Fifteen years of molecular EQA: progress and challenges

Transcription

1 Fifteen years of molecular EQA: progress and challenges Anton M van Loon 1, Stuart West 2 and Paul Wallace 2 1 Department of Virology, UMC Utrecht, The Netherlands 2 QCMD, Glasgow, Scotland

2 Key Issues In Molecular Diagnostics in the Mid-90 s New technologies, rapidly moving field,, many relevant targets, high expectations Sensitivity and specificity Genotypic variation Contamination Clinical significance of (low level) nucleic acids Need for quantitative results Large number of in-house assays Lack of robustness and standardisation Lack of international reference material

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4 False-Positivity in Molecular Diagnostics: the early years Valenthine-Thon, JCV, 2002 E Valentine-Thon et al 2001

5 Developments supporting improvements in molecular diagnostics Anti-contamination measures: physical separation, UNG system, real-time assays Technological developments: reagents, automation, real-time platforms Introduction of commercial assays WHO International Standards: few, mainly BBV s Use of universal internal controls External quality assessment programmes

6 External Quality Assessment in Microbiology in Europe : end of the 1990 s EQA organisations in many countries (NEQAS, INSTAND, EQUALIS, SKMM, Labquality, etc.), but mainly focusing on (virus)culture and serology. Lack of International Standards or reference reagents for molecular testing Molecular methods: high rate of false-positives ( > 40 % ); sensitivity often unclear 1998: 3 year EU grant for EQA program MDx of (viral) neurological diseases 2001: founding of QCMD, Quality Control for Molecular Diagnostics, an independent, not-for-profit organization ( based in Glasgow

7 The aim of QCMD programmes is To assist laboratories to.. Evaluate the sensitivity of their assays (various genotypes/ analytical sensitivity) Determine the specificity of their assays (contamination, crossreactivity with related pathogens) Assess precision of their quantitative assays Provide an international reference where none exists: EV, HPeV, HSV, VZV, JCV, BKV, etc. Compare performance with peer laboratories Comply with regulatory requirements/ accreditation process (ISO 15189) Panels of 8-12 samples with various genotypes and microbial loads: core and educational samples

8 Differences in sensitivity in MDx of enteroviruses: EU-QCCA panel 1998 Virus Viral load All datasets Commercial test X (cps/ml) (n = 70) (n = 16) CA CA CA CA CA

9 EVRNA11 panel : Overall Results

10 EV Ct value distribution: UMC Utrecht

11 Herpesvirus loads in neurological disease Aberle et al JCV 2002

12 HSV: Results per panel member HSV negative HSV x10 6 copies/ml HSV 2 HSV x10 2 copies/ml HSV 1 HSV x10 2 copies/ml HSV 1 HSV x10 7 copies/ml HSV 1 HSV negative HSV VZV copies/ml HSV x10 3 copies/ml HSV 2 HSV x10 2 copies/ml HSV 2 HSV x10 3 copies/ml HSV 1 HSV x10 3 copies/ml HSV 2 HSV x10 3 copies/ml HSV 1 0% 20% 40% 60% 80% 100% % Correct % Incorrect % Equivocal

13 % correct results in samples from QCMD HSV and VZV programmes Mean viral load (cps/ml) HSV-1 VZV % ND ND 85.5% ND 82.2% ND ND ND 63.1% 75.8% 72.7% ND ND 84.0% 78.5% <100 ND ND ND ND ND ND 46.4% 59.9%

14 % correct results in samples from QCMD JCV and BKV programmes Mean viral load (cps/ml) JCV BKV % 57.1% 82.0% 58.7% ND 96.0% ND 64.8% * 74.8% 52.0% 72.1% ND <100 ND ND 45.0% ND 54.3% 68.3% * Clinical strain

15 HSVDNA 2011: % correct results vs target gene HSV Strain Load (cps/ml) DNA Pol (n=67) Target gene gb (n=56) US7/US2 (n=25) HSV-1 McIntyre % 98.2% 96% % 53.6% 96% HSV-2 MS % 100% 100% % 57.1% 16%

16 Influenza virus detection and typing 2006 A collaboration between EISS, QCMD and ESCV Subtype Dilution % correct results (n=60) Detection Typing (A, B) Subtyping (H) A, H % 88% 53% A, H3 2 x % 85% 50% 2 x % 65% 28% A, H % 73% 67% 2 x % 43% 30% A, H7 4 x % 53% 22% B % 65% - Negative - 90% -* -* * : 7.5% reported INFA; 2.5% reported INFA, H5

17 Influenza virus detection and typing 2012

18 Sam ple Sam ple Sam ple conc. content Genotypic variation-pcr False-negatives: the Swedish variant of Ct. Copies/vial datasets Commercial In-house n=211 n=35 n=4 n % n % n % n % n % n % CTA10-02 Chlamydia trachomatis CTA10-05 Chlamydia trachomatis CTA10-01 Chlamydia trachomatis CTA10-04 Chlamydia trachomatis CTA10-03 C. trachomatis (Sw edish Variant) 2.27x10-2 Diln CTA10-06 Ct. negative urine CTA10-08 Chlamydia trachomatis CTA10-10 Chlamydia trachomatis CTA10-07 Chlamydia trachomatis CTA10-09 Ct. negative sw ab PCR Total Conventional Real time Commercial n=91 In-house n=28 Other n=53 C. trachomatis (Swedish Variant): this strain of the pathogen is lacking 377 base pairs of the cryptic plasmid, which is commonly used as a target for molecular assays. Other: SDA, TMA. Diln: Dilution of a stock.

19 Genotypic variation-pcr False-negatives: the Swedish variant of Ct (2012) C. trachomatis (Swedish Variant): this strain of the pathogen is lacking 377 base pairs of the cryptic plasmid, which is commonly used as a target for molecular assays. Other: SDA, TMA.

20 QCMD 2010 Legionella distribution: sensitivity and contamination issues

21 QCMD 2012 Legionella distribution: sensitivity and contamination issues

22 QCMD EV/ HPeV EQA : False-positivity General Performance EV-A EV03 EV05 EV07 EV09 EV11 EV12 EV Enterovirus Participant Numbers No Lab reporting Total No of datasets Participant countries % False positives 3,6 6,5 3,7 7,5 5,7 3,0 1,1 1.5 Parechovirus Total No of datasets % False positives 13,0 0,0 1,5 0,0 1.1

23 False-positivity rate in QCMD EQA panels

24 QCMD 2011 Aspergillus distribution: sensitivity and contamination issues

25 Observed VL variation in the presence WHO IS and usage of commercial assays (2008) Virus International Standard SD range of geometric mean (log10) % of commercial assays HIV Y > 95% HCV Y > 95% HBV Y ~ 70% BKV N ~ 30% HSV N ~ 25% EV N >1.0 < 10%

26 Spread of commercial kit quantitative results from the mean, BBV programmes

27 Spread of in-house assay quantitative results from the mean, BBV programmes

28 Overall precision in selected QCMD distributions Virus WHO Int St % IVD SD WHO % SD Median Range Int St IVD Median Range CMV N Y EBV N Y HSV N N

29 Paired sample analysis QCMD CMV programmes Paired sample precision in the CMVDNA EQA % of results 100,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10, log10 units Within 0.3 log10 units 0,0 CMVDNA10 CMVDNA11 CMVDNA12

30 Paired sample analysis QCMD EBV programmes Paired sample precision in the EBVDNA EQA % of results 100,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10, log10 units Within 0.3 log10 units 0,0 EBVDNA10 EBVDNA11 EBVDNA12

31 EBV quantification QCMD 2010 proficiency programme

32 Technology variation: Results of the 2009 HHV6 EQA (HHV6-B) Technology Consensus (log10 Copies/ml) y = 0.994x R² = y = x R² = Real time Commercial PCR Real time In-house PCR Consensus (log10 Copies/ml)

33 Use of QCMD panels as reference in assay evaluation

34 Summary and Conclusions Significant improvements have been achieved in MDx over the past 15 years with regard to sensitivity, specificity and precision QCMD programmes provide a reference when International Standards or reference materials are lacking A need for further improvement still exists, particularly in the non-viral area and where reference material is lacking (JCV, BKV, HHV6, ADV, respiratory/gastro pathogens) EQA programmes will also need to address new technological developments, such the increasing use of multiplex assays, Maldi-Tof and NGS