NRL EQAS for NAT: Assessing the variability and performance of molecular assays for clinical pathogens

Transcription

1 NRL EQAS for NAT: Assessing the variability and performance of molecular assays for clinical pathogens Vincini GA*, Cabuang LM, Land S, Best SJ NRL London, 12 January 2011

2 NRL EQAS for NAT HCV Genotyping Scheme HIV Drug Resistance Genotypic Testing HCV viral load CTNG and HSV Schemes 2

3 NRL EQAS for Nucleic Acid Testing Blood Screening 3 NATA4315 Multimarker HIV HCV HBV 15 samples Designed for Ultrio, AmpliScreen, TaqScreen, etc Clinical Diagnostics HIV & HCV Genotyping CT/NG HSV 1&2 HCV viral load & qualitative HIV viral load HAV/Parvovirus B19 HBV viral load CMV

4 Results of Global Collaboration Table 1: Number of participants enrolled in NRL EQAS for NAT Scheme (% ) HIV VL (45) HCV VL (54) HCV Qualitative (-21) HCV Genotyping (-12) HBV (17) HAV & Parvovirus B (61) HSV 1& (46) CTNG (22) CMV (23) HIV DR Genotyping (8) Multimarker 4 Blood Screening (65)

5 Summary of Global Collaboration 5

6 HCV Genotyping

7 HCV Genotyping One aim for HCV genotyping programme in 2010: reporting strategy when discriminating between genotypes 1 and 6 5 -UTR primers traditionally used No discrimination between 1 and 6 Incorporation of core primers improves discrimination 7

8 HCV Genotyping TE1 TE3 No. of participants Removed from analysis 2 2 Incorrect 11 8 Correct 13 (54%) 16 (67%) Assay Type No. of Participants Designed In-house 1 5'UTR only 6 5'UTR + Core 17 8

9 HCV Viral Load

10 HCV Viral Load In 2008, NRL HCV Viral Load EQAS was composed of samples containing different genotypes of HCV These panels were designed to assess quantification of different HCV genotypes 10

11 11 HCV Viral Load

12 HIV Drug Resistance Genotypic Testing

13 Goal of QA for HIVDR Genotypic Testing Use quality assessment to build capacity in laboratories by assuring the quality of HIV genotyping processes ensuring parity of test outcomes between participating laboratories developing a collaborative laboratory network to facilitate regional dissemination of laboratory know-how and expertise troubleshooting of testing problems support for laboratories setting up HIV genotyping 13

14 Process (1) Registered Laboratory tests 2 x 5-member panels per annum Samples are plasma or low level passaged virus Results returned edited nucleotide sequences, drug resistance mutations (DRMs) and ARV resistance interpretation Preliminary report 14 Issued prior to data analysis Final report

15 Lab mistake detected in Preliminary report * Labs that did not reported DRMs as NMs did not report resistance to NNRTIs 15

16 Process (2) Data Analysis 16 Phylogenetic analysis Sequence quality, cross contamination, sample mix up Alert laboratory to potential problem in their process Clean-up sequence data prior to QA analysis Nucleotide Sequence Alignments The Target Genotype (TG) is derived from the alignment of the edited nucleotide sequences returned by all labs, including the reference lab. Purpose..to compare edited nucleotide sequences generated by all labs What is it?...most likely consensus sequence Labs should inspect to see if their sequences differ from others. If necessary re-edit raw nucleotide sequences, then their call on DRMs and interpretation of ARV resistance.

17 Process (3) Final Report Level of agreement between the laboratories nucleotide sequences sequence (~5,000 bp) from 5 panel samples >98% is acceptable in a panel of plasma samples >99% is expected in a panel using electropherogram sample-format Detection of DRMs and NMs DRMs associated with drug resistance and level of drug resistance reported 17

18 Nucleotide sequence agreement (total) (%) Nucleotide Sequence Agreement Panel 1 Panel 2 Panel 3 Panel 4 Panel 6 Panel Lab 13 Lab

19 Detection of Nucleotide Mixtures Nucleotide Mixtures (NMs) reflect the evolution of resistant virus under the pressure of ARV drug treatment or re-emergence of sensitive virus when ARV drug pressure is removed Ability to detect NMs has correlated with detection of DRMs in past EQAS Indicator of the quality of the HIV genotyping outcome 19

20 No. of NMs and DRMs as per The Stanford Database analysis Correlation between detection of Drug resistance mutations [DRMs] and mixtures [NMs] 200 NMs DRMs All mutations Laboratory ID 20 Pearson s r =0.93, p =0.001

21 DRMs detected compared to the target genotypes (TG) (%) Detection of Drug Resistance Mutations Mutation Mixture of WT and Mutation Wildtype (WT) w hen TG mutation Mutation w hen TG WT 100% 95% 90% 85% 80% 75% 70% 65% 1 st Time Participant 21 60% Lab ID Under reporting of mixtures

22 Labs under-report drug resistance due to suboptimal detection of mixtures 22 Labs that did not reported DRMs as NMs did not report resistance to NNRTIs * * * * * * *

23 CT/NG and HSV

24 24 CTNG and HSV-1/2 Swabs

25 CT DNA - Baseline Baseline Cp Replicate RT C -20 C Mean Median

26 CT DNA Time 3 Time 3 Cp Replicate RT C -20 C Mean Median