what’s hot in diagnostic microbiology? · arch pathol lab med 2012 (early online release) •...

What’s Hot in DIAGNOSTIC MICROBIOLOGY?

Susan M. Poutanen, MD, MPH, FRCPCMicrobiologist/ID Consultant, UHN/MSH

Assistant Professor, U. of Toronto

AMMI Canada – CACMID 2013

Objectives1. To highlight a selection of the most

important and interesting literature in diagnostic microbiology from the last year

2. To review how these developments may influence diagnostic microbiology practice

Disclosures• Advisory Board/Consultant

– Optimer– Sunovion

• Speakers Bureau– Merck– Hoffman La Roche

• Research Support– Biomérieux– Bio-Rad– Copan– Pathogenica

The Most Important and Interesting Literature in Diagnostic Microbiology

in the Last Year

MALDI-TOF

Next Generation Sequencing

Random Access Automated PCR

Automation

Fecal Transplants? – be prepared to be asked!

MALDI-TOF

• Matrix-assisted laser deionization-time of flight

Detection

Separation

Acceleration

Ionization

Desorption MALDI-TOF

FEMS Microbiol Rev 2012;36:380-407

• Easy, rapid, high throughput, low cost, efficient identification tool of bacteria and yeast with potential applications to filamentous fungi, epidemiology, and resistance determinant identification

study reference

organism/ # tested

% identified to genus/species

% misidentified/no ID


Arch Pathol Lab Med 2012 (early online release)

• Implemented MALDI-TOF, rapid antimicrobial susceptibility, and near-real-time antimicrobial stewardship 24/7 in patients with positive blood cultures with Gram-negative organisms for a 3 m period and compared to a 3 m pre-intervention period


• Results:– Significant ↓ TAT to ID and susceptibility testing– Significant ↓ mean hospital length of stay– Significant ↓ costs


Results

Clin Chem Lab Med 2013;51(2):257-270

• “Mass spectrometry is the future of microbiology…accurate identification …timely decision-making … optimal antibiotics, decrease multi-drug resistance ... reduce length and costs of hospitalization”

Next Generation Sequencing

• also referred to as deep sequencing

NEJM 2012;366:2267-75Lancet Infect Dis 2013;13:130-36PLOS One 2013;8(1):e54898

BMC Inf Dis 2013, 13:110PLOS Medicine February 12, 2013

Lancet 2013, published online March 29 2013

Sci Trans Med 2012;4:148ra116PNAS 2012;109(8):3065-70

Whole Genomic Sequencing

• Challenge is translation from the research laboratory to the clinical laboratory given associated costs and technical and bioinformatics expertise

www.pathogenica.com; Poster ASCP 2012

• Multiplexed deep sequencing assay that will:– Rapidly identify multiple pathogens (<12 hours)– Document epidemiologic relationship

Random Access Automated PCR

• Sensitive• Low risk of

contamination• Fast turn-around-

time (~45-70 min)• Low hands-on-time

Journal of Infection 2013;64:580-8.

• A meta-analysis assessing the ability of the Xpert MTB/RIF (Cepheid) assay to diagnosis tuberculosis and RIF-resistance

• 18 studies (10,224 suspected specimens) • To detect pulmonary TB:

– Pooled Sn 90.4% (95%CI 89.2%-91.4%) – Pooled Sp 98.4% (95%CI 98.0%-98.7%)

• To detect extrapulmonary TB:– Pooled Sn 80.4%– Pooled Sp 86.1%


Results

Results (cont’d)• Detection of RIF-resistance

– Sn 94.1%– Sp 97.0%

• Subgroup analysis– Performance better in smear-positive specimens– Sn higher in pulmonary TB in adults than children

(90.8% versus 74.3%)• Conclusions: TB and RIF-resistance can be

rapidly and effectively diagnosed with XpertMTB/RIF assay.


Cochrane Database of Systematic Review 2013, Issue 1.

• A meta-analysis assessing the ability of the Xpert MTB/RIF (Cepheid) assay to diagnosis tuberculosis and RIF-resistance

• 18 studies

Results

• Detect TB – Pooled Sn 88%

• smear pos 98%, smear neg 68%• HIV pos 80%, HIV neg 89%

– Pooled Sp 98%• Detect rifampin resistance

– Pooled Sn 94%– Pooled Sp 98%


• Xpert used as an initial diagnostic test for TB detection and rifampin resistance detection in patients suspected of having TB, MDR-TB, or HIV-associated TB is sensitive and specific

• Xpert may also be valuable as an add-on test following microscopy for patients who have previously been found to be smear-negative

Conclusions


http://www.reuters.com/article/2010/12/08/us-tuberculosis-test-idUSTRE6B71RF20101208

Future Microbiol. 2011;6(9):1067-82

• Described as potential “game changer” for TB control• Strong recommendation to use it as the initial

diagnostic test for ?MDR-TB and HIV-ass TB• Otherwise, given resource implications, recommend

to use it as a follow-up to microscopy

J Virol Methods 2012;186:137-150

• Archived frozen NP swabs tested by RT-PCR • Sn (%) to detect influenza A/B

– Xpert 97.3/100, DFA 95.9/100– BinaxNOW 62.2/54.5, BD Directigen 71.6/48.5

• Sp 100% except DFA for influenza A 99.2%

JCM 2012;50(5):1704-10

• Tested archived and prospective NP swabs or nasal aspirates comparing to culture and PCR as reference

• Sn to detect influenza A (90-100%), A 2009 H1N1 (98.4-100%), and influenza B (93.8-10%)

• Sp all high 99.7-100%

JCM 2013;51(1):352-53

• Nanosphere Verigene RV+ test – random access kit that does not require pre-extraction;

detects influenza A/B (2009 H1N1, H1 and H3) RSV A/B• Focus Diagnostics Simplexa kit

– Kit assessed requires pre-extraction and batched testing but direct kit which does not require pre-extraction is available; detect influenza A/B, RSV A/B

Results

• Tested NP swabs prospectively and retrospectively using Cepheid, Luminex, and RT-PCR LDTs as reference

• Sn/Sp (%) Nanosphere Simplexa– Influenza A 96.6/100 82.8/99.7– Influenza B 100/99.7 76.2/100– RSV 100/100 94.6/100

JCM 2013;51(1):352-53

Random Access Automated PCR Platforms

• Xpert (Cepheid)• Verigene (Nanosphere)• Simplexa Direct Testing (Focus Diagnostics)• BDMax (BD)• Liat (IQuum)

Automation

• Time efficient• Personnel efficient

WASP, Copan Diagnostics

Inoqula, Kiestra

Previ-Isola, Biomérieux

Innova, BD

• Compared Previ Isola polyurethane swabs to standard swabs on 112 paired samples

• Noted 2.6 hours labour savings • Noted 71% concordance with culture and 55%

concordance with gram smear; where discordant, predominantly higher CFU and better gram stain characteristics with Previ Isola

JCM 2012;50(8):2732-36

JCM published online ahead of print on March 20 2013

• Summary overview of automation • Overview of currently available automated

specimen processors

Clin Microbiol Infect 2011;17:655-60

• Another useful summary overview of automation

• Overview of currently available automated specimen processors

Fecal Transplant

• If your microbiology laboratory has not yet been asked to process fecal transplants, it may soon

NEJM 2013 (published online January 16, 2013)

Results• RCT for patients with recurrent C. difficile• assessed fecal transplant by NG tube after

vancomycin vs vancomycin alone or vancomycin bowel prep

• Cures:– 81% of fecal transplants were cured (94% if a

second donor was found)– 31% vancomycin for 2 weeks– 23% vancomycin with bowel lavage


What Does this Mean to You?

• Donor screening• Good manufacturing procedures

– microbiology or pharmacy?• Advice regarding optimal administration

– Work with infectious disease colleagues/gastroenterologists

• Still a lot of unknowns


• Guidance documentation regarding donor selection, screening tests, transplant preparation

• Caveat – mostly expert opinion

Clin Gastro and Hep 2011;9(12):1044-9

What’s Hot -Take Home Messages

• MALDI-TOF– Low cost, low tech time, accurate identification– If you have not already considered MALDI-TOF, it is

time to do so• Next Generation Sequencing

– Better resolution at differentiating relatedness– Be on the lookout for novel diagnostics instruments

using next generation sequence technologies– Expect possible requests next generation

sequencing in specific outbreak situations

• Random Access Automated PCR– Random access, fast TAT, low tech time– Evaluate your needs and consider moving to random

access PCR when results are needed in a timely fashion (TB, influenza, C. difficile, ARO screening)

• Automation– Labour savings, better quality smears/plates– If you haven’t considered this, it’s time to do so

• Fecal Transplants? – be prepared to be asked!– Be prepared to answer!

What’s Hot -Take Home Messages

Questions?

what’s hot in diagnostic microbiology? · arch pathol lab med 2012 (early online release) •...

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