overview of diagnostic tools · overview of diagnostic tools dr. gülay korukluoğlu public health...

Overview of diagnostic tools

Dr. Gülay Korukluoğlu

Public Health General Directorate

Ankara,Turkey

WHO Blueprint meeting on clinical trial design, 31.10.2019, Geneva-Switzerland

Plan of presentation

•Diagnostic tools for confirmation of CCHF cases

•Pathogenesis of CCHF

•Assays for vaccine studies

•Experience of Turkey

Diagnostic tools for confirmation of CCHF cases

Diagnostic methods for CCHF

Mazzola LT, Kelly-Cirino C. Diagnostic tests for Crimean-Congo haemorrhagic fever: a widespread tickborne disease. BMJ Glob Health 2019;4:e001114.

In-house NAT tests for CCHFMethod Authors Reference Target

qRT-PCR Drosten C, Göttig S, Schilling S, et al. J. Clin Microbiol 2002;40:2323–30. 24 genomic targets

qRT-PCR Yapar M, Aydogan H, Pahsa A et al. Jpn J Infect Dis. 2005;58:358–62. 19 strains worldwide

qRT-PCR Duh D, Saksida A, Petrovec M, et al. J Virol Methods 2006;133:175–9. Kosovo Hoti and Drosdov strains

qRT-PCR Garrison AR, Alakbarova S, Kulesh DA Am J.Trop Med Hyg 2007;77:514–20. 18 strains worldwide

qRT-PCR Midilli K, Gargılı A, Ergonul O, BMC Infect Dis 2007;7:54.

qRT-PCR Wölfel R, Paweska JT, Petersen N, et al. Emerg Infect Dis 2007;13:1097–100. 17 strains worldwide

qRT-PCR Papa A, Drosten C, Bino S et al. Emerg Infect Dis 2007;13:805–6.

qRT-PCR Kondiah K, Swanepoel R, Paweska JT, et al. J Virol Methods 2010;169:34–8. 19 African strains

qRT-PCR Atkinson B, Chamberlain J, Logue CH, et al. Vector Borne Zoonotic Dis 2012;12:786–93.All known worldwide strains, including

the AP92 strain

qRT-PCR Jääskeläinen AJ, Kallio-Kokko H, Ozkul A, et al. Vector Borne Zoonotic Dis 2014;14:870–2

nested PCR Schwarz TF, Nsanze H, Longson M, et al. Am J Trop Med Hyg 1996;55:190–6.

nested PCR Rodriguez LL, Maupin GO, Ksiazek TG, et al. Am J Trop Med Hyg 1997;57:512–8.

nested PCR Deyde VM, Khristova ML, Rollin PE, et al. J Virol. 2006;80:8834–42.

nested PCR Aziz TAG, Ali DJ, Jaff DO. J Biosci Med 2016;04:36–42.

conventional RT-PCR Burt FJ, Leman PA, Smith JF, et al. J Virol Methods 1998;70:129–37. 7 geographically diverse strains

Isothermal RPA Bonney LC, Watson RJ, Afrough B, et al. PLoS Negl Trop Dis 2017;11:e0006013 7 geographically diverse strains

Low density array Wölfel R, Paweska JT, Petersen N, et al. J Clin Microbiol 2009;47:1025–30. strains worldwide

High density array Filippone C, Marianneau P, Murri S, et al. Clin Microbiol Infect 2013;19:E118–28. Nigerian strain

Multiplex RT-PCR/NGS Brinkmann A, Ergünay K, Radonić A, et al. PLoS Negl Trop Dis 2017;11:e0006075.

Mazzola LT, Kelly-Cirino C. Diagnostic tests for Crimean-Congo haemorrhagic fever: a widespread tickborne disease. BMJ Glob Health 2019;4:e001114

Syndromic multiplex approach

Mazzola LT, Kelly-Cirino C. Diagnostic tests for Crimean-Congo haemorrhagic fever: a widespread tickborne disease. BMJ Glob Health 2019;4:e001114

Researchers Title Journal /Year SummaryO'Hearn AE et al. Serosurveillance of viral pathogens

circulating in West Africa. Virol J 2016;13:163. A multiplexed MAGPIX® assay to detect IgG antibodies against Lassa, Ebola,

Marburg, Rift Valley fever, and Crimean-Congo hemorrhagic fever viruses as well as pan-assays for flaviviruses and alphaviruses.

Liu J et al. Development of a taqman array card for acute-febrile-illness outbreak investigation and surveillance of emerging pathogens, including ebola virus.

J Clin Microbiol2016;54:49–58

A real-time PCR-based TaqMan array card (TAC) that can test six to eightsamples within 2.5 h from sample to results and can simultaneously detect26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyovirus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiellaburnetii, Leptospira spp., Rickettsia spp., Salmonella enterica andSalmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa(Leishmania spp., Plasmodium spp., and Trypanosoma brucei).

Das S et al. A Multiplex PCR/LDR assay for the simultaneous identification of category a infectious pathogens: agents of viral hemorrhagic fever and variola virus

PLoS One2015;10:e0138484

The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). .

Drosten C et al. Rapid Detection and Quantification of RNA of Ebola and Marburg Viruses, Lassa Virus, Crimean-Congo Hemorrhagic Fever Virus, Rift Valley Fever Virus, DengueVirus, and Yellow Fever Virus by Real-Time Reverse Transcription-PCR

J Clin Microbiol2002;40:2323–30.

a PCR system comprising six one-step, real-time reverse transcription-PCR (RT-PCR) assays for MBGV and EBOV (MBGV/EBOV), LASV, CCHFV, RVFV, DENV, and YFV.

Commercial kits

Vanhomwegen J et al. Diagnostic assays for Crimean-Congo hemorrhagic fever. Emerg Infect Dis. 2012 Dec;18(12):1958-65.


Rapid diagnostic tests• RDTs can leverage the same antibody/antigen capture agents as an ELISA but in a lateral

flow strip format, with minimal specimen processing (blood, plasma, swabs). This enablesa faster time to result (10–30 min, however with a lower detection sensitivity than ELISA,due in part to reduced sample volume).

• RDTs are ideal screening tests, suitable for field testing and low infrastructure Settingsalthough follow-up confirmatory testing is often required.

• RDTs have been used to effectively screen and triage suspected high-risk cases ofdiseases such as Ebola and dengue ;BUT , the literature shows no evidence of CCHF RDTdevelopment.

• The primary challenge is detection sensitivity, as the IgG/IgM serological response istypically detectable only 5 days postinfection, and often undetectable in severe and fatalinfections.

Sequencing &NGS

• CCHFV displays a high degree of sequence diversity, with divergence of 20%, 31%

and 22% for the S, M and L segments among isolates in GenBank. Based on an

analysis of the viral S segment, seven viral lineages/clades have been identified,

suggesting a lengthy history of geographical dispersion of the virus from Africa to

Europe, the Middle East and then Asia. The extensive sequence diversity of

CCHFV is likely due to genetic reassortment, enhanced by circulation and

adaptation of strains into new geographical regions.

• NGS is important tool for to monitor circulating strains and viral mutations,

particularly to assess the sensitivity of probe design used in molecular

diagnostics.

Distribution of CCHF virus genotypes(S Segment)

Challenges on diagnostic tools

• There is high genetic diversity within the different CCHFV strains, whichconsequently hampers the performance of molecular tests. As a result,a range of different methods employing varied primer/probecombinations have been developed and a truly universal assay has beendifficult to devise. LOD of the methods is very important.

• All CCHFV genotypes belong to one serogroup but IgM can detectearliest on fifth day of the disease. Beside of this; patients with fataloutcome rarely develop measurable antibody responses.

• A CCHFV seroneutralisation test is not normally performed fordiagnostic purposes; it requires work with an infectious virus,necessitating a BSL4 laboratory, and is difficult to perform.

• There is no RTD/POC test yet( false negative results???)

Pathogenesis of CCHFV(still very complicated..)

The interaction of the virus with host cells is most likely responsible for the pathogenesis of CCHF. The main contributors are endothelial cells (ECs) and immune cells. There are 2 theories underlying the CCHF pathogenesis:

1.virus interacts with the ECs directly

2.Virus interacts indirectly via immune cells with subsequent release of soluble mediators. ECs are activated upon infection by the upregulation of soluble molecules and proinflammatory cytokines. Probably, in severe cases, deregulation and excessive release of the cytokines accompanied by endothelial activation have toxic effects, leading to increased vascular permeability, vasodilatation, and subsequently hypotension, multiple organ failure, shock, and death.

Akıncı E. Et al.Pathogenesis of Crimean-Congo hemorrhagic fever.Vector Borne Zoonotic Dis. 2013 Jul;13(7):429-37. .

Akıncı E. Et al.Pathogenesis of Crimean-Congo hemorrhagic fever.Vector Borne Zoonotic Dis. 2013 Jul;13(7):429-37.

Innate Immunity and Hyper-Inflammatory Cytokine Responses

• Innate immunity is the first line of defense against viruses before adaptive immunity develops and it is characterized by the production of type I IFN, which is crucial for limiting the early replication and spread of viruses. CCHFV is one of the viruses sensitive to type I IFN, and it delays the IFN response*. Furthermore, CCHFV challenge of type I IFN receptor-knockout mice results in a fatal outcome and higher CCHFV titers

*during CCHFV infection, RIG-I mediated a type I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression and increased viral replication.

Kaya s et al.Sequential determination of serum viral titers, virus-specific IgG antibodies, and TNF-α, IL-6, IL-10, and IFN-γ levels in patients with Crimean-Congo

hemorrhagic fever.BMC Infect Dis. 2014; 14: 416.

Spengler JR, Patel JR, Chakrabarti AK, et al.: RIG-I Mediates an Antiviral Response to Crimean-Congo Hemorrhagic Fever Virus. J Virol. 2015; 89(20): 10219–29.

• There is some correlation with the levels of the inflammatory cytokinesinterleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α with fatal/severeCCHF.

• Furthermore, high levels of the monocyte chemokine MCP-1 (CCL2) alsocorrelated with severe human disease .

• the host inflammatory response(s) may play an important role in viralpathogenesis.

• Retrospective analyses of CCHF human cases have identifiedpolymorphisms in toll-like receptors (TLR), TLR8/9 and TLR3 are reportedto play a role in acute disease . These epidemiological data suggest amodel whereby some TLR polymorphisms limit IFN-I activation inresponse to CCHFV allowing for enhanced viral replication that in turnenhances disease severity.

Innate Immunity and Hyper-Inflammatory Cytokine Responses

The effects of cytokines

• TNF-α : has anti-fibrinolytic effects by inhibiting the physiological anticoagulation pathways, and the production of plasminogen activators and the plasminogen activator inhibitor (PAI)-I are induced by this cytokine. These effects facilitate DIC development in the host.

• IL-6: is the actual mediator for the activation of the coagulation system in the host. Furthermore, tissue factor (TF)-mediated thrombin formation which is initiated by the IL-6 and TF/activated factor VII complex, has an important role in the development of DIC

• IFN-γ, which is secreted by activated T-cells and natural killer (NK) cells, is responsible for the inflammatory response and viral immunity via macrophage activation

Kaya s et al.Sequential determination of serum viral titers, virus-specific IgG antibodies, and TNF-α, IL-6, IL-10, and IFN-γ levels in

patients with Crimean-Congo hemorrhagic fever.BMC Infect Dis. 2014; 14: 416.

• IL-10, a potent anti-inflammatory mediator of vascular damage, controlscoagulation by inhibiting TF expression on the monocyte surface. IL-10,which inhibits cell-mediated immunity by downregulating IL-12expression, with high levels of proinflammatory cytokines IFN-γ and TNF-α might play an important role in the pathogenesis of CCHF. It washypothesised that CCHF could be the result of a delayed anddownregulated immune response caused by IL-10, which leads to anincreased replication and spread of CCHFV throughout the body

Saksida A.et al. Interacting Roles of Immune Mechanisms and Viral Load in the Pathogenesis of Crimean-Congo Hemorrhagic Fever. Clinical And Vaccine Immunology, July 2010, p. 1086–1093

• Human IgM and IgG antibody responses can be detected against the glycoproteins(GP38, GN and GC), and nucleocapsid (N) protein in CCHF survivors.

• The role for adaptive immune responses against CCHFV in human pathogenesis is lessclear. Low-to-absent anti-CCHFV antibody responses have been found to correlate withsevere disease and death, and levels of antibody may serve as a predictor of diseaseoutcome. However, whether antibody responses contribute to the control of primaryCCHFV infection is unknown, and neutralizing antibody responses even in survivors aretypically low

• However, non-neutralizing antibodies were protective against lethal CCHFV challenge,demonstrating that antibodies can be protective via mechanisms other thanneutralization.

• The role for T cells in controlling primary CCHFV infection is unclear.Levels of circulatingCD3+CD8+ T cells in peripheral blood were found to positively correlate with fataloutcome , and human CCHF survivors have been shown to exhibit long-lived CD8+ T-cellresponses to CCHFV.

Hawman DW. Recent advances in understanding Crimean–Congo hemorrhagic fever virüs.Version 1. F1000Res. 2018; 7: F1000 Faculty Rev-1715. 2018 Oct 29.

Adaptive Immune Response

As a conclusion…

Studies indicate that CCHF virus (CCHFV) can impair the innateimmune system and cause a delay in adaptive immune response, which iscritical for the clearance of CCHFV.

The virus has many different ways to block the immune response,leading to uncontrolled viral replication followed by systemic spread of thevirus throughout the body. Partial activation of dendritic cells andmacrophages, delayed induction of interferons, weak antibody response,apoptosis of lymphocytes, and hemophagocytosis are some of these tactics.However, there are many points waiting for clarification about thepathogenesis of CCHF.

Akıncı E. Et al.Pathogenesis of Crimean-Congo hemorrhagic fever.Vector Borne Zoonotic Dis. 2013 Jul;13(7):429-37. .

Important prognostic factors

• Higher CCHFV titers

• The absence of CCHFV specific immunity (no antibody response)

• Overproduction of cytokines (TNF-α and IL-6, IL-8, IL-10)

(a phenomenon called cytokine storm)

• Elevated ALT/AST levels

• The presence of DIC

• Hemorrhages

Akıncı E. Et al.Pathogenesis of Crimean-Congo hemorrhagic fever.Vector Borne Zoonotic Dis. 2013 Jul;13(7):429-37.

Vaccine targets

CCHFV is a tri-segmented virus, consisting of small (S), medium (M) andlarge (L) RNA segments which encode the viral nucleoprotein, theenvelope spike proteins and RNA polymerase, respectively. As the keystructural components of the virus, the nucleoprotein andglycoproteins are identified as potential antigenic targets for inclusionin a vaccine against CCHFV.

Dowall SD. Et al. Development of vaccines against Crimean-Congo haemorrhagic fever virus. Vaccine. 2017 Oct 20;35(44):6015-6023.

Mechanisms of protection• Non-neutralising monoclonal antibodies have been found to be effective at

protecting mice from a lethal CCHFV challenge, and interestingly, thosespecific against Gn were more effective than those against Gc .

• Additionally, the use of convalescent plasma as a post-exposure prophylactichas not been particularly effective .

• Recent vaccine studies have also indicated that the induction ofneutralisation antibodies does not correspond to vaccine efficacy . Thus cell-mediated immunity looks likely to play a major role in developing effectiveprotection against CCHFV.

• Interferon response may be critical it appears that the kinetics of theresponse and the activation of multiple pathways may be the mostimportant factors in the outcome of infection.

• Protection correlated with T-cell responses, but not antibody induction,although further studies are required to determine exactly which is theprotective component


Approach for human vaccines against to CCHF


Assays for vaccine efficacy

• Humoral immunity• Antibody analyses/IgG subclass determination

• Celluler immunity• Serum cytokine levels/ELISPOT tests

• Viral RNA detection• Neutralization assay• Histopathology • immunohistochemistry • in situ hybridization • Animal models

Animal models for CCHF

Garrison AR et al. Animal Models for Crimean-Congo Hemorrhagic Fever Human Disease.Viruses. 2019 Jun 28;11(7).

Animal models of CCHFVMice Cynomolgus macaques

Type I interferon-deficient mice• Either genetic knock out or antibody blockade

Develop viremia, inflammatory immuneresponses, liver failure and rapid-onset terminal disease

• Multiple CCHFV strains can be used. • Valuable for studying therapeutic interventions

against CCHFV • Limited for studying host immune responses to

CCHFV owing to innate immune deficiencies anddeath prior to adaptive immune responses

Humanized mice• Mice engrafted with human CD34+ hematopoietic stem cells• Develop a neurological-type disease

• Strain-specific virulence observed

• Adult, immunocompetent macaques infectedwith CCHFV strain Hoti• Exhibit a spectrum of disease outcomes fromasymptomatic to severe, lethal disease• Develop viremia, inflammatory immuneresponses, elevated liver enzymes, and increasedclotting times• Major sites of viral replication are liver and spleen• Valuable pre-clinical model for therapeuticinterventions against CCHFV • Can be used to study host and viral determinants

of disease outcome

Haddock E et al. A cynomolgus macaque model for CCHF. Nat Microbiol . 2018 May ; 3(5): 556–562.

Animal model

• A cynomolgus macaque model of CCHF may provide insight into therole of the adaptive immune response and disease outcome. In thismodel, neither antibody titers nor neutralizing activity of theantibodies correlated with disease outcome. However, studiesevaluating the contribution of the T-cell response to disease outcomein this model are still needed.

Haddock E et al. A cynomolgus macaque model for CCHF. Nat Microbiol . 2018 May ; 3(5): 556–562

Human

Cynomolgus macaquesHaddock E et al. A cynomolgus macaque model for CCHF. Nat Microbiol . 2018 May;3(5): 556–562

https://www.who.int/emergencies/diseases/crimean-congo-haemorrhagic-fever/introduction.pdf

• In a study the animals with the most elevated cell-mediated Th1 response combined with a good humoral Th2 type immune response resisted weight loss and lethal challenge. All CCHFV antigen-immunized mice developed detectable levels of neutralizing serum antibodies. However, animals with high neutralizing antibody titers and a Th2-type immune profile (tc-VLPs; group C) were only poorly protected. Thus, one may suggest that the protective immunity correlated strongly with a dominant Th1-type immune profile with a balanced Th2-type biomarker response, and, as determined by serum IgG subclass analysis, a strong IgG2a response was detected in the best-protected animals

Hinkula J.et al.Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice.J.of Virology May 2017 Volume 91 Issue 10 e02076-16

CCHFV Vaccine MCMs in humans and laboratory animals

Garrison AR et al. Animal Models for Crimean-Congo Hemorrhagic Fever Human Disease.Viruses. 2019 Jun 28;11(7).

Experience of Turkey

Highlights• Crimean-Congo hemorrhagic fever is an emerging infectious

disease in Turkey, with nearly 10,000 cases since 2002.

• Average of fatality rate is 4,6%

• Early supportive treatment is essential for the managementof CCHF.

• A robust surveillance system, training and communityengagement are keys to finding cases early and improvingoutcomes.

CCHF Laboratory Algorithm( Turkey)

SAMPLE(Serum)

REALTIME rtPCR*

NEGATIVE

POSITIVE

REPORT

NEGATIVE

POSITIVE

SEROLOGY

2nd sample****:If symptoms are compatible

*:within 24 hours

CCHF Laboratory Diagnosis

SAMPLE(1094)

REALTIME rtPCR

NEGATIVE

(882)

POSITIVE

(212)

REPORT

NEGATIVE(840)

POSITIVE(42*)

SEROLOGY

*: average of sampling time is appr. 9.5 days after onset of symptoms

Total no. of samples / Positive samples

1075

796

910 967

718

432

328

2244

1946

1898

2427

1847

1265 1192

0

500

1000

1500

2000

2500

3000

2011 2012 2013 2014 2015 2016 2017

POSITIVE SAMPLES TOTAL NO.OF SAMPLES

Conclusion-I(Awareness & Early warning)

• Sustainable training is crucial for two groups :➢The people who work with high risk occupation and live

in endemic area➢Clinicians and healthcare workers.

• Monitoring of indicators( enviromental studies such as ticksurveillance etc..)

Conclusion-II (diagnosis)

• Diagnosis of CCHF moleculer and serological assays should be combined.

• Sampling time should be considered during evaluation of results.• When the symptoms are compatible with CCHF; second

sample is important for to follow up the patient if the firstsample has been resulted as negative by moleculer/serologicalassays.

• Lack of RTD/POC tests is important challenge for early diagnosisof patients

Conclusion-III(clinical management)

• Awareness of clinicans / HCWs is very high;• There should be well defined case definition• All suspected cases should be investigated• The incidence rate of the nosocomial infection is very low in Turkey.

• Case management

• Waste management

• Supportive care ( blood bank, biochemistry laboratory service etc)

• Treatment• Antiviral agents; there are different approaches for ribavirin using• Early stage supportive treatment

CASE MANAGEMENT ALGORITHM

Visited the endemic area in last two

weeks

Lives in endemic area

Has tick contact on

body

SUDDEN ONSET

At least two or more symptoms; as followingFever, headache, myalgie, arthralgie, malaise

diarrhea and hemorrhage

YES

CBC

Thrombocyte < 150.000 and/or Leucosyte < 4000

Hemogram(Daily)

Refer the patient to 2nd level hospitals

Hospitalize the patient and take the sample forlaboratory confirmation and performed hemogram ( as

daily), biochemistry,hemostazis panel, support treatment

1. Older than 60 years old2. The patients who has rapidly deteriorating clinical status and laboratory values3.The patient who has confusion4. Thrombocyte <50.000, 5.The patient who has secondary chronicle disease such as organ failure etc..

Refer the patient to 3rd level hospitals

NOyes

Discharge criteria

• No fever and hemorrhage,

• Improvement in clinical findings,

• Platelet count of either >100,000/mm3 or >50,000/mm3 with a tendency to increase

• Normal bleeding tests

• Transaminase levels

• A negative CCHFV RT-PCR result is not feasible in endemic overcrowded settings.

Leblebicioğlu H. Et al. Multi-center prospective evaluation of discharge criteria for hospitalized patients with Crimean-Congo Hemorrhagic Fever. Antiviral Research Volume 133, September 2016, Pages 9-13

THANKS FOR YOUR ATTENTION….

overview of diagnostic tools · overview of diagnostic tools dr. gülay korukluoğlu public health...

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