National Institutes of Health, United States

15:45 High Levels of HIV Drug Resistance in Treatment-Naïve Children in Lagos, Nigeria: 22 Original Data and a Systematic Review in Sub-Saharan Africa Ragna Boerma Amsterdam Institute for Global Health and Development, the Netherlands

16:00 Drug Resistance Compromises Second-Line ART in Mozambican Children 26 Failing First-line Paula Vaz F. Ariel Glaser, Mozambique

16:15 Predictors of Persistent HIV Viraemia Among Treatment Experienced Children 23 and Adolescents at an Urban Clinic in Uganda Rita Atugonza Baylor-Uganda, Uganda

16:30 Transmitted Drug Resistance and First-Line ART Treatment Outcomes 21 in Ugandan Children Cissy Kityo Joint Clinical Research Centre, Uganda

16:45 – 18:45 Poster Session

Sunday, February 21, 2016

09:00 Introduction of Keynote Presentation Annemarie Wensing University Medical Center Utrecht, the Netherlands

09:05 Keynote Presentation: Second Line and Future Regimens in Sub-Saharan Africa K2 Willem Daniel Francois Venter University of the Witwatersrand, South Africa

09:35 Discussion

Clinical Implications of Resistance for Treatment and Prevention StrategiesChairs: Annemarie Wensing University Medical Center Utrecht, the Netherlands Roger Paredes IrsiCaixa AIDS Research Institute, Spain

09:45 Resistance Analyses of E/C/F/TAF in Phase 3 Clinical Studies 32, 33, 28 Christian Callebaut Gilead Sciences, United States

10:05 No Effect of HIV-1 Subtype C on Virological Failure Rate with First-Line TDF Regimens 35 Ellen White University College London, United Kingdom

10:20 The Co-Presence of Specific HIV-1 CRF02_AG Polymorphisms Correlates with a 31 Lower Response to PI-Based First Line HAART Daniele Armenia University of Rome Tor Vergata, Italy

International HIV Drug Resistance Workshop viiGlobal Antiviral Journal Volume 12 Supplement 1


Sunday, February 21, 2016 (cont.)

10:35 Protease Inhibitor Resistance at 2nd-Line HIV Treatment Failure in Sub-Saharan Africa 38 Tamara Sonia Boender Amsterdam Institute for Global Health and Development (AIGHD), the Netherlands

10:50 Is Resistance Testing of Value after First-Line ART Failure in Resource-Limited Settings? 37 Insights from ACTG 5273 Carole Wallis BARC-SA and Lancet Laboratories, South Africa

11:05 Break

11:30 Impact of Transmitted Thymidine Analogue Mutations on Responses to First-Line ART 29 Anna Maria Geretti University of Liverpool, United Kingdom

11:45 The Dynamics of Drug Resistance Detected During Acute HIV Infection 20 Ruth Kanthula University of Washington/Seattle Children's Research Institute, United States

12:00 Immunologic Criteria are Poor Predictors of Virologic Outcomes: 54 Implications for HIV Treatment Monitoring in a Large Treatment Program in Nigeria Nicaise Ndembi Institute of Human Virology, Nigeria

12:15 Resistance in PBMCs Can Predict Virological Rebound after Therapy Switch in 42 cART-Treated Patients with Undetectable HIV-RNA Maria Mercedes Santoro University of Rome Tor Vergata, Italy

12:30 – 14:00 Lunch

Epidemiology of ResistanceChairs: Elliot Raizes Centers for Disease Control and Prevention, United States Deenan Pillay University College London, United Kingdom

14:00 Transmission of HIV Drug Resistance Mutations Varies Regionally in Europe 73 Marije Hofstra University Medical Center Utrecht, the Netherlands

14:15 Prevalence of HIV-1 Drug Resistance in Treated Patients with 62 Viral Load > 50 copies/mL in 2014: A French Nationwide Study Sandrine Reigadas Hôpital Pellegrin CHU de Bordeaux, France

14:30 Rate of Transmitted Integrase Inhibitor Resistance Mutations Remains 72 Very Low in the SHCS Alexandra Scherrer University Hospital Zürich, Switzerland

14:45 Large Cluster Viral Lineages Fueling the MSM Epidemic Show Accelerated Development 58 of Resistance to Integrase Inhibitors Bluma Brenner Lady Davis Institute, Canada

15:00 Identifying Growing HIV Clusters Among Persons Who Inject Drugs - United States 59 Alexandra Oster Centers for Disease Control and Prevention, United States

15:15 Closing Remarks John Mellors University of Pittsburgh, United States

Abstract

International HIV Drug Resistance Workshop viiiGlobal Antiviral Journal Volume 12 Supplement 1


Abstract Listing session title and author abstract

Keynote Abstracts

Modelling the Spread and Impact of Resistance under the New WHO ART Initiation Guidelines

A Phillips

K1

Second Line and Future Regimens in Sub-Saharan Africa

WDF Venter

K2

Preclinical Resistance

Second Generation HIV-1 Maturation Inhibitor BMS-955176: Antiviral Optimization and Cross-Resistance Profiling

I Dicker

1

Resistance Pathways for Potent and Broadly Active HIV-1 Maturation Inhibitors

E Urano

2

Minimal Phenotypic Drug Susceptibility Effect of E529D RNase H Mutation of HIV-1 Subtypes on the Reverse Transcriptase Inhibitors

N Mkhwanazi

3

In Vitro Sensitivity of HIV-2 Isolates and Integrase Mutants to Cabotegravir

RA Smith

4

In Vitro Selection of HIV-1 Subtype C Resistant to Integrase Strand Transfer Inhibitors

MA Papathanasopoulos

5

New Resistance Technologies and Interpretations

Spatio-Temporal Dynamics of Drug Resistance Evolution and Persistence of RT-SHIV

A Feder

6

Genome-Wide Association Study of HIV Whole Genome Sequences Provides Insights into Drug Resistance

RA Power

7

International HIV Drug Resistance Workshop ixGlobal Antiviral Journal Volume 12 Supplement 1


session title and author abstract

Dual Infection by HIV-1 Among Newly Diagnosed Patients from the Spanish Cohort of Antiretroviral Naïve Adults (CoRIS)

F García

8

Low Frequency of Resistance Associated Mutations by Ultra-Deep Sequencing in HIV-1 Primary Infected Patients

C Rodriguez

9

Performance Evaluation System for Next Generation Sequencing-Based HIV Drug Resistance Genotyping Assay

D Liang

10

Comparison of HIV DNA Genotyping Par UDPS to Cumulative HIV RNA Genotypes in Pretreated Patients with Previous ARV Virological Failures

A Si-Mohammed

11

Dynamics of Integrase Inhibitors Multiresistant Variants Using Ultra Deep Sequencing in HIV-1 Infected Children

K Stefic

12

Simplified Platform for Detection of HIV Drug Resistance by the Oligonucleotide Ligation Assay

N Panpradist

13

Novel Isothermal Ligation Reaction for HIV Drug Resistance Testing

N Panpradist

14

Rare Linked Drug Resistance Mutations Detected by New Ultrasensitive SGS Assay

VF Boltz

15

The Impact of Changes over Time in the Stanford Resistance Algorithm

SA Hart

16

Capabilities for Management and Analysis of HIV Drug Resistance Survey Data Using a Central Data Repository

S Macauley

17

Quantitation of the HIV Proviral and 2-LTR DNA and Their Relation to Proviral Tropism

LE Soto Ramírez

18

International HIV Drug Resistance Workshop xGlobal Antiviral Journal Volume 12 Supplement 1



Clinical Implications of Resistance for Treatment and Prevention Strategies

HIV Drug Resistance Testing Among Patients New to HIV Care in the United States

AM Oster

19

The Dynamics of Drug Resistance Detected During Acute HIV Infection

R Kanthula

20

Transmitted Drug Resistance and First-Line ART Treatment Outcomes in Ugandan Children

C Kityo

21

High Levels of HIV Drug Resistance in Treatment-Naïve Children in Lagos, Nigeria: Original Data and a Systematic Review in Sub-Saharan Africa

RS Boerma

22

Predictors of Persistent HIV Viraemia Among Treatment Experienced Children and Adolescents at an Urban Clinic in Uganda

R Atugonza

23

In Cameroonian Children under Five Years, CCR5-Variants Prevail, Implying Vertical Transmission with CCR5-Tropic Viruses

J Fokam

24

Impact of Exposure to Lopinavir-Ritonavir in HIV-1 Infected Children and Adolescents in Madrid, Spain during 2000-2014

Á Holguín

25

Drug Resistance Compromises Second-Line ART in Mozambican Children Failing First-Line

P Vaz

26

Influence of Transmitted Drug Resistance on CD4 Decline Among ART Naïve HIV Patients

A Schultze

27

Pre-Existing Drug Resistance Mutations in Treatment-Naive Subjects Do Not Affect Response to Tenofovir Disoproxil Fumarate (TDF) or Tenofovir Alafenamide (TAF) Containing Regimens

MD Miller

28

International HIV Drug Resistance Workshop xiGlobal Antiviral Journal Volume 12 Supplement 1



Impact of Transmitted Thymidine Analogue Mutations on Responses to First-Line ART

AM Geretti

29

Transmitted Drug Resistance in ART-Naive Recently and Chronically-Infected Individuals: The ANRS 12249 Cluster-Randomised Trial of HIV Treatment as Prevention (TasP)

A Derache

30

The Co-Presence of Specific HIV-1 CRF02_AG Polymorphisms Correlates with a Lower Response to PI-Based First Line HAART

D Armenia

31

Resistance Analyses of E/C/F/TAF in Phase 3 Clinical Studies

C Callebaut

32

Pooled Week 48 Analysis of HIV-1 Drug Resistance in E/C/F/TAF Phase 3 Studies

ME Abram

33

Pre-ART HIV-DNA Correlates with Viro-Immunologic Status and Outcome in Patients with 1st-Line ART

F Ceccherini-Silberstein

34

No Effect of HIV-1 Subtype C on Virological Failure Rate with First-Line TDF Regimens

E White

35

Impact of Baseline Genotypic Resistance and Tropism on cART Outcomes in HIV-Positive Subjects Diagnosed during Primary HIV Infection

S Rusconi

36

Is Resistance Testing of Value after First-Line ART Failure in Resource-Limited Settings? - Insights from ACTG 5273

CL Wallis

37

Protease Inhibitor Resistance at 2nd-Line HIV Treatment Failure in Sub-Saharan Africa

TS Boender

38

HIV-1 Drug Susceptibility to Newer Second- and Third-Line Antiretroviral Regimens in Cameroon

AJ Nanfack

39

International HIV Drug Resistance Workshop xiiGlobal Antiviral Journal Volume 12 Supplement 1



Drug Resistance and Tropism as Markers of the Dynamics of HIV-1 DNA Quasispecies in Blood Cells of Heavily Pre-Treated Patients who Achieved Sustained Virological Suppression

J Ghosn

40

HIV RNA/DNA Drug Resistance in Naïve and Tenofovir-Treated Patients in Chennai, India

R Kantor

41

Resistance in PBMCs Can Predict Virological Rebound after Therapy Switch in cART-Treated Patients with Undetectable HIV-RNA

MM Santoro

42

Ultra Deep Sequencing Detect Minority Resistance Variants Archived in HIV-Cellular DNA in Antiretroviral Treatment Well-Suppressed Patients

C Rodriguez

43

Low-Level Viremia Can Indicate the Evolution of HIV Drug Resistance

J Verheyen

44

Impact on Residual Viremia of Switching to Elvitegravir-Based Single-Tablet Regimen

B Visseaux

45

HIV Drug Resistance Test at Low Viremia Levels: Light and Shadow in the Clinical Practice

D Mileto

46

Features of HIV Persistent Viremia after the Start and Restart of Antiretroviral Treatment Regimens

M Widera

47

Analysis of HIV Resistance Development in Long-Term Low Viremic Patients

P Braun

48

Ultra-Deep Sequencing in Pro-Viral DNA of PBMC in Patients with Low Level Viremia

JL Blanco

49

HIV-1 Non Group M Phenotypic Susceptibility to Integrase Inhibitors (Dolutegravir, Elvitegravir and Raltegravir)

C Charpentier

50

International HIV Drug Resistance Workshop xiiiGlobal Antiviral Journal Volume 12 Supplement 1



Low Frequency of the R263K Mutation in HIV-1 Integrase in Patients of the AREVIR Cohort Related to Raltegravir or Elvitegravir Therapy Failures

R Kaiser

51

The R263K Resistance Substitution Decreases Levels of Integrated HIV DNA over Time

T Mesplède

52

Durability and Virological Response to Dolutegravir (DTG)-Containing Regimens after Failing to Raltegravir (RAL) or Elvitegravir (EVG)

S Rusconi

53

Immunologic Criteria are Poor Predictors of Virologic Outcomes: Implications for HIV Treatment Monitoring in a Large Treatment Program in Nigeria

N Ndembi

54

HIV Resistance in Pregnant Women with Detectable HIV-1 RNA at Delivery in Mozambique

R Paredes

55

Strengthening HIV Therapy and Care in Rural Tanzania may Affect Rates of Viral Suppression

AJ Ntamatungiro

56

Epidemiology of Resistance

ARV Resistance and Clusters Among HIV-Positive Individuals in Washington State, 2005-2014

JR Reuer

57

Large Cluster Viral Lineages Fueling the MSM Epidemic Show Accelerated Development of Resistance to Integrase Inhibitors

BG Brenner

58

Identifying Growing HIV Clusters Among Persons Who Inject Drugs – United States

AM Oster

59

HIV Drug Resistance in Persons Who Fail to Achieve or Maintain Viral Suppression

AL Hernandez

60

International HIV Drug Resistance Workshop xivGlobal Antiviral Journal Volume 12 Supplement 1



Polymorphic Mutations Increase NNRTI and II Resistance in Primary HIV-1 Infected Patients

ML Chaix

67

Integrase Inhibitors-Transmitted Drug Resistance Detected by UltraDeep Sequencing

E Todesco

68

National Molecular Surveillance of Recently Acquired HIV Infections, Germany 2013-2014

N Bannert

69

Trends in Transmitted Drug Resistance and Subtype Distribution in Spain in the Period 2007 to 2015

F García

70

Presence, Persistence and Effects of Pre-Treatment HIV-1 Drug Resistance Variants Detected at a Low Frequency using Next Generation Sequencing: A Retrospective Longitudinal Study from Rural Coastal Kenya

AS Hassan

71

HIV-1 Non-B Subtype Associations and Emerging Resistance Mutations in First-Line ART Failure in Resource Limited Countries

CJ Villabona-Arenas

61

Prevalence of HIV-1 Drug Resistance in Treated Patients with Viral Load > 50 copies/mL in 2014: A French Nationwide Study

S Reigadas

62

Receipt and Timing of Genotypic HIV Drug Resistance Testing in the United States

AM Oster

63

HIV Pre-Treatment Drug Resistance in Mexico: A Nationally Representative WHO Survey

S Ávila-Ríos

64

Prevalence of Transmitted HIV-1 Drug Resistance in Tel-Aviv, Israel, and Antiretroviral Strategy in a Potential Transmitter Population from 2010-2015

D Turner

65

HIV-1 Variants and Drug Resistance in Pregnant Women from Equatorial Guinea: 2012-2013

Á Holguín

66

International HIV Drug Resistance Workshop xvGlobal Antiviral Journal Volume 12 Supplement 1



Rate of Transmitted Integrase Inhibitor Resistance Mutations Remains Very Low in the SHCS

AU Scherrer

72

Transmission of HIV Drug Resistance Mutations Varies Regionally in Europe

LM Hofstra

73

Dynamics of Transmitted HIV-1 Drug Resistance According To Subtype in Italy over the Years 2000-2014

L Fabeni

74

Transmitted HIV Integrase Inhibitor Resistance Prevalence in Canada During 2002-2012

H Ji

75

Longitudinal Evaluation of Coreceptor Use in Human Immunodeficiency Virus 1 Subtype A in Western Kenya

L Ledingham

76

Potential Drug Resistance Outcomes of Dapivirine Vaginal Ring Pre-Exposure Prophylaxis Scale-up in South Africa

UL Abbas

77

More Efficacious Drugs Lead to Hard Selective Sweeps in HIV-1 Drug Resistance Evolution

PS Pennings

78

Prevalence of Minority Resistant Variants in HIV-2 Naïve Patients: ANRS CO5 Cohort

C Charpentier

79

Correlation between HIV-2 RNA and HIV-2 Total DNA Levels

C Charpentier

80

HIV-2 Group A in France Displayed Two Clades with Distinct Geographical Origins

B Visseaux

81

International HIV Drug Resistance Workshop 1Global Antiviral Journal Volume 12 Supplement 1


Keynote Abstracts



effectiveness and cost-effectiveness of potential future policy responses.

ABSTRACT K2

Second Line and Future Regimens in Sub-Saharan AfricaWDF Venter

University of the Witwatersrand, South Africa

Sub-Saharan Africa routinely has to factor in issues such as pregnancy and concomitant TB therapy, when designing sequential antiretroviral regimens. In addition, issues such as refrigeration and cost are given high consideration, due to the widespread poverty across the region. Protease inhibitor-based second-line therapy is effective, and can be used even without virological or resistance monitoring. However, it is expensive, may require refrigeration in some cases, and carries a high pill burden, with significant toxicity and drug interactions. However, current non-nucleoside reverse transcriptase therapy is very vulnerable to even slight drops in adherence, and South Africa alone has 200 000 patients on second line protease-inhibitor based regimens, and around 400 on third line regimens. Many countries in Africa have begun implementing third line programmes, although these are inhibited by the lack of access to viral load or genotype resistance testing. With a move to ‘’test and start’’ approaches, more healthy patients will be started on first line, and there will predictably greater numbers moving into second and third line. However, new antiretrovirals, as well as new combinations of existing drugs, offer exciting options for the ‘’public health approach’’ that has saved millions of lives. This presentation will focus on potential candidate drugs that may be more robust and cheaper, for use in first, second and subsequent regimens.

ABSTRACT K1

Modelling the Spread and Impact of Resistance under the New WHO ART Initiation GuidelinesA Phillips

UCL, London on behalf of the Working Group on

Modelling Transmitted HIV Drug Resistance

As more countries in sub-Saharan Africa begin to adopt a policy of offering treatment for all with HIV diagnosed and ART coverage levels become over 80% in some countries it becomes of increasing importance to monitor levels of transmitted drug resistance. The studies and surveys that have been performed in the region to date suggest that prevalence of transmitted drug resistance has been almost universally below 15% and generally closer to 5%. This has been somewhat reassuring, and contributes to accumulating knowledge on the critical parameters of transmissibility and persistence of drug resistant virus. However, most data on prevalence of transmitted drug resistance still come from periods in which only the minority of people living with HIV were on ART, and the recent trajectory of ART scale-up is such that there could plausibly be recent or imminent substantial increases in prevalence of transmitted drug resistance. It is important to consider how we should recommend that countries respond if and when prevalence of transmitted drug resistance reaches certain elevated levels. Changes in standard drug regimen in ART naïve initiators, with dolutegravir a possible alternative first line drug to efavirenz, and individual level resistance testing, are among the possibilities considered. Modelling provides a basis for understanding and projecting the emergence, spread and effects of transmitted drug resistance. It also enables cost-effectiveness analysis, which provides an approach to evaluating potential new interventions in response to a high prevalence of transmitted drug resistance in the light of the opportunity costs of adopting them. In this talk we will review findings from relevant modelling studies and present results from an updated evaluation from the HIV Synthesis model on projecting future transmitted drug resistance levels and on informing



Preclinical Resistance



ABSTRACT 1

Second Generation HIV-1 Maturation Inhibitor BMS-955176: Antiviral Optimization and Cross-Resistance ProfilingB Nowicka-Sans1, T Protack1, Z Lin1, N Ray2, T Li1, PM van Ham3, C Hwang2, M Nijhuis3, A Regueiro-Ren1, M Cockett1, M Krystal1, M Lataillade1 and I Dicker1

1BMS Research & Development; Wallingford CT, USA; 2Bristol-Myers Squibb (BMS) Research & Development, Princeton NJ, USA; 3UMC Utrecht, Utrecht, Netherlands

BACKGROUND: Assays designed to optimize target specificity, serum binding and virologic potency against viruses with Gag polymorphisms were used to identify an improved clinical candidate. BMS-955176 is a second-generation MI with low human serum binding that has improved coverage of Gag polymorphisms and a wider spectrum compared with earlier MIs. As HIV-1 isolates resistant to protease inhibitors reportedly have a higher frequency of Gag substitutions, susceptibility of clinical PI-resistant (PIR) viruses to BMS-955176 was also extensively assessed.

METHODS: BMS-955176 was optimized against recombinant viruses with defined Gag polymorphs and libraries of viruses with gag/Pr genes from subtypes B, C, and CRF01_AE using multiple (MC) and single cycle (SC) assays. Inhibition of Gag cleavage and specific binding to Gag virus-like particles was used to assess specific MI targeting. Cross-resistance was evaluated with a panel of antiretroviral-resistant viruses. PI cross-resistance was further assessed with a panel of longitudinal clinical isolates (n=21) from 15 PI-treatment failure subjects with major Pr resistance-associated mutations, with 17/21 containing secondary changes in Gag associated with PIR. This was evaluated using the PhenoSense Gag/Pr (PS) assay (Monogram Biosciences) or Bristol-Myers Squibb (BMS) gag/Pr SC or MC assays. In addition, seven non-longitudinal highly PIR clinical viruses were evaluated for BMS-955176 susceptibility in the MC assay.

RESULTS: BMS-955176 exhibits potent activity (EC50 3.9±3.4) toward a library (N=87) of gag/pr recombinant viruses representing 96.5 % of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site (FC-EC50s=1-6.8, BMS MC assay). In the BMS SC assay, the mean ± SD FC-EC50s of subtype B (N=21), C (N=32), and CRF01_AE (N=16) viruses were 1.4±1.4, 4.2±2.3, and 3.5±4.1, respectively. BMS-955176 inhibits HIV-1 protease cleavage at Gag CA/SP1 and binds tightly and reversibly to HIV-1 Gag VLPs. Activity was maintained against reverse transcriptase, protease and integrase inhibitor-resistant viruses. In the PS assay, 19/21 samples had fold changes from baseline (FCFB) within the no-effect level (< 3), while the two outliers had FCFBs in the BMS SC assay of 2.2 and 4.2, respectively. Both outlier samples also remained susceptible in the BMS MC assay (FCFB=2.1 and 1.5, respectively). Further, secondary Gag changes were not associated with a greater median FCFB to BMS-955176. Finally, the panel of 7 highly PIR non-longitudinal clinical viruses remained susceptible (FC=0.16-0.68) to BMS-955176.

CONCLUSIONS: BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity towards a range of Gag polymorphisms associated with reduced susceptibility to a first-generation MI and possesses broad coverage of HIV-1 subtypes B, C and CRF01_AE. BMS-955176 exhibits no cross-resistance to existing antiretrovirals and maintains activity towards treatment failure PIR isolates with emergent Pr and Gag mutations. These findings support the continued development of BMS-955176 in treatment-experienced subjects.



ABSTRACT 2

Resistance Pathways for Potent and Broadly Active HIV-1 Maturation InhibitorsE Urano1, SD Ablan1, J Kaplan1, N Kuruppu1, DE Martin2, TJ Nitz2, CT Wild2, and EO Freed1

1HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD; 2DFH Pharma, Inc., Gaithersburg, MD

BACKGROUND: A betulinic acid-based compound, bevirimat (BVM), the first-in-class HIV-1 maturation inhibitor, acts by blocking a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown to be safe and effective in reducing viral loads in HIV-1-infected patients. However, single-amino-acid polymorphisms in the SP1 region of Gag reduced HIV-1 susceptibility to BVM in patients, leading to the discontinuation of BVM’s clinical development.

METHODS: We carried out an extensive medicinal chemistry campaign to identify BVM derivatives that demonstrate increased potency against consensus clade B strains of HIV-1 and are active against primary isolates with polymorphisms in SP1. Compound activity was tested in assays that measure CA-SP1 processing and virus replication kinetics. Selection experiments were performed to identify mutations that confer resistance to these novel compounds and a variety of virological, structural, and molecular approaches were applied to elucidate the mechanism of resistance for each mutant. To evaluate the effect of Gag polymorphisms and resistance mutations on the kinetics of Gag processing, pulse-chase radiolabeling assays were performed.

RESULTS: We identified a set of BVM derivatives that are more potent than BVM against WT HIV-1 and show robust antiviral activity against SP1 polymorphic strains and clinical isolates. The best of these analogs retain significant activity against BVM-resistant mutants. Selection experiments identified an Ala-to-Val mutation at SP1 residue 1 (SP1-A1V). The SP1-A1V

mutation was previously identified in our selections for BVM resistance. In addition, we also selected for the mutation CA-P157A, located in the major homology region (MHR) of CA. Remarkably, the P157A mutant was resistant to not only BVM and the second-generation BVM analogs but also to the structurally distinct maturation inhibitor PF-46396. Pulse-chase data demonstrate that CA-SP1 processing kinetics for P157A are similar to those of the WT. Analysis of the HIV-1 database reveals that Ala1 of SP1 and Pro157 of CA are conserved in ~99.95% of available sequences.

CONCLUSIONS: This study identifies a panel of BVM derivatives that display marked improvements relative to BVM in antiviral potency and breadth of activity. The characterization of resistant mutants provides novel insights into the structure of the maturation inhibitor-binding site and the role of SP1 and the CA MHR in virus assembly and maturation. In addition, mutations that confer resistance to these novel compounds arose at highly conserved residues, suggesting a high genetic barrier to resistance. The identification of BVM analogs that are highly potent against a wide range of HIV-1 isolates containing polymorphic SP1 sequences supports ongoing clinical development of this class of inhibitors.

ABSTRACT 3

Minimal Phenotypic Drug Susceptibility Effect of E529D RNase H Mutation of HIV-1 Subtypes on the Reverse Transcriptase InhibitorsN Mkhwanazi and M Gordon

College of Health Science School of Laboratory Medicine and Medical Science HIV Pathogeneses Programme, Doris Duke Medical Research Institute, Nelson R Mandela School of Medicine, University of KwaZulu-Natal

BACKGROUND: HIV-1 antiretroviral drugs prevent deaths that are associated with HIV-1 and AIDS, however the prolonged used of these drugs is limited by the development of HIV-1 drug resistance mutations. Reverse transcriptase inhibitors are a key component



ABSTRACT 4

In Vitro Sensitivity of HIV-2 Isolates and Integrase Mutants to CabotegravirRA Smith1, DN Raugi1, VH Wu1, S Masoum1, SS Leong1, F Sall2, P Salif Sow2, S Ba2, M Seydi2, and GS Gottlieb1,3

for the University of Washington-Dakar HIV-2 Study Group

1Department of Medicine, Division of Allergy and Infectious Diseases and 3Department of Global Health, University of Washington, Seattle, Washington, USA; 2Clinique des Maladies Infectieuses Ibrahima DIOP Mar, Centre Hospitalier Universitaire de Fann, Universite Cheikh Anta Diop de Dakar, Dakar, Senegal

BACKGROUND: A growing body of evidence suggests that integrase strand transfer inhibitors (INSTI) might help fill the need for safer, more effective antiretrovirals (ARV) for HIV-2 infection. Cabotegravir (GSK1265744) is a second-generation INSTI that has advanced to Phase II clinical trials in HIV-1–infected subjects. To assess the potential utility of cabotegravir for HIV-2 treatment, we examined its in vitro activity against HIV-2 isolates from ARV-naïve individuals, and evaluated the resistance profile of the drug using INSTI-resistant HIV-2 variants that harbor clinically-relevant changes in the integrase enzyme.

METHODS: HIV-1 and HIV-2 isolates from ARV-naïve individuals were obtained from the NIH AIDS Reagent Program. Site-directed mutants of HIV-1 and HIV-2 integrase were constructed using full-length pNL4-3 and pROD9 molecular clones, respectively. All strains were evaluated for cabotegravir susceptibility using a HeLa-CD4 indicator cell-based assay that quantifies drug activity in a single cycle of infection.

RESULTS: Cabotegravir inhibited HIV-2 isolates from groups A and B with 50% effective concentrations (EC50) of 1.8 ± 0.7 and 2.9 ± 1.1 nM (mean ± SD for n=7 and n=5 isolates, respectively). Similar EC50 values were observed for HIV-1, including group M subtype A, B and D viruses and a group O strain (mean EC50 = 1.7 ± 0.3 nM; n=5). Relative to the parental wild-type clone, HIV-2ROD9 integrase variants E92Q+N155H,

of antiretroviral treatment. Previous data indicated that isolates that did not develop any classical NRTI mutations in the RT domain harbored the E529D RNase H mutation. We therefore aimed to study the phenotypic effect of the E529D RNase H mutation on replication capacity assay and drug susceptibility.

METHODS: The RT region was amplified from the patient isolate and cloned into a TOPO vector using the TOPO TA cloning kit (Invitrogen). The E529D mutation was introduced into the TOPO+RT recombinant by Quick Change II XL site directed mutagenesis (Stratagene), and sequenced using BigDye Terminator v2.1 in order to confirm the presence of the mutant RNase H insert. Plasmid pNL4.3 ΔRT was co-transfected with the TOPO+RT plasmid using Fugene 6. Replication capacity and phenotypic assays were performed using the reporter TZM-bl cell line. The phenotypic drug susceptibilities to 2’, 3’-Dideoxyinosine, stavudine, zidovudine and Nevirapine were measured by Bright-Glo luciferase assay.

RESULTS AND CONCLUSIONS: The viral replication capacity of the mutant virus was measured by TZM-bl assay comparing to the wild type. These results suggest that E529D RNase H mutation has a lower replicative capacity as compare with the pNL4.3 wild type. The E529D RNase H mutation did alter susceptibility to 2’,3’-Dideoxyinosine as compared to the wild type however it alters the susceptibility to stavudine (d4T), zidovudine (AZT) and Nevirapine (NVP).



METHODS: Two HIV-1 subtype C primary virus isolates (FV3 and FV6) from antiretroviral-naïve patients were propagated in peripheral blood mononuclear cells in the presence of increasing concentrations of RAL (0.26-266.24nM), EVG (0.26-66.56nM) and DTG (0.26-66.56nM). Viral RNA was extracted after each passage and the entire pol coding region (PR-RT-IN) was amplified and sequenced by Sanger and Illumina MiSeq. Full-length IN sequences from the WT and resistant viruses were cloned into an HIV-1LAI backbone and phenotyped for RAL, EVG, DTG, cabotegravir, MK-2048 and BMS-707035 susceptibility.

RESULTS: Sequence analysis revealed that in the FV6 isolate RAL selected for Q148R and EVG selected for T66I and R263K. Q148R and T66I are primary mutations associated with clinical resistance to RAL and EVG, respectively. No resistance to DTG emerged in the FV6 isolate, whereas the E92Q mutation was selected in the FV3 isolate at passage 44. Phenotypic analyses revealed that the E92Q conferred ~ 4-fold resistance to DTG and significant cross resistance to RAL and EVG. However, cabotegravir and MK-2048 retained activity against the E92Q mutation.

CONCLUSIONS: This is the first report of in vitro selection of DTG resistance (E92Q) in HIV-1 subtype C primary virus isolates. DTG will be evaluated as a first line agent in South Africa, whereas RAL is currently used in salvage therapy. This raises the concern that individuals failing a first line DTG-containing regimen may not respond to RAL based salvage therapy.

I84V+E92Q+A153G+N155H and G140S+Q148R were 15-, 71-, and 255-fold resistant to cabotegravir, and drug concentrations as high as 10 µM failed to reduce infection with T97A+Y143C HIV-2ROD9 to 50% of untreated controls. In contrast, HIV-1NL4-3 integrase mutants T97A+Y143C, E92Q+N155H, and G140S+Q148R showed no change, 4-fold, and 21-fold resistance to cabotegravir, respectively.

CONCLUSIONS: Cabotegravir is an effective inhibitor of HIV-2 isolates from ARV-naïve individuals, with EC50 values comparable to those seen for HIV-1. As previously described for dolutegravir, mutations in all three of the canonical INSTI resistance pathways can confer cross-resistance to cabotegravir in HIV-2ROD9. Although these findings support future clinical studies of cabotegravir for initial treatment of HIV-2 infection, additional studies are needed to assess the potential usefulness of the drug in HIV-2 patients failing INSTI-based regimens.

ABSTRACT 5

In Vitro Selection of HIV-1 Subtype C Resistant to Integrase Strand Transfer Inhibitors M Mphahlele1,2, N Giacobbi3, R Hewer2, SAA Travers4, S Mosebi2, K Steegen1, F Venter5, S Carmona6, WS Stevens1,6, N Sluis-Cremer3, and MA Papathanasopoulos1

1University of the Witwatersrand Medical School, Johannesburg, South Africa; 2Mintek, Johannesburg, South Africa; 3University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; 4University of the Western Cape, Western Cape, South Africa; 5 Wits Reproductive Health and HIV Institute, Johannesburg, South Africa; 6National Health Laboratory Services, Johannesburg, South Africa

OBJECTIVES/AIM: Resistance to integrase strand transfer inhibitors (INSTIs) has not been defined in non-subtype B HIV-1. In this study we selected in vitro resistance to raltegravir, (RAL), elvitegravir (EVG) and dolutegravir (DTG) in HIV-1 subtype C primary virus isolates.



New Resistance Technologies and Interpretations



in gut vDNA compared to vDNA other compartments. We find that RT-SHIV does not acquire detectable drug resistance immediately after treatment initiation, but takes more than one week. Upon treatment removal, plasma vRNA does not change significantly over the following 6 weeks, suggesting that drug resistance has limited fitness cost.

CONCLUSIONS: Drug resistance evolution depends on the interactions between partially independent compartments, some of which have SHIV populations that show more persistence than others or respond more quickly to pressures imposed by treatment. Characterizing the differences and relationships between the compartments could be important in understanding how multidrug resistance emerges, particularly outside of the blood, and could inform the administration of treatment. That many of these findings are reliant on the sampling scheme of both vDNA and vRNA, across compartments and time demonstrates the importance of varied sampling to obtain a more complete picture of how intrahost evolution proceeds.

ABSTRACT 7

Genome-Wide Association Study of HIV Whole Genome Sequences Provides Insights into Drug ResistanceRA Power1, S Davaniah1, A Derache1,2, E Wilkinson1, F Tanser1, RK Gupta3, D Pillay1,3, T Oliveira1

1Africa Centre for Health and Population Studies, University of KwaZulu-Natal, South Africa; 2Sorbonne Universités, UPMC Univ Paris 06, Inserm, Institut Pierre Louis d’Epidémiologie et de Santé Publique (IPLESP UMRS 1136), Paris, France; 3Division of Infection and Immunity, University College London, London, UK

BACKGROUND: Genome-wide association studies (GWAS) have considerably advanced our understanding of human traits and diseases. With the increasing availability of whole genome sequences (WGS) for pathogens, it is important to establish whether GWAS

ABSTRACT 6

Spatio-Temporal Dynamics of Drug Resistance Evolution and Persistence of RT-SHIV A Feder1, C Kline2, B Keele3, S-L Hu4, D Petrov1, P Pennings5, and Z Ambrose2

1Stanford University, Stanford, CA; 2University of Pittsburgh, Pittsburgh, PA; 3Leidos Biomedical Research, Inc., Frederick, MD; 4University of Washington, Seattle, WA; 5San Francisco State University; San Francisco, CA

BACKGROUND: To better design therapeutic approaches that prevent the evolution of drug resistance in HIV-1, it is important to understand how drug resistance spreads and establishes within a patient. There is mounting evidence that intrahost viral evolution is a non-homogenous process within the body and therefore must be understood spatially and temporally.

METHODS: We examined >3300 single-genome sequences from four macaques infected with RT-SHIV, a SIV with an HIV reverse transcriptase (RT). Macaques were given monotherapies to induce the emergence of drug resistance within RT between weeks 12-20 post-infection. Both viral RNA and DNA (vRNA and vDNA) were sampled from four different compartments (lymph node, vagina, gut and PBMC) in addition to plasma vRNA between 13 and 30 weeks post-infection. Compartmentalization was assessed using the variance partitioning statistic ΦST from population genetics.

RESULTS: Acquisition of drug resistance is a highly dynamic process with periods of stasis. Although compartmentalization is observed, both natural selection within and migration between compartments are important for establishing drug resistant lineages. We observe differential dynamics between compartments, with the gut being particularly notable. In all four macaques, gut vRNA has the greatest rate of turnover, both compared to gut vDNA and to vRNA in other tissues. In addition, drug resistance increases in frequency more quickly



ABSTRACT 8

Dual Infection by HIV-1 Among Newly Diagnosed Patients from the Spanish Cohort of Antiretroviral Naïve Adults (CoRIS)M Pernas1*, JÁ Fernandez-Caballero2*, C Casado1, I Olivares1, S Martínez-Arbas1, C Vidal3, I Viciana4, M Alvarez2, I Arkaitz5, J Portilla6, AB Lozano7, Ce López-Galíndez1*, F Garcia2* on behalf of CoRIS

* contributed equally to this paper

1 CNM. ISCIII. Majadahonda. Madrid, Spain; 2 Servicio de Microbiología. Complejo Hospitalario Granada. Hospital Universitario San Cecilio. Instituto de Investigación Ibs.Granada. Spain; 3 H. Son Espases, Mallorca, Spain; 4H. Virgen de la Victoria, Malaga, Spain; 5H. Bellvitge, Barcelona, Spain; 6 H General de Alicante, Alicante, Spain; 7 H. Poniente, El Ejido, Almaería, Spain

BACKGROUND AND AIM: Dual infection (DI) by HIV-1 has been related to a higher pathogenicity and progression of infection. Cornelissen et al., (2007) described how a high number of ambiguous positions in RT and Pro sequences can be a marker of DI. In our study, using this method, we investigated the prevalence of DI in a cohort highly representative of IV-1 epidemic in Spain (CORIS) and, in addition we used massive parallel sequencing (UDPS) to confirm cases presumptively identified with Sanger sequencing.

PATIENTS & METHODS: We tested 2490 Sanger RT & Pro sequences form patients screened for baseline resistance in CoRIS through the period 2004-2012. We selected the samples with a high number of ambiguities, excluding those with ambiguities in resistance associated codons. We also amplified RT & Pro by means of an in house modification of the commercial resistance UDPS assay for GS-Junior (Roche Diagnostics). To confirm DI, phylogenetic trees were built by the “Neighbour Joining” method, using UDPS Pro & RT sequences from each patient, along with 1778 sequences (88% subtype B) from CoRIS database and subtype B HXB2 reference.

of viral genomes could reveal important biological insights. Here we perform the first proof of concept analysis examining the selection of antiretroviral therapy (ART) associated variants.

METHODS: We performed a GWAS of drug resistance (DR) in a sample of 343 HIV subtype C patients failing 1st line treatment in rural KwaZulu-Natal, South Africa. The majority and minority variants within each sequence were called using GATK and PILON, and GWAS was performed within PLINK. HIV WGS from patients exposed to different antiretroviral drugs (zidovudine, stavudine, tenofovir, efavirenz, nevirapine and lopinavir) were compared to sequences derived from individuals naïve to the respective treatment.

RESULTS: GWAS methodology was validated by identifying five associations on a genetic level that led to amino acid changes known to cause DR. Further, we identified two variants within amino acid 68 of the reverse transcriptase protein associated with tenofovir exposure (p-value=5.38E-06 & 1.45E-05; Odds Ratio=11.9 & 2.89) previously described as potential fitness compensatory mutations. We replicated these associations in the Stanford University HIV Drug Resistance Database (488 exposed vs. 9,357 unexposed, p<0.001). We also identified a possible additional DR variant for tenofovir within amino acid 91 of the matrix region of the Gag protein. Replication in publicly available datasets was not possible here due to the lack of Gag sequences.

CONCLUSION: These results validate the applicability of GWAS to HIV WGS data with respect to phenotypes with large genetic effects such as DR. The sample size required was also relatively small. The data also highlight how GWAS can provide novel and possibly clinically relevant insights into pathogen genomes in an era of high throughput sequencing.



ABSTRACT 9

Low Frequency of Resistance Associated Mutations by Ultra-Deep Sequencing in HIV-1 Primary Infected PatientsC Rodriguez1, C Goujard2, M Mercier-Darty1, ML Nere3, V Demontant1, M Splittgerber4, C Delaugerre3, J Ghosn5, P De Truchis6, M Obadia7, C Rouzioux8, L Meyer2, ML Chaix3,4 on behalf the French PRIMO Study Group

1Virology Dpt CHU Henri Mondor, INSERM U955 Team 18, NGS Platform IMRB Paris-Est University, Créteil; 2University Paris Sud, INSERM CESP U1018, APHP Bicêtre Hospital, Paris; 3INSERM U941, Université Paris Diderot, AP-HP, Laboratoire de Virologie, Hôpital Saint-Louis, Paris ; 4CNR VIH associé Primo infection, Paris ; 5

UF de Thérapeutique en Immuno-Infectiologie, Hôpital Hôtel Dieu, APHP, Paris, and Université Paris Descartes, EA 7327, Faculté de Médecine site Necker, Paris ; 6Service de Maladies infectieuses et tropicales, CHU Raymond Poincare, Garches ; 7Service de Maladies infectieuses et tropicales, CHU Purpan, Toulouse ; 8EA 7327, Université Paris Descartes, AP-HP, Laboratoire de Virologie, Hôpital Necker – Enfants malades, Paris, France

BACKGROUND: Minor drug resistant variants may increase the risk of treatment failure. The aim of our study was to evaluate the frequency of resistant associated mutations (RAMs) using Ultra-Deep Sequencing (UDS) in patients with HIV-1 primary infection.

METHODS: Protease (PR), reverse transcriptase (RT), integrase (IT) and V3 loop of envelope genes were Sanger sequenced using ANRS protocol. Same amplicons were used for UDS by Roche/454 GS FLX+. Sequences were analyzed using: i) AVA software (Roche) and in house software PyroPack® for RT, PR and IT, ii) geno2pheno[454] (Max Plant Institute) and in house software PyroTrop® for tropism. Variability of mutations were analyzed using two cut off of presence of mutation (>1% and >20%): i) RAMs were characterized using both the 2009 WHO list of mutations and the 2014 French ANRS algorithm (including rilpivirine-, etravirine- and integrase inhibitors (INI) related RAMs reported in the 2014

RESULTS: Thirty-seven out of 2490 patients (2%) had more than 19 ambiguous positions in RT and Pro sequence. Protease has been UDPs amplified in all patients, whilst RT has been amplified in 7. Phylogeny of UDPS sequences in Pro detected DI in 10 patients (27%); all of them were confirmed in RT region. Median number of ambiguities in this confirmed DI cases was 50±12. Of note, all the patients with confirmed DI had more tan 30 ambiguous positions in RT and Pro. Viruses from different HIV subtypes infected two of the patients with DI.

CONCLUSIONS: The method based on the number of ambiguous positions to detect HIV-1 DI lacks specificity but could be valid above 30 ambiguities. This method can be used on large-scale studies as a screening method to enrich in potential DI cases, prior to using more specific tools, as massive parallel sequencing. HIV-1 Dual Infection seems to have a low prevalence in Spanish HIV patients. This work alerts diagnostic laboratories doing baseline HIV resistance about the need to rule out Dual Infection in cases with a high number of ambiguous positions.

-Cornelissen, M., Jurriaans, S., Kozaczynska, K., Prins, J. M., Hamidjaja, R. A., Zorgdrager, F., Bakker, M., Back, N. & van der Kuyl, A. C. (2007). Routine HIV-1 genotyping as a tool to identify dual infections. AIDS (London, England) 21, 807-811.



ABSTRACT 10

Performance Evaluation System for Next Generation Sequencing-Based HIV Drug Resistance Genotyping AssayD Liang1, BL Cooper1, L Yan1, T Taylor2, M Nykoluk2, P Sandstrom2, H Ji2

1MOgeneDx, LLC, St. Louis, MO, USA; 2National Microbiology Laboratory at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, Canada

BACKGROUND: Next Generation Sequencing (NGS)-based HIV DR assays outperform conventional Sanger sequencing approach in scalability, cost-effectiveness and especially the quantitative detection for low abundance drug resistance mutations (DRMs). Thus far, it has been applied primarily in research but rarely in clinical settings. One main obstacle is the lack of a standardized evaluation system that allows regulatory agencies to benchmark and accredit such new assays for clinical use.

METHODS: By revisiting the existing principles for molecular assay validation, we have defined a new performance assessment system that helps to qualitatively and quantitatively evaluate the performance of NGS-based HIV DR assays. Illumina MiSeq platform was taken as exemplar NGS platform in this study. A comprehensive proficiency test panel including pedigreed plasmids, plasmid mixtures at known ratios, well-characterized virus isolates, and clinical specimens were constructed. An in-house, MiSeq-based HIV DR assay coupled with automated data analysis pipeline was applied for testing the panel in four separate runs. The exact panel was re-tested in a second lab independently using different protocol. The results from all runs were then compared and assessed against the newly established assay validation criteria.

RESULTS: Known HIV DRMs present in all the examined samples were detected at expected frequencies. A limit of detection at 1% on DRM can be achieved reliably and accurately. The performance

IAS-USA resistance mutations list), ii) all mutations reflecting the diversity.

RESULTS: Samples from the 42 patients consecutively enrolled in the French ANRS PRIMO Cohort between 07/2014 and 10/2014 (95% men, 71% homo/bisexual) were studied. At inclusion, median viral load and median CD4 cell count were 5.5 log10 copies/ml [range: 3.2-7.0] and 543 cells/ml [179-1074] respectively. More than 7000 sequences/target/samples (mean length 488bp for RT, PR, IT and 380bp for V3) were obtained for 40 samples and analyzed with cut off validated at 1% with controls. RAMs were identical between Sanger sequencing and UDS with the cut off at 20% (NRTI RAMS: M41L (n=1, 93%), NNRTI RAMs: K103N (n=2, >98%), E138A (n=3, >99%)). When using a cut-off at 1%, we found 1 patient who harbored a resistant virus with a mutation M46I (1.06%) in the PR gene. Tropism was X4/DM in 7.7% of patients including 2 patients with X4/DM population below 20% (1 of them was X4/DM in Sanger). No virus was resistant to INI. Independently of resistant mutations, the UDS revealed a diversity higher for the RT and IT genes (9.0% and 10.6% respectively) compared to the PR gene (6.6%). This could suggest a greater adaptability and ability of the RT and IT genes to allow for the emergence of resistant variants under antiretroviral pressure.

CONCLUSION: In this study, few differences were evidenced in the rate of transmitted RAMs using UDS compared to classic Sanger sequencing. These results strongly support the establishment of a clonal viral population at the time of primary HIV-1 infection.



ABSTRACT 11

Comparison of HIV DNA Genotyping Par UDPS to Cumulative HIV RNA Genotypes in Pretreated Patients with Previous ARV Virological FailuresA Si-Mohammed1, C Truntzer2, M Blot3, C Briandet4, H Giraudon1, A Fillion3, P Ducouroy2, M Duong3, A Waldner3, S Mahy3, P Chavanet3, P Pothier1, L Piroth3

1Laboratoire de Virologie; Centre Hospitalier Universitaire François Mitterrand, Dijon, France; 2Plateforme Protéomique – CLIPP; Université de Bourgogne, Dijon, France; 3Service des Maladies Infectieuses; Hôpital du Bocage, Centre Hospitalier Universitaire François Mitterand, Dijon, France; 4Service de Pédiatrie; Hôpital du Bocage, Centre Hospitalier Universitaire François Mitterrand, Dijon, France

BACKGROUND: Cumulative HIV-RNA resistance test results are used to identify potentially active drugs in heavily pretreated patients. However, HIV-RNA resistance tests may not be informative when was not performed at the time of virological failure and under selection pressure of drugs. The potential interest of UDPS technology drug resistance genotyping of PR-RT regions of viruses derived from PBMC in pretreated patients was thus assessed in the present pilot study.

METHODS: Samples used from 12 pretreated patients were derived from routine genotyping resistance testing. Since drug-resistant HIV-1 quasi-species in peripheral blood mononuclear cells are highly diversified, PCR were performed in quadruplets for each DNA sample. Amplicons Library was obtained by purifying, quantifying and pooling PCR products. For UDPS, emulsion PCR was performed according to manufacturer instructions. Roche AVA software was used for decoding the MIDs and generating sequence contigs for individual patients using HXB-2 as the reference. The DNA archived resistance mutations were then compared to cumulative genotype of all available plasma RNA previously obtained by Sanger technology.

RESULTS: Overall, the number of drug resistance mutations in HIV-1 proviral DNA by UDPS exceeds

characteristics, definitions and validation output are summarized as below:

Performance Characteristics

Definitions specific to NGS HIV DR assay

Validation output

Limit ofDetection

The lowest actual percentage of a DRM that can be consistently detected with acceptable precision, sensitivity and specificity.

≥1%

LinearRange

The percentile range of actual DRM frequencies within which linear correlation is achievable accurately between the expected and observed values.

1%~100%

Precision

The extent to which repeated testing on identical samples renders comparable results with acceptable intra-run repeatability and inter-run reproducibility.

Combined%CV≤25%

AccuracyThe extent to which the detected DRM frequency is in agreement with reference materials.

%CV≤20%

SystemError

The compounding error from all experimental procedures and data processing.

≤0.4%

AnalyticalSensitivity

The probability that the assay detects known DRM (measured as 1- False Negative Rate).

≥99%

AnalyticalSpecificity

The probability that the assay does NOT detect a DRM when it is absent (measured as 1- False Positive Rate).

≥95%

Limit ofViral Load

The lowest viral load level at which the test can positively identify all known DRMs from a sample at a defined input volume.

≥1000cp/mL

Robustness

The capability of the assay to reliably genotype clinical samples comprised of any major HI V subtypes.

A l l m a j o r subtypes

CONCLUSIONS: A new validation system for NGS-based HIV DR assay has been established, which will help to routinize such tests for close HIV DR monitoring in clinical settings. It is applicable for MiSeq or any other NGS technologies to be applied in HIV DR testing.



deep sequencing (UDS) is a powerful tool able to detect minority resistant variants (MRV) with a threshold of 1% and could be useful to identify variants harboring single or multiple drug-resistance mutations (DRMs). Our aim was to analyze longitudinally the integrase gene region using UDS in a two-year-old boy failing rapidly a raltegravir based-regimen.

METHODS: A treatment containing abacavir, lamivudine and raltegravir was initiated in a HIV-1 subtype C infected-child. Plasma viral load decreased slowly from 6.22 log to 5.69 log at week 17. Longitudinal plasma samples at baseline, week (W) 4, W8, W13 and W17 were obtained, as well as a mother’s baseline plasma sample. Integrase gene was Sanger sequenced using the Viroseq assay. Specific primers covering the subtype C-integrase gene region including the DRMs were designed, and sequencing was performed on amplicons using Roche/454 GS-Junior. An in-house workflow was developed to identify major DRMs (Y143X, N155H, Q148H), and accessory mutations at the threshold of 1% and to identify the linkage between mutations.

RESULTS: In Sanger and UDS, no MRV was detected in mother and child at baseline. Sanger sequencing identified the selection of mutations E92Q and N155H at W8 and an additional mutation Y143R at W17. Using UDS, mutation N155H was detected at W4 alone in 4% of the sequences. At W8, 90% of the viral population harbored N155H, including 16% with E92Q accessory mutation. The double mutant E92Q+N155H became the major variant at W13 (57%). On the last time point W17, Y143R emerged leading to different resistant mutations patterns: single mutants N155H (47%) and Y143R (24%) and double mutants E92Q+N155H (13%), Y143R+N155H (2%) and E92Q+Y143R (2%).

CONCLUSION: The major DRM N155H conferring resistance to raltegravir and elvitegravir was detected rapidly by W4 using UDS whereas it was missed by conventional genotyping. The double mutant E92Q+N155H conferring resistance to the whole integrase inhibitors class, including dolutegravir, emerged at W8 and became rapidly dominant. The selective pressure exerted by raltegravir also led to the emergence of Y143 pathways including different

what observed by cumulative HIV-RNA resistance test. The median number of resistance mutations in HIV DNA (UDPS) and RNA (cumulative test) were 3 and 2 for NRTIs (p>0.05), 1 and 0 for NNRTIs (p<0.05),, and 3 and 1 for PIs (p<0.05), respectively. Major drug resistance mutations were detected only in DNA in 4 patients out of 12 (33%) for NRTIs, in 5 patients (42%) for NNRTIs, and in 6 patients (50%) for PIs. Although statistically not significant, when compared to cumulative HIV RNA genotypes, HIV DNA genotyping by UDPS shows a high level of resistance in accordance with previous ARV drugs failure.

CONCLUSION: In this pilot study in patients with past ARV failures, UDPS of HIV archived DNA was more sensitive than classical cumulative RNA resistance genotyping to detect drug resistance mutations. Our strategy, based on high amount of DNA included in PCR assay (each DNA extract tested in quadruplets), may allow us to encircle the fluctuations of archived drug resistance mutations in DNA compartment. Large study is needed to confirm the interest of such approach in clinical practice.

ABSTRACT 12

Dynamics of Integrase Inhibitors Multiresistant Variants Using Ultra Deep Sequencing in HIV-1 Infected ChildrenK Stefic1,2, M Capitao3,4, M Splittgerber3,4, Z Maakaroun5, M-L Néré3,4, M Salmona3,4, L Bernard6, M-L Chaix3,4, F Barin1,2, and C Delaugerre3,4

1Université François Rabelais, Inserm U966, Tours France; 2Laboratoire de Bactériologie-Virologie & Centre National de Référence du VIH, CHU Bretonneau, Tours, France; 3Université Paris Diderot, Inserm U941, Paris, France; 4Laboratoire de Virologie, Hôpital Saint-Louis, APHP, Paris, France; 5Pédiatrie, CHU Bretonneau, Tours, France; 6Médecine Interne et Maladies Infectieuses, CHU Bretonneau, Tours, France

BACKGROUND: Population based sequencing (as Sanger) has low sensitivity to detect drug resistance mutations present below 20% of the viral population and cannot reveal the link between mutations. Ultra



of nevirapine-resistant/nevaripine-susceptible HIV plasmid DNA, followed by 5-cycles of ligation. The ligation efficiency of the preserved, dried mixture was compared to fresh (non-freeze-dried) ligation mixtures in the presence and absence of the preservatives. The ligation efficiency was quantified based on the optical density generated using plate-based OLA detection.

(2) Characterization of engineered DNA tag sequences for multiplexed paper-based detection. To enable the detection of multiple mutations on a single device, six different engineered DNA tag sequences were designed and tested for their pairwise specificity, using synthetic oligonucleotide targets containing the sequence of a mutant-specific ligation probe conjugated to each of the DNA tags. Each target was tested for correct binding to its engineered DNA tag, as well as for non-specific binding to the other five engineered tags. In each case, the amount of synthetic probe captured was measured using plate-based OLA detection.

RESULTS: (1) The newly-developed preservative blend effectively stabilized the activity and specificity of the ligase enzyme after the freeze-drying process and storage at 21°C with <15% relative humidity (Fig.1A). The dried reagent platform demonstrated comparable ligation efficiency to fresh ligation solution with no preservatives (standard) and with added preservatives, respectively. Increased ligation efficiency could be due to polyethylene glycol (a known PCR enhancer) in the preservative blend.

(2) The six pairs of engineered DNA tags showed highly specific pairwise binding (Fig.1B). These novel DNA tags can potentially be used in conjunction with the chemistry previously developed for paper-based OLA detection.

CONCLUSIONS: This lyophilized reagent platform simplifies and reduces the time for ligation set-up from typically 30 minutes to 1 minute. Also, the reduction of reagent and amplified DNA transfer between tubes compared to the standard protocol could reduce the chance for PCR-carry-over contamination. The multiplexed DNA capture system will be adapted to paper-based detection to allow

double mutants with unknown phenotypic impact. This case study illustrates the usefulness of UDS to detect early MRV and to determine HIV-1 patterns of resistance in longitudinal analysis, considering its potential benefit in clinical practice for virological failure management.

ABSTRACT 13

Simplified Platform for Detection of HIV Drug Resistance by the Oligonucleotide Ligation Assay N Panpradist1, N Higa1, A Wong-On-Wing1, I Andrews1, B Atkinson1, J Lai1, I Beck2, L Frenkel1,2, B Lutz1

1University of Washington, Washington, United States of America; 2Seattle Children’s Research Institute, Washington, United States of America

BACKGROUND: To ensure effective treatment in infected individuals, screening for drug-resistant HIV prior to initial administration of anti-retroviral treatment (ART) has been recommended. The oligonucleotide Ligation Assay (OLA) is an economical approach, compared to consensus sequencing, to sensitively identify known point mutations that are linked to 1st-line ART resistance. However, the current OLA is labor-intensive and requires trained personnel and equipment; for each mutation, 4.5 hours of ligation and detection are needed in addition to RNA extraction and reverse-transcription/amplification. To increase access to OLA-based drug-resistant HIV testing, our ultimate goal is to develop an inexpensive, point-of-care device while maintaining high sensitivity and specificity of the OLA. Progress includes novel chemistry and platform for the ligation, and multiplexed detection on a paper-based format.

METHODS: (1) Preserved, dried reagents and platform for ligation. The ligation mixture containing a thermostable ligase, mutation-specific probes, cofactors, and salts in the presence of a newly developed preservative blend (polyethylene glycol and trehalose) were loaded in the pipette tip and freeze-dried. To evaluate the ligation efficiency, the preserved, dried mixture was rehydrated in mixtures



based detection. This OLA modification eliminated the need for a plate reader. Here, we further simplify the assay by utilizing a novel isothermal ligation assay without using a thermal cycler.

METHODS: The OLA ligation mix – containing a thermostable ligase, mutation-site targeting probes, cofactors, and ligase buffer – was mixed with amplified HIV pol plasmids containing either nevirapine-resistant (mutant) or nevirapine-sensitive (wild-type) sequences. The ligation mixture was then incubated for 45 minutes at different temperatures (25°C, 37°C, 45°C, 49°C, 55°C, 65°C, and 70°C) to identify an optimal temperature for isothermal ligation. The mutant signal of the isothermal ligation products was quantified using standard plate-based enzyme-linked immunoassay detection, and compared to 1 cycle of ligation (30 seconds at 94°C and 4 minutes at 37°C).

RESULTS: Previous data on paper-based detection showed OLA’s capability to detect as low as 2% of nevirapine-resistant HIV after 1-cycle ligation. As a result, we used the signal from 1-cycle ligation as the baseline for comparison to isothermal ligation. For samples containing mutant sequences, isothermal ligation yielded ligated products at 49-65°C, with maximal ligation efficiency at 55 °C after a 45-minute incubation (Fig.1A, black bars); the mutant signal of isothermal ligation at 55°C was comparable to the signal of one-cycle ligation. We hypothesized that ligation product at 49-65°C was generated via spontaneous DNA breathing, which would allow probe invasion and subsequent ligation (Fig.1B). On the other hand, the mutant signal of isothermal ligation from the samples containing wild-type sequences was almost zero, which indicated a specific ligation. (Fig.1A, red bars) Our next step is to test the efficiency of isothermal cycling using clinical specimens.

CONCLUSIONS: OLA using isothermal ligation produced signal intensities compatible with the simplified paper-based detection step, while maintaining high specificity. Successful isothermal ligation coupled with isothermal amplification technologies such as LAMP, SDA, and RPA would

detection of six mutations associated with current World Health Organization 1st-line ART resistance testing recommendations in a single device. These developments could improve global access to OLA in labs that have the capability for PCR but do not yet have the capability for the complete OLA.

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Figure 1. (A) Comparison of the performance for detecting a HIV mutations using novel dried reagents and a dispensing platform (preloaded, dried reagents in a pipette tip) for ligation (red bar) to the standard protocol (black bar) and the fresh condition with preservatives (gray bar). (B) An example showing a high specificity of our engineered DNA sequences for multiplexed detection in OLA. The engineered tag sequence 1 only bound specifically to its own pair (complement1) and not the others.

ABSTRACT 14

Novel Isothermal Ligation Reaction for HIV Drug Resistance TestingN Panpradist1, A Wong-On-Wing1, J Lai1, I Beck2, L Frenkel1,2, B Lutz1

1University of Washington, Washington, United States of America; 2Seattle Children’s Research Institute, Washington, United States of America

BACKGROUND: Compared to consensus sequencing, the oligonucleotide ligation assay (OLA) is a more economical approach to sensitively identify a set of known point mutations that are linked to 1st-line ART resistance in HIV. However, the OLA procedure is complex and requires advanced equipment (e.g. a thermal cycler and a plate reader), limiting its use in low-resource settings. The current OLA involves four main steps: HIV RNA extraction, amplification, ligation, and detection. To increase access to OLA-based HIV drug-resistance testing, our ultimate goal is to develop an inexpensive point-of-care device while maintaining the high sensitivity and specificity of this assay. Previously, we presented a simplification of the OLA detection step by successfully converting the labor-intensive plate-based detection into a paper-



Libraries were sequenced with paired-end Illumina MiSeq technology. A modified version of the algorithm of Zhou, et. al. was used to remove sequences whose primer IDs contained PCR/sequencing errors. The uSGS pipeline further eliminated PCR recombinants and PCR/sequencing errors by applying a “>80% majority rule” to each site in alignments of reads with the same primer ID. uSGS data were analyzed for unique linkage patterns and for phylogenetics using neighbor-joining (NJ) analyses.

RESULTS: Using the uSGS assay, a median of 1227 SGS were obtained from each of 3 plasma samples from 2 ART-experienced donors. The presence of clusters of resistant variants on independent nodes of NJ trees implied that resistant variants emerged independently and diversified primarily due to stochastic changes rather than from in vivo recombination with other variants. Within clusters of variants, rare, linked resistance mutations were detected in each sample. In the 1st sample from one donor, a single variant was detected with linked mutations in RT at codons 106, 108, and 101 (0.06% frequency) on a background of 67N (98.9% aac). In a 2nd sample from the same donor obtained 2 weeks later, a different 67N mutation (aat, 1.1%) was linked to the previously detected rare mutations at codons 106 and 101. Similarly, in plasma from the second donor, rare (00.06% frequency) linked mutations were detected at codons 70, 108, and 184.

CONCLUSION: The new ultrasensitive SGS assay described here can detect rare, linked mutations at drug resistance sites and permits accurate phylogenetic analyses of HIV variants. This capability will improve the understanding of resistance evolution and could help identify individuals at risk of treatment failure because of linked resistance mutations.

eliminate the need for a thermal cycler and thus facilitate access to OLA-based HIV drug resistance testing in low-resource laboratories.

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Figure 1. (A) Performance of isothermal ligation at different temperatures compared to 1-cycle ligation. Optical density of ligated products was normalized to that of the standard 1 cycle ligation. The highest yield of ligation from the sample containing mutant sequence (black bars) was observed at 55°C after 45-minute incubation. Conversely, the mutant signal of isothermal ligation from the sample containing wild-type sequence was almost zero (red bars), indicating specific detection. (B) Schematic of hypothesized isothermal ligation event. Probe invasion and subsequent ligation happens during the spontaneous, transient DNA breathing.

ABSTRACT 15

Rare Linked Drug Resistance Mutations Detected by New Ultrasensitive SGS Assay VF Boltz1, J Hattori1, W Shao2, JM Coffin3, F Maldarelli1 and MF Kearney1

1National Cancer Institute, Frederick, Maryland, USA; 2Leidos Biomedical Research, Frederick, Maryland, USA; 3Tufts University, Boston, Massachusetts, USA

INTRODUCTION: Targeted next generation sequencing (NGS) is a powerful tool for detecting low frequency, HIV-1 drug-resistant mutations but frequent in vitro recombination prevents accurate detection of linked mutations and assessment of phylogenetic relationships. We developed a new NGS-based ultrasensitive single-genome sequencing (uSGS) assay that eliminates in vitro recombinants and reduces PCR errors to investigate linkage of resistance mutations and the phylogenetics of variants in plasma samples.

METHODS: NGS uSGS libraries were generated by tagging cDNA molecules from plasma RNA with primer IDs, optimizing PCR, and generating overhangs on amplicons for efficient ligation of Illumina adaptors.



Sequences were subtyped by their genetic distances from ~150 reference sequences from Los Alamos National Laboratory, representing pure subtypes and circulating recombinants.

Sensitivity analysis was performed considering only naïve, only experienced, only subtype B, and only subtype C. The distribution of sequences was: naïve 45%, experienced 52%, unclassifiable 3%; subtype B 53%, C 34%, other 13%.

RESULTS: Table 1 presents the percentage of all sequences that showed high-level resistance (≥60 points) for each version/drug combination.

For many PIs and NRTIs there were clear differences in results by version, mostly with recent versions calling more resistance. Some differences were large enough that if they had been actual increases in resistance they could have had policy implications. The most striking case was TDF, where all versions through 2009 scored ≤1% of sequences as having high-level resistance, while the 2013-14 algorithms, on the same sequences, found high-level resistance in >10%. TDF rules have been substantially revised since 2002 with some mutations (65R, 67 deletion) given more points in 2014, and 19 new rules in 2014 not in 2002 giving points for mutations (e.g. 115F) or combinations of mutations (e.g. 65R and 62V). NNRTI resistance was more consistent across algorithm versions.

Naïve sequences had low levels of resistance under all versions and for all drugs. Analysis of experienced

ABSTRACT 16

The Impact of Changes over Time in the Stanford Resistance AlgorithmSA Hart1, S Vardhanabhuti2, LJ Harrison2, SA Strobino1

1Frontier Science and Technology Research Foundation, Amherst, NY, USA; 2Harvard T.H. Chan School of Public Health, Boston, MA, USA

BACKGROUND: The Stanford Resistance Algorithm evolves over time. Over 60 versions have been released since 2002. When analyzing sequences or mutations on http://hivdb.stanford.edu, the version current at the time of analysis is used. Published results from different populations, or from similar ones at different time points, may stem from different versions, making comparisons problematic.

Our goal was to determine whether different algorithm versions produce systematically different results.

METHODS: We selected a single HIV-1 sequence, covering all or part of pol, from 5,604 sequenced participants in 74 AIDS Clinical Trials Group protocols. Draw dates were 1992-2015. A sample of algorithm versions was chosen by selecting the one released nearest to 7/1 in 2002-2003 and 2005-2014 (none was available for 2004). Each sequence was analyzed, using technology developed by Frontier Science, under each version.



METHODS: In 2014, CDC reviewed current approaches for survey data management with a focus on opportunities to reduce the level of effort for individual survey data management and to enable cross-survey analysis using participant-level data to reconstruct results with a high degree of data quality and completeness. This review identified the need to establish a central data repository for the management and analysis of HIVDR survey data where CDC is involved.

The central data repository is based on review of the variables collected for the last 10 CDC-supported surveys and the variables defined in the new generic protocol for HIVDR . Fundamental to the establishment of a central data repository was recognition of the variability in how survey data is captured across surveys, timing of different survey data availability, and compliance with CDC information and security requirements based on the Federal Information Security Management Act.

RESULTS: The establishment of the central repository includes defined specifications and standards for the import of different types of HIVDR survey data, automated integration with the Stanford University genotypic resistance interpretation algorithms, support for updating survey data based on feedback to data suppliers, predefined reports to continuously assess data quality and completeness, and security controls to ensure data confidentially. Referred to as the CDC HIV Drug Resistance Data Warehouse (cHIVDR DW), CDC completed establishment of the capabilities for the central management and analysis of Pre-Treatment Drug Resistance (PDR), Acquired Drug Resistance, and Transmitted Drug Resistance survey data in September 2015.

CONCLUSIONS: As a result of this work, CDC now has the capability to reduce the effort of individual HIVDR survey data management and to enable cross-survey analysis with a high degree of data quality and completeness, as well as assess, moving forward, that the benefits of cHIVDR DW continue being achieved. With this CDC will be using cHIVDR DW for the management of multiple in-process PDR surveys, incorporating historical HIVDR survey, and educating

sequences yielded results similar to those in Table 1. Subtype C sequences appeared to be more affected by algorithm version than subtype B ones.

CONCLUSION: Version matters. The specific algorithm version used should be stated whenever reporting resistance results interpreted by the Stanford algorithm. One should use caution when comparing results from different versions of the algorithm. If raw sequences or original mutation lists are available, reanalysis using a common algorithm version is preferable. However, this is often not possible. We plan to construct a calibration tool to compare results across Stanford algorithm versions.

ABSTRACT 17

Capabilities for Management and Analysis of HIV Drug Resistance Survey Data Using a Central Data RepositoryS Macauley1, L Mattocks2, C Yang2, E Raizes2

1InductiveHealth Informatics, Atlanta, Georgia, USA, 2Centers for Disease Control and Prevention, Atlanta, Georgia, USA

BACKGROUND: Hundreds of pieces of data and metadata are generated across the lifecycle of an HIV Drug Resistance (HIVDR) survey including participant data captured at clinical facilities, laboratory genotyping, and drug resistance interpretations. Ministries of Health, laboratories, the Centers for Disease Control and Prevention (CDC), and partner organizations use disparate approaches to manage HIVDR survey data, including capturing data in multiple formats and utilizing different methods for uniquely identifying records. As a result, it requires a labor intensive approach to gather and consolidate survey data to support determination of survey outcomes and return of drug resistance findings to facilities. These disparate data management approaches also limit cross-survey analysis to meta-analysis and literature review.



Ministries of Health on the role of cHIVDR DW in HIVDR survey data management.

ABSTRACT 18

Quantitation of the HIV Proviral and 2-LTR DNA and Their Relation to Proviral TropismM Vergara Mendoza1,2, LL Fuentes Romero1, M Viveros Rogel1, ML Cabrera-Ruiz3, LE Soto Ramírez1

1Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico; 2Universidad Autónoma Metropolitana, Unidad Iztapalapa, México City, Mexico; 3Hospital Médica Sur, México City, Mexico

BACKGROUND: The existence of a HIV reservoir despite long-lasting suppressive antiretroviral treatment (ART) is responsible of the inability to cure HIV infection. Besides the provirus, several episomal forms of viral DNA (circular and lineal) have been detected, being the most easily quantifiable the circular 2-LTR form; episomal DNA has been related to ongoing viral replication. Several factors have been related to the size of the reservoir, proviral and 2 –LTR, as the time on treatment and the amount of viral load before treatment, but no correlation with the tropism of the infecting virus has been performed. Our objective was to correlate the presence of proviral R5/X4 tropic viruses with the amount of proviral DNA and 2-LTR forms in Mexican patients suppressed on ART.

METHODS: We determined the proviral tropism in 123 samples from Mexican patients virologically suppressed with ART, sent to our reference lab from clinical care centers all over Mexico. Proviral tropism was determined genotipically with env sequences interpreted with the geno2pheno algorithm. Proviral and episomal (2-LTR) DNA were quantified using an in-house method with real-time PCR and expressed in copies per million cells.

RESULTS: One hundred and twenty three samples were quantified, 67(54%) were previously determined with a genotypic test as having R5-tropic viruses and

56 (46%) with X4-tropic viruses. Quantitation of proviral DNA was not obtained in 5(7.4%) and 8(14%) of the R5 and X4-tropic samples respectively, while episomal DNA was not quantified in a higher number of samples, 23 (18.6%) for R5 samples and 37 (66%) of the X4 samples.

In those samples that were quantified, samples with R5-tropic viruses had a mean proviral DNA load of 380 (11-4133) copies per million cells in comparison to a mean of 132 (10-687) copies per million cells in samples with X4-tropic viruses, p=0.027. On the other hand, quantitation of 2-LTR episomal DNA showed a mean of 3,949 (58-145,000) copies per million cells in samples with R5-tropic viruses, and a mean of 471 (96-7750) copies in X4-tropic viruses, p=0.000081.

CONCLUSIONS: Quantitation of proviral and episomal HIV DNA, showed higher levels of proviral DNA and 2-LTR circles in patients virologically suppressed with R5-tropic viruses than in those with X4-tropic viruses. These significant differences could be related to changes in replication capacity, viral fitness or the long duration of the episomal forms in R5-virus infected cells. It is also possible that survival of the X4-virus-infected cells could be shorter than those infected by R5 viruses reducing markers of replication and the amount of the viral reservoir.



Clinical Implications of Resistance for Treatment and

Prevention Strategies



ABSTRACT 19

HIV Drug Resistance Testing Among Patients New to HIV Care in the United StatesMCB Ocfemia1, AM Oster1, E Valverde1, Y Tie 2, L Beer1, A Hernandez1, J Weiser1

1Centers for Disease Control and Prevention (CDC), Atlanta, GA, United States; 2 ICF International, Atlanta, GA, United States

BACKGROUND: To guide antiretroviral therapy (ART), the U.S. Department of Health and Human Services and the International Antiviral Society–USA recommend baseline genotypic HIV drug resistance testing for HIV-infected persons at entry into care or as soon as possible after diagnosis. We assessed reported HIV genotype testing of patients new to HIV care among HIV care providers in the United States.

METHODS: We used data collected during 06/2013–01/2014 through the Medical Monitoring Project HIV Provider Survey, which was administered to a nationally representative sample of HIV care providers in the United States. Providers were asked for what proportion of patients new to HIV care they order HIV genotype testing as part of the initial evaluation; we included responses from 1,193 providers who answered this question. We weighted the data to account for unequal selection probabilities and non-response. We performed bivariate analyses and calculated prevalence ratios (PR) and 95% confidence intervals (CI) to examine differences in genotype testing for all patients by provider, practice, and medical care characteristics, including qualifications as an HIV specialist as defined by the HIV Medicine Association or the American Academy of HIV Medicine.

RESULTS: In all, 84.5% (CI=80.4%‒88.5%) of providers reported ordering genotype testing for all patients; 8.8% (CI=5.9%‒11.7%) for more than half, but not all patients; and 6.7% (CI=2.8‒10.6%) for half of patients or fewer. Ordering genotype testing for all patients was significantly less common among:

non-specialists (PR=0.89, CI=0.80–0.99) compared with HIV specialists; providers in private practices (PR=0.91, CI=0.82–1.00) compared with those at other facility types; and providers who first prescribe ART based on CD4 count (PR=0.83, CI=0.75–0.91) compared with providers who prescribe ART regardless of CD4 count.

CONCLUSIONS: Most providers in the United States reported ordering genotype testing for all patients new to HIV care. Providers in private practice and non-specialists were less likely to order genotype testing for all patients and may benefit from additional support to implement drug resistance testing guidelines. As providers move toward adopting guidelines for universal ART prescription, we may also see increasing adoption of baseline genotype testing recommendations.

ABSTRACT 20

The Dynamics of Drug Resistance Detected During Acute HIV Infection R Kanthula1,2, J Weis3, C Warth3, N Mugo1, L Frenkel1,2, C Celum1, J Overbaugh3, F Matsen IV3, J Baeten1, and D Lehman3

1University of Washington, Washington, United States; 2Seattle Children’s Research Institute, Washington, United States; 3Fred Hutchinson Cancer Research Center, Washington, United States

BACKGROUND: HIV drug resistance detected during acute infection may be a result of transmission from an infected partner, selection by pre-exposure prophylaxis (PrEP), or error prone replication. Here we describe the long-term dynamics of resistance detected during early acute HIV infection.

METHODS: The Partners PrEP Study was a randomized trial in which serodiscordant partners were assigned to emtricitibine co-formulated with tenofovir (FTC/TDF) or TDF alone compared to placebo, and monitored for HIV infection monthly. In a previous study, baseline samples from 137 HIV serocovnverters were tested



for drug resistance using 454 ultra deep sequencing at the time seroconversion was first detected. Here, we conducted resistance testing by 454 on plasma samples at 6, 12 and 24 months following seroconversion from individuals with resistance mutations detected during acute infection at frequencies ≥1% that had a Stanford score ≥30 and cause resistance to non-nucleoside or nucleoside reverse-transcriptase inhibitors (NNRTIs or NRTIs), including both PrEP and non-PrEP antiretrovirals.

RESULTS: There were 35/137 (26%) individuals with resistance detected at frequencies ≥1% at the time seroconversion was first identified: 30 had resistance to non-PrEP antiretrovirals (not TDF or FTC) likely due to transmitted resistance or error-prone replication and 11 individuals had PrEP-related mutations (K65R, K70E and M184IV); of whom 6 had both PrEP-related and non-PrEP mutations. Of the 35, 31 individuals had resistance results available through 12-24 months. Resistance faded to frequencies <1% in 16/31 (52%) individuals by 6 months after seroconversion and in 21/31 (68%) by 24 months. PrEP-selected mutations did not persist. In 5 individuals, resistance persisted at low frequencies between 1-10% of the viral population and in 3 individuals, resistance mutations K103N (n=2) or Y181C (n=1) persisted at frequencies ≥ 99% throughout follow-up.

CONCLUSIONS: Among individuals who acquired HIV in a PrEP trial, non-PrEP (non TDF or FTC) mutations accounted for the majority of resistance detected during acute infection. Persistence of high frequency resistance was limited to 2 NNRTI mutations that were most likely transmitted. Most of the resistance present during acute infection faded below detection by 24 months regardless of whether it was selected or transmitted. These data suggest that in the absence of antiretroviral treatment, onward transmission of resistance is most likely to occur early after HIV infection.

ABSTRACT 21

Transmitted Drug Resistance and First-Line ART Treatment Outcomes in Ugandan Children C Kityo1, R Boerma2,3, KC Sigaloff2,3, E Kaudha1, C Job 3,4, V Musiime1,5, S Balinda1, TS Boender2,3, TF Rinke De Wit2,3, PN Mugyenyi1

1Joint Clinical Research Centre(JCRC), Kampala, Uganda; 2Amsterdam Institute for Global Health and Development, Amsterdam, The Netherlands; 3Department of Global Health, Academic Medical Center of the University of Amsterdam, The Netherlands; 4Emma Children’s Hospital, Academic Medical Centre, Amsterdam, The Netherlands; 5Department of Paediatrics and Child Health Makerere University College of Health Sciences

BACKGROUND: Transmission of drug-resistant (TDR) HIV-1 can impair virologic response to antiretroviral therapy (ART) especially in children. This study evaluated the effect of TDR on virologic and acquired drug resistance (ADR) outcomes among children initiating first-line ART.

METHODS: Children ≤12 years initiating first-line ART were enrolled at 3 sites in Uganda between 2010 and 2011. Blood was taken at baseline and 6-monthly in 24 months of ART for later determination of viral load (VL) and pol genotypic testing if VL>1,000 copies/ml at JCRC Kampala laboratories. The 2014 IAS-USA mutation list and Stanford algorithm were used to score drug resistance mutations (DRMs) and susceptibility. Patients were classified into two groups: fully active (no TDR or TDR without reduced susceptibility) and patients with TDR and partially active ART. Virological failure (VF) was defined as 2 consecutive VLs >1000 copies/ml, at least 6 months from ART initiation. Factors associated with VF and acquired drug resistance (ADR) were assessed in multivariate logistic regression analysis and evolution of resistance was described.

RESULTS: 317 children median age 4.9 years were enrolled on mainly NNRTI based regimens



(91.2%). TDR mutations were detected in 47(16.9%) participants of whom 22(46.8%) initiated a partially active ART. 256(80.8%) participants were still on first line ART and in care at 24 months of follow-up of whom 32%(92/287) had VF. Children with TDR and partially active ART had significantly higher risk of VF (aOR:15.25, 95%-CI:3.77-61.7,p<0.001) and ADR (aOR:3.47, 95%-CI:1.31-9.22,p<0.012). A single VL >1000 copies/ml at 6 months on treatment was strongly associated with VF (OR:22.09, 95%-CI:9.68-50.42,p<0.001) and ADR (OR:9.89, 95%-CI:5.16–18.94;p<0.001). Other factors associated with VF in the adjusted model were higher baseline VL (aOR:2.28,p<0.001) and WHO stage 2 vs Stage 1 (aOR:10.3,p=0.022). Among the 66 children with prolonged viraemia, there was a high rate of acquisition of DRMs.

CONCLUSIONS: In this pediatric cohort, TDR was found to be high and strongly associated with VF and ADR. Accumulation of DRMs was high and may jeopardize future therapeutic options. Efforts are needed to incorporate affordable drug resistance testing in developing countries to prevent the use of suboptimal ART. In the context of universal access of VL monitoring, programmatic use of a 6 month VL on treatment would detect majority of future VFs early and allow for treatment adjustment to prevent ADR.

ABSTRACT 22

High Levels of HIV Drug Resistance in Treatment-Naïve Children in Lagos, Nigeria: Original Data and a Systematic Review in Sub-Saharan AfricaTS Boender1,2*, RS Boerma1,2*, KC Sigaloff 1,3, TF Rinke de Wit 1, M Boele van Hensbroek 1,2, N Ndembi4, T Adeyemo5 , EO Temiye5, A Osibogun5, P Ondoa1, JC Calis2*, AS Akanmu5*

*Both first and both senior authors contributed equally to the work

1Amsterdam Institute for Global Health and Development & Department of Global Health, Academic Medical Center of the University of Amsterdam, the Netherlands; 2Global Child Health Group, Emma Children’s Hospital, Academic Medical Center of the University of Amsterdam, the Netherlands; 3Department of Internal Medicine, Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; 4Institute of Human Virology Nigeria, Abuja, Nigeria; 5University Teaching Hospital, University of Lagos, Lagos, Nigeria.

BACKGROUND: Since the roll-out of antiretroviral treatment (ART) in sub-Saharan Africa in the beginning of this millennium, ART coverage has increased substantially. The increased usage of ART, however, is likely to come at a cost, as the levels of HIV drug resistance (HIVDR) are expected to rise. Children who have been exposed to antiretroviral drugs (ARV) for the prevention of mother-to-child transmission are at increased risk of HIVDR prior to ART initiation. Monitoring pre-treatment HIVDR is especially important in children as they have fewer ART options than adults, and will require ART for more years. Data on pediatric HIVDR prevalence, especially from sub-Saharan Africa, are scarce.

METHODS: HIV-1 infected ARV-naïve children ≤12 years were enrolled in the Monitoring Antiretroviral Resistance in Children (MARCH) cohort at the Lagos University Teaching Hospital, Nigeria. Pre-treatment viral load and population based pol genotypic testing was performed retrospectively. HIVDR mutations



were identified using the World Health Organization list for transmitted drug resistance. Additionally, we conducted a systematic review and meta-analysis on pre-treatment HIVDR prevalence in children in sub-Saharan Africa to put our findings into context.

RESULTS: Ninety ARV-naïve children were enrolled in Nigeria, of whom genotypic resistance testing was successful in 82 children. Thirteen of 82 (15.9%) children had pre-treatment HIVDR. All 13 harbored non-nucleoside reverse-transcriptase inhibitor mutations, of whom seven (8.5%) also had nucleoside reverse-transcriptase inhibitor resistance. No protease inhibitor mutations were detected. G190A/S (n=7) and M184V/I (n=6) were the most prevalent mutations (figure 1). All 13 children with HIVDR had resistance against one or more drugs of their first-line regimen.

Our systematic review included 16 studies from 11 different African countries, including 2,057 children, and the pooled HIVDR prevalence was 28.1% (95%CI 18.5-37.7) (figure 2). Among ARV-naïve children, the pooled HIVDR prevalence was 10·8% (95%CI: 4·4-17·1). Meta-regression showed an increase in prevalence in these children from 0·6% (95%CI 0·6-5·4) in 2004 to 36·2% (95%CI 25·5-46·9) in 2011 (p=0·05).

CONCLUSION: One in six Nigerian children is started on a sub-optimal ART regimen. Our systematic review confirmed these high figures throughout the African continent and showed a worrisome increase of pre-treatment HIVDR in ARV-naïve children over the past decade. These findings stress the importance of rapid implementation of protease inhibitor-based regimens in all children under three years of age. Overcoming practical barriers to implement protease inhibitor-based regimens, and introduction of a population-based HIVDR surveillance system among children should receive priority to ensure optimal treatment for HIV-infected children in sub-Saharan Africa.

Figure 1: HIV drug resistance mutations detected in this cohort (n=82). Mutations are based on the 2009 World Health Organization list for surveillance of transmitted drug resistance. NNRTI: non-nucleoside reverse transcriptase inhibitor; NRTI: nucleoside reverse transcriptase inhibitor

Figure 2: Pooled proportions of pre-treatment HIV drug resistance in children in sub-Saharan Africa, by year of treatment initiation. Red box indicates the current study in Nigeria. HIVDR: HIV drug resistance. Meta-analysis of studies of the systematic review was conducted to pool the reported HIVDR prevalence using a random-effects model, because of expected heterogeneity among studies. The variance of the raw proportions was stabilized using a Freeman-Tukey arcsine square root transformationand was subsequently back-transformed to the original scale. I2=97.7%



ABSTRACT 23

Predictors of Persistent HIV Viraemia Among Treatment Experienced Children and Adolescents at an Urban Clinic in UgandaR Atugonza, D Damba, JB Kanywa, A Kekitiinwa

Baylor College of Medicine Children’s Foundation Uganda (Baylor-Uganda), Kampala, Uganda

INTRODUCTION: In May 2013, Uganda updated the national ART treatment guidelines to include recommendations for viral load as the preferred monitoring approach to diagnose and confirm ARV treatment failure. Baylor–Uganda adapted these guidelines in August 2014 and with support from the National Central Public Health Laboratories (CPHL) systematically ensured that all children who had been on ART for six months or more had at least one viral load test performed. Little is known about the predictors of HIV suppression among treatment experienced children and adolescents in low income settings. We describe factors associated with persistent HIV viraemia among this age group after at least 6 months of adherence interventions at an HIV clinical center of excellence in Uganda.

METHODS: We reviewed records of participants aged <18 years, on 1st line ART for at least 6 months, with an index detected plasma viral load above 1000 cp/ ml and a repeat viral load test after 6 months of intensified adherence support; monthly clinic visits with counseling sessions, home health visits and caretakers meetings. A logistic regression model was applied to analyze the association between subsequent HIV viral load suppression with primary caretaker, age at ART initiation, PMTCT exposure, duration on ART, ART regimen, baseline CD4, baseline and current WHO stage, nutrition status, TB status, age at time of index detected viral load and adherence to ART. Data was analyzed using Stata/SE 13.0.

RESULTS: We studied 311 children and adolescents. Their median age was 9.2 [IQR 5.6; 13.2] years, 173

(55.6%) were males, the median CD4% was 25% [IQR 18; 33] and the median duration on ART was 3.4 [1.6–10.4] years. Only 72/311 (23.2%) participants suppressed on their 1st line regimen. There was a positive correlation between unsuppressed viral load and duration on ART; participants on ART for ≥ 2 years were more likely to have persistent Viraemia OR 2.47 [95%CI 1.42; 4.30]. Participants who had persistent HIV viraemia were also more likely to have mild–moderate malnutrition OR 3.06 [95%CI 1.26; 7.45]. There were no other statistically significant associations.

CONCLUSIONS: Malnutrition and duration on 1st line ART for more than 2 years are predictors of persistent viraemia with possible resistance and could be considered as indicators for early switch to second line ART.

ABSTRACT 24

In Cameroonian Children under Five Years, CCR5-Variants Prevail, Implying Vertical Transmission with CCR5-Tropic VirusesJ Fokam1,2,3,4, MC Bellocchi2, D Armenia2, AJ Nanfack1,5, L Carioti2, F Continenza6, D Takou1, E Temgoua1, C Tangimpundu1, JN Torimiro1,3,4, CN Fokunang3,7, G Cappelli1,8, A Ndjolo1,3, V Colizzi1,2,9, F Ceccherini-Silberstein2, CF Perno1,2, and MM Santoro2

1Chantal Biya International Reference Centre for research on HIV/AIDS Prevention and Management, Yaounde, Cameroon; 2University of Rome Tor Vergata, Rome, Italy; 3University of Yaounde I, Yaounde, Cameroon; 4National HIV Drug Resistance Prevention and Surveillance Working Group, Yaounde, Cameroon; 5New York University School of Medicine, New-York, USA; 6“L Spallanzani” National Institute of Infectious Diseases, Rome, Italy; 7University of Bamenda, Bamenda, Cameroon; 8National Research Council, Rome, Italy; 9UNESCO Board of Multidisciplinary Biotechnology, Rome, Italy

BACKGROUND: Despite increasing coverage in prevention of mother-to-child transmission (PMTCT), vertical-transmission remains consistent in sub-Saharan Africa (SSA). With use of low-genetic



barrier drugs, poor-adherence and HIV-1 variability in SSA, vertically transmitted HIV-1 drug-resistance (HIVDR) is a concern and could jeopardise paediatric antiretroviral therapy (ART). We then sought to ascertain majority and minority HIVDR and viral-tropism among vertically infected-children in SSA, as well as factors potentially associated with resistance.

METHODS: A comparative analysis was conducted among 17 Cameroonian HIV-infected children: seven from mothers with PMTCT-exposure (with reverse transcriptase inhibitors [RTI]), versus 10 from mothers without PMTCT-exposure (control-group: antiretroviral-naïve). CD4-count, viremia, Sanger-sequencing and ultra deep-454-pyrosequencing (UDPS) were performed. The cut-off of UDPS detection was set at 1%. Phylogeny was performed for subtyping. Protease/RT drug-resistance mutations (DRMs) and HIV-1 co-receptor usage were interpreted using Stanford HIVdb.v7.0 and geno2pheno.v2.5, respectively. Viruses were considered CXCR4-tropic (X4-variants) by UDPS when ≥2% viral species had a false positive rate (FPR) ≤3.5%, or by Sanger-sequencing, when FPR was ≤10%. HIV-1 variants in children were compared between both PMTCT-groups. Viral-tropism was also explored according to age and CD4-count.

RESULTS: Median [interquartile range, IQR] age, viremia and CD4-count were 6 [3-10] years, 5.5 [4.9-6.4] log10 copies/ml, and 523 [282-621] cells/mm3, respectively. Median UDPS coverage was 1642 [IQR: 1269-5193] reads. CRF02_AG (60%) and F (35.3%) were prevailing subtypes. All children had wild-type viruses at both Sanger-sequencing and UDPS. The only exception was 1/7 PMTCT-exposed children, where minority NNRTI-DRMs (K103E[2.74%]; K103N[8.31%]) were found. This infant was born from an RTI-treated mother harboring multiple-DRMs (T69A[5.80%]; T69N[56.48%]; L74V[2.54%]; K103E[2.20%]; Y181C[96.67%]; K219N[30.13%]). Five out of 15 children (33.3%) having both V3 UDPS- and Sanger-data harboured X4-variants; no X4-variants prevalence difference was found between the two PMTCT-groups. Concordance between UDPS and Sanger V3-sequencing was 86.7%. Indeed, by

UDPS, two children had minority X4-variants with a prevalence of 3.9% and 36.2%. Of relevance, X4-variants were found only in children aged >5 years, compared to younger ones (5/9 [55.6%] vs. 0/6 [0%]; p=0.040), suggesting that MTCT CXCR4-transmission is limited/absent, and appears later during chronic infection and immunological impairment of the child. Not surprisingly, very high percentage of children with CD4≤200 cell/mm3 harboured X4-variants (3/4 [75%] vs. 2/11 [18.2%], p=0.077).

CONCLUSIONS: In Cameroonian children, minority NNRTI-DRMs are likely present with PMTCT-exposure while no PI-DRMs are found. This suggests the potential advantage of starting ART with protease inhibitor-based regimens, in case of documented, failing PMTCT. In children under five years, CCR5-variants prevail, implying vertical transmission with CCR5-tropic viruses (and possible suitability of anti-CCR5 at younger ages). These preliminary evidences, generated from a population mainly CRF02_AG infected, should be further explored for improved pediatric-HAART strategies in resource-limited settings.



ABSTRACT 25

Impact of Exposure to Lopinavir-Ritonavir in HIV-1 Infected Children and Adolescents in Madrid, Spain during 2000-2014PR Sánchez1, L Prieto2, SJ De Ory3, EF Cooke4, M Navarro3, JT Ramos5 and Á Holguín1, for the Madrid Cohort of HIV-1 Infected Children and Adolescents Integrated in the Pediatric Branch of the Spanish National AIDS Network (CoRISPe)

1HIV-1 Molecular Epidemiology Laboratory, Microbiology and Parasitology Department, Hospital Ramon y Cajal-IRYCIS and CIBERESP, Madrid , Spain; 2Infectious Diseases Department, Hospital Universitario de Getafe, Madrid, Spain; 3Molecular InmunoBiology Laboratory, Hospital General Universitario Gregorio Marañón-IISGM, Madrid, Spain. Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain; 4Infectious Diseases Department, Hospital Universitario Doce de Octubre, Madrid, Spain; 5Infectious Diseases Department, Hospital Clínico Universitario and Universidad Complutense, Madrid (28040), Spain

BACKGROUND: Lopinavir-ritonavir (LPV/r) is the most-used protease-inhibitor (PI) in children, providing durable suppression of viral load (VL) and increasing CD4+T-counts. Our study describes the baseline features of HIV-1-infected children and adolescents receiving LPV/r during 2000-2014 in Madrid, Spain and the virological-immunological response after LPV/r exposure as part of first-second or third or more-line antiretroviral regimens.

METHODS: Patients from the Madrid Cohort of HIV-1-infected children and adolescents exposed to LPV/r during 2000-2014 were selected, recording the baseline epidemiological-clinical features and the mean VL, CD4 and CD8 values before and during LPV/r exposure until December 2014. Those with available pol sequence, genotypic resistance profile or sample availability by January 2011 were selected, identifying drug-resistance mutation (DRM) and predicting susceptibility to 19 antiretrovirals using the Stanford s HIVdb Algorithm.

RESULTS: A total of 199 (37.3%) of the 534 patients from the Cohort were exposed to LPV/r during 2000-2014 as first (16.6%, group 1), second (18.6%, group 2) or third or more-line therapy (63.3%, group 3), being unknown line in 1.5% subjects. Patients were mainly Spaniards (81.9%), perinatally infected (96.5%) with subtype-B (65.3%), and mainly HIV-diagnosed before year 2000 (67.8%). The mean age at first LPV/r exposure was 9.7 years, with mean time of LPV/R exposure of 37.3, 41.9 and 57.5 months in groups 1, 2 and 3, respectively. Good clinical status at the last clinic visit during LPV/r treatment was observed, with undetectable viraemia (below 50 HIV-1-RNA-copies/ml) in 47.8% patients and high CD4+T cells counts (median 911). After LPV/r exposure, VL reduction was significantly higher in groups 1 and 2 than in group 3. The CD4% and CD8% gain was significantly higher only in group 1. DRM to PI were presented by 42.6% of 64 patients with available resistance data, all belonging to groups 2 and 3. The most common DRM were D30N, M46IL, I54V, V82A and L90M in protease. Patients from group 3 reported the highest number of DRM to PI (52.2%), to nucleoside reverse transcriptase inhibitor or NRTI (62.5%) and to non nucleoside reverse transcriptase inhibitors or NNRTI (46.9%). Darunavir-ritonavir and tipranavir/ritonavir presented the highest susceptibility and nelfinavir the lowest among those with resistance data at PR.

CONCLUSIONS: We describe the epidemiological-clinical-virological features of children and adolescents who received LPV/r during 14 years in Madrid, Spain, observing higher VL reduction and better CD4 and CD8 recovering when LPV/r was taken as part of a first-line regimen. We identified the antiretrovirals less compromised despite the presence of DRM in the LPV/r-exposed-cohort, mainly new PIs. This study will provide new data to improve clinical management of LPV/r exposed children and adolescents.



ABSTRACT 26

Drug Resistance Compromises Second-Line ART in Mozambican Children Failing First-LineP Vaz1, WC Buck2, D Bila1,3, N Bhatt3, K Jobarteh2, L Cossa3, C Alfredo2 , J Houston4, A Sousa3, C Yang4

1Fundação Ariel Glaser, Maputo, Moçambique; 2Centers for Disease Control and Prevention, Maputo, Moçambique; 3Instituto Nacional da Saúde, Maputo, Moçambique; 4Centers for Disease Control and Prevention, Atlanta, GA, USA

BACKGROUND: Mozambique’s pediatric antiretroviral treatment (ART) program grew from 9,393 patients in 2007 to 41,400 in 2013. This rapid and ongoing expansion has made assurance of quality care a challenge and has occurred without viral load monitoring. In this context, we assessed the prevalence of virologic failure (VF) and HIV drug resistance mutations (DRM) in a cohort of ART-experienced children to understand implications for the current pediatric second-line regimen of LPV-r/3TC/ABC or TDF.

METHODS: A cross-sectional study was conducted at six clinics providing pediatric ART in Maputo. Children aged 1-14 years and active on ART for at least 12 months were enrolled from August 2013 to March 2014. Clinical and demographic information were collected and viral load was performed. Dried blood spots were prepared from samples with VF (>1000 copies/mL) for genotyping. DRM interpretation and drug susceptibility were assessed using HIVdb (Stanford v7.0). Statistical analysis was done with SAS (v9.2).

RESULTS: In total 713 children were enrolled; mean age of 102.9 months (95%CI: 81.0-124.7), mean time on ART of 60.3 months (95%CI: 38.8-81.2), 73.2% (95%CI: 43.5-90.6) were on d4T/3TC/NVP, 85.8% (95%CI: 75.2-92.3) had no immune suppression and 20.2% (95%CI: 15.0-26.6) had PMTCT exposure. VF was observed in 256 patients (35.9%) (95%CI: 26.9-46.2%), and 96.9% (n=248) were successfully

genotyped, with DRM found in 94.8% (n=235). High levels of NRTI and NNRTI mutations were observed with M184V (90.7%) and Y181C (49.6%) most common in each class. K65R and major PI DRMs occurred in 2.4% and 1.6% of sequences, respectively. Thymidine analog mutations (TAMs) were observed in 33.5% (n=83), all in children ≥5 years. TAM-2 was the main pathway observed (n=69; 83.1%) with a mixture of both pathways in 12 patients. Second-line ART had three, two, and one-drug efficacy in 5.4%, 82.4%, and 12.2% of patients with DR, respectively, age-stratified results in Fig. 1.

CONCLUSIONS: VF was common (35.9%) in this large, treatment-experienced, older pediatric ART cohort, and was strongly associated with DRM (94.8%). Many of these children did not have immunologic failure and had likely been on failing regimens for some time, accumulating DRMs. These findings support the ongoing roll-out of viral load testing in Mozambique and have implications for pediatric second-line outcomes. Additional analysis of these data will look at differences in VF and DR by age group and associated risk factors.

Figure 1



ABSTRACT 27

Influence of Transmitted Drug Resistance on CD4 Decline Among ART Naïve HIV Patients A Schultze1, C Torti2, A Cozzi-Lepri1, A-M Vandamme3,4, M Zazzi5, H Sambatakou6, A De Luca7, AM Geretti8 , A Sonnerborg9 and G Lapadula10

1Department of Infection and Population Health, University College London, London, UK; 2Unit of Infectious and Tropical Diseases, Department of Medical and Surgical Sciences, University “Magna Graecia”, Catanzaro, Italy; 3KU Leuven – University of Leuven, Rega Institute for Medical Research, Leuven, Belgium; 4Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal; 5University of Siena, Siena, Italy; 6Hippokration General Hospital, University of Athens, Athens, Greece; 7University Division of Infectious Diseases, Siena University Hospital, Siena, Italy; 8University of Liverpool, Liverpool, UK; 9Karolinska Institute, Stockholm, Sweden; 10Ospedale S. Gerardo, Monza, Italy

BACKGROUND: The presence of transmitted drug resistance mutations (TDRM) may influence the natural history of HIV infection. We evaluated the effect of TDRM on CD4 count decline in a large European cohort collaboration.

METHODS: Data from several European HIV clinics (ViroLAB, EuResist and EuroSIDA contributing clinics; Royal Free and St Mary’s Hospital, London; University of Bari) were merged. Individuals were included if they were aged ≥18 and had ≥1 CD4 count and ≥1 genotypic resistance test before starting antiretroviral therapy (ART). Baseline was defined as the date of the first available CD4 count. TDRM were identified using the WHO 2009 surveillance list, and were presumed to have been present since the time of infection. Linear mixed models with a random intercept and slope were used to estimate the effect of TDRM on CD4 slopes.

RESULTS: 6326 individuals were included: 74% were male and 65% infected with a subtype B virus. The median follow-up was 1.2 (IQR=0.07-3.4) years. Overall, 623 individuals (9.9%) had at least 1 TDRM (NRTI:7.0%, NNRTI:3.0% and PI:2.5%). The most common mutations were thymidine analogue

mutations for the NRTIs (5.9%), K103N for the NNRTIs (1.8%) and L90M for the PIs (0.9%). The median baseline CD4 count was 418 (IQR=284-580) cells/mm3, and there was no evidence that this differed according to the detection of TDRM (426 in those with TDRM v. 417 in those without, p=0.14). The viral set point (median of the mean distribution of all pre-ART RNA measurements) was 4.4 log10cp/ml among individuals with TDRM and 4.5 among those without (p=0.07). In unadjusted models, the overall estimate of CD4 decline was 54 cells/year in the whole population; 56 cells/year among those with TDRM and 54 cells/year among those without (difference=-2.30 cells/year, 95%CI=-9.67; +5.03, p=0.54). After adjustment for potential confounders (Table 1), there was no evidence to suggest that the rate of CD4 decline differed according to TDRM presence (p=0.29). There was also no evidence to suggest that CD4 slopes differed according to the class of resistance present (Table 1). 71 (1.1%) individuals had M184V, and we could not find any evidence that this was associated with baseline CD4 counts (p=0.15) or CD4 slopes (p=0.68).

CONCLUSIONS: In one of the largest European datasets of resistance tests results from ART-naive individuals, we were not able to find any evidence supporting the hypothesis that the rate of CD4 decline in the absence of ART is different between patients with and without TDRM.



Table 1. Annual CD4 decline (cells/mm3) according to presence of TDRM

Unadjusted Adjusted1

Slope (95% CI)

Slope (95% CI)

Difference (95% CI)

P- value

Any TDRM

Wild-type (N=5703)

-53.96 (-56.30; -51.63)

-57.39 (-60.20; -54.57)

Any TDRM (N=623)

-56.27 (-63.25; -49.29)

-62.16 (-70.55; -53.77)

-4.78 (-13.63; +4.07)

0.29

NRTI2Wild-type (N=5703)

-54.02 (-56.37; -51.68)

-57.41 (-60.23; -54.59)

≥1 NRTI (N=442)

-55.32 (-63.34; -47.31)

-61.09 (-70.74; -51.44)

-3.68 (-13.73; +6.37)

0.47

NNRTI2Wild-type (N=5703)

-53.87 (-56.17; -51.58)

-57.45 (-60.25; -54.65)

≥1 NNRTI (N=189)

-65.58 (-78.89; -52.27)

-72.25 (-88.02; -56.48)

-14.80 (-30.81; +1.22)

0.07

PI2Wild-type (N=5703)

-53.87 (-56.17; -51.58)

-57.34 (-60.12; -54.57)

≥ 1 P I (N=158)

-45.29 (-58.72; -31.86)

-50.39 (-66.56; -34.22)

6.95 (-9.46; +23.36)

0.41

1. Adjusted for baseline age, gender, mode of infection, subtype, cohort, viral set point, calendar year of the resistance test and baseline CD4 counts. Individuals with missing covariate values (N=749) were excluded from multivariable analyses.2. Individuals with dual and triple-class resistance could contribute data to all the relevant analyses.

ABSTRACT 28

Pre-Existing Drug Resistance Mutations in Treatment-Naive Subjects Do Not Affect Response to Tenofovir Disoproxil Fumarate (TDF) or Tenofovir Alafenamide (TAF) Containing RegimensNA Margot, R Kulkarni, K White, D Porter, M Abram, C Callebaut, and MD Miller

Gilead Sciences, Inc., Foster City, CA, USA

BACKGROUND: Pre-existing mutations conferring resistance to antiretroviral (ARV) drugs in treatment-naive individuals may adversely affect the outcome of ARV therapy. Such mutations exist because of natural variation in the HIV sequence or because of transmission of HIV with acquired resistance to ARVs. Genotypic testing is widely used to assess the presence of resistance mutations prior to initiating ARV therapy. In this analysis, we assessed the presence and impact of pre-existing ARV resistance mutations not specifically associated with study drugs on the response to TDF- or TAF-based therapy in treatment-naive subjects.

METHODOLOGY: Pre-treatment HIV-1 reverse transcriptase (RT) and protease genotypic data from treatment-naive subjects in 9 Phase 3 studies of tenofovir-based regimens initiated at Gilead Sciences between 2000 and 2013 were included (Studies 903, 934, 236-0102, 236-0103, 216-0114, 216-0130, 264-0110, 292-0104, and 292-0111). Study regimens also included emtricitabine or lamivudine, with efavirenz, protease inhibitors, rilpivirine, or elvitegravir. Studies excluded subjects with pre-existing resistance mutations to study drugs, however other resistance mutations were permitted including thymidine analog mutations (TAMs).

RESULTS: RT and protease genotypic data were obtained for 6705 subjects prior to enrollment in the studies. Among these, 1071 subjects (16%) had NNRTI resistance mutations (most frequently K103N/S, see Table 1). NRTI-associated mutations were found in



606 subjects (9%); 183 subjects (2.7%) had TAMs (most frequently M41L) and 183 subjects (2.7%) had non-canonical mutations at position T215 indicative of reversion from the TAM T215Y/F. Seven subjects (0.1%) had M184V/I, and 1 subject (0.01%) had K65R and was excluded from enrollment.

Table 1. Drug resistance mutations detected in treatment-naïve subjects prior to study enrollment

Drug Resistance MutationNumber of Subjects (%)N=6705

Any NNRTI 1071 (16)

K103N/S 302 (4.5)

Any Primary PI 199 (3)

M46I/L 62 (0.9)

L90M 57 (0.9)

Any NRTI 606 (9)

V118I 353 (5.3)

Any TAMa 183 (2.7)

M41L 100 (1.5)

T215F/Y 6 (0.1)

T215(other) 183 (2.7)

M184I/V 7 (0.1)

K65R 1 (0.01)

a TAMs include M41L, D67N, K70R, L210W, T215Y/F or K219Q/E/N/R.

A total of 5990 subjects were enrolled in the studies, of which 4586 subjects received a tenofovir disoproxil fumarate (TDF)-based regimen and 865 subjects received a tenofovir alafenamide (TAF)-based regimen. Response to these regimens at Week 48 (FDA Snapshot or TLOVR of <50 copies/mL) was similar regardless of the presence of pre-existing resistance mutations. Among subjects with pre-existing TAMs, 87% and 95.2% had virologic success in the TDF and TAF groups, respectively, versus 85.7% and 92.4% without pre-existing TAMs.

CONCLUSIONS: The prevalence of pre-existing/transmitted resistance to tenofovir (K65R) or emtricitabine/lamivudine (M184V/I) in treatment-naïve subjects was extremely rare despite the prevalent use of TDF and FTC. The presence of other pre-existing resistance mutations, notably TAMs in RT, had no impact on treatment response with either TDF- or TAF-based regimens.

ABSTRACT 29

Impact of Transmitted Thymidine Analogue Mutations on Responses to First-Line ARTAM Geretti1, E White2, A Beloukas1, C Orkin3, A Tostevin2, P Tilston4, D Chadwick5, C Leen6, C Sabin7, D Dunn2 on behalf of UKHDRD and UKCHIC

1Institute of Infection and Global Health, University of Liverpool, Liverpool, UK; 2MRC Clinical Trials Unit at UCL, London, UK; 3Barts and The London NHS Trust, London, UK; 4Department of Clinical Virology, Manchester Royal Infirmary, Manchester, UK; 5South Tees Hospitals NHS Trust, Middlesbrough, UK; 6University of Edinburgh and Western General Hospital, Edinburgh, UK; 7University College London Medical School, London, UK

BACKGROUND: Thymidine analogue mutations (TAMs; RT codons 41, 67, 70, 210, 215, 219) are a prevalent form of transmitted drug resistance (TDR) in Europe and North America, commonly occurring as singleton revertants of T215Y/F (e.g., T215E), and thought to often represent onward transmission from ART-naive subjects. Although PI/r-based therapy is recommended for patients with transmitted TAMs, it is not known whether alternative regimens carry an increased risk of virologic failure (VF). The study aim was to analyze ART outcomes in subjects with ≥1 TAM (and no other resistance) vs. subjects without evidence of resistance.

METHODS: Subjects underwent genotypic resistance testing in 1998-2012 prior to starting TDF or ABC + 3TC or FTC + PI/r (ATV, DRV, FPV, LPV) or NNRTI (EFV, NVP, RPV). VF definition: confirmed viral load >50 (or 200) cps/mL after ≥6 months of ART, or one viral load >50 (or 200) cps/mL followed by a treatment change. Time to VF was analyzed using Kaplan Meier plots and Cox models adjusted for age, ethnicity, risk group, pre-ART viral load and CD4 count, and ABC use.

RESULTS: Of 6926 patients evaluated before ART initiation, 6345 (92%) had no resistance; 271 (4%) had ≥1 TAM, including 204/271 (75%) with singleton TAMs, most commonly T215 revertants (112/271, 41%). VF risks at the 50 cps cut-off were 808/6345



(13%) in subjects with no resistance vs. 33/271 (12%) in subjects with ≥1TAM (P=0.53, log rank test). VF risks in subjects with no resistance were 304/1591 (19%) for PI/r use vs. 504/4754 (11%) for NNRTI use (adjHR=2.2; 95% CI 1.9-2.5; P<0.001). The same direction of effect was observed with ≥1TAM: 16% (21/131) for PI/r vs. 9% (12/140) for NNRTI (adjHR=1.7; 0.8-3.4, P=0.15). At the 200 cps cut-off, VF risks were 401/6345 (6%) in subjects with no resistance vs. 12/271 (4%) in subjects with ≥1TAM (P=0.14, log rank test). VF risks in subjects with no resistance were 149/1591 (9%) for PI/r use vs. 252/4754 (5%) for NNRTI use (adjHR=1.9; 1.6-2.4, P<0.001). With ≥1TAM, VF risks were 6/131 (5%) for PI/r vs. 6/140 (4%) for NNRTI (adjHR=0.9; 0.3-2.8, P=0.87).

CONCLUSIONS: This cohort analysis supports the hypothesis that in patients with ≥1 TAM as the sole form of TDR (predominantly singleton T215 revertants), there was no apparent virologic advantage of starting ART with a PI/r-based regimen. As the influence of confounding factors cannot be excluded, the data should be regarded as providing a framework for designing a controlled trial.

ABSTRACT 30

Transmitted Drug Resistance in ART-Naive Recently and Chronically-Infected Individuals: The ANRS 12249 Cluster-Randomised Trial of HIV Treatment as Prevention (TasP)A Derache1,2, C Iwuji1,3, S Danaviah1, AG Marcelin3, V Calvez3, T De Oliveira1, F Dabis4 and D Pillay1,3

1Africa Centre for Population Health, Durban, South Africa; 2University of Paris 6, Paris, France; 3University College London, London, UK; 4School of Public Health, Bordeaux, France

BACKGROUND: The recent scale-up of antiretroviral therapy (ART) in resource-limited countries may increase the transmission of drug resistance (TDR) to similar levels in rich settings. In the context of

treatment as prevention (TasP), with earlier initiation of ART, it is plausible that an increasing proportion of individuals seroconverting to HIV would be infected with resistant virus. In the ANRS 12249 TasP trial, we assessed the frequency of TDR in individuals recently- and chronically-infected ART-naïve individuals.

METHODS: Individuals who enrolled in the trial clinics from March 2012 to September 2015 were included in the analysis. Sanger sequencing of the POL gene was performed for 185 recently-infected individuals. HIV whole genome sequences were generated (WGS) on Illumina MiSeq on available plasma samples from a subset of recently-infected (n=31) and chronically-infected ART-naïve individuals (n=89) who linked to care. WGS were assembled using Geneious software, and a 2% threshold was used to assess the presence of minority DR variants (MDRV), defined as representing less than 20% of the viral population. TDR levels were assessed according to the WHO 2009 list of mutations for TDR surveillance. Only descriptive statistics are presented.

RESULTS: Median age in recently-infected individuals was 21.5 years, IQR (18.7, 28.1) and 159/185 (86%) were female. For chronically-infected individuals, median age was 35 years, IQR (28, 45) and 63/89 (71%) were female. Prevalence of TDR was estimated at 4.9% (9/185) and 3.4% (3/89) among recently- and chronically-infected individuals, respectively. All had a single DRM, associated with NNRTI resistance (mainly K103N); only one had a NRTI resistance mutation (T215S). In recently-infected individuals, TDR was higher in males than females (11.5% vs. 3.8%) and more frequent in higher HIV-1 prevalence clusters (7.6% for prevalence >30% vs. 1.2% for prevalence ≤30%). A similar finding was seen in chronically-infected individuals. However, due to few TDR observations, none of the results were statistically significant.

MDRV were found in 25.8% (8/31) and 6.7% (6/89) of recently- and chronically-infected individuals respectively. Only one participant had MDRV associated with DRM found at level >20% (K103N 50% + K101E 12 % + G190A 2.5%).



Among participants with TDR only (n=4), MDRV only (n=10) or both (n=1) with documented follow up viral load, 13/15 were suppressed (VL<400 copies/mL) after median ART duration (IQR) of 1.78 years (0.81, 2.06)

CONCLUSION: The prevalence of TDR estimated in TasP was not different between recently- and chronically-infected individuals and is similar to what was described in the same area in 2012. This is despite an increase in ART coverage from earlier ART initiation in the trial. The presence of TDR seems to have minimal impact on response to first-line ART.

ABSTRACT 31

The Co-Presence of Specific HIV-1 CRF02_AG Polymorphisms Correlates with a Lower Response to PI-Based First Line HAARTD Armenia1, D Di Carlo1, C Gori2

, AB Pérez3, V Borghi4, A Bertoli1, M Alvarez3, A Latini5, G Sterrantino6, S Lambert7,8,9, AP Callegaro10, M Milesi11, V Ghisetti11, L Fabeni2, N Coppola12, P Scognamiglio2, R Bellagamba2, A Ammassari2, S Cicalini2, C Cerva13, F Maggiolo10, G Di Perri11, A Cristaudo5, E Girardi2, AG Marcelin7,8,9, V Calvez7,8,9, M Andreoni13, C Mussini4, F Garcia3, A Antinori2, F Ceccherini-Silberstein1, CF Perno2 and MM Santoro1

1University of Rome Tor Vergata, Rome, Italy; 2L. Spallanzani Hospital, Rome, Italy; 3Complejo Hospitalario Universitario de Granada, Granada, Spain; 4Polyclinic of Modena, Modena, Italy; 5IRCSS San Gallicano, Rome, Italy; 6Azienda Ospedaliera Universitaria Careggi, Florence, Italy; 7UPMC University Paris 06-UMR_S 1136 Pierre Louis Institute of Epidemiology and Public Health, Paris, F-75013 France; 8INSERM-UMR_S 1136 Pierre Louis Institute of Epidemiology and Public Health, Paris, F-75013 France; 9AP-HP, Groupe hospitalier Pitié Salpêtrière, Paris, F-75013 France; 10AO Papa Giovanni XXIII Bergamo, Bergamo, Italy; 11University of Turin, Turin, Italy; 12Second University of Naples, Naples, Italy; 13University Hospital Tor Vergata, Rome, Italy

BACKGROUND: The presence of natural protease polymorphisms characteristic of HIV-1 non-B subtypes might be predictive of a poorer response to protease inhibitors (PIs). Thus, we evaluated the virological

response to first-line HAART containing a ritonavir-boosted PI (PI/r) among patients from Italy, Spain and France infected with HIV-1 subtypes CRF02_AG, C or F compared with those infected with subtype B.

METHODS: The impact of HIV-1 subtype on virological success (VS, HIV-RNA<50 copies/mL after therapy starting) and virological rebound (VR, 2 consecutive HIV-RNA>50 copies/mL after VS) was evaluated by survival analysis. The baseline prevalence of subtype associated protease polymorphisms and the resistance at failure were explored.

RESULTS: 1283 patients were analyzed: 938 infected with B subtype and 345 with non-B subtype (CRF02_AG: 50.8%; F: 29.8%; C: 19.4%).

At baseline, stratification based upon subtypes shows a different distribution of the proportion of patients with viremia >500,000 copies/mL (C: 31.3%; F: 21.4%; CRF02_AG: 21.1%, B: 15.8%, p<0.001) or CD4<200 cells/mm3 (C: 55.2%; F: 54.4%; CRF02_AG: 44.0%, B: 43.6%, p=0.063).

By 12 months of treatment, CRF02_AG infected patients showed a lower probability of VS compared to others (C: 91%; B: 90%; F: 84%; CRF02_AG: 80%; p=0.001).

CRF02_AG infected patients also showed a trend to a higher probability of VR by 24 months after VS, compared to other patients (CRF02_AG: 23%; F: 19%, B: 17%; C: 17%; p=0.088). Adjusting for gender, age, pre-therapy CD4/HIV-RNA, treatment and transmitted drug-resistance, CRF02_AG infected patients showed both a lower relative hazard of achieving VS (RH [95%C.I.]: 0.81[0.68-0.98], p=0.028) and a higher relative hazard of experiencing VR (RH [95%C.I.]: 1.66[1.11-2.49], p=0.013) compared with subtype B infected patients.

At baseline, among 188 natural polymorphisms detected in CRF02_AG infected patients, 25 showed a significantly higher prevalence in CRF02_AG compared to B subtype. Among them, the co-presence of the 3 mutations K20I, K70R and L89M (typical of



CRF02_AG) in the overall population was associated with both the lowest probability of VS by 12 months of treatment and the highest probability of VR by 24 months from VS (0 vs.1 vs. 2 vs. 3 mutations: VS, 90% vs. 82% vs. 84% vs. 78%, p<0.001; VR: 17% vs. 20% vs. 19% vs. 30%, p<0.001).

Among 105 patients treated with PI/r containing HAART regimens (all including 3TC/FTC) and with an available genotype at failure, CRF02_AG infected patients showed a higher prevalence of M184V mutation compared to subtype B infected patients (19.0% vs. 3.6%,p=0.028).

CONCLUSION: Patients infected with CRF02_AG show a poorer response to a first line PI/r based treatment compared to other subtypes (including B). The co-presence of polymorphisms K20I, K70R and L89M correlates with this phenomenon. Further investigations are needed to clarify these observations that may modulate the use of certain, less-effective, PI/r-based regimens.

ABSTRACT 32

Resistance Emergence in Treatment-Naive Patients Receiving E/C/F/TAF for 96 WeeksC Callebaut, NA Margot, M Das, MW Fordyce, and MD Miller


BACKGROUND: Tenofovir alafenamide (TAF) is a novel prodrug of the NtRTI tenofovir (TFV) that loads lymphocytes with TFV-diphosphate more efficiently than tenofovir disoproxil fumarate (TDF). The single-tablet regimen (STR) composed of elvitegravir/cobicistat/emtricitabine/TAF (E/C/F/TAF) has demonstrated non-inferiority to the STR of E/C/F/TDF in clinical studies, with high proportions of patients achieving HIV-1 RNA <50 copies/mL at week 48 (92.5% vs. 90.8%, for E/C/F/TAF and E/C/F/TDF groups, respectively) that were maintained through

week 96 (86.6% vs. 85.2%, for E/C/F/TAF and E/C/F/TDF groups, respectively). With 91% lower plasma levels of TFV, an improved renal and bone safety profile was observed for E/C/F/TAF group as compared to E/C/F/TDF. An integrated resistance analysis across 3 Phase 2/3 clinical studies is described.

METHODOLOGY: HIV-1 genotypic testing was conducted at screening using commercial assays to assess HIV-1 PR/RT/IN sensitivity to study drugs. For patients with HIV-1 RNA >400 copies/mL at time of virologic failure (VF) or early discontinuation, genotypic and phenotypic susceptibility to ARVs was evaluated (Monogram Biosciences).

RESULTS: Most of the 1903 patients had HIV-1 subtype B (87%), followed by subtype AE (6.7 %). Pre-existing primary resistance-associated mutations (RAMs) at baseline were observed: 7.5% had NRTI-RAMs, 18.2% had NNRTI-RAMs, and 3.4% had PI-RAMs. HIV-1 subtype or the presence of baseline NRTI or NNRTI-RAMs did not influence E/C/F/TAF treatment response at Week 96 (p values > 0.05); a small numerical difference was noted for PI-RAMs (72% vs. 87%, p=0.04), likely driven by the small number of patients (n=29). Through week 96, VF resistance analyses were conducted for 27 patients in each group (2.8%, 27/978; and 2.9%, 27/925; for E/C/F/TAF and E/C/F/TDF, respectively). Resistance development was rare, with 10 patients in each group (E/C/F/TAF: 1.0%, 10/978; E/C/F/TDF: 1.1%, 10/925) developing primary resistance to ARVs of the regimen (E/C/F/TAF: M184V/I, n=9; INSTI-RAMs, n=8; K65R/N, n=2; E/C/F/TDF: M184V/I, n=8; INSTI-RAMs, n=6; K65R/N, n=3; K70E, n=1). Overall, there were similar patterns of emergent mutations in each treatment group, with M184V/I being the most frequent.

CONCLUSIONS: E/C/F/TAF achieved a high level of virologic suppression in HIV-1 treatment-naïve patients through 96 weeks of treatment. Resistance development was rare (E/C/F/TAF 1.0 % vs. E/C/F/TDF 1.1%), with comparable genotypic changes.



ABSTRACT 33

Pooled Week 48 Analysis of HIV-1 Drug Resistance in E/C/F/TAF Phase 3 StudiesME Abram, NA Margot, S Cox, R Ram, DP Porter, KM Kitrinos, MW Fordyce, S McCallister, MD Miller, and C Callebaut


BACKGROUND: Seven ongoing Phase 3 studies are evaluating the efficacy and safety of the elvitegravir (E)/cobicistat (C)/emtricitabine (F)/tenofovir alafenamide (TAF) fixed dose combination (E/C/F/TAF) in ART-naive adult (GS-US-292-0104 and GS-US-292-0111) and adolescent (GS-US-292-0106) subjects, virologically suppressed subjects with (GS-US-292-0119) or without (GS-US-292-0109) ≥2 class resistance, subjects with mild to moderate renal impairment (GS-US-292-0112), and subjects with HIV/HBV co-infection (GS-US-292-1249). Virologic success rates of E/C/F/TAF at Week 48 using FDA snapshot analysis and HIV-1 RNA < 50 copies/mL was high and similar among all studies (86.6-97.2%) and showed non-inferiority to comparator arms. Here we present a pooled Week 48 resistance analysis for these Phase 3 studies across the different treatment populations.

METHODS: Genotypic analyses were performed at screening to assess HIV-1 protease (PR), reverse transcriptase (RT) and integrase (IN) susceptibility to study drugs. Confirmed virologic failure visits through Week 48 or at discontinuation with ≥400 copies/mL HIV-1 RNA were analyzed for emergent genotypic and phenotypic resistance.

RESULTS: A total of 2308 subjects were enrolled in these E/C/F/TAF studies. Among ART-naive adults, 16 of 866 were analyzed; 7 (0.8%) developed NRTI RAMs (M184V/I, n=7; K65R, n=1) and also primary INSTI RAMs (T66I/A, n=2; E92Q, n=2; Q148R, n=1, N155H, n=1). Among ART-naive adolescents, 2 of 50 subjects were analyzed and did not develop RAMs. Among virologically suppressed subjects, 4 of 959

were analyzed; 1 developed resistance (M184M/I) and resuppressed to < 50 copies/mL before treatment discontinuation. Among virologically suppressed subjects with prior ≥2 class resistance, none of the 110 subjects met the analysis criteria. Among renally impaired subjects, 2 of 248 were analyzed; both subjects had multi-class resistance detected: 1 pre-existing and 1 due to possible re-infection followed by resuppression to < 50 copies/mL. Among HBV co-infected subjects, 0 of 75 subjects met the analysis criteria.

CONCLUSIONS: In these 7 Phase 3 studies, E/C/F/TAF achieved high rates of virologic suppression through 48 weeks of treatment. The presence of PI, NRTI, or NNRTI RAMs at baseline did not affect treatment response. Resistance development to ≥1 components of E/C/F/TAF was rare in all studied populations, even in highly treatment-experienced subjects switching to E/C/F/TAF.

ABSTRACT 34

Pre-ART HIV-DNA Correlates with Viro-Immunologic Status and Outcome in Patients with 1st-Line ARTF Ceccherini-Silberstein1, A Cozzi Lepri2, E Merlini3, M Surdo1, G Marchetti3, MR Capobianchi4, A De Luca5, N Gianotti6, G Viale7, A Antinori4, CF Perno4, A D’Arminio Monforte3 for the ICONA Foundation

1University of Rome “Tor Vergata”, Rome, Italy; 2University College London, UK; 3San Paolo Hospital, University of Milan, Milan, Italy; 4INMI “L.Spallanzani”, Rome, Italy; 5University Hospital of Siena, Siena, Italy; 6San Raffaele Scientific Institute, Milan, Italy; 7University of Bologna, Bologna, Italy

BACKGROUND: The clinical relevance of archived

resistance is still on debate. This depends also on the

quantity of HIV DNA and how this is measured. HIV DNA

remains the most sensitive measure of viral burden and

residual infection. By using a commercial assay, we aimed

to investigate the correlation of pre-ART (baseline, BL) HIV

DNA with the BL viro-immuno-clinical status and on the



viro-immuno-clinical response to ART in patients starting

their first regimen.

METHODS: HIV+ patients of the ICONA cohort, starting

1st-line ART, for whom PBMC or blood sample was stored at

BL were analysed. Total HIV-DNA was quantified by using

a modified version of the Cobas HIV-1 test (Surdo 2015).

Results were normalized by CD4+ cells number. Associations

between BL DNA levels and patients BL characteristics were

evaluated using chi-square/signed-rank test and Pearson

correlation. In a subset of patients starting “modern”

ART (after 2004), standard survival analysis was used to

examine the association between BL HIV-DNA and pre-

defined time to event endpoints: viral load (VL) ≤50 cp/

mL, virological rebound defined by a confirmed VL>50 cp/

mL after VL≤50 cp/mL, gain in CD4 count >200 cells/mm3

after ART and AIDS diagnosis or serious non-AIDS or death.

Kaplan-Meier curves and Cox regression models were also

used.

RESULTS: We included 607 patients (23% female, 38

years median age, median [IQR] CD4 = 288 [144-401],

who started ART on median [IQR] = 2010 [2002-2011]. BL

median [IQR] HIV-DNA and HIV-RNA was 10574 [3208-

38218] cp/106 CD4+ and 4.83 [4.31-5.33] log cp/mL,

respectively. According to BL HIV-DNA levels (divided in

3 groups: 10-1000 cp/106 CD4+, n=69, 1000-10000 cp/106

CD4+, n=224, and >10000 cp/106 CD4+, n=314), a strong

significant correlation (p<0.001) was observed with BL HIV-

RNA (Pearson rho=+0.41), CD4 (-0.48) and CD4/CD8 ratio

(-0.40) and less strong (p<0.03) with IL-6 (+0.15), sCD14

(+0.11) and CD8 (-0.09).

By week 48, 393/607 (65%) achieved HIV-RNA ≤50 cp/

mL (290/395, 73% in patients starting ART after 2004).

Within this last subset, median (95% CI) times to a HIV-RNA ≤50 cp/mL were 7 (4-11) months in people with 10-

1000 HIV-DNA cp/106 CD4+, 8 (5-11) months in those with

1000-10000 cp/106 CD4+ vs. 10 (7-15) months in those with

>10000 cp/106 CD4+ (p=0.0008). Unadjusted and adjusted

hazard ratios of the pre-defined outcomes from fitting the

Cox models are shown in Table.

CONCLUSIONS: HIV DNA can be quantified by using CAP/

CTM HIV-1 test in both whole-blood and PBMC samples.

Pre-ART HIV-DNA normalized for CD4+ cells strongly

correlated with BL viro-immunologic parameters and

inflammation markers. BL HIV-DNA was also found to

predict virological and clinical outcome of 1st-line ART,

although not independently of HIV-RNA and CD4 count.

Other studies are warranted to understand whether

measuring HIV reservoirs could help individualizing

therapy for initiation and maintenance.

Table hazard ratios of various outcomes per log10 106/CD4 HIV-DNA higher levels in patients starting 1st line ART after 2004

Hazard Ratio (95% CI)p-value

OutcomesUnadjusted Adjusted

(a)Adjusted(b)

Adjusted(c)

Vi ra l load ≤50 cp/mL

0.71 (0.62, 0.82)

0.73 (0.63, 0.85)

0.86 (0.72, 1.02)

0.89 (0.74, 1.06)

P<.001 P<.001 P=0.074 P=0.197

CD4 count ga i n >20 0 cells/mm3 above pre-ART

1.01 (0.88, 1.16)

1.00 (0.86, 1.16)

0.88 (0.74, 1.05)

0.87 (0.73, 1.04)

P=0.896 P=0.994 P=0.156 P=0.133

Viral rebound >50 cp/mL

2.14 (1.41, 3.25)

1.99 (1.29, 3.09)

1.45 (0.88, 2.39)

1.23 (0.71, 2.12)

P<.001 P=0.002 P=0.142 P=0.459

AIDS, serious non-A I D S a n d death

2.14 (1.13, 4.05)

2.08 (1.00, 4.33)

1.49 (0.69, 3.20)

1.53 (0.59, 3.95)

P=0.019 P=0.051 P=0.309 P=0.380

(a) Adjusted for calendar year of ART initiation and type of regimen started

(b) Adjusted for viral load and CD4 count at ART initiation

(c) Adjusted for all factors in footnotes a-b, + age, smoking, HCV status, IL-6 and sCD14.



ABSTRACT 35

No Effect of HIV-1 Subtype C on Virological Failure Rate with First-Line TDF RegimensE White1, E Smit2, D Churchill3, S Collins4, C Booth5, A Tostevin1, C Sabin6, D Pillay7, D Dunn1 on behalf of UKHDRD and UKCHIC

1MRC CTU at UCL, London, United Kingdom; 2Public Health England, Birmingham Heartlands Hospital, Birmingham, United Kingdom; 3Brighton and Sussex Hospitals NHS Trust, Brighton, United Kingdom; 4HIV i-Base, London, United Kingdom; 5Royal Free London NHS Foundation Trust, London, United Kingdom; 6Department of Infection and Population Health, UCL, London, United Kingdom; 7Division of Infection and Immunity, UCL, London, United Kingdom

BACKGROUND: In vitro and clinical studies have shown that subtype C viruses have a greater propensity to develop a K65R mutation due to polymorphisms at codons 64-66. This has potentially important public health implications given that subtype C infection accounts for around 50% of HIV infections worldwide and with the expanded use of tenofovir (TDF) as per WHO 2013 recommendations. We have exploited the wide diversity of viral subtypes within the UK to examine whether viral subtype influences the rate of virological failure (VF) on first-line TDF-containing regimens.

METHODS: Patients were included if HIV care was received at a participating clinic in the UK CHIC study; their first-line regimen was TDF+(XTC)+(EFV, NVP, LPV/r, DRV/r or ATV/r); and ≥2 viral loads (VLs) measured after 6 months following ART initiation. Subtypes were defined according to Rega-3, based on resistance tests conducted pre-therapy or at treatment failure. Time to VF (2 consecutive VLs >200 copies/ml after 6 months of ART) was analysed using Cox models, adjusting for demographic factors, baseline CD4 and VL, ART regimen, and year of initiation. Follow-up was censored at last VL or discontinuation of TDF. Multiple imputation was used to include patients with missing subtypes, taking advantage of the strong association with demographic factors. Tenofovir associated resistance was compared between

subtypes for patients with ≥1 major IAS mutation found at time of VF.

RESULTS: 8746 patients were included and followed for a median of 3.3 years; 5465 (4123 observed, 1342 average of imputed) were subtype B, 1455 (823, 632) subtype C, and 1826 (1203, 623) non-B/non-C. Subtype B patients were mostly white (83%) and MSM (85%) while subtype C mostly black (70%) and heterosexual (79%). Subtype non-B/non-C patients were demographically more mixed (35% white, 53% black; 26% MSM, 63% heterosexual). Risk of VF for subtype non-B/non-C (173, 9.5%) was similar to subtype C (142, 9.8%) (aHR=1.1, 95% CI 0.8-1.4). In unadjusted analyses, patients with subtype B infection had a much lower risk of VF (309, 5.7%) than subtype C (HR=0.5, 95% CI 0.4-0.7). However this difference was markedly reduced in adjusted analyses (aHR=0.9, 95% CI 0.6-1.2, P=0.41), largely mediated by the effects of exposure group and ethnicity (Figure). K65R was observed in 14% (6/43) of subtype B patients, 33% (13/39) of subtype C and 16% (4/25) of non-B/non-C (P=0.08). K70E was rarely observed in all subtype groups (5% overall).

CONCLUSIONS: Although patients infected with subtype C virus on a first-line TDF containing regimen experienced a higher rate of VF, this was explained by demographic factors rather than a subtype effect per se. This is a reassuring finding for expanded use of TDF in southern Africa, India, and other areas where subtype C virus predominates.



ABSTRACT 36

Impact of Baseline Genotypic Resistance and Tropism on cART Outcomes in HIV-Positive Subjects Diagnosed during Primary HIV InfectionP Tau1, M Fabbiani2, M Ripa3, E Focà4, S Nozza3, A Bandera2, A Muscatello2 and S Rusconi1 on behalf of the INACTION study group

1Divisione Malattie Infettive, DIBIC Luigi Sacco, Università degli Studi di Milano; 2Divisione Malattie Infettive, Ospedale San Gerardo, Università degli Studi di Milano - Bicocca; 3Divisione Malattie Infettive, Ospedale San Raffaele, Università Vita-Salute, Milano; 4Divisione Malattie Infettive, Spedali Civili, Università degli Studi di Brescia, Italy

BACKGROUND: Diagnosis and treatment during the early stages of HIV infection (stage I to V according to Fiebig classification), is crucial for the reconstitution of the immune system and the control of viral replication.

METHODS: Patients with primary HIV infection (PHI) were enrolled between 2008-2014 in an Italian Network AcuTe HIV InfectiON (INACTION). In this study we present an analysis including 54 patients from 3/24 centers with available genotypic sequence data. Immunological and virological data were collected since Baseline (BL). Patients that started cART within 3 months since PHI diagnosis were classified as early cART group, while other patients were included in late/no cART group. Characteristics of groups (X4 vs R5) were compared using Student T-test and continuous variables are described as median (IQR). Logistic regression analysis was performed to identify factors associated to early cART initiation. Cox regression analysis was performed to determine predictors of time to virological suppression after cART initiation and determine predictors of time to first-line regimen discontinuation. Moreover, the same analysis was performed to determine predictors of time to first CD4 >500 cells/mmc. Estimated scores of the total antiretroviral activity of a combination antiretroviral regimen was analyzed with GSS. Gp120 V3, INT and RTPR sequences were analyzed using the

Geno2pheno algorithm. P-value <0.05 was considered statistically significant.

RESULTS: The majority of patients were diagnosed in Fiebig V stage and symptomatic patients were 85.2%, which most common symptom was fever. Median plasma HIV-RNA was 5.80 log10copies/ml while median BL CD4 T-cell was 444 cells/µl. During follow-up, patients that started cART was 98.1%, of which 90.7% initiated during the first 3 months. The majority of patients was R5 (72.3%) than X4 (27.7%). At univariate analysis genotypic parameters (X4 vs R5) were evaluated in function of three outcomes: early cART initiation, time to virological suppression and time to first-line regimen discontinuation. Results demonstrated that X4 vs R5 tropism was not associated with these 3 outcomes. Moreover, univariate analysis showed that genotypic susceptibility scores for reverse transcriptase, protease and combined were not associated with these outcomes. As far as the immunological outcome, this was not associated to GSS and genotypic parameters but with the early cART initiation.

CONCLUSION: Our preliminary data show a predominant presence of R5-tropic viruses in the acute phase of HIV infection and suggest that the presence of resistance in pol gene does not correlate with cART outcomes. R5 vs X4 tropism is not associated with early cART initiation, time to virological suppression and time to first-line regimen discontinuation.



ABSTRACT 37

Is Resistance Testing of Value after First-Line ART Failure in Resource-Limited Settings? - Insights from ACTG 5273 L Harrison1, A La Rosa2, RV Viana3, B Taiwo4, EK Halvas5, L Zheng1, A Collier6, J Mellors5, and CL Wallis3

1Harvard TH Chan School of Public Health, Boston, Massachusetts, USA; 2Asociacion Civil Impacta Salud y Educacion, Lima, Peru; 3BARC-SA and Lancet Laboratories, Johannesburg, South Africa; 4Northwestern University, Chicago, USA; 5University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 6University of Washington, Seattle, Washington, USA

BACKGROUND: Over 15 million people have initiated antiretroviral treatment (ART) in resource-limited settings (RLS), the majority with a WHO-recommended regimen of two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) and a non-nucleoside reverse transcriptase inhibitor (NNRTI). We describe HIV-1 drug resistance characteristics among individuals in RLS from 3 continents with virologic failure on WHO-recommended first-line ART.

METHODS: Participants from India, Malawi, South Africa, Kenya, Zimbabwe, Tanzania, Brazil, Peru and Thailand failing first-line ART were enrolled into the ACTG 5273 study of second-line ART. Entry HIV-1 drug resistance and subtype were determined retrospectively from RT sequences generated by NIAID virology quality assessment (VQA)-certified laboratories and interpreted using the IAS-USA July 2014 mutations list. Univariable and multivariable logistic regression models assessed associations between clinical characteristics at first-line failure and 3 outcomes; presence of ≥3 NRTI mutations, ≥3 NNRTI mutations and K65R.

RESULTS: Of the 512 participants enrolled, 490 (96%) had available sequences, median CD4 was 135 cells/mm3, HIV-1 RNA was 4.5 log10copies/ml and time on first-line ART was 4.1 years (range 6 months-11.4

years). Initial first-line ART was d4T/3TC/NVP for 49%, ZDV/3TC/NVP for 13% and TDF/3TC/EFV for 12%. Many (50%) substituted drugs such that at study entry participants were on TDF/3TC/EFV (24%), ZDV/3TC/NVP (23%), d4T/3TC/NVP (21%) and TDF/3TC/NVP (12%); only 18% had been exclusively on TDF with 3TC/FTC. Subtype C was predominant (396, 81%), followed by A1 (47, 10%), B (18, 4%), D (11, 2%) and other (18, 4%). Overall, 465 (95%) had NRTI and NNRTI resistance; 257 (52%) had ≥3 NRTI mutations and 231 (47%) had ≥3 NNRTI mutations. Longer time on first-line ART, higher entry HIV-1 RNA and lower CD4 were independently associated with ≥3 NRTI mutations; whereas, younger age, TDF use and ≥3 TAMs were independently associated with ≥3 NNRTI mutations (Table). K65R was present in 107 (22%); 63 (70%) treated with TDF, 39 (38%) with both TDF and ZDV/d4T and 5 (2%) with ZDV/d4T. Shorter time on first-line ART, TDF exposure and <3 TAMs, but not subtype C, were independently associated with K65R (Table).

CONCLUSION: In contrast to previous studies, most (95%) individuals in the current study with first-line failure had NRTI and NNRTI resistance and half had extensive resistance (≥3 NRTI or NNRTI mutations). As previously reported, extensive NRTI resistance was associated with longer duration of NRTI use and more advanced HIV. By contrast, extensive NNRTI resistance was independently associated with TDF exposure, multiple TAMs, and shorter duration of ART. K65R was strongly associated with TDF exposure and fewer TAMs but not independently with HIV-1 subtype C. Although resistance profiles at first-line failure varied by NRTI exposure, dual-class resistance was nearly always present, suggesting that resistance testing is not needed to decide whether to switch to second-line ART.



ABSTRACT 38

Protease Inhibitor Resistance at 2nd-Line HIV Treatment Failure in Sub-Saharan Africa TS Boender1, RL Hamers1,2, P Ondoa1, M Wellington3, C Chimbetete3, M Siwale4, EEF Labib Maksimos5, SN Balinda6, CM Kityo6, TA Adeyemo7, AS Akanmu7, K Mandaliya8, ME Botes9, WS Stevens10, TF Rinke de Wit1, and KCE Sigaloff1, 2

1Amsterdam Institute for Global Health and Development, Department of Global Health, Academic Medical Center of the University of Amsterdam, Amsterdam, Netherlands; 2Department of Internal Medicine, Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; 3Newlands Clinic, Harare, Zimbabwe; 4Lusaka Trust Hospital, Lusaka, Zambia; 5Coptic Hospital, Lusaka, Zambia; 6Joint Clinical Research Centre, Kampala, Uganda; 7Department of Haematology & Blood transfusion, College of Medicine of the University of Lagos, Lagos, Nigeria; 8Coast Province General Hospital, Mombasa, Kenya; 9Muelmed Hospital, Pretoria, South Africa and 10Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg and the National Health Laboratory Service, South Africa

BACKGROUND: As antiretroviral therapy (ART) programs in sub-Saharan Africa mature, increasing numbers of HIV-positive people will experience treatment failure, and require second- or third-line ART. It is yet unclear how many patients will develop protease inhibitor (PI) resistance and need third-line ART regimens.

METHODS: HIV-1 positive adults were enrolled in the PanAfrican Studies to Evaluate Resistance Monitoring (PASER-M) cohort, at the time of switch to second-line PI-based ART, and included in the analysis if they received >180 days of second-line ART. We assessed risk factors for virological failure (viral load >400 cps/ml) after up to 3 years of second-line PI-based ART using Cox models. If viral load was ≥1,000 cps/ml, pol genotyping was performed. Drug resistance mutations were scored using the 2014 IAS-USA drug mutation list and genotype susceptibility was calculated using the Stanford algorithm Version 7.0.

RESULTS: Of 227 included participants, 25.0% (n=54/216) experienced virological failure at some point during follow-up at a rate of 138.9 failures (95%CI 106.4-181.3) per 1,000 person-years. In multivariable analysis the risk factors for virological failure were: failing a non-standard non-nucleoside reverse transcriptase inhibitor (NNRTI)-based first-line regimen (hazard ratio [HR] 7.10; 95%CI 3.40-14.83; p<0.001) or PI-based first-line regimen (HR 7.59; 95%CI 3.02-19.07; p=0.001) compared to ZDV/3TC/NNRTI, PI-resistance at switch (HR 6.69; 95%CI 2.49-17.98; p<0.001) and <95% adherence (HR 3.05; 95%CI 1.71-5.42; p=0.025). For 32/43 (74%) participants with VL≥1,000 cps/ml during follow-up, genotypic data was available. At least one drug resistance mutation was found among 22/32 (69%) participants. Major PI mutations were detected in 7 (21.9%) participants (table). The acquired mutations conveyed reduced susceptibility to all PIs (figure).

CONCLUSIONS: While over 85% of participants on 2nd-line ART had viral suppression after up to 36 months, major PI resistance was detected in 22% of those failing second-line ART. This represents approximately 3% of people initiating 2nd-line ART. Future treatment of these individuals require third-line drugs (i.e. darunavir/ritonavir, etravirine and raltegravir), which are currently unavailable in sub-Saharan Africa. To ensure long-term ART success, availability of third-line drug options, preferably guided by HIV drug resistance testing, is urgently needed.



ABSTRACT 39

HIV-1 Drug Susceptibility to Newer Second- and Third-Line Antiretroviral Regimens in CameroonAJ Nanfack1,2, D Takou1, J Fokam1,3, R Salpini3, MM Santoro3, G Cappelli4, M Baane5, SM Tetang5, FC Silberstein3, JN Torimiro1, V Colizzi1,3, CF Perno3, A Ndjolo1

1CIRCB, Yaoundé, Cameroon; 2New York University, New York, USA; 3University of Rome Tor Vergata, Rome, Italy; 4National Research Council, Rome, Italy; 5National Social Insurance Fund Hospital, Yaoundé, Cameroon

BACKGROUND: Scale-up of antiretroviral therapy (ART) and the growing number of long-term treated patients in low and middle income countries (LMIC) may favor multi-HIV drug resistance (HIVDR). Strategies to minimize its spread are essential in maintaining a successful ART Program. This requires a close monitoring of patients to ensure timely and appropriate regimen switch soon after virological failure, which in turn sustains the effectiveness of standard first and second-line treatment combinations. Understanding the burden of HIVDR with ART-exposure will provide new insights for an effective long-term management of infected patients in LMIC.

METHODS: Sixty-six HIV-infected individuals (18 ART-naïve, 24 failing first-line, 24 failing second-line ART) living in Yaoundé-Cameroon were evaluated by sequencing of protease-reverse transcriptase (PR-RT, n=62), envelope-V3 loop (V3, n=58) and integrase (IN, n=30) regions. Drug resistance mutations (DRMs) were interpreted using Stanford University HIV drug resistance database and geno2pheno, while viral tropism prediction was done using geno2pheno, position-specific scoring matrices (PSSM) and Net charge rule.

RESULTS: All our study subjects were infected with HIV-1 non-B subtypes with a predominance of CRF02_AG (58%). Mean viremia was 5.17 log10 copies/ml and median CD4 was 147 cells/µL. Among ART-naïve

patients, 6.7% had DRM to non-nucleoside reverse transcriptase inhibitors (NNRTIs), of note K103N was the only major mutation detected, 28.6% had integrase accessory-mutations (L74I, E157Q), and 26.7% carried CXCR4-tropic viruses. At first-line failure, 79.2% had DRMs to nucleoside and non-nucleoside reverse transcriptase inhibitors (with 50% multi-DRMs). 33.3% had IN accessory-mutations (L68I, L74I, T97A, E157Q), and 47.4% carried CXCR4-tropic viruses. At second-line failure, 91.3% had multi-DRMs to PR-RT inhibitors with 52.2% and 4.3% DRMs to second-generation NNRTIs (etravirine and rilpivirine) and darunavir/r, respectively; 27.3% had IN accessory-mutations (L74I, T97A, E157EQ), and 37.5% carried CXCR4-tropic viruses.

CONCLUSION: Rate of HIVDR increases with long-term ART exposure, especially from first- to second-line regimen. Integrase inhibitors (raltegravir and elvitegravir) and darunavir/r are suitable third-line drugs for an effective public health approach in LMIC, while the use of second generation NNRTIs or CCR5 antagonists requires genotypic testing.



ABSTRACT 40

Drug Resistance and Tropism as Markers of the Dynamics of HIV-1 DNA Quasispecies in Blood Cells of Heavily Pre-Treated Patients who Achieved Sustained Virological SuppressionP Gantner1,2, L Morand-Joubert3, C Sueur2, F Raffi4, C Amiel5, S Lambert-Niclot6, DB Fofana3, JP Viard1,7, S Fafi-Kremer2, C Rouzioux1,8, V Avettand-Fenoel1,8, J Ghosn1,7

1University Paris Descartes, EA7327, Paris, France; 2Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 3Sorbonne University, Paris, France; Hôpital Saint-Antoine, Paris, France; 4CMIT, Paris, France; 5University Paris 06, Paris, France; Tenon Hospital, Paris, France; 6UMR_S 1136, INSERM et Sorbonne Universités, UPMC Univ Paris 06 ; AP-HP, Department of Virology, Hôpital de la Pitié-Salpêtrière, Paris France; 7Centre Hospitalier Universitaire Hôtel-Dieu, Paris, France; and 8Centre Hospitalier Universitaire Necker Enfants Malades, Paris, France

BACKGROUND: Antiretroviral treatment modification is challenging in heavily treated patients harboring viruses with drug-resistance associated mutations (DRAMs) who are currently virologically controlled under treatment. Change over time of archived resistant quasispecies in cell-associated HIV-1 DNA in such a situation needs to be better studied.

METHODS: We analyzed frozen blood cells from heavily pretreated patients enrolled in the INNOVE and ANRS 123 ETOILE studies who achieved sustained viral suppression after salvage optimized therapy (SOT). Longitudinal ultra-deep 454/pyrosequencing (UDPS) analysis of reverse transcriptase-protease Pol and V3 Env regions was performed on blood cell-associated HIV-1 DNA samples (1% sensitivity). Protease inhibitors (PI), nucleoside (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors DRAMs and tropism were interpreted using the ANRS and Geno2Pheno algorithms.

RESULTS: Samples (n=29) were available at baseline, 6 and ≥12 months after SOT initiation in ten patients.

At the last sequencing time point, patients were virologically controlled for a median duration of 45 months (range, 9-69). Overall, we found 125 and 51 variants, including 67% and 27% of minority variants (<20% of quasispecies) for Env and Pol regions, respectively. V3 loop sequences displayed wide intra-individual dynamics over time.

Viral variants harboring DRAMs exhibited three non-exclusive scenarios (Figure). First, when SOT exerted the same selective pressure as previous failing regimens, some viral quasispecies still harbored the same DRAMs at the same level than at the time of prior virological failure (Patients n°1, 2, 4, 5). Thus, as DRAMs were mostly linked on the same viral variant, variants with a complete resistance pattern were maintained archived. Second, some viral variants harboring DRAMs were no longer detected over time when SOT consisted in new antiretroviral classes or with resistance profiles distinct from those of previous failing regimens (Patients n°3, 6, 8, 9, 10). Third, variants with new DRAMs associated with SOT emerged in blood cells during follow-up despite sustained virological control (Patients n°3, 5, 7, 10). For example, patient n°7 harbored a major L100I and L76V viral variant after 72 months on an etravirine and darunavir-containing SOT.

Given that most patients experienced an important decrease in cell-associated HIV-1 DNA level over time (median decreased at month 6 of -0.36 log10 copies/106 peripheral blood mononuclear cells; p=0.004), the archived resistant viral DNA load decreased as well.

CONCLUSION: Using longitudinal UDPS analysis and focusing on DRAMs and tropism as markers in HIV-1 DNA, we were able to evidence more than 25% minority variants in heavily pretreated patients who achieved sustained viral suppression. We demonstrated that, despite sustained virological control in blood, archived HIV-1 DNA quasispecies continued to evolve while decreasing. We also showed that archived virus with DRAMs can persist for lengthy periods of time, which should be taken into account when modifying treatment, even after many years of virological suppression.



phylogenetically within individuals. The Table compares drug resistance results using standard sequencing (Sanger) and a K65R ASPCR for RNA and DNA, among drug naïve (row 1; 64% female; median age 26 years; median viral load (VL) 62,053 copies/mL) and experienced (row 2; 54% female; median age 38 years; median VL 102,920 copies/mL) participants. AZT intermediate/high predicted resistance was seen in 22% and 15% of failing participants’ RNA and DNA, respectively. RNA/DNA mutation discordance in treatment experienced patients was seen in 65% (43% more mutations in RNA; 9% in DNA; 13% both). ASPCR identified 65R mutations not detected in Sanger in 17% RNA and 48% DNA failure samples at a >1% threshold, 7% and 30% at >2%, 2% and 9% at >5% and 0% and 7% at >10%.

CONCLUSION: In this study TDR was low, with high frequency of low level (<5%) K65R, suggesting ASPCR overestimation of K65R minority variants. In contrast ADR was high, though with low (26%) K65R occurrence for subtype C, and evidence for higher levels (>5%) of K65R minority variants, particularly in DNA. Data support the potential value of point mutation resistance assays to detect significant resistance, e.g. identify those who might benefit from AZT and/or TDF in subsequent regimens; as well as the importance of early identification of treatment failure before resistance accumulation and archival.

ABSTRACT 41

HIV RNA/DNA Drug Resistance in Naïve and Tenofovir-Treated Patients in Chennai, India S Saravanan1, TR Dinesha1, S Poongulali1, N Kumarasamy1, SS Solomon, L Ledingham2, A Derache3, M Coetzer2, D Katzenstein4, R Kantor2

1YRG CARE, Chennai, India; 2Brown University, Providence, RI, United States; 3 Africa Centre for Health and Populations studies, KwaZulu Natal,South Africa; 4Stanford University, Stanford, CA

BACKGROUND: Drug resistance poses a challenge among treatment naïve and experienced HIV-infected persons through transmitted (TDR) and acquired drug resistance (ADR) mutations. Treatment success may be further compromised by archived mutations and minor resistance variants, not detected by standard genotyping. More sensitive lower cost point mutation resistance assays may enhance resistance detection in resource limited settings.

METHODS: We examined reverse transcriptase TDR and ADR in drug naïve and tenofovir (TDF)-treated HIV-infected persons at YRG CARE (Chennai, India) using standard Sanger sequencing, and allele-specific-PCR (ASPCR; implemented at YRG-CARE) for K65R minority variants, in plasma (RNA) and peripheral blood mononuclear cells (DNA). TDR mutations were based on the World Health Organization surveillance drug resistance mutation list and ADR mutations were based on the Stanford Database.

RESULTS: Participants included 83 adults (≥18 years), 37 drug naïve and 46 on TDF-based 1st-line (n=26) or 2nd-line (n=20) regimens. All 166 RNA and DNA sequences were subtype C and clustered

RNA Sanger RNA ASPCR K65R

DNA Sanger DNA ASPCR 65R

% with Any Resistance

% with K65R

% with >1%

% with >2%

% with >5%

% with >10%

% with Any Resistance

% with K65R

% with >1%

% with >2%

% with >5%

% with >10%

Naïve (n=37) 5 0 38 14 3 0 3 0 35 8 3 0

Treated (n=46) 96 26 37 24 20 15 89 17 59 43 26 15



ABSTRACT 42

Resistance in PBMCs Can Predict Virological Rebound after Therapy Switch in cART-Treated Patients with Undetectable HIV-RNAD Armenia1, M Zaccarelli2, V Borghi3, W Gennari3, A Giannetti2, F Forbici2, C Pinnetti2, F Ceccherini Silberstein1, C Mussini3, CF Perno2, A Antinori2, and MM Santoro1

1University of Rome Tor Vergata, Rome, Italy; 2L. Spallanzani Hospital, Rome, Italy; 3Polyclinic of Modena, Modena, Italy

BACKGROUND: The clinical relevance of resistance detected in peripheral blood mononuclear cell (PBMC) compartment is still debated. Thus, we evaluated the impact of baseline resistance detected in PBMC genotypic-resistance-test (GRT) on maintaining virological suppression after therapy switching in combined antiretroviral therapy (cART) treated patients with undetectable HIV-RNA.

METHODS: cART-experienced patients switching therapy with a GRT from PBMCs available before therapy change (median [Interquartile-range, IQR]: 3 [1-7] months) were included. The prevalence of resistance in protease and reverse transcriptase was evaluated using IAS/Stanford resistance lists, and genotypic susceptibility score (GSS) was calculated according with HIVdb ver.7.1 algorithm. Survival analysis was used to assess probability of virological rebound (VR: two consecutive viremia >50 copies/mL after therapy switching) according to baseline PBMC GSS. Multivariable Cox-regression was performed according to age, gender, CD4 nadir, duration of virological suppression before switching, previous treatments, number and type of antiretrovirals administered at switch.

RESULTS: Overall, 130 cART-treated patients, with virological suppression lasting from a median (IQR) of 3.8 (1.3-7.0) years before switching were analyzed. Patients were on antiretroviral treatment since a median (IQR) time of 9 (3-16) years, with a CD4 nadir of 141 (50-289) cells/mm3. Patients had a median

(IQR) number of previous regimens of 7 (5-10); of them, 54.7% experienced ≥3 antiretroviral classes. 3.8% and 30.0% of patients switched to darunavir-monotherapy and dual therapy, respectively. At baseline, 51.5% of patients showed at least 1 resistance mutation (PI:17.7%; NRTI:36.9%; NNRTI:20.0%); 82.3%, 15.4% and 2.3% of patients showed fully susceptible, intermediate resistant and fully resistant GSS, respectively.

Twenty-four months after therapy switching, the overall probability of VR was 18%. Patients showing at baseline a GSS intermediate or fully resistant had a higher probability of experiencing VR compared to those carrying a fully susceptible virus (36% vs. 14%, p=0.018). Patients having ≤1 year of suppressed viremia before switching showed the highest probability of experiencing VR compared to those with a longer or unknown time of undetectability (≤1 years: 39%; unknown time: 29%; 2-4 years: 15%; >4 years: 4%, p=0.025). Multivariable Cox-regression confirmed that a higher hazard of experiencing VR was found in patients with intermediate or fully resistant GSS compared to those with fully susceptible GSS (AHR: 4.9, 95% CI:1.6-14.8, p=0.005) and in patients with ≤1 year of undetectable HIV-RNA before therapy switching (AHR: 8.9 95% CI: 1.6-49.2, p=0.012). CD4 nadir <100 cells/mm3 was also associated to VR (AHR: 3.0 95% CI:1.1-8.5, p=0.040).

CONCLUSIONS: In clinical practice, treatment switch with long-term undetectable HIV-RNA warrants a high rate of maintaining virological suppression. However, patients with GSS intermediate or fully resistant in PBMCs have a higher risk of rebound after therapy switching. In patients under virological suppression, PBMC genotyping might be a useful tool for tailoring antiretroviral treatment switch.



ABSTRACT 43

Ultra-Deep Sequencing Detect Minority Resistance Variants Archived in HIV-Cellular DNA in Antiretroviral Treatment Well-Suppressed PatientsC Rodriguez1,2, ML Néré3,4, V Demontant1,2, M Mercier-Darty1,2, M Splittgerber3,4, M Salmona3,4, I Charreau5, N de Castro6, ML Chaix3,4, JM Molina4,6, and C Delaugerre3,4

1Laboratoire de Virologie, Hôpital Henri Mondor, APHP, Créteil, France; 2Université Paris Est Créteil, UPEC, U955 Inserm, France; 3Laboratoire de Virologie, Hôpital Saint-Louis, APHP, Paris, France; 4Université Paris Diderot, Inserm U941, Paris, France; 5INSERM SC 10, Villejuif, France; 6Maladies infectieuses, Hôpital Saint-Louis, APHP, Paris, France

BACKGROUND: In patients on effective antiretroviral therapy with previously therapeutic failure with drug-resistance mutations (DRM) detected in plasma, standard genotypic test performed in HIV-cellular DNA detects fewer resistance mutations. The underestimation of drug resistance mutation has implications for patient management, particularly in case of switch. The low detection of DRM could be due to the lack of sensitivity of Sanger sequencing that cannot detects DRM present on variants below 20% of the viral population. Ultra deep sequencing (UDS) is a powerful tool able to detect minority resistant variants (MRV) with a threshold of 1%. Our aim is to compare HIV DNA resistance mutations using UDS at two thresholds (> 20% that is considered as the Sanger sequencing cut off and >1% for the detection of MRV) in well-controlled patients with multiple DRM detected in previous plasma.

METHODS: We included 169 extensively treated patients from the ANRS-EASIER. All of them had already received three antiretroviral drug classes (NRTI, NNRTI and PI) and had plasma HIV-1 RNA < 400 copies/ml at inclusion. Protease (PR) and reverse transcriptase (RT) genes were amplified on HIV DNA from whole blood using ANRS protocol. RT (804 bp)

and PR (548 bp) amplicons were sequenced using UDS by Roche/454 GS FLX+. Sequences were analyzed using AVA software (Roche) and in house software PyroPack® for RT and PR. Detection of mutations were analyzed using two cut off (20% and 1%). Drug resistance interpretation was according to the 2015 French ANRS algorithm. We present here the results provided from the 84 first patients.

RESULTS: We obtained 82 PR sequences (mean reads 5300, mean length 474 bp) and 78 RT sequences (mean reads 9000, mean length 496 bp). Median number of DRM at 20% and 1% respectively was 5 and 6 for NRTI mutations, 1 and 1 for NNRTI mutations and 8 and 11 for PI mutations. Using the 1% threshold, number of patients harboring resistance to at least one antiretroviral increased from 59 to 68 patients for NRTI, from 52 to 64 patients for NNRTI and from 56 to 69 patients for PI, respectively. In 15 patients (18%), we described at least one stop codon in RT (n=14) and/or PR (n=3) sequences. For 5 out of 15 patients, the stop codons were detected in more than 60% of the viral population.

CONCLUSION: In this study, archived DRM detection was observed more frequently for each resistance codon in RT and PR gene using UDS compared to classic Sanger sequencing. Resistance to RT and PI inhibitors increased for 13 % to 20% and could jeopardize antiretroviral regimen in case of treatment switch or sparing class. UDS sequencing may be use to detect archive DRM in HIV DNA in clinical practice.



ABSTRACT 44

Low-Level Viremia Can Indicate the Evolution of HIV Drug ResistanceJ Verheyen1, M Dirks1, M Widera1, R Jablonka2, H Walter3, M Däumer4, and S Esser2

1Institute of Virology, University Hospital, University of Duisburg-Essen, Essen, Germany; 2Clinic for Dermatology, University Hospital, University of Duisburg-Essen, Essen, Germany; 3Laboratory Berg, MIB - Medical Infectiology Center Berlin, Berlin, Germany; 4Institute for Immunology and Genetics, Kaiserslautern, Germany

BACKGROUND: The clinical implications of low-level viremia (LLV) during continuous antiretroviral therapy (cART) still remain a matter of debate. LLV might indicate either ongoing viral replication potentially paving the way for the emergence of drug resistance mutations (DRMs) or might just represent the mere production of viruses from activated T-cells evolving from the viral reservoir of HIV positive patients.

METHODS: In this study we analyzed HIV infected patients (n=32) with cART who showed up with LLV at the outpatient center of the University hospital from 2013 until 2015. Viral RNA and proviral DNA were sequenced using the Illumina platform for next generation sequencing (NGS). In addition, we determined drug levels during LLV around the time point of genotyping and analyzed immunograms of these patients. .

RESULTS: By comparing HIV genotypes (RNA/DNA) obtained during LLV with genotypes obtained before LLV additional drug resistance mutations were detected in 34,4% (n=11). Due to the presence of PR mutations, which were not related to the currently used drugs, two of these mutational patterns detected in RNA (n=1) or DNA (n=1) might be related to archived drug resistance variants. New mutations potentially selected by cART were either detected in RNA (n=5) or as minority variants in RNA (n=2) or only in DNA (n=2). Of note, specifically the newly

detected mutations in integrase were found as minor DRMs (G163R: n=1, E92Q: n=1, T66A: n=1) probably indicating the first steps of evolution towards HIV drug resistance.

An accompanying decline of CD4+ cells at the time of genotyping could be observed in 6 of these 9 patients with evidence of HIV evolution but only 3 of these 9 patients showed increased numbers of HLA-DR positive cells. All HIV drug levels were above their Cmin concentrations in the majority of patients, however, in 4 of nine patients with probably newly selected mutations at least one drug of the currently used regimen was below its specific cmin concentration. Overall, no specific patterns in terms of the cellular tropism were detected HIV variants during LLV.

Interestingly, patients without evidence of viral evolution towards HIV drug resistance experienced at least one episodes of LLV, blip or viremia prior to their LLV episode (16/21) and/or after its ending (10/21).

CONCLUSION: In some patients LLV can indicate viral evolution towards HIV drug resistance. Especially, in the context of suboptimal adherence LLV seems to be a risk factor for the evolution of drug resistance, which can be indicated by the emergence of minor and/or major DRM. However, there is another group of patients with LLV who experience multiple episodes of LLV in the absence of compliance problems and without the emergence of new DRM.



ABSTRACT 45

Impact on Residual Viremia of Switching to Elvitegravir-Based Single-Tablet Regimen C Charpentier1-3, G Peytavin1,2,4, M Lê1,2,4, B Visseaux1-3, V Joly1,2,5, A Pinto5, M Perrier1-3, S Matheron1,2,5, Y Yazdanpanah1,2,5, D Descamps1-3, and R Landman1,2,5

1INSERM, IAME, UMR 1137, F-75018 Paris, France; 2IAME, UMR 1137, Univ Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France; 3AP-HP, Hôpital Bichat-Claude Bernard, Laboratoire de Virologie, F-75018 Paris, France; 4AP-HP, Hôpital Bichat-Claude Bernard, Laboratoire de Pharmacologie, F-75018 Paris, France; 5AP-HP, Hôpital Bichat-Claude Bernard, Service de Maladies Infectieuses et Tropicales, F-75018 Paris, France

BACKGROUND: To assess, in a clinical cohort of virologically-suppressed patients, the impact on residual viremia of switching treatment to TDF/FTC/EVG/Cobi as a single-tablet regimen (STR).

METHODS: A prospective observational single-center cohort enrolling all patients with a plasma viral load (VL) < 50 c/mL initiating TDF/FTC/EVG/Cobi between April and December 2014. VL were performed using Cobas Taqman HIV-1 V2.0 assay. Negative PCR (PCRneg) was defined as an undetected PCR signal. Plasma drug concentrations (C24h) were measured using UPLC-MS/MS. Medians (IQR 25%-75%) are presented.

RESULTS: 188 patients were enrolled. At time of switch, CD4 cell count was 600/mm3 (476-780), time since first ARV therapy and time with VL < 50 c/mL were 6 years (3-11) and 2 years (1-4), respectively. Among the 142 patients with available historical RT genotypes, 15% displayed virus with at least one NRTI drug resistance mutation (DRM) and 11% displayed resistant viruses to TDF or FTC. In 6 cases of historical TDF/FTC resistance, a supplementary ARV (ABC or ATV) was added.

At W24, 8.5% of the patients had discontinued STR due to adverse events, mainly gastro-intestinal or neurological symptoms. The proportion of patients with VL < 20 c/mL was 89%, 90% and 91% at baseline,

W12 and W24, respectively. The proportion of those with PCRneg was 69%, 75% and 73% at baseline, W12 and W24, respectively. Median EVG C24h was 551 ng/mL (253-940, n=150), with 93% of patients exhibiting EVG C24h > 45 ng/mL. No association was observed between EVG C24h and residual viremia.

Eight patients (4%) displayed a VL > 50 c/mL (median=161 c/mL): 3 with virological failure; 4 with viral blips; and 1 with no further VL control. Among these 8 patients, 2 had historical NRTI RAMs (M184V; L74I/V-T215F-K219E-M184V). New NRTI DRMs were observed in 2 of the 5 RT sequences obtained (D67N-T69N-K70R; M184V) and no emergent integrase DRM was evidenced in the 2 available sequences. Among the 5 patients with a VL > 50 c/mL and paired C24h data, 3 displayed inadequate EVG C24h.

Regarding the 15 patients with TDF or FTC historical resistance, 12 maintained viral suppression (< 50 c/mL) at W24, 1 had a viral blip (ATV had been added concomitantly to the STR), 1 had a virological failure and 1 stopped the STR.

CONCLUSION: At W24 of EVG-based STR switch, suppression of viral replication was maintained in 96% of cases without significant changes in residual viremia. Most of the patients with VL rebound > 50 c/mL had historical NRTI RAMS or low plasma drugs concentrations.



ABSTRACT 46

HIV Drug Resistance Test at Low Viremia Levels: Light And Shadow in the Clinical PracticeD Mileto1, A Mancon1, D Cattaneo2, A Tamoni1, A Palazzin1, P Meraviglia3, S Rusconi3, MR Gismondo1, V Micheli1

1Clinical Microbiology, Virology and Bioemergency Diagnosis, L. Sacco Hospital, Milan; 2Unit of Clinical Pharmacology, L. Sacco Hospital, Milan; 3Infectious Diseases Department, L. Sacco Hospital, Milan

BACKGROUND: the genotypic HIV drug-resistance test (GRT) should be perform even at low-level viremia (LLV), according to the most recent Italian guidelines (suggested cut-off ≥ 200 copies/mL). Anyway, the significance of LLV could be related to a virological failure with/without the emergence of drug resistance mutations, poor adherence and/or the release of viruses from HIV reservoir. Objectives: to assess the usefulness of GRT at LLV to predict virological failure and to drive the subsequent therapeutic choice.

METHODS: a total of 441 plasma samples has been tested using the TruGene HIV-1 genotyping Assay (Siemens) in antiretroviral therapy experienced patients addressing to the Infectious Diseases Department of Sacco Hospital, since 2012. The GRT results have been expressed as the weighted GSS to the ongoing regimen, calculated according to the ARCA database (AntiRetroScan 2.0). Demographic, virological and therapeutic information have been recorded. The patients have been classified as either potentially failing (presence of at least a drug resistance mutation to one drug of the combination therapy) or harboring a susceptible virus to the on-going regimen. Statistical analysis: Φ2 test for trend (to compare different viremia groups); Fisher’s exact test (to compare 2 viremia groups at a time); Kruskal-Wallis test to compare all viremia groups; univariate and multiple regression analysis.

RESULTS: There was no difference in GSS among viremia groups (<200; 200-499; 500-999; 1,000-9,999; ≥10,000 cp/mL; p=0.27); higher GSSs and virological

rebounds without mutations correlated with female gender (p=0.02; p=0.004) and PI/r-based regimens (p<0.001), regardless the viremia levels and the HIV subtypes (B versus non-B). Low genetic barrier mutation M184V was more frequent in regimens excluding PI/r (p<0.001; OR: 2.7; 95% CI 1.7-4.3), being the administration of PI/r equally distributed in all viremia groups. A significant increase of the resistance rate (resistant versus susceptible viruses) correlated with a higher viremia, as chategorical variable, into the no PI/r-based regimen group (χ2 test for trend: p=0.04). The majority of virological failures resulted for 500-1,000 versus <200 cp/mL (p=0.02; OR 2.6 95%CI 1.2 – 6.0). For Atazanavir-administered patients, mutations for the PI and/or the backbone were detected only in 22% of plasma samples with an Atazanavir concentration <20 ng/mL.

CONCLUSION: GRT at LLV is useful in clinical practice even if a pharmacokinetic evaluation should be recommended to better depict the factors determining virological rebounds. The correlation between a higher GSS and virological rebound without mutation with female gender suggests a higher exposure to the drugs, probably due to the better adherence and a lower BMI of women.

ABSTRACT 47

Features of HIV Persistent Viremia after the Start or Restart of Antiretroviral Treatment RegimensM Widera1, M Dirks1, R Jablonka2, M Däumer3, H Walter4, S Esser2, and J Verheyen1

1Institute of Virology, University Hospital Essen, University Duisburg-Essen, Germany; 2Clinic of Dermatology, University Hospital Essen, University Duisburg-Essen, Germany; 3Institut für Immunologie und Genetik, Kaiserslautern, Germany; 4Laboratory Berg, MIB - Medical Infectiology Center Berlin, Berlin, Germany

BACKGROUND: After the start of antiretroviral therapy (ART) HIV RNA levels decrease in a multiphasic manner. Here, after a severe drop of



viral RNA levels in the blood within days, a phase of slower RNA declining below the detection limit of 50 copies/ml is observed at least within 26 weeks. Under certain circumstances, however, the drop in viral RNA may take longer than 26 weeks. A prolonged decline might be associated with the presence of drug resistant variants. Conversely, whether persistent viral replication itself can lead to the emergence of drug resistant mutations and thus to treatment failure is nowadays still unclear. Furthermore, the treatment regimen itself can influence the kinetic of HIV RNA decline. However, if persistent viremia might expire without clinical significance, or whether it indicates an increased risk of low-level viremia still remains unpredictable. Whether a therapy switch might be beneficial for clinical outcome of PV also has to be evaluated.

METHODS: In this study, we analyzed 19 patients with persistent viremia and evaluated the clinical outcome with respect to treatment failure, viral evolution, and the emergence of drug resistance mutations (DRM). To this aim, we analyzed viral RNA and DNA using next generation sequencing (NGS). In addition, we compared the drug levels, baseline viral loads, and CD4+ levels of each patient. Furthermore, the impact of therapy switches on persistent viremia was evaluated.

RESULTS: Using NGS we identified viruses with therapy associated drug resistance mutations (DRM) only in a single patient. While receiving FTC, TDF and LPV/r, new DRM (V82AV (2%), N88DN (1%), D30N (1%)) emerged. We were able to demonstrate that drug levels of LPV were below the Cmin concentration. In three other patients we found DRM, which were not related to the current therapy regimen and were not detected in previous genotypes. Some of these DRMs were only detected as minority variant (1% and 5%). Predominantly, according to each therapy regimen drug levels were found to be in range indicating that PV is a drug independent phenomenon. Furthermore, we observed that 31.6% (n=6) of patients with PV showed up with low-level viremia (LLV) during follow up while subsequent blips were observed only in 10.5% (n=2). When comparing patients with and without

therapy switch we did not find any clinical impact of ART switching on the duration of PV suggesting a drug independent phenomenon.

CONCLUSION: Managing persistent viremia remains challenging. According to our data persistent viremia was not related to adherence issues and ceased with and without ART switching. Thus, only in a minority of patients close monitoring using NGS might be helpful to detect ongoing viral evolution during persistent viremia after start of ART.

ABSTRACT 48

Analysis of HIV Resistance Development in Long-Term Low Viremic PatientsP Braun, F Wiesmann, G Naeth and H Knechten

PZB Aachen, Aachen, Germany

BACKGROUND: The aim of each antiretroviral therapy is to diminish HIV viral load below the detection limit of 50 copies/ml. However in some patients this goal is not always achievable and viral load maintains constantly on low viremic levels. Genetic barriers against resistance may also differ depending on the used antiviral drugs. The focus of this analysis was to assess the course of resistance development under different regimens in patients with long-term low level viremia.

METHODS: 266 patient samples were selected between 2013 and 2015 according to quantified viral loads between 50 and 1000 copies/ml between two relevant genotypic resistance tests. Results of genotypic resistance tests were classified into 4 groups: group A consisted of samples with viral loads of 40-100 copies/ml (n=53), group B 101-200 (n=73), group C 201-500 (n=86) and group D 501-1000 copies/ml (n=54) at time of resistance analysis, respectively. Newly developed resistance associated mutations (RAMs) were assessed according to the HIV-Grade algorithm and analyzed according to the corresponding therapy regimen under which they were selected.



RESULTS: In 18 patients of group A (34.0%) major RAMs could be detected at second resistance analysis, 26 were detected in group B (35.6%), 34 of group C (39.5%) and 19 of group D (35.2%), respectively. Beside pre-existing mutations, newly diagnosed RAMs were detected in 4/53 (7.5%) of group A, 10/73 (13.7%) of group B, 13/86 of group C (15.1%) and 11/54 of group D (20.4%). In group A, the newly developed RAMs were NRTI-related. M184V/I newly developed in 3/4 cases and M41L in two of cases, although quantified viral loads remained <200 copies/ml in these patients. In group B, 2 mutations were PI-based, 4 INI-based and 5 NRTI-based. In group C 9 newly RAMs were NRTI-related (M184V in all cases), 4 were related to INI-containing regimens, 3 were NNRTI-related, respectively. In group D, 8 cases showed new NRTI-related resistance, 6 included M184 substitutions. 6 were related to INI-treatment and 5 were NNRTI-related.

CONCLUSION: The risk to develop new RAMs increased with the level of viral load in this analysis even in a setting of long-term viral loads below 1000 copies/ml. Substitutions at position M184 were not only the most prevalent but also seemed to represent early events in resistance development under common 3TC- or FTC-based NRTI therapies. INI-relevant mutations also developed early in groups B-D in this retrospective evaluation.

ABSTRACT 49

Ultra-Deep Sequencing in Pro-Viral DNA of PBMC in Patients with Low Level ViremiaV Illanes1, J Rojas1, JA Fernandez-Caballero2, M Mosquera3, M Laguno1, B Torres1, M Martinez-Rebollar1, M Lonca1, A Gonzalez-Codon1, E Martinez1, J Mallolas1, JM Gatell1, F Garcia2, JL Blanco1

1Infectious Diseases Department, Hospital Clinic, Barcelona, IDIBAPS; 2Microbiology Department, Hospital Clinic, Barcelona; 3Microbiology Department, Complejo Hospitalario Granada, Hospital Universitario San Cecilio, Instituto de Investigación Ibs, Granada

BACKGROUND: Guideline based management of patients with Low Level Viremia (LLV) (>=50 and <200 copies/mL) are not conclusive. Tools that guide an active management are often insufficient given that the sensitivity of plasma Genotypic Resistance Testing (GRT) for detection of Drug resistant Mutations (DRM) is less as the viral loads are lower. We explore the role of ultra-deep sequencing of pro-viral DNA from peripheral blood mononuclear cells (UDS-pDNA-PBMC) as a potential tool that provides additional information to help an active management of patients with LLV.

METHODS: An observational prospective cohort (FRABA Cohort) of 96 patients with LLV defined by two consecutive samples with LLV is followed since 2012. Plasma was tested at the time of inclusion by population sequencing (PS) with Trugene assay (Siemens, NAD) and sample of pro-viral DNA from was stored and latter analyzed. From this sample 5x10E6 PBMCs isolated by Ficoll-Hyapque were used and were tested by ultra deep sequencing (UDS) using a modification of the Roche prototype for the 454 GS-Junior platform. Information regarding pooled previous genotyping was collected.

RESULTS: Twenty patients, ten of which had two or less previous virological failures were included in this analysis. The median viral load at inclusion was 59 copies/ml (IQR 51-99). The previous ARVs and the ones at inclusion, basal characteristics and the result



of both plasma PS and PBMC UDS is presented in table 1. Only in 5/20 of the patients PS was possible whereas in all of the patients UDS-pDNA-PBMC was amplifiable. The latter identified relevant mutations in 14/20 that were not identified by the conventional GRT and 13/20 not documented in the pooled GRT.

CONCLUSION: The results presented suggest a role for the pro-viral DNA from peripheral blood mononuclear cells (UDS-pDNA-PBMC) in the management of patients with LLV showing a higher rate of amplification than conventional RNA sequencing and a potential role to identify relevant DRM that influence the choice of ARVs.

ABSTRACT 50

HIV-1 Non Group M Phenotypic Susceptibility to Integrase Inhibitors (Dolutegravir, Elvitegravir and Raltegravir) E Alessandri-Gradt1,2, G Collin3,4, A Tourneroche1,2, M Bertine3,4, M Leoz1,2, C Charpentier3,4, D Descamps3,4 and JC Plantier1

1GRAM EA2656, UFR Médecine-Pharmacie, Université de Normandie, France ; 2Laboratoire de virologie associé au CNR du VIH, Hôpital Charles Nicolle, Rouen, France ; 3Laboratoire de virologie, Groupe Hospitalier Bichat-Claude Bernard AP-HP, Paris, France ; 4EA4409 PRES, Université Paris Diderot, Sorbonne Paris Cité, France

BACKGROUND: HIV-1 are classified into four distinct groups: HIV-1/M pandemic and HIV-1/O, HIV-1/N, HIV-1/P endemic in Cameroon. HIV-1 non-group M (HIV-1/non-M) show a high natural genetic polymorphism that limits options for therapeutic management, particularly for HIV-1/O. Evaluating efficacy of new drugs is therefore essential. Few data are available concerning HIV-1/non-M to integrase inhibitors; they are based on in vivo case reports or limited data on in vitro studies. Thus, we aimed to determine the natural susceptibility of HIV-1/non-M to Dolutegravir (DTG), Elvitegravir (EVG) and Raltegravir (RAL), and to associate these findings with potential genotypic determinants.

METHODS: Susceptibility of 35 clinical isolates representative of HIV-1/non-M diversity (23 HIV-1/O subgroup H and 9 subgroup T, 2 HIV-1/N and 1 HIV-1/P) was compared to that of HIV-1/M BRU-HXB2 reference strain. Peripheral blood mononuclear cells from healthy donors were infected with 100 TCID50 for 2 hours, washed and seeded at 2.105 cells/ml in a 96 micro-well plate containing serial increasing concentrations of each drugs (from 0 to 2000 nM) during 3 days. Fifty percent inhibitory concentration (IC50) was calculated from the relation between the diminution of replication in supernatant and level of drug concentrations. Susceptibility results were compared to genotyping data of the integrase region (codon 19 to 263), obtained for each isolate by Sanger method.

RESULTS: For DTG, the mean IC50 is 0.85nM and median 0.34nM. Compared to BRU-HXB2’s IC50

(2.09nM), 6 strains of group O have a fold change (FC) greater than 1, ranging between 1.0 and 1.9. For RAL, the mean IC50 and the median are 0.86nM and 0.55nM respectively. Whereas BRU-HXB2’s IC50 is 1.68nM, 5 HIV-1/O have FC>1 [1.1-2.3]. For EVG, the mean IC50 and median values are respectively 1.72nM and 0.48nM respectively, and BRU-HXB2’s IC50 is 0.20nM. Near half the panel (N=16) has FC>1 with mean FC at 17.5 [2.4-88.0]: 8 strains, with HIV-1/P, have FC between 1 and 10, 6 HIV-1/O between 10 and 18 and the highest FC values are observed in 2 HIV-1/O, respectively 88 (IC50=17.69nM) and 69 (IC50=13.90nM). There is no group or clade effect on IC50 results. Concerning the genotyping of the strains, we find natural genotypic polymorphism (L74I, S153A, V201I, T206S) already described in HIV1/O. Although there is no specific pattern dedicated to the highest IC50, punctual mutations have been identified among these strains.

CONCLUSION: Our results showed that HIV-1/non-M viruses are naturally susceptible to DTG and RAL. For EVG a wide range of IC50 are observed, with a potential lower susceptibility of 2 HIV-1/O strains. Further investigations are now needed to determine the impact of the mutations on these differences of drug susceptibility.



ABSTRACT 51

Low Frequency of the R263K Mutation in HIV-1 Integrase in Patients of the AREVIR Cohort Related to Raltegravir or Elvitegravir Therapy Failures N Lübke1, E Knops2, E Heger2, B Jensen3, T Kümmerle4,5, J Timm1, and R Kaiser2

1Institute of Virology, University of Düsseldorf, Düsseldorf, Germany; 2Institute of Virology, University of Cologne, Cologne, Germany; 3Department of Gastroenterology, Hepatology and Infectiology, University of Düsseldorf, Germany; 4First Department of Internal Medicine; 5University Hospital of Cologne, Cologne, Germany

BACKGROUND: The substitution R263K in integrase (IN) has been identified as dolutegravir (DTG)-associated mutation selected in cell culture. Meanwhile, the R263K has been detected in treatment-experienced patients after therapy failure on the first INI-containing salvage therapy with DTG and in combination with the M50I polymorphism in patients who failed raltegravir (RAL) treatment. Since the emergence of R263K mutations in vivo was only rare reported, we screened the IN sequences of the AREVIR cohort for the existence of the R263K mutation and the possible factors for its appearance.

METHODS: The study included 1914 IN sequences of plasma viral RNA, cellular proviral DNA or total nucleic acid from whole blood of treatment-naive and –experienced patients of the AREVIR cohort in Germany. The sequences were obtained by classical Sanger sequencing or since 09/2014 by NGS analyses performed by the Illumina MiSeq technology.

RESULTS: The screening of the IN sequences presented the R263K substitution in 9/1914 samples (0.47%) of different patients infected with subtype B (n=8) or F1 (n=1). All samples with detected R263K in integrase were obtained by total nucleic acid analyses. The majority of the patients (8/9) were treatment-experienced. While 6/8 samples presented the R263K after therapy failure with a median VL of 11,362

cop/ml (range 122-92621 cop/ml), two samples (#14278 and #14334) presented the R263K with suppressed viral load, almost certainly in proviral DNA, which could be attributed to APOBEC3G/3F mediated hypermutations. Noticeable, 5/6 patients with therapy failures and a detected R263K in the integrase gene were treated with a RAL- or elvitegravir (EVG)-containing therapies (p=0.048). While the resistance profiles after failure on EVG-containing therapies presented the R263K combined with the INI-associated resistance mutations N155H or H51Y (#12245, #13183), the samples analysed after failure on an RAL-containing regimen presented either the R263K mutation alone (#10981, #12003) or combined with the polymorphism L234V present in >99% of the sequenced amplicons (#14278). In contrast, the R263K mutation at baseline (#11987) seems to have no critical influence on DTG-therapy response in case of a fully active backbone (Triumeq®), which was started subsequent to baseline screening, and is still active 16 months after initiation confirmed by suppressed VL.

CONCLUSION: The frequency of the R263K mutation in the RESINA cohort with <0.5% is quite low. Its incidence can be related to treatment-experience, significantly to failures on integrase-inhibitor containing regimens. Nevertheless, the R263K could also be attributed to cellular APOBEC3G/3F mediated hypermutation, indicated by their detection in proviral DNA. However, a single R263K substitution at baseline seems to have no critical impact on therapy success of a DTG-containing therapy with an active backbone, indicating only a minor impact on DTG susceptibility in vivo.



Sample-Id

INI-associated mutations

Subtype Sequencing Method

VL (cop/ml)

Therapy

10918 R263KR B Sanger 22000 DRV/r, RAL

11770 R263KR B Sanger 39777 KVX, ATV/r

11987 R263KR B Sanger 89564 naiv

12003 R263KR B Sanger 92621 DRV/r, RAL

12061 R263K B Sanger <40 TZV

12245 N155H, R263KR B Sanger 709 STB

13183 H51HY, R263KR B NGS 7203 STB

14278 L234V, R263KR F1 NGS 122 TVD, RAL

14334 L74I, R263KR B NGS <20 KVX,

ATV/r

KVX = Kivexa (3TC+ABC); TZV = Trizivir (AZT+3TC+ABC); STB = Stribild

(FTC+TDF+EVG+COBI)

ABSTRACT 52

The R263K Resistance Substitution Decreases Levels of Integrated HIV DNA over TimeT Mesplède, J Liang, K Anstett, N Osman, H Thi Pham, MA Wainberg

McGill AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, QC, Canada

BACKGROUND: Integrated HIV DNA persists for decades within the genome of HIV-positive individuals, even when they are successfully treated with antiretroviral therapy (ART) with undetectable HIV RNA levels. The persistence of integrated HIV DNA within reservoir cells contributes to our inability to achieve viral eradication in infected individuals who are thus obligated to take life-long treatments.

Although rare, treatment failure in treatment-experienced, integrase inhibitor-naïve individuals treated with dolutegravir (DTG) is commonly associated with the emergence of the R263K substitution in integrase and plasma viral loads that

are lower than those observed when treatment failure occurs with ART regimens that do not contain DTG. This is likely due to the fact that R263K confers low-level resistance against DTG and also decreases viral replication capacity and viral integrase activity in short-term infectivity assays.

We sought to determine the effect of the DTG-specific R263K resistance substitution on integration during long-term infections.

METHODS: We measured HIV integration by Alu-mediated QPCR over 5 weeks of infection of Jurkat cells with WT, R263K and H51Y/R263K viruses. Levels of integration were measured every week and expressed relative to integration of the WT virus after week 1. Means ± standard deviations were calculated and Student’s t-test was used to evaluate significance of differences.

RESULTS: The R263K substitution impaired HIV integration over time and was associated with a progressive decline in levels of integrated HIV DNA. Even further impairments were noted if both the R263K and H51Y substitutions were simultaneously present. Differences were statistically significant.

CONCLUSIONS: Our study raises the possibility that emergence of the R263K substitution in individuals who experience treatment failure with DTG might result in a progressive decline in the size of the viral reservoir. Further studies are needed to study this hypothesis.



ABSTRACT 53

Durability and Virological Response to Dolutegravir (DTG)-Containing Regimens after Failing to Raltegravir (RAL) or Elvitegravir (EVG)S Rusconi1, F Adorni2, P Tau1, A Mancon3, V Micheli3 on behalf of ARCA (Antiviral Response Cohort Analysis)

1Divisione Malattie Infettive, DIBIC Luigi Sacco, Università degli Studi di Milano; 2ITB-CNR, Segrate, MI; 3 Laboratorio Microbiologia Clinica - Virologia - Bioterrorismo, Ospedale L. Sacco, Milano, Italy

BACKGROUND: Dolutegravir (DTG) is a next-generation HIV-1 and HIV-2 integrase inhibitor (INI) with an increased genetic barrier to resistance with respect to first-generation INIs such as raltegravir (RAL) or elvitegravir (EVG). Few data are available on the durability of DTG-containing regimens after failure to RAL or EVG in a clinical cohort setting.

METHODS: From the Antiretroviral Resistance Cohort Analysis database, we selected 52 triple-class-experienced subjects who started DTG after being treated with RAL or EVG, including those who were still receiving DTG. Selection criteria included HIV-RNA, CD4 count and HIV genotype within 3 months of DTG initiation. Factors associated with virological response were analysed, including weighted genotypic sensitivity scores (wGSS). Univariate and multivariate (Cox regression model) analyses were performed.

RESULTS: Forty-eight of 52 subjects were still on DTG, with a median duration of 9.4 (6.5-11.2) months. The 4 subjects who discontinued DTG had a median duration of 19.1 (0.8-54.1) months. Over 48 subjects, 10 had a wGSS of 0, 10 had a wGSS between 0.25-1.75 and 28 had a wGSS of 2; all 4 DTG-discontinuing subjects had wGSS of 0. No subjects had positive HIV-RNA after suppression during DTG regimen among those on an ongoing regimen, whereas this was evident in 2/4 subjects who discontinued DTG. The number of subjects with isolated HIV-RNA blips was 13/48 in those who have an ongoing regimen and 0/4 in those who discontinued DTG. As far as the multivariate

analysis, at least one HIV-RNA blip during follow-up (on treatment) was less frequent in male sex (AHR 0.15, p 0.049) and more frequent in non-B subtype (AHR 50.27, p 0.022). The probability of having a HIV-RNA positive value at the last follow-up (ITT) was significantly increased in patients with a detectable HIV-RNA at DTG start (AHR 12.08, p 0.036) and significantly decreased in patients aged 40-49 (AHR 0.16, p 0.009), with a diagnosis of HIV infection ≥ 10 years (AHR 0.11, p 0.007) and with a CD4 nadir of 200-499/µL (AHR 0.07, p 0.007) or <200/µL (AHR 0.13, p 0.028).

CONCLUSION: After previous exposure to first-generation INIs, treatment with DTG showed a good durability and did not cause virological rebound after reaching viral suppression although 27% of subjects still on DTG experienced a HIV-RNA blip. Subjects infected with a non-B subtype had a greater risk of having a HIV-RNA blip during follow-up and a detectable HIV-RNA value at the beginning of DTG was related to a HIV-RNA positive value at the last observation.

ABSTRACT 54

Immunologic Criteria are Poor Predictors of Virologic Outcomes: Implications for HIV Treatment Monitoring in a Large Treatment Program in Nigeria N Ndembi1, F Murtala-Ibrahim1, J Okuma1, A Aliyu1, S Peters1, C Adebamowo1,2, C Mensah1, A Abimiku1,2, M Charurat1, P Dakum1

1Institute of Human Virology Nigeria, Abuja, Nigeria; 2Institute of Human Virology, University of Maryland School of Medicine, Baltimore MD, USA

BACKGROUND: Nigeria has made significant gains in scaling-up access to HIV prevention, treatment and support services and by the end of 2014, provided antiretroviral therapy (ART) to over 747,382 individuals and care and support to almost 1.5 million people.



The main objective of this study was to determine predictors of immunologic failure in the absence of routine viral load monitoring.

METHODS: This was a retrospective cohort study of 12,456 HIV-infected patients enrolled into HIV care at the University of Abuja Teaching Hospital (UATH) between February, 2005 and December, 2014.

Immunologic failure defined as having a decrease in CD4 cell count to pre-therapy baseline level (or below) or persistent CD4 levels <100 cells/mm3 after 6 months on therapy. HIV genotyping was performed on a subset of patients with two consecutive Viral Load (VL) measurements >1,000copies/ml after at least 6 months of ART. The incidence of immunologic failure was determined as the percentage of cases among all patients enrolled in the treatment program. To identify predictors of immunologic failure, univariate and multivariate analyses were performed using log binomial models to estimate relative risks (RR) and confidence intervals. All available plausible predictors were included in the multivariate models if they were significant at a P value of <.20. The criterion for significance for all analyses was a 2-sided P value of <.05. All statistical analyses were performed with the statistical software package SAS release 9.3 (SAS Institute Inc, Cary, North Carolina).

RESULTS: A total of 5,928 patients who initiated ART were included in the analysis. The entry point for 3,924 (66.2%) was through (Voluntary Counseling and Testing (VCT)), 3,468 (58.5%) were initiated on NVP-based containing regimen and 2,140 (36.1%) initiated on TDF, baseline CD4 was 268±23.7 cells/ul, and mean VL was 3.3±1.3.log10copies/ml. Among 2,602 patients with immunologic failure, 868 (33.3%) had VL measurements and 381 (43.9%) of these had a detectable VL. Fifty six samples (56/198; 28%) had no resistance; 160 (80%) harbored NRTI resistance; 151 (76.3%) M184I/V; 29 (14.6%) had ≥ 3 TAMs, and 37 (18.7%) had K65R, of which all were on TDF. One hundred and sixty-two samples (81.8%) harbored NNRTI resistance; 72 (36.4%) Y181C and 68 (34.3%) K103N with 53 % having ≥ 2 etravirine associated mutations. Service

entry point [RR (95%CI): 0.79 (0.64–0.91); p<0.001]; being on NVP containing regimen [RR (95%CI): 1.21 (0.99 – 1.45); p=0.023]; WHO stage III or IV [0.76 (0.60 – 0.96); p=0.013]; baseline CD4 cell count <200cells/ul [0.19 (0.16 – 0.22); p<0.001]; and male gender [1 (1.07 – 1.40); p=0.005] were associated with immunologic failure.

CONCLUSIONS: Immunological criteria for failure erroneously classified patients without virological replication as failing therapy in our program. Patients with both virologic and immunologic failures had extensive accumulations of drug resistance mutations. This erroneous classification is exacerbated by lack of diagnosis testing that limits provider’s ability to rule out tuberculosis, opportunistic infection or other potential causes of depressed CD4 counts.

ABSTRACT 55

HIV Resistance in Pregnant Women with Detectable HIV-1 RNA at Delivery in MozambiqueM Rupérez1,2, M Noguera-Julian3,5, R González1,2, R Bellido3, A Vala1, C Rodríguez3, E Sevene1,6, E Macete1, R Paredes3,4,5, C Menéndez1,2

1Manhiça Health Research Center (CISM), Manhiça, Maputo, Mozambique; 2Barcelona Centre for International Health Research (CRESIB, Hospital Clinic-Universitat de Barcelona), ISGlobal, Barcelona Institute for Global Health, Barcelona, Spain; 3IrsiCaixa AIDS Research Institute, Badalona, Catalonia, Spain; 4Lluita Contra la Sida Foundation, HIV Unit, Hosp Univ Germans Trias i Pujol, Badalona, Catalonia, Spain; 5Universitat de Vic-Universitat Central de Catalunya, Catalonia, Spain. Faculdade de Medicina; 6Universidade Eduardo Mondlane (UEM) Maputo, Mozambique

BACKGROUND: Few data on HIV resistance in pregnant

women are available from Mozambique, one of the countries

with the highest HIV toll in the world. The HIV resistance

implications of reaching delivery with detectable HIV-1

RNA despite prevention of mother to child transmission

of HIV (pMTCT) are not fully understood.

METHODS: We analyzed stored plasma samples from HIV-

infected pregnant woman participating in a randomized



Table 1. Drug susceptibility of samples from 1st antenatal care and delivery visits

Drug class

1st ANC visit (N=90) Delivery (N=60)

Drug Intermediate Resistant Intermediate Resistant

NRTI ABC 8 (8.9%) 0 5 (8.3%) 1 (1.7%)

AZT 3 (3.3%) 0 2 (3.3%) 0

DDI 0 4 (4.4%) 1 (1.7%) 3 (5.0%)

D4T 7 (7.8%) 0 3 (5.0%) 1 (1.7%)

FTC 4 (4.4%) 4 (4.4%) 2 (3.3%) 3 (5.0%)

TDF 0 4 (4.4%) 0 2 (3.3%)

3TC 4 (4.4%) 4 (4.4%) 2 (3.3%) 3 (5.0%)

NNRTI EFV 0 6 (6.7%) 2 (3.3%) 4 (6.7%)

ETR 3 (3.3%) 0 1 (1.7%) 2 (3.3%)

NVP 2 (2.2%) 6 (6.7%) 3 (5.0%) 5 (8.3%)

RPV 9 (10.0%) 2 (2.2%) 7 (11.7%) 2 (3.3%)

PI ATV/r 1 (1.1%) 0 0 0

DRV/r 1 (1.1%) 0 0 0

FPV/r 3 (3.3%) 1 (1.1%) 0 0

IDV/r 4 (4.4%) 0 0 0

LPV/r 4 (4.4%) 0 0 0

NFV 20 (22.2%) 1 (1.1%) 12 (20.0%) 0

SQV/r 4 (4.4%) 0 0 0

TPV/r 2 (2.2%) 0 0 0

controlled trial on intermittent preventive treatment of

malaria in pregnancy (IPTp) at the Manhiça district hospital

(MDH) in a semi-rural area in southern Mozambique.

Women attending their 1st antenatal (ANC) visit between

2009 and 2013 were followed prospectively through 1

month post-partum. Women with HIV-1 RNA levels>400 c/

mL at delivery were included in our HIV resistance analysis.

HIV drug resistance mutations (HIVDRM) were determined

using MiSeq® (limit of detection 1%) at the first ANC

visit and at the time of delivery.

RESULTS: 150 plasma samples from 113 pregnant women

were analyzed. Ninety and 60 samples were available at the

1st ANC and delivery visits, respectively. Women attended

the first ANC visit with a mean of 25 years of age and a

median gestational age of 22 weeks. Of them, 96% had

HIV-1 RNA>400 c/mL, 39% had CD4+ counts <350 c/mm3

and 20% were on antiretroviral therapy (ART). Thirteen

women (14%) had at least 1 HIVDRM at the 1st ANC, of

whom 2/3 were not on previous ART. The number of women

with at least 1 HIVDRM to nucleoside reverse-transcriptase

inhibitors (NRTI), non-nucleoside reverse-transcriptase

inhibitors (NNRTI) and protease inhibitors (PIs) in the 1st

ANC visit was 8 (9%), 6 (7%) and 2 (2%), respectively. Eight

women (13%) had at least 1 HIVDRM at delivery, 6 (10%),

5 (8.3%) and 0 (0%) to NRTI, NNRTIs and PIs, respectively.

Table 1 summarizes the predicted susceptibility to the

different antiretrovirals (HIVdb) in both visits. Of the 37

women with longitudinal data available from the two time

points, 5 (13.5%) developed at least 1 new HIVDRM during

pMTCT; 2 (5.4%) to NNRTI, 2 (5.4%) to NRTI and 1 (3%)

to PI.

CONCLUSIONS: Even with ultrasensitive HIV-1 genotyping,

less than 15% of women with detectable viremia at delivery

had HIVDRM before initiating pMTCT with 1st line ARVs.

This suggests that other factors beyond pre-existing

resistance, such as lack of adherence or interruptions of

the ANC chain, are relevant to explain lack of virological

suppression in women receiving pMTCT at the time of

delivery.



could not be detected, despite the fact that one-third of patients with detectable HIV-DRM had been on tenofovir-based regimen for a minimum of a year. CD4 cell count <200 cells/µl at the time of recruitment was independently associated with increased risk of virologic failure (odds ratio [OR] 6.36; 95% CI 2.51-16.07; P <0.001).

ABSTRACT 56

Strengthening HIV Therapy and Care in Rural Tanzania may Affect Rates of Viral SuppressionAJ Ntamatungiro1, L Muri, T R Glass2, S Erb3, M Battegay3, H Furrer4, C Hatz2, M Tanner2, I Felger2, T Klimkait5* and E Letang1, 2, 6* on behalf of the KIULARCO Study Group

* Equal contribution

1Ifakara Health Institute, Ifakara branch, Tanzania; 2Swiss Tropical and Public Health Institute of Basel, Switzerland; 3Division of infectious Diseases and Hospital Epidemiology, Departments of Medicine and Clinical Research, University Hospital Basel, Switzerland; 4Department of Infectious Diseases, Bern University Hospital, University of Bern, Switzerland; 5Molecular Virology, Department Biomedicine Petersplatz, University of Basel, Switzerland; 6ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic - Universitat de Barcelona, Barcelona, Spain

BACKGROUND: Observations from rural HIV clinics in developing countries have shown that inadequately managed HIV treatment and care programs may results in high levels of virologic failure and widespread drug resistance. We aimed to investigate the rate of viral suppression, prevalence of acquired HIV-drug resistance, and to characterize the spectrum of HIV-drug resistance mutations in HIV-infected subjects in a rural Tanzanian HIV clinic.

METHODS: Cross-sectional study was nested into the Chronic Diseases Clinic of Ifakara, implemented since 2005 in rural Tanzania.

RESULTS: The median time on ART was 3.5 (IQR 1.7-5.3) years. Viral suppression, defined as HIV-1 RNA<50 copies/mL, was observed in 277/304 patients (91 %). Among Virologic failure, 21 samples were successfully genotyped; the prevalence of ≥ 1 clinically relevant HIV-drug resistance mutation was 5.5 % (17/304). Of these, 14/17 (82 %) had a HIV-1 plasma viral load >1000 copies/mL. 9 % of patients had been switched to 2nd-line ART regimen based on immunological and clinical criteria of treatment failure and none of these patients had detectable viral-load. K65R mutation



Epidemiology of Resistance



category differences were seen in individuals in clusters compared to individuals not in clusters. High level NNRTI resistance was present in 12% of cases overall, including 16% of individuals in clusters and 9% of individuals who were not members of a cluster (p <.0001, see table). Likelihood of being in a cluster was lower for other and multi-class drug resistance.

Table. Washington state residents diagnosed with HIV 2005-2014 with a PR/RT sequence reported, by cluster and resistance status.

In cluster Not in cluster X2

N (%) N (%) Totals p-value

ANY High level resistance

Yes 283 (67%) 141 (33%) 424 <.0001

No 1,323 (52%) 1,205 (48%) 2,528

PI resistance

Yes 32 (59%) 22 (41%) 54 0.47

No 1,574 (54%) 1,324 (46%) 2,898

NRTI resistance

Yes 28 (35%) 52 (65%) 80 0.0004

No 1,578 (55%) 1,294 (45%) 2,872

NNRTI resistance

Yes 250 (69%) 112 (31%) 362 <0.0001

No 1,356 (52%) 1,234 (48%) 2,590

Multi-class drug resistance (MDR)

Yes 19 (32%) 41 (68%) 60 0.0004

No 1,587 (55%) 1,305 (45%) 2,892

CONCLUSION: Differences in genotype sequence reporting appear to be influenced by a number of factors in Washington State. While reporting is not complete, a high proportion of cases do have a sequence and cluster analysis has been able to provide information on potential transmission pathways. High level resistance, most commonly NNRTI resistance, was correlated with an increased likelihood of being in a cluster, while PI, NRTI, and multi drug resistance are correlated with a decreased likelihood. These results could be related to differences in viral fitness. Further analysis will be needed to clarify these findings.

ABSTRACT 57

ARV Resistance and Clusters Among HIV-Positive Individuals in Washington State, 2005-2014JR Reuer1, KMG Toren2, SE Buskin2,3

1Washington State Department of Health, Tumwater, WA, USA; 2Public Health – Seattle & King County, Seattle, WA, USA; 3University of Washington, Seattle, WA, USA

BACKGROUND: The use of HIV genotype sequence data has rapidly expanded within HIV surveillance programs. Cluster analyses, based on genotypic sequences, provide insight into potential transmission dynamics and the transmission of drug resistance allowing for expanded epidemiologic characterization of HIV transmission. In this analysis we examine the characteristics of individuals diagnosed with HIV in Washington State (WA) 2005-2014, with and without reported sequences, including those identified as belonging to transmission clusters.

METHODS: We analyzed genotypic sequences collected from CDC (Centers for Disease Control and Prevention) supported Molecular HIV Surveillance (MHS) that were reported for WA HIV cases diagnosed 2005-2014,. Transmission network analysis was performed at CDC. Protease/Reverse Transcriptase (PR/RT), PR/RT/Integrase (PR/RT/IN), and RT sequences were used to calculate pairwise genetic distance and identify linked pairs with distances ≤1.5%. We examined the comprehensiveness of sequence surveillance, comparing population characteristics by cluster and drug resistance status.

RESULTS: Between 2005 and 2014, 5,012 WA residents were diagnosed with HIV; 2,953 (59%) had a genotypic sequence. Differences in reported genotype results were seen by age, sex, transmission risk group, and viremia (viral load of ≥10,000). A total of 512 clusters, ranging in size from 2 to 97 individuals, were identified and 1,607 (54% of 2,953) individuals with genotype results were members of these clusters. Forty-one percent of clusters had 10 members or more; 20% had 20 members or more. Race and transmission



to 1235 transmissions as compared to 1375 solitary “dead-end” transmissions. PHI/EHI (0-0.44% genetic diversity) accounted for 57% and 26% of transmissions in large cluster and solitary transmission groups, respectively. Viruses from seven representative large clusters often harbored X4/R5 dual tropic viruses (n=5/7) as compared to dominant R5 tropism in the unique transmission group (0/6). Overall, 18% of transmitted viruses associated with large clusters harbored drug resistance mutations (G190A, K103N, T215 revertants). Tissue culture studies showed accelerated development of resistance under DTG, (R263K, H51Y or S153Y), elvitegravir (EVG), 3TC (M184I/V), and DTG/3TC dual pressure (R263K +M184V). Resistance mutations for DTG arose within 6-8 weeks in large cluster lineages while solitary transmission variants retained wild-type genotype at 26 weeks.

CONCLUSION: Failure to control early stage transmissions is leading to worrisome trends towards the selection of super-viruses showing prolonged high viremia, dual tropism, rapid tropism shift, and/or facilitated escape from drug pressure.

ABSTRACT 59

Identifying Growing HIV Clusters Among Persons Who Inject Drugs – United StatesAM Oster, S Xu, PJ Peters, R Galang, M Patel, W Switzer, E Campbell, A Siddiqi, AL Hernandez, HI Hall

Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Georgia, United States

BACKGROUND: A recent, large HIV transmission cluster among persons who inject drugs in Indiana has prompted concerns that similar clusters may exist or develop in other U.S. areas. We analyzed HIV-1 sequence data reported to the U.S. National HIV Surveillance System to identify clusters that have transmission attributable to injection drug use

ABSTRACT 58

Large Cluster Viral Lineages Fueling the MSM Epidemic show Accelerated Development of Resistance to Integrase InhibitorsBG Brenner, M Oliveira, R-I Ibanescu, O Golubkov, I Hardy, M Roger, and MA Wainberg

Lady Davis Institute, Montréal, QC, Canada

BACKGROUND: Despite advances in antiretroviral treatment paradigms, MSM epidemics have remained unchecked. This has raised concerns that primary/early-stage infection (PHI/EHI), frequently undiagnosed, may offset the benefit of Treatment-as-Prevention. The Quebec MSM epidemic, concentrated in Montreal, has shown a shift towards large cluster outbreaks, averaging 38 linked transmissions/cluster. Overall, 40 viral lineages accounted for half of the growth of the MSM epidemic from 2002-2014. This study characterized the distinct genotypic and phenotypic features of large cluster viral variants favoring their selective advantage.

METHODS: Phylogenetic analysis of RT/protease sequences from all newly diagnosed MSM (n=4319, 2002-2014), assessed transmission dynamics (cluster frequency, size, periodicity). Viruses were amplified from representative large clusters (n=7, 20+ linked transmissions/cluster) and solitary transmissions (n=6). Viruses from both groups were compared for tropism, drug susceptibility and emergent resistance to integrase inhibitors. Amplified viruses were serially passaged in CBMCs in the presence of increasing concentrations of (DTG), elvitegravir (EVG), and DTG +3TC dual pressure. Genotyping was performed at select passages to evaluate time to the development of drug resistance.

RESULTS: Phylogenetic analysis revealed a significant rise in the relative contribution of large clusters (10+ linked transmissions/cluster) in transmission dynamics, accounted for 29%, 34%, and 46% of new infections over the 2002-2005, 2006-2009 and 2010-2014 periods. Overall, 40 viral lineages contributed



as syringe exchange and interventions to improve engagement in care and viral suppression among members of these clusters.

ABSTRACT 60

HIV Drug Resistance in Persons Who Fail to Achieve or Maintain Viral SuppressionAL Hernandez1, MCB Ocfemia1, AM Oster1, N Saduvala2, AS Johnson1, H. Irene Hall1

1Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention; 2 ICF International

BACKGROUND: Since 2012, the U.S. Department of Health and Human Services recommends antiretroviral therapy (ART) for all HIV-infected persons in the United States. ART aims to slow HIV disease progression and reduce transmission by achieving viral suppression. Approximately half of persons in the United States with HIV diagnosed by 2011 who were alive in 2012 achieved viral suppression. We assessed the presence of drug resistance-associated mutations (DRAMs) to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors at baseline (i.e., sequence <3 months of HIV diagnosis) and on any subsequent HIV sequence (i.e., >2 months after baseline sequence) among persons diagnosed with HIV who never achieved or failed to maintain viral suppression.

METHODS: We analyzed data reported to the U.S. National HIV Surveillance System (NHSS) on persons aged >13 years diagnosed with HIV infection in 2010 and alive at year-end 2013. We included 4 states with complete laboratory reporting of CD4/viral load (VL) results as of December 2014 and >20% completeness of baseline sequences for each year for 2010–2013. We assessed DRAMs to any drug class at baseline and on any subsequent sequence among persons with at least one VL test within each 12-month follow-up period who were virally suppressed (i.e., VL <200 copies/

(IDU) and have grown recently and determine which of these clusters exhibit transmitted drug resistance mutations.

METHODS: We included partial HIV-1 pol sequences (one per person) reported through March 2015 for persons diagnosed in 2012 or later. We aligned each sequence to a reference sequence, then calculated Tamura-Nei 93 genetic distance between each pair of sequences in the data set. Pairs of sequences with genetic distance ≤1.5% were considered linked, and we used these linkages to construct transmission clusters. Among clusters with at least three persons, we identified clusters that included at least two persons with infection attributable to IDU and had grown by at least two persons during 2014–2015. For these clusters, we described the size and growth of the cluster and the transmission category, geographic location, viral load, recency of infection (as determined by BED or Bio-Rad Avidity testing), and drug resistance mutations of persons in the cluster.

RESULTS: Among 26,093 sequences, we identified 1,244 clusters of 3–355 persons, of which 8 included at least two persons with infection attributable to IDU and had grown by at least two persons during 2014–2015. These eight clusters ranged in size from 5 to 28 persons, experienced growth of 2 to 13 persons during 2014–2015, and were focused in medium-sized and large cities in three geographic regions (Table). The percentage of persons in these clusters without evidence of recent viral suppression ranged from 10% to 65%. Six clusters included persons who were determined to be recently infected at the time of diagnosis. In two clusters, the K103N drug resistance mutation was widespread, and in one cluster, the T215C drug resistance mutation was widespread.

CONCLUSIONS: Molecular HIV surveillance data can be used to identify transmission clusters of concern, including clusters with widespread drug resistance mutations. The demographic, geographic, and clinical characteristics of these clusters can help to guide prevention interventions. CDC is collaborating with the state and local health departments where clusters have been identified to determine the need for additional epidemiologic investigation as well



emergence of other DRMs in patients failing first-line antiretroviral therapy (ART) (AZT/d4T+3TC+NVP/EFV) with limited biological monitoring in RLC.

METHODS: Partial Reverse Transcriptase (RT) sequences from 1,367 patients failing first-line RTI based ART (VL>1,000 copies/ml) after >6 months mainly from West and West Central Africa, were analyzed for subtype association with DRMs from the International AIDS Society (IAS) list. The most prevalent subtype/CRF (excluding URFs) were used (>60 occurrences). Phylogeny and recombination analysis determined subtype/CRF. To identify potential new mutations, comparisons by amino acid position from all subtype/CRF were done with 2,214 sequences from ART-naïve patients from the same RLC; we further selected significant mutations with frequency ≤0.05% in naïve and ≥1% in treated patients. Fisher exact tests and corrections for multiple comparisons (Bonferroni and Benjamini–Hochberg procedures) were used to assess significance of any association (p< 0.05).

RESULTS: Median time on ART was 26 months (IQR=22-51); 90.7% and 98.2% patients had at least one NRTI and one NNRTI DRM, respectively. Predominant viral subtypes/CRFs were CRF02(43.6%), A(6%), CRF06(6%), CRF01(5.2%), C(4.8%) and G(4.7%) for treated and C(24.8%), CRF02(24.4%), A(8.3%), CRF01(8.2%), CRF06(7.4%) and D(3.2%) for naïve patients. All IAS-DRMs were observed except F227C; some of these mutations do not confer resistance to the administered regimen. Three NRTI-DRM differed by subtype/CRF: K65R was more frequent in C, M41L in CRF06; T215Y was less frequent in CRF01. For NNRTI–DRM V106M was more frequent in C, V90I and K103N in CRF02; V179D and G190A were less frequent in CRF02. Compared to naïve patients, nine accessory DRMs present in ANRS or Stanford DRM’s lists were observed at 1.39% to 5.19% (D67G, T69D, T69N, L74I, V75M, I132L, D218E, F227L and K238T) as well as four additional mutations (I94L (1.02%), L109I (2.27%), T139R (2.34%) and T165L (2.56%)). I94l and T165L were frequently observed with other NRTI-DRM; L109I and T139R were associated with both NRTI and NNRTI-DRM. Overall, all TAMs,

mL each year), never achieved suppression (i.e., VL >200 copies/mL each year), or who failed to maintain suppression (i.e., viral load >200 copies/mL after achieving viral suppression).

RESULTS: In all, 2,042 persons met the inclusion criteria. Of the 1,266 persons who achieved and maintained viral suppression, 195 (15%) had baseline DRAMs; of 187 persons who never achieved viral suppression, 37 (20%) had baseline DRAMs. Persons who failed to maintain viral suppression (589) were more likely to have baseline DRAMs (116, 20%; p=0.02) compared with those who achieved and maintained viral suppression. Evidence of distinct DRAMs on any subsequent sequence was low (1%–3%) in all groups.

CONCLUSIONS: NHSS data can be used to monitor viral suppression longitudinally and other factors such as DRAMs. We found an association between baseline DRAMs and failure to maintain viral suppression. Few sequences with distinct DRAMs were identified. HIV drug resistance testing and reporting are necessary to monitor DRAMs at a population level.

ABSTRACT 61

HIV-1 Non-B Subtype Associations and Emerging Resistance Mutations in First-Line ART Failure in Resource Limited CountriesCJ Villabona-Arenas1,2, N Vidal1, E Guichet1, L Serrano1, A Dangra3, E Delaporte1, O Gascuel2, M Peeters1, 2

1UMI233, Institut de Recherche pour le Développement (IRD), INSERM U1175, Université de Montpellier, Montpellier, France; 2l’Institut de Biologie Computationnelle (IBC), LIRMM and CNRS, Université de Montpellier, Montpellier, France; and 3Université de Lomé, Laboratoire de Biologie Moléculaire et d’Immunologie (BIOLIM/FSS/UL), Lomé, Togo

BACKGROUND: Knowledge on the impact of HIV-1 subtype/CRF on drug resistance mutations (DRM) remains incomplete, especially in the context of access to antiretroviral drugs (ARVs) in resource-limited countries (RLC). We studied the frequency of known DRMs according to subtype/CRF and eventual



ABSTRACT 62

Prevalence of HIV-1 Drug Resistance in Treated Patients with Viral Load > 50 copies/mL in 2014: A French Nationwide StudyL Assoumou1, C Charpentier2, M Grudé1, C Pallier3, L Morand-Joubert4, S Fafi-Kremer5, B Montes6, V Ferré7, M Bouvier-Alias8, J-C Plantier9, M-A Trabaud10, S Yerly11, C Alloui12, H Le Guillou-Guillemette13, A Vabret14, F Barin15, C Delaugerre16, D Descamps2, S Reigadas17 and the ANRS AC-11 Resistance Study Group.

1Sorbonne Universités, UPMC Univ Paris 06, INSERM, Institut Pierre Louis d’épidémiologie et de Santé Publique (IPLESP UMRS 1136), F75013, Paris, France ; 2INSERM UMR1137, Université Paris Diderot Sorbonne Paris Cité, AP-HP, Laboratoire de Virologie, Hôpital Bichat-Claude Bernard, CNR VIH associé Résistance aux Antirétroviraux, Paris, France; 3HU Paris sud, Hôpital Paul Brousse, Laboratoire de Virologie, Villejuif, France; 4Sorbonne Universités, UPMC Univ Paris 06, INSERM, Institut Pierre Louis d’épidémiologie et de Santé Publique (IPLESP UMRS 1136), AP-HP, Laboratoire de Virologie, Hôpital Saint-Antoine, F75012, Paris, France; 5Laboratoire de Virologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 6Laboratoire de Virologie, Hôpital Saint-Eloi, CHU Montpellier, France; 7EA 4271, Nantes Université UFR Pharmacie, Laboratoire de Virologie, CHU Nantes, France; 8INSERM U955, National Reference Center for Viral Hepatitis B, C et delta, Department of Virology; Henri Mondor Hospital, University of Paris-Est, Créteil, France; 9Laboratoire de Virologie et COREVIH Haute-Normandie, CHU de Rouen, Rouen, France; 10Hôpital de la Croix-Rousse, Hospices Civils de Lyon, Lyon, France; 11 Laboratoire de Virologie, hôpitaux universitaires de Genève, Genève, Suisse; 12Laboratoire de Virologie, Hôpital Avicenne, APHP, HU Paris Seine Saint Denis, Bobigny, France; 13Laboratoire de Virologie, CHU Angers et HIFIH Laboratory, UPRES 3859, SFR 4208, LUNAM University, Angers, France; 14Laboratoire de Virologie, CHU Caen, Caen, France; 15Laboratoire de Virologie, CHU Bretonneau, & INSERM U966, Tours, France; 16Laboratoire de Virologie, APHP, Hôpital Saint Louis, INSERM U941, Université Paris Diderot, Paris, France; 17Laboratoire de Virologie, Hôpital Pellegrin, CHU de Bordeaux; UMR 5234 MFP CNRS, Université de Bordeaux, 33076 BORDEAUX cedex, France

BACKGROUND: Surveillance of resistance in treated patients with detectable viral load (VL) is important

certain NNRTI-DRMs (A98G, K101H, K103S, V108I and P225H) and I94L, L109I and K238T were more common in ART >2 years leading to cross-resistance to other RTI, and compromising standardized second-line regimens.

CONCLUSION: We found associations of DRMs to certain subtypes/CRFs in patients on standardized 1st-line ART failure in RLC and reported additional mutations. Understanding mutational patterns aid determining better standardized regimens in RLC. Also, in the absence of biological monitoring, patients continue on failing ART, accumulate DRMs, which consequently limit the RTIs options for second-line therapy, and potentially cumulate DRMs and select new DRMs, which can be transmitted.



ABSTRACT 63

Receipt and Timing of Genotypic HIV Drug Resistance Testing in the United StatesS Dasgupta1, HI Hall1, AL Hernandez1, MC Bañez Ocfemia1, N Saduvala2, AM Oster1

1Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA; 2ICF International, Atlanta, GA

BACKGROUND: The U.S. Department of Health and Human Services recommends genotypic HIV drug resistance (DR) testing upon entry to care; however, receipt and timing of DR testing has not been well characterized. We examined DR testing at and after initiation of HIV care in the United States.

METHODS: We analyzed data from the U.S. National HIV Surveillance System (NHSS) for persons aged > 13 years with HIV infection diagnosed in 2013 who were linked to care (i.e., had a CD4 count or viral load test) within 3 months of diagnosis and resided in a jurisdiction with complete laboratory reporting and high reporting of nucleotide sequence data from DR testing (Los Angeles County, Michigan, New York, South Carolina, Texas, and Washington). We assessed the proportion of individuals who received DR testing at or after linkage and the distribution of time between linkage to care and DR testing. Among those who received DR testing, we conducted Mantel-Haenszel chi-square tests to identify factors associated with testing at the same time (i.e., in the same month) as linkage to care.

RESULTS: Of 11,351 persons in these jurisdictions who received a diagnosis of HIV infection during 2013, 9,435 (83%) were linked to care within 3 months of diagnosis. Among those linked to care, 6,106 (65%) ever received DR testing and 5,996 (64%) received DR testing within 12 months of linkage to care. Of those tested within 12 months of linkage, 4,195 (70%) received DR testing in the same month as linkage and an additional 1,153 (19%) within 1 month of linkage. The proportion of individuals who received testing at the time of linkage differed significantly across racial/

to evaluate both the risk of spreading resistant viruses and the proportion of patients for which new drugs with minimal cross-resistance are needed.

METHODS: The protease and reverse transcriptase (RT) genes of plasma viruses were systematically performed in samples from 782 consecutive HIV treated patients failing with a VL >50 copies/mL on two consecutive measurements, seen in 37 specialized centers between September and December 2014. The integrase gene was also sequenced by centers which perform it routinely. The genotype results were interpreted using the ANRS algorithm v24 pooling possible resistance and resistance. Weighted analyses were used to derive representative estimates of the percentage of patients. Prevalence rates were compared to those obtained during the same study conducted in 2009.

RESULTS: The protease and RT sequences were obtained for 566 patients and the integrase sequence for 382 patients. Sequencing was successful in 60% (n=206/346), 78% (n=95/122), 78% (n=59/76), and 87% (n=206/238) of samples with VL of 51-200, 201-500, 501-1000, and >1000 copies/mL respectively. The median CD4 cell count was 375/mm3 (IQR: 203-575).

Resistance to at least one ARV was observed in 56.3% of samples, to at least one NRTI in 36.0%, one NNRTI in 32.1%, one PI in 20.2%, and one II in 12%. The percentage of patients with viruses with no sensitive drugs in the NRTI, NNRTI, PI, and II family was 3.9%, 8.7%, 1.5%, and 3.4% respectively. All resistance prevalence rates were significantly lower in 2014 than in 2009, except for rilpivirine and etravirine. Moreover, resistance was evidenced in 48.5% of samples in patients with VL between 51 and 200 copies/mL.

CONCLUSION: In France, in 2014, 90% of HIV-diagnosed patients received cART and only 12.0% had a detectable VL above 50 copies/mL at least once a year, suggesting that at least 6.7% of treated patients could potentially transmit resistant viruses. This study also suggests that resistance testing may be recommended in all patients with a VL above 50 copies/mL, including those with a VL between 51 and 200 copies/mL



clinics were selected to contribute with participants from 136 Ministry of Health facilities by probability-proportional-to-size sampling, according to the number of ART initiators observed in each clinic during 2013. PDR was assessed from plasma virus, based on the WHO surveillance HIVDR mutation list, using the Stanford CPR tool. All samples were processed in a WHO-accredited lab by Sanger sequencing using the software RECall and by next generation sequencing (NGS) using the software HyDRA.

RESULTS: A total of 274 participants were included in the study; 84% were men. The median CD4+ T cell count was 257 cells/uL reflecting the known late presentation to clinical care in the country. PDR to any antiretroviral (ARV) drug was 12.0% (95% CI: 8.4-16.5%). NNRTI PDR was highest (6.9%), followed by NRTI PDR (5.1%) and PI PDR (2.6%). The most frequent PDR mutations were RT K103N (4.0%), M41L (1.1%) and PR L90M (1.8%). The prevalence of PDR to any ARV drug estimated with NGS at a 20% DR mutation frequency threshold was 12.2%, but increased considering lower thresholds: 14.2% at 10%; 17.3% at 5%; 30.3% at 2%. The most frequent minority variants (<5% of the viral population) included PR M46I, N88D and RT D67G, K70E. At the 2% DR mutation frequency threshold, intermediate levels of NNRTI (7.8%), NRTI (10.9%) and PI (10.1%) PDR were observed. Three putative transmission clusters were found, one including a male and a female from Baja California with M41L, one with two MSM from Sonora with Y181C and one with a female and an MSM from Veracruz with DR to two drug classes.

CONCLUSIONS: PDR in Mexico remains at the intermediate level, but individual PDR level to NNRTI has also reached intermediate level with high frequency of K103N, consistent with the wide use of efavirenz-containing first-line regimens in the country. Low frequency DR mutations mainly to NRTI and PI were observed. Evidence of DR mutation transmission was found in specific geographic areas involving both heterosexuals and MSM. These observations warrant continuous PDR surveillance in the country.

ethnic groups (p = .01) and age groups (p = 0.03). The proportion receiving DR testing at linkage was lower among blacks/African Americans (66%) compared to whites (71%) and Hispanics/Latinos (70%), and among those aged < 35 years (67%) compared to older individuals (70-71%).

CONCLUSIONS: NHSS data indicate that almost two-thirds of HIV-infected persons linked to HIV care received DR testing within 12 months of initiation of care, which may be an underestimate if not all DR tests were reported to surveillance. The timing of DR testing suggests that most providers order DR testing at entry to care as recommended. Increasing DR testing among blacks/African Americans and in younger age groups may help ensure appropriate ART selection and thus reduce disparities in viral suppression.

ABSTRACT 64

HIV Pre-Treatment Drug Resistance in Mexico: A Nationally Representative WHO SurveyS Ávila-Ríos1, C García-Morales1, D Tapia-Trejo1, M Matías-Florentino1, V Quiroz-Morales1, J Casillas-Rodríguez2, J Sierra-Madero3, E León4, C Magis-Rodríguez4, G Reyes-Terán1, for the HIVDR MexNet Group

1Centre for Research in Infectious Diseases, National Institute of Respiratory Diseases, Mexico City, Mexico; 2Condesa Specialised Clinic, Mexico City, Mexico; 3National Institute of Medical Sciences and Nutrition Salvador Zubirán, Mexico City, Mexico; 4National Centre for HIV/AIDS Prevention and Control, Mexico City, Mexico

BACKGROUND: To ensure sustainabil ity of antiretroviral treatment (ART) programmes after ART scale up, the WHO has proposed a global standardized HIV drug resistance (DR) monitoring and surveillance strategy, including the assessment of pre-treatment HIVDR (PDR).

METHODS: We present the f irst national ly representative WHO HIV PDR survey performed in Mexico from February to July 2015. Twenty-five



the TDR rate by class was 22.7%, 32.8%, 28.7% and 10.6% for nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and more than one class, respectively. Phylogenetic analysis in subtype B viruses among MSM patients supported clustered transmission. Among the potential transmitter population, the rates of ARV-treated patients increased from 66.1% in 2010 to 85% in 2015 (p<0.05). Treatment by PIs decreased from 53.1% in 2010 to 29% in 2015 (p<0.05). Treatment by NNRTIs decreased from 40.5% to 23.1%, respectively, and treatment by integrase inhibitors increased from 12.2% to 54%.3%, respectively (p<0.05). The treatment failure rate was 35.6%, 32.3%, 22.3%, 13.2%, 8.9 % and 6.1% in 2010, 2011, 2012, 2013, 2014 and 2015, respectively (p<0.05).

CONCLUSIONS: There was a drop in TDR between 2010 and 2013 among patients followed in Tel-Aviv. It then increased in 2014 and 2015, although there were significantly lower treatment failure rates and changes in treatment strategy, mainly to integrase inhibitors, in the potential transmitter population. This supports the important role of clusters in TDR. Integrase region was not routinely tested, but the rate of TDR for these drugs is reportedly low. Regular assessment of resistance in the ARV-treated and naïve populations is necessary to understand the potential epidemiological effect of new ARV strategies and drugs on TDR.

ABSTRACT 65

Prevalence of Transmitted HIV-1 Drug Resistance in Tel-Aviv, Israel, and Antiretroviral Strategy in a Potential Transmitter Population from 2010-2015D Turner1, S Girshengorn1, A Braun1, L Tau1, R Cohen-Poradisu1, T Finn 1, A Leshno1, D Alon1, T Pupko2,, E Katchman1, I Zeldis1, S Ahsanov1, L Giladi1, N Matus1 and B Avidor1

1Tel Aviv Crusaid Kobler AIDS Center, Tel Aviv Sourasky Medical Center , Israel, affiliated to the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; 2Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

BACKGROUND: The prevalence of transmitted drug resistance (TDR) mutations is assessed regularly in treatment-naïve patients in Tel-Aviv. We now evaluated the antiretroviral (ART) strategy among all patients followed in Tel-Aviv who were considered as comprising a potential transmitter population.

METHODS: All blood samples obtained from treatment-naïve patients between 2010 and 2015 (October 31) were analyzed for reverse transcriptase and protease resistance-associated mutations. TDR was defined according to the 2009 criteria of Bennett et al. A phylogeny was inferred using pol sequences. The number of patients treated with antiretroviral drugs (ARVs) was tallied. Treatment failure was defined as a viral load >200 copies/ml. The c2 test for trend was applied to compare the different parameters between the years.

RESULTS: Sequences from 626 patients were tested. The resistance rate decreased from 15% in 2010 to 6.9% in 2012 and to 5.8% in 2013 (p<0.05). It increased again to 11.6% in 2014 and to 11.7% in 2015. Among men who have sex with men (MSM), which is the major exposure risk category in Tel-Aviv, the rate of TDR was 19.4% in 2010, 14.2% in 2011, 8.9% in 2012, and 9.2% in 2013 (p<0.05), and it increased again to 15.6% in 2014 and to 12.5% in 2015. Overall,



RESULTS: Among the 69 HIV-1 infected DBS collected from Equatoguinean women, a total of 38 specimens were successfully amplified and sequenced. Twenty-nine (76.3%) were ART-experienced. Fifteen had been exposed to nucleoside reverse transcriptase inhibitors (NRTI), mainly to monotherapy with zidovudine (14 cases). Fourteen had received highly active ART (HAART) as first regimen, based on two NRTI and one non-nucleoside reverse transcriptase inhibitor (NNRTI) or one protease inhibitor or PI (boosted Lopinavir, LPV/r). No TDR were found in NRTI o NNRTI-naive women, but the TDR rate for PI was 3.4%. The D30N at PR was detected in a PI-naive woman carrying CRF02_AG recombinant. The ADR rate for NRTI was 7.7% (M41L or M184V), to NNRTI was 40% (V90I, V106I or G190A) and absent for PI among treated women with the corresponding drug family. No case of NNRTI+NRTI resistance was found. HIV-1 group M non-B variants caused most (97.4%) infections, being mainly (78.9 %) recombinants: CRF02_AG (55.2%), CRF22_A101 (10.5%), subtype C (10.5%), unique recombinant forms (5.3%), and A3, D, F2, G, CRF06_cpx and CRF11_cpx (2.6% each).

CONCLUSIONS: The present study performed during 2012-2013 is the first one analyzing drug resistance and the molecular epidemiology among HIV-infected pregnant woman from Equatorial Guinea. We confirmed the predominance of group HIV-1 recombinant variants, causing CRF02_AG nearly 55% of infections. The presence of DRM to PI among naive women and the high DRM rate to retrotranscriptase inhibitors (mainly NNRTIs) observed among pretreated, reinforce the importance of systematic DRM monitoring in Equatorial Guinea. It would permit to reduce HIV-1 resistance transmission, to optimize first and second-line ART regimen when DRM are present, and to preserve future ART options in infected patients carrying resistant viruses.

ABSTRACT 66

HIV-1 Variants and Drug Resistance in Pregnant Women from Equatorial Guinea: 2012-2013 P Alvarez1, C Fernández McPhee2, L Martín1, L Prieto3, J Obiang4, A Vargas5, P Rojo6, A Benito5, JT Ramos7 and Á Holguín1

1Hospital Ramón y Cajal, IRYCIS-Microbiology Department, Madrid, Spain; 2Hospital Universitario Gregorio Marañón, Madrid, Spain; 3Hospital Universitario de Getafe, Madrid, Spain; 4Hospital Provincial de Bata, Ministerio de Sanidad y Bienestar Social, Equatorial Guinea; 5Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III-Madrid, RICET, Spain; 6Hospital Universitario Doce de Octubre, Madrid, Spain; 7 Hospital Universitario Clínico San Carlos, Madrid, Spain.

BACKGROUND: The World Health Organization (WHO) recommends population-based surveys to monitor the drug resistance mutations (DRM) to antiretroviral therapy (ART) in HIV-1 infected patients. Despite its high HIV prevalence (6.2%), Equatorial Guinea (GQ) lacks of systematic surveillance studies on DRM prevalence and HIV-1 variants. This is the first study describing the presence of DRM and the current circulating HIV-1 variants among pregnant women from Bata, Equatorial Guinea.

METHODS: The study was conducted among 69 HIV-1 infected naive and pretreated women who participated in a prevention of mother-to-child HIV transmission program at Hospital Provincial de Bata and Primary Health Care Center María Rafols, in Equatorial Guinea. Dried blood spots were collected from November 2012 to December 2013. The transmitted (TDR) or acquired (ADR) antiretroviral drug resistance mutations among naive or ART-exposed pregnant women was defined following the lists provided by WHO or IAS-USA 2014, respectively. HIV-1 variants were identified by phylogenetic analyses (protease-PR and/or retrotranscriptase-RT), taking as reference GenBank sequences from all subtypes and 66 circulating recombinant forms (CRFs) available at study time.



was sequenced and HIV tropism was determined using Geno2Pheno algorithm (FPR 10%). HIV-1 subtype was determined after phylogenetic analysis of the RT sequence.

RESULTS: Patients were mainly men (92%), having sex with men (MSM) (69%), living in Paris area in 43% of cases. At enrollment, median viral load and CD4 cell count were 5.65 (range: 1.38-8.25) log10 copies/mL and 483 (range: 6-1871) cells/mL respectively. Non-B variants were identified in 39% of patients. RAMs were identified in 9.2% strains, 95% CI [6.5-12.7] using the WHO 2009 list and in 14.1%, 95% CI [10.7-18.1] using both the WHO list and the ANRS 2014 algorithm definition. The prevalence of PI-, NRTI-, first-generation NNRTI-associated RAMs was 2.4% (M46L in 4/9 cases), 4.3% (mostly TAMs in 15/16 cases, T215D/E/C/S revertant mutations in 10 cases, M184V in 1 case) and 3.0% (K103N in 7 cases, G190A in 3 cases) respectively. RAMs to second-generation NNRTI (RPV and ETR) were observed in 6.0% (E138A (18), E138K (1), E138G (1), E138R (1), K103N, Y188L (1)). Overall, resistance to at least one NNRTI was 8.4%. Viruses with single and dual class resistance were isolated in 48 (12.8%) and 4 (1.1%) patients, respectively. Additionally, 6/223 (2.7%) strains had II-related RAMs (E157Q mutation in 4 cases (2 subtype B, 2 CRF02_AG) conferring resistance to raltegravir (RAL) and elvitegravir (EVG) and R263K in 2 cases (1 subtype B, 1 subtype F) conferring resistance to dolutegravir (DTG). None of these 6 viruses harbored any other mutation in RT or PR genes. Phenotypic resistance tests are in progress. At enrolment, 33 out of 216 (15.3%) harbored a X4/DM-tropic virus.

CONCLUSIONS: The prevalence of transmitted drug-resistant variants according to the WHO list was 9.2% in France in 2014, stable since 1996. However, we describe a high level of NNRTI resistance (8.4%) including ETR and RPV. Moreover, 4-naïve subjects (1.8%) had a drug resistance mutation to both RAL and EVG and 2 (0.9%) to DTG.

ABSTRACT 67

Polymorphic Mutations Increase NNRTI and II Resistance in Primary HIV-1 Infected PatientsML Chaix1,2, L Assoumou3, N Mahjoub1, M Wirden4, J Cottalorda5, V Schneider6, K Saune7 V Avettand-Fenoël8, G Collin9, M Grude3, S Vallet10, H Pere11, L Bocket12, C Goujard13, L Meyer13, D Descamps9 on behalf the AC11 ANRS Resistance Group and the French PRIMO Cohort Study Group

1INSERM U941, Université Paris Diderot, Sorbonne Paris Cité, CNR VIH associé Primo infection, Paris; 2AP-HP, Laboratoire de Virologie, Hôpital Saint-Louis, Paris; 3Sorbonne Universités, UPMC Univ Paris 06, INSERM, Institut Pierre Louis d’épidémiologie et de Santé Publique (IPLESP UMRS 1136), F75013, Paris; 4Sorbonne Universités, UPMC Paris 06, INSERM, IPLESP UMRS 1136, AP-HP, Service de Virologie, Hôpital Pitié-Salpêtrière, Paris; 5Laboratoire de Virologie, Hôpital de l’Archet, CHU Nice; 6AP-HP, Service de Virologie, Hôpital Tenon, Paris; 7Department of Virology, Federative Institute of Biology, CHU Toulouse, Department of Physiopathology Toulouse Purpan, Inserm Unit U563, Toulouse; 8EA7327, Université Paris Descartes, AP-HP, Laboratoire de Microbiologie Clinique, Hôpital Necker – Enfants malades, Paris; 9INSERM UMR1137, Université Paris Diderot Sorbonne Paris Cité, AP-HP, Laboratoire de Virologie, Hôpital Bichat-Claude Bernard, CNR VIH associé Résistance aux Antirétroviraux, Paris; 10Laboratoire de Virologie, CHU Brest, Brest; 11AP-HP, Service de Virologie, HEGP, Paris; 12Laboratoire de Virologie, CHU Lille, Lille; 13University Paris Sud, INSERM CESP U1018, APHP Bicêtre Hospital, Paris, France

BACKGROUND: Our study describes the prevalence of transmitted drug-resistance (TDR) among 368 naïve patients diagnosed at the time of primary HIV-1 infection (PHI) in France in 2014.

METHODS: Genotypic resistance studies were performed at the time of PHI on protease, reverse transcriptase and integrase genes. HIV-1 resistance-associated mutations (RAMs) were characterized using both the 2009 WHO list of mutations and the 2014 French ANRS algorithm. The ANRS algorithm includes all the rilpivirine-, etravirine- and integrase inhibitors (II) related RAMs reported in the 2014 IAS-USA resistance mutations list. The HIV envelope gene



harbored MRV detected by UDS only: two R263K (at a rate of 9.7%, mutational load: 7099 copies/mL; and 13.5 %, 8345 copies/mL) and one E138K mutations (at 4.8%, 111 copies/mL). All these mutations were retrieved among B subtype viruses.

CONCLUSIONS: None of the three classical ISTIs signature resistance mutations (at positions 143, 148 and 155) were retrieved. However, in this population of MSM naive-treatment patients, the prevalence of ISTI-resistance mutations, mainly related to polymorphisms, seems to be relatively high (9.2% by Sanger and 13.8% by UDS). In conclusion, with the increase use of ISTIs in clinical practice, TDR for this therapeutic class should be carefully monitored in the future, as well as the impact of these MRV on the virological response.

ABSTRACT 69

National Molecular Surveillance of Recently Acquired HIV Infections, Germany 2013-2014A Hauser1, A Hofmann2, K Hanke1, V Bremer2, B Bartmeyer2, C Kuecherer1, N Bannert1

¹Division of HIV and Other Retroviruses, Robert Koch Institute, Berlin, Germany; ²Division of HIV/AIDS, STI and Blood-borne Infections, Robert Koch Institute, Berlin, Germany

BACKGROUND: Continuous molecular HI V surveil lance provides valuable public health information concerning the transmission of drug resistant viruses and the dynamics of currently circulating variants. To enable an up-to-date molecular analysis of HIV-genotypes circulating in Germany, the Robert Koch Institute (RKI) has established a surveillance system based on recently acquired HIV infections. The aim is to assess the current prevalence of transmitted drug resistant (TDR) variants and HIV-1 subtypes in the main HIV-transmission groups: men who have sex with men (MSM), women/men with heterosexual contacts (HET) and persons with intravenous drug use (PWIDs) with respect to their origin and place of infection.

ABSTRACT 68

Integrase Inhibitors-Transmitted Drug Resistance Detected by UltraDeep SequencingE Todesco1, J Jaffré1, C Soulié1, D Armenia2, M Wirden1, S Lambert1, C Katlama1,3, F Ceccherini-Silberstein2, V Calvez1, AG Marcelin1

1Sorbonne Universités, UPMC Univ Paris 06, INSERM, Institut Pierre Louis d’épidémiologie et de Santé Publique (IPLESP UMRS 1136), F75013, Paris, France.

Department of Virology, Hôpital Pitié-Salpêtrière, AP-HP, F75013, Paris, France; 2Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy; 3Sorbonne Universités, UPMC Univ Paris 06, INSERM, Institut Pierre Louis d’épidémiologie et de Santé Publique (IPLESP UMRS 1136), F75013, Paris, France. Department of Infectious Diseases, Hôpital Pitié-Salpêtrière, AP-HP, F75013, Paris, France

BACKGROUND: Transmitted Drug Resistance (TDR) can impair first-line antiretroviral therapy response. Moreover, HIV-1 minority resistant variants (MRV) can be a source of virological failure if they are present before antiretroviral treatment: it was mainly shown for non nucleoside reverse transcriptase inhibitors first line based regimens. Few data are available for TDR Integrase Strand Transfer Inhibitors (ISTIs). In this work, we have studied resistance mutations in integrase gene by Sanger sequencing and UltraDeep Sequencing (UDS) in ISTI-naive patients.

METHODS: Integrase genotypic analysis was performed by Sanger sequencing and by UDS. Plasma samples of 65 treatment-naïve Men having Sex with Men (MSM) patients were analyzed from the amino acid 53 to 281. GS Amplicon Variant Analyzer was used to analyze the UDS data, with a detection threshold of MRV of 1% (forward and reverse). Resistance was interpreted according to the last version of ANRS algorithm (www.hivfrenchresistance.org).

RESULTS: Among the 65 patients, 60% of them were infected by B subtype. Viruses of six patients harbored majority resistant mutations by Sanger sequencing (four L74I and two E157Q mutations). Three viruses



ABSTRACT 70

Trends in Transmitted Drug Resistance and Subtype Distribution in Spain in the Period 2007 to 2015M Alvarez1, AB Pérez1, R Camacho-Luque 1, N Chueca1, JA Iribarren2, JL Gómez-Sirvent3, M Masiá4 , A Aguilera5, J Peraire6, E Bernal7, S Monge8, F García1 on behalf of Coris

1CHU Granada. PTS-San Cecilio. Instituto de Investigación ibs.Granada; 2HU Donostia; 3HU de Canarias; 4Hospital de Elche; 5 CHUSantiago de Compostela; 6HU Joan XXIII; 7HU Reina Sofía; 8Universidad Alcalá de Henares, Spain

BACKGROUND: We aim to characterize temporal trends in transmitted drug resistance mutations (TDR), resistance to first line reverse transcriptase and protease inhibitors, and non-B subtypes in CoRIS, a cohort with wide territorial representation in Spain.

METHODS: CoRIS is a multicenter cohort of adult HIV naïve patients. By 2015, 27 sites from Spanish centers participating in the cohort contributed with 4734 patients to the study. Transmitted Drug Resistance associated mutations were evaluated following the WHO 2009 update. HIV Subtype and resistance to first line drugs were investigated using Stanford HIV Db algorithm (v.7.0). The association of sex, age, transmission category, educational level and country of origin, CD4 count and Viral Load (VL), CDC stage, and delayed diagnosis (CD4 count <350 cells/mm3 at diagnosis), with resistance and subtype distribution was evaluated.

RESULTS: Throughout the study period, TDR mutation prevalence was 7.6% (6.8-8.4). Similar prevalence was found for NRTIs [3.5% (2.9-4.0)] and NNRTIs [3.5% (3.0-4.1)]. Resistance to PIs was 1.8% (1.4-2.2). TDR mutation prevalence remained stable throughout the study period, being 6.2% (4.2-8.3) in 2008 and 8.5% (6.0-10.8) in 2014 (3.0% to 4.3% for NRTIs; 2.3% to 4.6% for NNRTIs, and 0.7% to 2.5% for PIs). On the other hand, when resistance, using Stanford algorithm interpretation, was evaluated, we found an overall prevalence of resistance to first line drugs of

METHODS: Newly diagnosed cases are reported to the RKI as a statutory duty for anonymous notification. Diagnostic laboratories provide dried serum spot (DSS) of ~60% of all newly diagnosed HIV infections reported. DSS serologically classified as “recently acquired infections” (<140 days; BED-CEIA, Sedia) were genotyped in the HIV-pol-region to identify TDR and to determine the HIV-1 subtype. The results are linked to notification data from the report.

RESULTS: In 2013 and 2014 1,963 of 6,371 DSS originated from a recent infection. Of these recent infections, 881 were successfully sequenced and analysed. Total TDR was 10.7%, comprising 4.3% with mono resistance to nucleotide reverse transcriptase inhibitors (NRTIs), 2.7% to non-NRTIs, 2.7% to protease inhibitors and 0.6% and 0.3% with dual and triple class resistances, respectively. HIV-subtype B was most prevalent with 76.2%. Non-B infections were identified more often in HET compared to PWIDs or MSM (79%; 39%; 12%, all p<0.05). Non-B subtypes were also more frequently found in patients originating in countries other than Germany (49% vs. 15%; p<0.05) and in patients infected outside of Germany (65% vs. 14%; p<0.05).

CONCLUSION: TDR prevalence in recent HIV infections among notified newly diagnosed HIV patients in Germany remained high (>10%) in 2013/2014 and is comparable to other European countries, including with regard to the proportions of resistance classes. Therefore, genotypic resistance testing of HIV prior to first-line treatment should be continued. Our data also demonstrate that subtype B infections remains the most frequently transmitted subtype in the country based on its high prevalence in MSM.



infected antiretroviral treatment naïve participants from rural Coastal Kenya.

METHODS: In a retrospective longitudinal study, samples from HIV-1 infected participants enrolled for care between 2008 and 2013 were analysed. Individual longitudinal samples from three time-points were considered: i) at least 6 months prior to treatment initiation; ii) at most 3 months prior to treatment initiation; and iii) at least 6 months after treatment initiation. An amplicon-based next generation sequencing assay, calling for nucleotide variants at >2.0% frequency of viral population, was used. Suspected virologic failure (sVF) was defined as a one-off HIV-1 viral load of >1000 copies/ml whilst on antiretroviral therapy.

RESULTS: Fifty participants were included (females, n=36 [72.0%]; subtype A1, n=32 [64.0%]; median age, 34.7 [IQR, 28.9 – 42.0] years). Of these, 12 (24.0% [95% CI: 13.1 – 38.2]) had at least one detectable pre-treatment HIVDR variant against Protease Inhibitors (PIs, n=6 [12%]), Nucleoside Reverse Transcriptase Inhibitors (NRTIs, n=4 [8.0%]) and Non-NRTIs (NNRTIs, n=3 [6.0%]). Exclusively, 3 (6.0%) had majority (frequency, >20%) resistance variants whilst 10 (20.0%) had minority (frequency, >2% and <20%) resistance variants. Overall, 15 pre-treatment variants were detected (mutation frequency, range: 2.3 – 92.0%). A positive correlation was observed between mutation frequency and absolute mutation load for NRTI (r=0.919 [p=0.080]) and NNRTI (r=1.000 [p<0.001]), but not for PI (r= -0.185 [p=0.726]) variants (figure). Participants with pre-treatment NRTI and/or NNRTI resistance had increased odds of sVF (OR [95% CI], p=value: 6.0 [1.0 – 36.9], p=0.054).

10.9% (10-11.8), with a significant (p=0.048) decrease throughout the study period in resistance to the first line NNRTIs Tenofovir, Abacavir, Lamivudine and Emtricitabine. TDR mutations to more than one ARV family were uncommon (0.9%; 0.6-1.1). 750 patients had non-B subtypes [15.8%, (14.8-16.9)], with a significant increase (p<0.001) from 2007 [10.2%, (7.4-13.0)] to 2014 [17.4%, (14.2-20.7)]. CRFs accounted for 40.3% (36.7-43.8) (n=302) of non-Bs (6.4% prevalence)

CONCLUSIONS: Transmitted Drug Resistance (TDR) in Spain remains low and with no trend over time during the period 2007 to 2015. Although resistance to first line NRTIs decreased in Spain, baseline resistance to Efavirenz and Nevirapine still remains a problem of concern for the selection of first line regimens. Non-B subtype infections are on the rise with a majority of CRFs among the non-Bs.

ABSTRACT 71

Presence, Persistence and Effects of Pre-Treatment HIV-1 Drug Resistance Variants Detected at a Low Frequency using Next Generation Sequencing: A Retrospective Longitudinal Study from Rural Coastal KenyaAS Hassan1 , DF Bibby2 , SM Mwaringa1 , CA Agutu1 , KK Ndirangu3 , EJ Sanders1,4 , PA Cane2 , JL Mbisa2 and JA Berkley1,4

1KEMRI/Wellcome Trust Research Programme, Kilifi, Kenya; 2Virus Reference Department, Public Health England, London, UK; 3Kilifi County Hospital, Kilifi, Kenya; 4Centre for Tropical Medicine & Global Health, University of Oxford, UK

BACKGROUND: The epidemiology of HIV-1 drug resistance (HIVDR), determined by conventional genotypic resistance testing, has been widely studied. However, much less is known about HIVDR detected at lower frequencies. We aimed to determine the presence, persistence and effect of HIVDR variants detected at a low frequency in HIV-1 chronically



ABSTRACT 72

Rate of Transmitted Integrase Inhibitor Resistance Mutations Remains Very Low in the SHCSAU Scherrer1, J Böni2, WL Yang1, R Kouyos1, S Yerly3, T Klimkait4, V Aubert5, HF Günthard1 and the Swiss HIV Cohort Study

1University Hospital Zürich, University of Zürich, Switzerland; 2University of Zürich, Switzerland; 3Geneva University Hospitals, University of Geneva, Switzerland; 4University of Basel, Switzerland; 5Lausanne University Hospital, Switzerland

BACKGROUND: Integrase inhibitors (INSTIs) are increasingly used in first-line and salvage treatment of HIV-1 infected patients in Switzerland. The broader use of INSTIs might lead to a higher prevalence of INSTI transmitted drug resistance (TDR).

METHODS: We studied the prevalence of INSTI TDR mutations (as defined by IAS-USA 2014) among INSTI-naïve patients in the Swiss HIV Cohort Study (SHCS) who had a genotypic resistance test done before 1 July 2014. We performed logistic regression models to analyse the association of INSTI TDR mutations with the calendar year and the approval of the first INSTI in Switzerland (28 February 2008). Models were adjusted for subtype (B vs. non-B infections). To estimate the risk of transmission of INSTI TDR, we calculated the population viral load (PVL, sum of patients yearly area under curve of log10 HIV-RNA) after the first treatment failure on INSTI and compared it to the PVL of patients who failed a treatment with PIs and NNRTIs during the time those regimens were introduced.

RESULTS: In 1,362 INSTI-naïve patients, we detected four major mutations (T66I, Q148K and 2 N155H) (0.3%) and 39 minor INSTI TDR mutations (2.9%). T66I was the only major mutation detected before the introduction of INSTI in Switzerland. Minor mutations were more frequently among non-B subtype infections (26/462 [5.6%] vs. 13/900 [1.4%], p-exact<0.001). No difference in prevalence was found

Figure: Graph illustrating the distribution and relationship between HIV-1 low frequency pre-treatment resistance variants frequency and absolute load observed amongst participants from a rural HIV clinic in Coastal Kenya.

0.0

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CONCLUSION: At a low frequency, pre-treatment resistance variants were common, though observed PI variants were unlikely transmitted, but rather probably generated de novo. Even at low frequencies, pre-treatment NRTI and/or NNRTI variants may adversely affect treatment outcomes. Further studies are needed to delineate the independent pathways of low frequency variants, and to empirically assess the performance of mutational frequency and absolute load on treatment outcomes.



ABSTRACT 73

Transmission of HIV Drug Resistance Mutations Varies Regionally in Europe LM Hofstra1,2, N Sauvageot2, J Albert3,4, I Alexiev5, D Struck2, DAMC Van de Vijver6, B Åsjö7, D Beshkov5, S Coughlan8, D Descamps9, F Garcia10, P Gomes11,12, A Griskevicius13, O Hamouda14, A Horban15, T Kolupajeva16, LG Kostrikis17, K Liitsola18, M Linka19, O Mor20, C Nielsen21, D Otelea22, D Paraskevis23, M Parczewski24, R Paredes25, M Poljak26, E Puchhammer-Stöckl27, J Ruelle28, A Sönnerborg3,4, D Staneková29, M Stanojevic30, K Van Laethem31, T Yalcinkaya32, M Zazzi33, SZ Lepej34, J-C Schmit2, CAB Boucher6 and AMJ Wensing1 on behalf of the SPREAD program

1University Medical Center Utrecht, Virology, Utrecht, the Netherlands; 2Luxembourg Institute of Health, Luxembourg, Luxembourg; 3Karolinska Institute, Solna, Sweden; 4Karolinska University Hospital, Stockholm, Sweden; 5National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria; 6Erasmus MC, University Medical Center, Rotterdam, the Netherlands; 7University of Bergen, Bergen, Norway; 8University College Dublin, Dublin, Ireland; 9AP-HP Groupe hospitalier Bichat-Claude Bernard, IAME INSERM UMR 1137, Univ Paris Diderot Sorbonne Paris Cité, Paris, France; 10Complejo Hospitalario Universitario de Granada; Instituto de Investigación IBS Granada; on behalf of Cohorte de Adultos de la Red de Investigación en SIDA (CoRIS), Spain; 11Laboratório de Biologia Molecular, Centro Hospitalar Lisboa Ocidental, Lisboa, Portugal; 12Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Monte de Caparica, Portugal; 13Lithuanian AIDS Center, Vilnius, Lithuania; 14Robert Koch Institute, Berlin, Germany; 15Hospital of Infectious Diseases, Warsaw, Poland; 16Infectiology Center of Latvia, Riga, Latvia; 17University of Cyprus, Nicosia, Cyprus; 18Department of Infectious Diseases, National Institute for Health and Welfare, Helsinki, Finland; 19National Reference Laboratory for HIV/AIDS, National Institute of Public Health, Prague, Czech Republic; 20National HIV Reference Laboratory, Public Health Services, Chaim Sheba Medical Center, Tel-Hashomer, Israel; 21Statens Serum Institute, Copenhagen, Denmark; 22National Institute for Infectious Diseases “Prof. Dr. Matei Bals”, Bucharest, Romania; 23National Retrovirus Reference Center, University of Athens, Athens, Greece; 24Pomeranian Medical University, Szczecin, Poland; 25IrsiCaixa Foundation, Badalona, Spain; 26Slovenian HIV/AIDS Reference Centre, University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia; 27Medical

before and after approval of INSTIs (4/153 [2.6%] vs. 35/1209 [2.9%], p-exact=0.810). The odds ratio (OR) adjusted for subtype was 1.2 (95% CI 0.5-3.1). Thus, minor mutations are most likely polymorphic. Despite broader use of INSTIs, the prevalence of INSTI TDR mutations did not increase over time (OR per calendar year adjusted for subtype: 1.0 [95% CI: 0.9-1.1]). Patients failing a treatment (VL>500 HIV-RNA copies/mL) containing INSTIs have the highest risk to transmit INSTI TDR. The number of these patients steadily increased from 13 patients in 2008 to 37 in 2013. The median yearly PVL after failure on INSTI treatment was 491 log10 HIV-RNA between 2008 and 2013. This number is very small compared to the PVL measured after the introduction of PIs (1996-2001) and NNRTIs (1997-2002). The median yearly PVL was 5,011 and 17,300 log10 HIV-RNA after treatment failure on PI or NNRTI in the mentioned time periods, respectively.

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�

CONCLUSION: Major INSTI TDR mutations are very rare in the SHCS. The increasing use of INSTI did not yet influence the INSTI TDR rate. One explanation is the careful administration of INSTI and early treatment changes of failing regimens as reflected by the low PVL after treatment failure on INSTI. However, baseline resistance testing on integrase is warranted for early detection of a potential increase as the number of patients receiving and failing treatments containing INSTIs is increasing



4.5% in Central, Mediterranean and Northern Europe respectively (p0.0096).

CONCLUSION: The prevalence of TDRM varies regionally in Europe and is higher in Western Europe, due to a higher TDRM to NRTI and NNRTIs. To inform clinicians and public health authorities with up-to-date figures from their region, ESAR developed an interactive tool with maps of regional surveillance data from the SPREAD program.

Table 1. Baseline characteristics of study population

University Vienna, Vienna, Austria; 28AIDS Reference Laboratory, UCLouvain Bruxelles, Brussels, Belgium; 29Slovak Medical University, Bratislava, Slovakia; 30University of Belgrade Faculty of Medicine, Belgrade, Serbia; 31University Hospital Leuven, Rega Institute for Medical Research, AIDS Reference Laboratory, Leuven, Belgium; 32Refik Saydam National Public Health Agency, Ankara, Turkey; 33University of Siena, Siena, Italy; 34University Hospital for Infectious Diseases “Dr. Fran Mihaljevic”, Zagreb, Croatia

INTRODUCTION: The SPREAD surveillance program, carried out by the European Society for translational Antiviral Research, has been in place for over 10 years, to monitor transmitted HIV drug resistance mutations (TDRM) in Europe.

METHODS: Clinical and virological data of >14,000 patients diagnosed in 2002 to 2013 has been collected from 28 European countries (Table 1). TDRM were defined using the WHO surveillance of drug resistance mutations list. Prevalence of TDRM was assessed by region in Europe (Central: Bulgaria, Croatia, Czech Republic, Latvia, Lithuania, Poland, Romania, Serbia, Slovakia, Slovenia; Mediterranean: Cyprus, Greece, Israel, Italy, Portugal, Spain, Turkey; Northern: Denmark, Finland, Norway, Sweden; Western: Austria, Belgium, France, Germany, Ireland, Luxembourg, Netherlands). Wald tests were used to compare prevalence over the regions.

RESULTS: Overall in Europe the prevalence was stable from 2002 to 2013, with 9.0% in 2011-2013. In these last three years, the lowest prevalence was observed in Northern Europe (5.7%), followed by Central and Mediterranean Europe (6.7% and 7.4% respectively). In Western Europe the prevalence was highest (12.1% in 2011-2013; p<0.0001), driven by a higher prevalence of TDRM to NRTIs (7.4%, p<0.0001) and NNRTIs (4.4%, p0.025). TDRM to PIs was generally low in all regions. The prevalence of TDRM in MSM was higher in Western Europe (2011-2013: 13.5%, p<0.0001) than the other regions (Central: 7.8%, Mediterranean: 8.5%, Northern: 8.0%), but has remained stable over 2002-2013. The prevalence in heterosexuals was lower than in MSM in all regions, but was again highest in Western Europe, 9.6%, compared to 5.6%, 7.7% and



transmission route (42.2%), followed by heterosexual (36.4%) and drug use (4.1%). Non-B infected patients accounted for 30.8% (N=1,331) of the overall population (CRF02_AG: N=330; F: N=248; C=222; other=531). Of note, non-B subtypes progressively increased over time (<2005 to 2014: 19.5% to 38.5%, p<0.001), particularly in Italians (<2005-2014: 6.5%-28.8%, p<0.001). Overall, TDR prevalence was 8.8% (N=382) (B subtype: 9.7%; non-B subtype: 6.9%, p=0.003), and did not differ according to age, sex, nationality, risk-exposure, year of diagnosis, recent infection, CD4 and viremia. By evaluating TDR to any class over time, TDR increased in non-B subtypes (<2005-2014: 2%-7.1%, p=0.018), while decreased with a slightly trend toward significance in B subtype (<2005-2014: 13.4%-8.1%, p=0.137).

Regarding cluster analysis, 364 TCs were identified (74 pairs and 25 large clusters in non-B subtypes; 204 pairs and 61 large clusters in B subtype). A high proportion of recently diagnosed individuals (2011 [2010-2013] vs. 2010 [2008-2012], p<0.001), women (22.9% vs. 9.1%, p<0.001), and heterosexuals (34.7% vs. 23.1% p<0.001) was found in non-B vs. B subtype TCs.

By analysing resistance, 10.4% of TCs involved subjects with TDR, also in non-B subjects (non-B TCs: 6; B TCs: 32). These TDR-TCs were mainly represented by pairs both in non-B (83.3%) and B subtypes (68.7%). The six TDR non-B-TCs were mainly characterized by NNRTI resistance, followed by PI and NRTI resistance (75.0%, 12.5% and 6.3%). Among the 32 TDR B-subtype-TCs, NRTI, NNRTI, and PI TDR was 37.6%, 24.8%, 17.8%, respectively.

CONCLUSIONS: HIV-1 non-B diagnoses have been increased in Italy over the years 2000-2014. Even if TDR is mainly accounted for B infections, a TDR increase over time is found in non-B subtypes, and is also related to TCs. These findings highlight the epidemiological changes in HIV-1 infection occurred in Italy over the last 15 years. This requires an improvement of HIV-1 prevention strategies and screening activities.

ABSTRACT 74

Dynamics of Transmitted HIV-1 Drug Resistance According To Subtype in Italy over the Years 2000-2014L Fabeni1, C Alteri2, D Di Carlo2, L Carioti2, A Bertoli2, C Gori1, F Forbici1, MC Bellocchi2, F Continenza1, S Cicalini1, C Pinnetti1, R Bellagamba1, G D’Offizi1, V Borghi3, M Giuliani4, G De Carli1, N Orchi1, FM Fusco1, P Scognamiglio1, A Pennica5, C Mastroianni6, F Montella7, A Cristaudo4, C Mussini3, E Girardi1, M Andreoni8, A Antinori1, F Ceccherini-Silberstein2, CF Perno1 and MM Santoro2

1National Institute for Infectious Diseases L Spallanzani, Rome, Italy; 2University of Rome Tor Vergata, Rome, Italy; 3Modena University Hospital, Modena, Italy; 4IRCSS San Gallicano, Rome, Italy; 5S.Andrea Hospital, Rome, Italy; 6Sapienza University, Latina, Italy; 7S. Giovanni Addolorata Hospital, Rome, Italy; 8University Hospital Tor Vergata, Rome, Italy

BACKGROUND: We evaluated the dynamics and phylogenetic relationships of transmitted-drug-resistance (TDR) and subtype among individuals with HIV-1 infection confirmed in different counselling and testing centres in North and Central Italy from 2000 to 2014.

METHODS: 4,323 HIV-1 pol sequences from drug-naïve patients (1 per patient) were analysed. TDR was evaluated over time by considering WHO-2009 list with the additional reverse-transcriptase mutations 215N/138GKQR/179L/221Y/227C/230I listed in IAS/Stanford 2014. Phylogeny was generated using GTR model and 1,000 bootstrap with maximum-likelihood method using RAxML. Transmission clusters (TCs) were recognized by Cluster Picker based on bootstrap values >90% and genetic distance ≤0.02; TCs included large TCs (≥3 sequences) and pairs (2 sequences). Factors associated with TDR (in both overall population and TCs) were evaluated by uni-multivariable logistic regression analysis.

RESULTS: The majority of individuals was male (80.2%) and Italian (72.1%), with a median age of 37 (IQR:30-45) years. Homosexual was the main



and Miscellaneous INI-Associated Mutations). The presence and prevalence of primary and accessory INI DRMs were determined and reported by sampling year. With matching sequences available for the protease (PR) and reverse transcriptase (RT) genes, the potential linkage of the DRMs in PR and RT and those INI DRMs was also evaluated.

RESULTS: Among all examined specimens, only a single major primary DRM, S147G, was detected in one subtype C specimen from 2008. In contrast, several major accessory mutations, including L74M, T97A, E138K,V151I, S153YF and R263K, were observed at varied but low frequencies in different years and no trend of accumulation observed over time. Similar trend was also noted for the detected miscellaneous INI-associated mutations such as V54I, L68V, Q95K, A128T and E157Q. No association between INI DRMs and those in PR and RT genes was observed.

CONCLUSIONS: The prevalence of transmitted major INI DRMs in Canada is rare. INI therapy in ART-naïve patients in Canada is expected to be safe and effective. However, ongoing monitoring on both major and accessory INI DRMs may be required.

ABSTRACT 76

Longitudinal Evaluation of Coreceptor Use in Human Immunodeficiency Virus 1 Subtype A in Western KenyaM Coetzer1, L Ledingham1, L Diero2,3, E Kemboi2, M Orido2, W Emonyi2, R Kantor1

1Brown University, Providence, RI, USA; 2 Academic Model Providing Access to Healthcare (AMPATH), Eldoret, Kenya; 3Moi University, Eldoret, Kenya

BACKGROUND: HIV-1 coreceptor switching from CCR5 to CXCR4 is not well understood, particularly in globally predominant non-B subtypes, yet is important for pathogenesis and advanced treatment options. We hypothesized that treatment failure mimics a disease progression environment, and longitudinally investigated CXCR4 use and NRTI/NNRTI resistance

ABSTRACT 75

Transmitted HIV Integrase Inhibitor Resistance Prevalence in Canada During 2002-2012T Taylor1, A Patterson1, Y Li1, H Merks1, J Brooks1, P Sandstrom1, 2, H Ji1, 2, 3

1National Microbiology Laboratory at JC Wilt Infectious Disease Research Center, Public Health Agency of Canada, Winnipeg, Canada; 2 Dept. of Medical Microbiology, University of Manitoba, Winnipeg, Canada; 3 Dept. of Biology, University of Winnipeg, Winnipeg, Canada

BACKGROUND: The first HIV integrase inhibitor (INI), Raltegravir, was approved by Health Canada in late 2007 and INI has since been administered as a component in combined antiretroviral therapy (ART). Currently, five drugs either containing INI alone (Isentress by Merck & Co., Tivicay by ViiV Healthcare, and Vitekta by Gilead Sciences) or drug combinations of INI and drugs from other categories (Stribild by Gilead Sciences and Triumeq by ViiV Healthcare) are available in Canada. Consistent with other antiretrovirals, treatment failure and associated genotypic patterns of drug resistance (DR) have been identified for INIs and some DR mutations (DRMs) even lead to cross resistance against multiple INIs. This study aims to examine the transmitted INI DR prevalence among ART-naïve subjects in Canada over time and to predict INI efficacy when administered clinically in Canada.

METHODS: A total of 935 ART–naïve HIV specimens collected during 2002-2012 from the Canadian HIV Strain and Drug Resistance Surveillance Program were included in this study. These samples were randomly but proportionally selected SDR specimens from each and every sampling year. HIV integrase gene sequences from all subjects were analysed using conventional Sanger sequencing approach by following a well-established in-house protocol. INI DRMs were identified using Stanford HIV Genotypic Resistance Interpretation Algorithm, in which all INI relevant mutation were divided into three groups (Major Primary Mutations, Major Accessory Mutations



no proportion differences between NRTI/NNRTI resistance and CXCR4-use.

CONCLUSION: In the AMPATH dataset, CXCR4 use was not associated with treatment status possibly due to higher prevalence of CXCR4 in subtype A naïves than previously reported, suggesting late diagnosis when CXCR4 use has evolved. Statistical significance in the expanded dataset suggests the need for further, larger investigations. Concordance between prediction tools was lower than expected and additional subtype A validation is needed. Findings suggest entry inhibitors might still be applicable for a proportion of treatment experienced patients in this setting.

ABSTRACT 77

Potential Drug Resistance Outcomes of Dapivirine Vaginal Ring Pre-Exposure Prophylaxis Scale-up in South AfricaR Glaubius1, KJ Penrose2, G Hood3, UM Parikh2, UL Abbas1

1Cleveland Clinic, Cleveland, OH, United States; 2University of Pittsburgh, PA, United States; 3Pittsburgh Supercomputing Center, Pittsburgh, PA, United States

BACKGROUND: A vaginal ring (VR) containing dapivirine (DPV) is being studied for pre-exposure prophylaxis (PrEP) for HIV prevention among women. However, cross-resistance is common between DPV and first-line antiretroviral therapy (ART) in resource-limited settings. Further, while low systemic DPV exposure has been observed in safety trials, variable genital drug concentrations could potentially promote localized resistance emergence among infected PrEP users.

METHODS: We modeled the HIV epidemic in KwaZulu-Natal, South Africa, and compared the combined scale-up of ART (initiated at CD4 ≤ 500 cells/µL), male medical circumcision (MMC) and DPV VR PrEP to a baseline scenario of just ART and MMC. We simulated four strategies of PrEP scale-up among women during 2017-2027: unprioritized (to 15-54 year-olds) or age-

in viruses from treatment naïve and experienced patients.

METHODS: Coreceptor usage was predicted in HIV-1 subtype A infected patients at different treatment statuses (naïve or failing 1st/2nd-line therapy) from one clinical setting in western Kenya (Academic Model Providing Access to Healthcare; AMPATH). env V3 was amplified and coreceptor use predicted with Geno2Pheno (G2P; http://coreceptor.geno2pheno.org/) and PhenoSeq (http://tools.burnet.edu.au/phenoseq/). V3 single-genome sequencing (SGS) was performed on select samples to investigate undetected minor CXCR4-using variants. Proportion differences between CXCR4/CCR5 use and NRTI/NNRTI resistance determined by Stanford database (HIVdb.stanford.edu) were tested using Fisher’s exact test.

RESULTS: V3 and pol sequences were obtained for 47 patients: 19 naïve (median viral load (VL) 144,619 copies/mL; CD4 140 cells/mL), 14 1st-line failures (median VL 120,267 copies/mL; CD4 83 cells/mL) and 14 2nd-line failures (median VL 84,665 copies/mL; CD4 116 cells/mL). G2P predicted 29% use CXCR4 (26% of naïves, 36% of 1st-line, 28% of 2nd-line); and PhenoSeq predicted 40% use CXCR4 (37% of naïves, 28% 1st-line, 57% of 2nd-line); with 63% concordance. Both models predicted 8 (17%) CXCR4-using samples (of these, 38% were naïve, 12% 1st-line, 50% 2nd-line), and 22 (46%) CCR5-using. CXCR4 prevalence in 1st and 2nd-line failures was not different than in naïves (Fisher exact test). SGS on seven R5 and two X4 samples yielded 92 sequences and indicated 3/7 (43%) R5 samples had minor CXCR4 variants at/below Sanger detection level. All clones from X4 samples were CXCR4-using. Inclusion of 211 Kenyan V3 sequences downloaded from the Los Alamos Database (64 naïve and 147 experienced; of which 35 were predicted by both models as CXCR4-using, 7% naives and 17% experienced) suggested a higher CXCR4 prevalence in treatment-experienced compared to naïve patients (p=.05). Prevalence of NRTI mutations in CCR5- and CXCR4-using viruses in the AMPATH dataset was 32% (12/38) and 38% (3/8), and of NNRTI mutations 50% (19/35) and 38% (3/8), respectively. There were



old women, with minimal selection of drug resistance, despite modest adherence. However, blood-based resistance monitoring may underestimate resistance from DPV VR PrEP scale-up.

ABSTRACT 78

More Efficacious Drugs Lead to Hard Selective Sweeps in HIV-1 Drug Resistance EvolutionAF Feder1, S-Y Rhee2, RW Shafer2, DA Petrov1, PS Pennings3

1Department of Biology, Stanford University, Stanford CA; 2Department of Medicine, Stanford University, Stanford, CA; 3Department of Biology, San Francisco State University, San Francisco, CA

BACKGROUND: In the early days of HIV treatment, drug resistance occurred rapidly and predictably in all patients, but under modern treatments, resistance arises slowly, if at all. The probability of resistance should be controlled by the rate of generation of resistance mutations. If many resistance mutations arise simultaneously, evolution of resistance proceeds by soft selective sweeps in which multiple adaptive mutations spread concomitantly, but if resistance mutations occur rarely in the population, then a single mutation can spread alone in a hard selective sweep. We look for genetic signatures to test the hypothesis that the transition from fast to slow evolution of drug resistance was accompanied by a transition from soft to hard selective sweeps.

METHODS: We examine 6,717 HIV-1 direct PCR sequences from patients treated with first-line

prioritized (to 15-24 or 20-29 year-olds) reaching 15% overall population-level coverage; or prioritized to female sex workers (FSWs) (~0.1% overall coverage). We examined scenarios of low (50%) or high (95%) average adherence in base-case analysis, assuming 90% PrEP efficacy against wild-type and drug-resistant HIV, and 80% cross-resistance between ART and PrEP, and modeled HIV drug resistance dynamics in genital and blood compartments. We performed multivariate sensitivity analysis using 10,000 simulations to identify key drivers of drug resistance.

RESULTS: At low (50%) adherence, unprioritized DPV VR PrEP scale-up prevented about 58,400 new infections over ten years. Impact improved modestly with scale-up among women aged 15-24 (62,000 infections prevented) but more substantially when focused to women aged 20-29 (93,100). Scale-up among FSWs prevented 30,500 infections despite their small group size. In high (95%) compared to low adherence scenarios, HIV prevention almost doubled (to 108,500, 116,200, 175,200 and 62,800 infections prevented, respectively). All strategies decreased the total number of prevalent drug-resistant infections at 2027 in base-case analysis (Figure). PrEP prioritized to women aged 20-29 decreased resistance most (by 7.4% at low adherence), while resistance fell by ~5% when PrEP was unprioritized or focused to women aged 15-24. PrEP scale-up among FSWs decreased resistance least (by 1.6%). The decreases in resistance were augmented (4.4%-14.8%) at high adherence. However, decreases in total resistance diminished (to 1.5%-6.5% and 4.4%-14.3% in low and high adherence scenarios, respectively), and resistance from PrEP increased by up to 128%, when resistance was tracked in the genital compartment in addition to blood. Sensitivity analysis identified PrEP efficacy against wild-type (range: 50%-99% efficacy) and cross-resistant (range: 50%-100% efficacy relative to wild-type) HIV as the principal drivers of resistance decreases. Unprioritized and age-prioritized PrEP strategies decreased drug resistance in ~75% of these simulations, while scale-up among FSWs increased resistance in 72% of simulations.

CONCLUSIONS: DPV VR PrEP could have considerable impact on HIV prevention if prioritized to 20-29 year-



ABSTRACT 79

Prevalence of Minority Resistant Variants in HIV-2 Naïve Patients: ANRS CO5 CohortA Storto1, B Visseaux1-3, G Collin1-3, C Fagard4, M Naudin4, F Damond1-3, MA Khuong5 S Matheron1,2,6, D Descamps1-3 and C Charpentier1-3

1AP-HP, Hôpital Bichat-Claude Bernard, Laboratoire de Virologie, F-75018 Paris, France; 2INSERM, IAME, UMR 1137, F-75018 Paris, France; 3IAME, UMR 1137, Univ Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France; 4INSERM, ISPED, Centre INSERM U897-Epidemiologie-Biostatistique, F-33000 Bordeaux, France Université Bordeaux, France; 5Hôpital Delafontaine, Service de Maladies Infectieuses et Tropicales, St-Denis, France; 6AP-HP, Hôpital Bichat-Claude Bernard, Service de Maladies Infectieuses et Tropicales, F-75018 Paris, France

BACKGROUND: To assess the prevalence of minority resistant variants (MRV) and X4-minority variants in antiretroviral-naïve HIV-2-infected patients included in the French HIV-2 Cohort (ANRS CO5).

METHODS: Antiretroviral-naïve HIV-2-infected patients with detectable plasma viral load (VL) (>100 c/mL) were assessed. We performed UltraDeep Sequencing (UDS) (Roche 454® Life Sciences) in protease (PR) and reverse transcriptase (RT) regions issued from plasma viruses. Only mutations >1% were considered and interpreted with HIV-2 ANRS list (Charpentier et al., 2015). HIV-2 tropism was assessed by UDS of V3 loop region. Tropism of each sequence read was interpreted with HIV-2 major determinants of CXCR4 co-receptor use (L18X, V19K/R, V3 global net charge, insertions at position 24).

RESULTS: 47 patients were assessed (median age: 48 years [IQR=36-57], 61% women, 68% originating from West Africa, 12% at CDC-C stage). At time of sampling, median CD4 cell count was 326/mm3 (IQR=215-438) and median VL was 2130 c/mL (IQR=816-4495). 67% of patients were infected with HIV-2 group A and 33% with group B. Protease and RT UDS was successful in 41 (87%) and 38 (81%) samples, respectively. Prevalence of virus with PR or RT drug resistance mutations (DRM) using 1% and 20%

therapies between 1989 and 2013 from the Stanford HIV Drug Resistance Database and determine genetic diversity and the number of drug resistance mutations for each sequence. We fit generalized linear models for each type of treatment to measure how a drug resistance mutations taking over the virus population in a patient affects viral population diversity. This effect should be small if resistance establishes via soft sweeps, but large and negative if hard sweeps predominate.

RESULTS: We find that resistance to treatments with low clinical efficacy generates patterns consistent with soft sweeps, as marked by the relatively small decrease in diversity associated with resistance. In contrast, populations receiving treatments with high clinical efficacy showed large decreases in diversity associated with a drug resistance mutation taking over the population within a patient, a pattern more consistent with hard sweeps. Among patients given treatments with 30% efficacy, sequences with 3 DRMs are predicted to have marginally fewer ambiguous calls as those with 0 DRMs (0.5 fewer ambiguous reads over 1000 bases), but among those patients given treatments with 80% efficacy, sequences with 3 DRMs are predicted to have 10 fewer ambiguous calls than those with 0 DRMs over 1000 bases, a substantial decrease in genetic diversity.

CONCLUSION: We confirm that the transition from fast to slow evolution of drug resistance was indeed accompanied with the expected transition from soft to hard selective sweeps. These results suggest that effective drugs may push HIV-1 populations into a hard sweep regime in which populations must wait long periods of time for the correct mutation.



ABSTRACT 80

Correlation between HIV-2 RNA and HIV-2 Total DNA Levels M Bertine1-3, G Collin1-3, F Damond1-3, V Avettand-Fenoel4, JC Plantier5, A Storto3, M Naudin6, S Matheron1,2,7, D Descamps1-3, C Charpentier1-3, and the HIV-2 ANRS CO5 Cohort

1INSERM, IAME, UMR 1137, F-75018 Paris, France; 2Université Paris Diderot, IAME, UMR 1137, Sorbonne Paris Cité, F-75018 Paris, France; 3AP-HP, Hôpital Bichat-Claude Bernard, Laboratoire de Virologie, F-75018 Paris, France; 4Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, EA 7327, Paris France; AP-HP, Service de Virologie, Hôpital Necker-Enfants Malades, Paris, France; 5Laboratoire de Virologie, CHU Charles Nicolle, Rouen, France; EA 2656 GRAM, Université de Rouen, Rouen, France; Laboratoire associé au Centre National de Référence du VIH, CHU Charles Nicolle, Rouen, France; 6INSERM, ISPED, Centre INSERM U897-Epidemiologie-Biostatistique, F-33000 Bordeaux, France Université Bordeaux, France; 7AP-HP, Hôpital Bichat-Claude Bernard, Service de Maladies Infectieuses et Tropicales, F-75018 Paris, France

BACKGROUND: Few data are available regarding HIV-2 reservoir. The aim of the study was to assess the size of reservoir in different populations of HIV-2-infected patients.

METHODS: HIV-2 total DNA was assessed in ARV-naïve and ARV-treated patients in virological failure (VF) included in the HIV-2 ANRS CO5 cohort. HIV-2 total DNA and RNA quantifications were performed using “in-house” real-time PCR assays: adapted from the HIV-2 RNA Biocentric® kit with a LOQ=7.5 c/PCR for DNA and with a LOQ=100 c/mL for RNA.

RESULTS: Among the 57 ARV-naïve patients, median CD4-cell-count was 475/mm3 (IQR=381-659), plasma viral load (pVL) and HIV-2 total DNA were below the LOQ in 74% and in 12% of the patients, respectively. Median pVL was 1,458 c/mL (IQR=313-3,574); median HIV-2 total DNA was 2.7 log10c/106PBMC (IQR=2.5-3.2). No difference was observed in HIV-2 total DNA or RNA values between patients infected with group A and with group B (p=0.22; p=0.06).

detection threshold was 17.1% (95%CI=5.5-28.7) and 7.3% (95%CI=0.0-15.4), respectively. DRM detected at the 20% detection threshold were M184V in one case and N69S in two cases. MRV exhibiting at least 1 NRTI DRM were detected in 1 patient (2.6%, 95%CI=0.0-6.8), showing the mutation N69S in a proportion of 1.4%. MRV exhibiting at least 1 PI DRM were detected in 4 patients (9.8%, 95%CI=0.7-18.9): (i) two I50V-mutated MRV (1.6% and 1.0%); (ii) one V47A (1.0%); and (iii) one with both I50V and I54L (1.2% and 1.1%, respectively). Tropism was assessed in 19 samples (mean number of reads=7591) showing 2 patients (11%) exhibiting X4-tropic viruses in more than 50% of the reads. Among the 17 remaining samples, X4-minority variants were detected in 11 (65%) in a median proportion of 0.41% (IQR=0.33-0.76).

CONCLUSION: In this first study assessing the prevalence of MRV in HIV-2 infection, we observed a two to three-fold higher prevalence of DRM in antiretroviral-naive patients when 1% detection threshold of mutations was used compared to 20% threshold. In addition, X4-minority variants were detected in the majority of patients.



ABSTRACT 81

HIV-2 Group A in France Displayed Two Clades with Distinct Geographical OriginsB Visseaux1-3, C Charpentier1-3, M Bertine 1-3, A Besseghir4, C Fagard4, F Damond1-3, S Matheron1,2,5, S Hué6, D Descamps1-3 , on the behalf of the French ANRS CO5 HIV-2 cohort

1INSERM, IAME, UMR 1137, Paris, France; 2Université Paris Diderot, IAME, UMR 1137, Sorbonne Paris Cité, Paris, France; 3AP-HP, Hôpital Bichat, Laboratoire de Virologie, Paris, France; 4Université Bordeaux, ISPED, Centre INSERM U897- Epidemiologie-Biostatistique, Bordeaux, France; 5AP-HP, Hôpital Bichat, Service de Maladies Infectieuses et Tropicales, Paris, France; 6Department of Infectious Disease Epidemiology, Faculty of Epidemiology and Public Health, London School of Hygiene and Tropical Medicine, London, United-Kingdom

BACKGROUND: Damond et al. 2000 showed that HIV-2 group A can be divided in two distinct genotypes. We analysed all the HIV-2 pol, env and vif sequences available in France to better characterise genetic variations between these two genotypes, analyse their relative prevalence in France and to explore a potential link with patient’s country of birth.

METHODS: Maximum likelihood phylogenetic trees were reconstructed from 446 HIV-2 partial pol (PR and RT; 1350 nt), 155 vif (655 nt) and 154 partial env (525 nt) sequences sampled from 386 patients followed up in France, using FastTree 2.1 under the GTR evolutionary model. Publically available sequences sampled outside of France were included for pol and vif fragments (207 and 22 sequences, respectively). Recombination analyses were conducted with the RDP4 software. Patients’ country of birth was retrieved for the patients included in the French ANRS CO5 HIV-2 cohort (n=272).

RESULTS: The group A formed two distinct and strongly supported clusters, herein called A1 and A2, in all trees (cf. Figure 1), suggestive of a past founder effect. Overall, 72% and 28% of the group A sequences belonged to cluster A1 and A2 respectively, with 20% of the latter being sampled in France. Among the 193

Among the 50 patients with VF, treated since a median of 8 years (IQR=4-13), median CD4-cell-count was 232/mm3 (IQR=137-361), 14% and 2% had pVL and HIV-2 total DNA below the LOQ, respectively. Median pVL was 832 copies/mL (IQR=192-4,011) and median HIV-2 total DNA was 3.2 log10c/106PBMC (IQR=2.6-3.5). HIV-2 total DNA and RNA values were significantly higher in patients infected with group A than with group B (3.3 vs. 2.7 log10c/106PBMC, p=0.03; 1,658 vs. 659 c/mL, p=0.04). We observed a strong positive correlation between HIV-2 RNA and DNA levels: r=0.74, CI95%=0.57-0.85.

In ARV-treated patients, a trend to a lower proportion of patients with HIV-2 total DNA below the LOQ and a significant higher median of HIV-2 total DNA were observed than in ARV-naïve patients (p=0.06; p=0.04; respectively).

CONCLUSIONS: This is the first description of correlation between HIV-2 RNA and DNA levels. The size of reservoir was significantly higher in ARV-treated patient in VF than in ARV-naïve patients, as described in HIV-1 infection.



HIV-2 A sequences obtained from public databases 19 (10%) belong to clade A2. Inter-cluster median genetic distances were 0.12 [IQR=0.11-0.14], 0.12 [0.11-0.13] and 0.15 [0.12-0.18] substitutions/site for pol, vif and env, respectively. For the 163 viruses with more than one genetic region available, 17 (10%) presented inconsistent clade assignments across trees, suggesting potential recombination events. A1 viruses were most prevalent amongst patients born in coastal Western African countries (i.e. Senegal, Gambia, Guinea Bissau and Guinea) with 40 A1- and 8 A2-infected patients. Inversely, A2 strains were predominantly found among patients originating from inland Western countries such as Mali and Burkina Faso with 8 A1- and 29 A2-infected patients, suggesting distinct origins of the two clades. Sequences issued from patients born in Ivory-coast displayed a balanced prevalence of these clades with 13 A1- and 16 A2-infected patients).

DISCUSSION: This study provides an enhanced understanding of the geographical and genetic diversity of HIV-2 group A. It highlights the co-circulation of two distinct clades in France that likely appeared from an ancient divergent event, followed by a founder effect explaining the distinct geographical patterns in Western Africa.

gaj global - hivrdb.org.uk€¦ · insights from actg 5273 carole wallis barc-sa and lancet...

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