WHO/BS/2012.2209 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012
Report on an International Collaborative Study to Establish the 2
ndWHO International Subtype Reference panel for HIV-1 NAT Assays
C L Morris1 E Wigglesworth and A B Heath2
Divisions of Retrovirology1 and Biostatistics2, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK.
This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 01 October 2012 and should be submitted electronically to the Responsible Officer: Dr Ana Padilla at email:
[email protected], with a copy to Dr David Wood at email: [email protected].
© World Health Organization 2012
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Summary
Twenty one laboratories were invited to participate in a collaborative study to evaluate the proposed 2nd WHO HIV-1 RNA Subtype Reference Panel for use with nucleic acid-based tests (NAT). The Reference Panel consisted of 10 heat inactivated, lyophilised coded samples representing HIV-1 subtypes; A, B, C, D, AE, F, G, AG-GH, groups N and O. The 3rd HIV-1 NAT International Standard (IS) was also included in the study as a blinded panel member.
Each laboratory assayed the blinded panel members which included the 3rd HIV-1 IS on at least two separate occasions and the data were collated and analysed at NIBSC. In total nineteen laboratories returned data. Some laboratories returned data using more than one assay type. Eighteen data sets were received from quantitative assays and nine from qualitative assays. The results showed that panel members gave a mix of tightly grouped results in the more common representation of subtypes such as A, B, C and D, through to a more mixed response with F, G and outliers form groups N and O. It is therefore considered that this is a valuable panel that continues to provide a means by which to challenge assay development. It is proposed that this panel is established as the 2nd International Reference Subtype Panel for HIV-1 NAT. No unit is to be assigned to any member of the panel.
Introduction
HIV-1 exhibits substantial genetic diversity and several different subtypes of HIV-1 exist.
There is a major group (group M), consisting of subtypes A-K and a more diverse collection of outliers have been referred to as groups N, P and O. Group P being the most recently identified outlier (Plantier JC et al).
Many of the early nucleic acid-based tests (NAT) had a fairly narrow band of specificity, targeting mainly subtype B viruses, as these predominated in the developed world. Greater awareness of HIV genetic diversity and the desire to detect as broad a range of HIV strains as possible following the global of once regional viruses has led to a number of improvements in assay design.
The 1st WHO HIV-1 panel was produced in 2001 in recognition that some assays were poor at detecting certain subtypes/groups, possibly giving sub-optimal or even negative results for samples that are clearly positive in other assays. Representatives of the WHO Collaborating Centres involved in the Working Group on Reference Preparations for testing HBsAg, HCV and HIV Diagnostic Kits as well as the WHO International Working Group on Standardisation of Gene Amplification Techniques for the Virological Safety Testing of Blood and Blood Products (SoGAT) agreed that there was a need for a well characterised reference panel of different HIV-1 subtypes/groups. Such a reference panel would be of particular use in regions of the world where non-B subtypes of HIV-1 predominate or are frequently encountered by laboratories involved in molecular (NAT) diagnosis and patient monitoring as well as by kit manufacturers. NIBSC agreed to formulate a candidate reference panel and evaluate it in an international collaborative study. The 1st panel has been a fundamental tool in the ongoing development of HIV-1 NAT assays, the panel is now depleted and the WHO ECBS approved the replacement of the panel in 2010. It was agreed that the panel should contain the same viral isolates as was used for the production of the first panel, however in line with other HIV International Standards, the panel members would be heat inactivated and lyophilised preparations (Holmes et al.). Heat inactivation has the advantage of (i) being safer to handle during aliquoting and freeze-drying and (ii) simplifies shipping of the material as they are no longer treated as infectious. All viral stocks therefore
were heat inactivated at 60C for 1 hour and an appropriate dilution of heat inactivated stocks were made into human plasma prior to freeze drying. Approximately 2000 vials of each panel member were prepared, each containing a volume of 1ml, when reconstituted,. This panel (labelled S01-S10) was evaluated in an international collaborative study; the study included a coded sample of the 3rd HIV-1 IS (labelled S11).
Materials
Candidate standards
The initial isolates used for the preparation of the 1st panel were, where possible, viral isolates at low passage. All viruses were propagated on human PBMC’s and after clarification by centrifugation, a large stock of cell-free culture supernatant was stored under liquid nitrogen vapour as 1ml aliquots. In order to maintain continuity between the first and second panels these viral stocks were used.
Selection of virus strains
Table 1 shows the selection of viruses used for this panel. The viral strains and subtypes their corresponding study code, geographic origin and source through which they were obtained.
Where possible, viral isolates with a published full length genomic sequence were chosen (accession numbers given where known), thus establishing that they were non-recombinant, representative viruses. An exception was 92TH001, which is an AE recombinant (no pure subtype E virus has ever been isolated), and V1525, which was subsequently shown to be a complex mixture of two recombinant viruses, one a GH and the other an AG recombinant.
Source
All viruses were sourced from the Centre for AIDS reagents based at NIBSC.
Heat inactivation of virus
1ml aliquots of virus were placed in a water bath adjusted to 60oC for a period of 1 hour. The heat-treated virus was pooled, re-aliquoted and stored at ≤-80oC in a laboratory where infectious material is not propagated to avoid any contamination, viruses were then tested using a commercial assay capable of detecting viral reverse transcriptase activity. During the heat inactivation step the reverse transcriptase enzyme of the virus is denatured rendering it impossible for the virus to replicate. Heat inactivated samples vs the live specimens were tested in parallel. Where no RT activity is seen in the heat treated sample, inactivation is considered successful.
Freeze-drying
This was undertaken at the Centre for Biological Reference Materials (CBRM), NIBSC, Potters Bar, UK, during 2011/2012. The virus subtypes were freeze dried in batches of three, separated by an empty shelf. The freeze dried material consisted of viruses shown in the table 1, diluted in human plasma that had been tested and found negative for HBsAg, HIV antibody and HCV RNA by PCR. For the purpose of traceability during the fill process each candidate was assigned an individual NIBSC code number. Once the panel is established a single,
separate, code number will be applied. 1 ml volumes of each subtype member were filled into screw-cap glass freeze-drying vials and the vials freeze-dried.
Post-fill testing
Assessments of residual moisture and oxygen content, as an indicator of vial integrity after sealing, were determined for twelve vials of each of the freeze-dried batches. Residual moisture was determined by non-invasive near-infrared (NIR) spectroscopy (MCT 600P, Process Sensors, Corby, UK). NIR results were then correlated to Karl Fischer (using calibration samples of the same excipient, measured using both NIR and Karl Fischer
methods) to give % w/w moisture readings. Oxygen content was measured using a Lighthouse Infra-Red Analyzer (FMS-750, Lighthouse Instruments, Charlottesville, USA). The final number of vials filled along with the residual moisture and oxygen measurements can be found in table 2.
Design of collaborative study
Twenty one laboratories in sixteen countries were invited to take part in the collaborative study. Each laboratory received up to 4 heat-inactivated, lyophilised coded panels, each panel member (including the 3rd HIV-1 IS) was assigned a number chosen at random between S01- S11. As many automated assays now require different minimum input volumes each laboratory was sent the protocol in advance and asked to state how many vials they would require to complete the requested testing. Laboratories were requested, where possible, to assay the panel members in duplicate to facilitate the evaluation of intra and inter lab variability. The 3rd HIV-1 NAT IS was also included as a coded panel member. It was requested that all vials were frozen at -20oC on receipt and participants were requested to return an acknowledgement form sent with the package to report the safe receipt of the samples. Participants were asked to reconstitute each vial with 1ml of deionised, nuclease- free, molecular biology-grade water and left for a minimum of 20 minutes with occasional agitation before use; a visual check after this time was requested to ensure all vials were fully reconstituted. The collaborative study was conducted between December 2011 and March 2012. A copy of the study protocol can be found in appendix 1. All panels were sent with an accompanying Information for Use sheet (IFU), a copy of the IFU can be found in appendix 2.
Statistical Methods
Quantitative Assays: Results from quantitative assays submitted by participants were used directly. Quantitative assays provide estimates in “copies/ml” based on the calibration of the particular assay kit or method. To obtain a single estimate of copies/ml per sample for each laboratory, a single estimate for each assay was obtained by taking the mean of the log10 estimates across replicates and dilutions (after correcting for dilution factor). A mean of the assay means was then calculated to give a single laboratory mean value of log10 copies/ml.
The linearity of the assay response across the dilutions range was assessed and in some cases, estimates that fell outside the apparent linear range were excluded from subsequent calculations.
Qualitative Assays: Qualitative assays were analysed by pooling the assay data to give a dilution series of number positive out of number tested. A single estimate of ‘detectable units per ml’ in the undiluted sample was calculated using the method of maximum likelihood for
‘dilution assays’ (Collet, 1991). This model assumes that the probability of a positive result at a given dilution follows a Poisson distribution (with mean given by the expected number of
“copies” in the sample tested), and that a single “copy” will lead to a positive result. After correction for the equivalent volume of sample amplified, the result is given in “PCR detectable units/ml” (or NAT detectable units) and expressed as a log10 value. As a single value is obtained for each laboratory and sample no assessment of within laboratory, i.e between assay variability can be made.
Results
Data were received from nineteen laboratories of the initial twenty one that were invited to participate. Each member was identified by a code number, allocated at random, and not necessarily representing the order of listing in appendix 3. Where laboratories returned data using more than one method, each data set were analysed separately and treated as if from different laboratories, and coded, for example, laboratories 1A and 1B. The results were from a mixture of commercial and in-house methods. In total, there were eighteen data sets from quantitative assays, and nine data sets from qualitative assays.
The scoring of the 10 panel members plus the International Standard, coded as samples S01 to S11, are shown in table 4. The quantitative and qualitative assays are grouped together in the table, as are results from laboratories using the same assay method. Samples found positive are marked ‘P’, while samples found negative are marked ‘N’. Where a sample had both positive and negative results from repeat assays, or replicates within an assay, they are marked ‘P/N’. Some of the samples tested by laboratory 16 (16A and 16B) with qualitative assays were negative at a 1 in 10 dilution, but were not tested neat. These are marked ‘N 1/10’.
The laboratory mean estimates for the panel members are shown in figures 1-10. The overall means for the different assay methods and overall for qualitative and quantitative assays, are shown in tables 5 and 6. The laboratory mean estimates (log10 copies/ml) are based on the geometric mean estimates of copies/ml across dilutions and across assays for the quantitative assays, or calculated from the series of number positive out of number tested for the qualitative assays.
The majority of participants returned results as estimates copies/ml. The in-house assays of laboratories 3 and 4 were returned as IU/ml. Most (6) laboratories using the Abbott RealTime (ART) returned results as copies/ml, but one laboratory using this kit returned results as IU/ml. Where a sample was found both positive and negative across repeat assays or replicates, the mean of the positive results is given. Where all results are negative, they are coded ‘neg’.
For the two most commonly used assays, the Abbott Real Time (ART) and the Roche COBAS Ampliprep/COBAS Taqman (CAP), the overall means of those laboratories reporting in copies/ml are also shown.
Where laboratories reported data from qualitative assays a log10 PCR detectable units/ml was estimated from the dilution series of positive or negative scores for each sample and laboratory, using the statistical methods described earlier in this report
The estimates from each laboratory and assay method were converted to log10 IU/ml, by direct comparison with the concurrently tested HIV-1 IS, panel member S11, which has an assigned value of 5.27 log10 IU/ml (Morris and Heath). The results are shown in table 7 for quantitative assays, and table 8 for qualitative assays. They are also shown in histogram form in figures 1-10. In the figures, each box represents the mean log10 IU/ml for an individual laboratory and assay method. The boxes are labelled with the laboratory code number and a code indicating the assay method. Results from the qualitative assays are shaded in grey.
Negative results are plotted on the extreme left of the scale. The laboratories that obtained negative results at a 1 in 10 dilution, but did not test the samples neat, are plotted next to the negatives, and labelled as Neg.
Stability studies on the product in the final container
Accelerated degradation studies have been initiated at NIBSC. To date, the results of accelerated degradation studies at 4 and 8 months are available, shown in tables 3a and 3b.
Samples were incubated at the temperatures and times indicated and were evaluated using the Roche HIV Ampliprep V2 assay.
It is evident that each freeze-dried virus preparation was stable at -20oC, +4oC and +20oC at the latest time point tested (8 months). Samples that had been stored at +37oC and +45oC proved difficult to reconstitute, this has been observed in previous studies where samples are based on pooled human plasma. Rather than actual loss of RNA activity, the lower viral copy number seen at elevated temperatures (+37 and +45oC) may reflect an inability to fully reconstitute the freeze-dried pellet. However, in line with other studies, it is expected that this material will show good long term stability (Holmes et al).
Subsequent testing will take place at a further 12, 24 and 36 months
Real time stability studies are also carried out. The panel is included in NAT assays used on site at NIBSC.
Discussion
Overall all panel members performed well in this study. There were however some differences in detection ability by the different assays.
Quantitative assays
For the quantitative assays, all samples were scored positive with the following exceptions.
Sample S05 (Group O) was negative in two in-house assays from laboratories 6 and 16B.
Sample S06 (Group N) was negative in the Abbott RealTime assays from laboratories 13 and 16A and both positive and negative in the same assay from laboratory 12. For the qualitative assays, the in-house assays from laboratory 12 (12A and 12B) showed low sensitivity for all panel members except sample S11, the current IS, which would suggest that the assays have initially been calibrated against the IS. The in-house assays from laboratory 14 finds samples S05 and S06 negative, with sample S10 (subtype F) both positive and negative. Laboratory 5 also finds S06 negative. As samples 05 and 06 are groups O and N respectively, it could be concluded that further optimisation of the primer/probe design was required.
The 3rd HIV-1 IS (study code sample 11) was included in this study to allow an estimation of potency for each subtype member. From table 7, it can be seen that the results from participants using the Roche Ampliprep assay (CAP) are generally reporting higher values than for the other laboratories reporting in copies/ml. The mean log10 copies/ml for the Roche assay is 5.21, compared to 4.94 log10 copies/ml for the Abbott assay (ART), and between 4.73 and 4.97 for the other laboratories reporting in log10 copies/ml. The difference between the means for the CAP and ART assays is 0.27 log10, close to 2-fold. This difference is marginally statistically significant (p=0.016).
From table 5 it can also be seen that the Roche Ampliprep assay (CAP) also yields a higher mean estimate of log10 copies/ml for the majority of the panel members. For samples S01, S02, S03, S04, S07 and S10, the CAP assay gives estimates around 0.3 – 0.4 log10 higher than obtained using the ART assay. These differences are all statistically significant (p<0.01).
For S05 (Group O), the CAP is a little lower than ART (difference not significant). For S06 (Group N) the ART assay is generally much lower than the CAP assay, with one laboratory (16A) getting a negative result. The mean difference between assays is 1.27 log10. The Roche High Pure (RHP) assay from laboratory 17 was seen to give estimates for all panel members that are generally lower than from the other laboratories and assays, and has particularly low estimates for S05 and S06. Such a difference could, in part, be due to the different genome regions targeted by the assays.
Figures 1-10 illustrate that expressing results relative to the concurrently tested HIV-1 IS leads to generally good agreement between the quantitative assays. The in-house assays from laboratory 4 are giving lower estimates than the majority of assays for panel members S01, S03, S04 and S07. For panel members S05 and S06 (groups O and N respectively) there is more variability, this is not wholly unexpected due to the diversity within the groups. For panel member 6 in particular, there is an apparent bi-modal distribution, with differences of around 1 log10 IU/ml between the Roche Ampliprep (CAP) and the in-house assays of laboratories 3 and 6, and the other assays. There are also several negative results for panel members 5 and 6. It should be observed that there is a distinct pattern of detection with panel member 6 (Group N) whereby the Abbott real time assay consistently reports values at least 1.0 Log10 IU/ml lower than then Roche COBAS Taqman Ampliprep v2 assay, this pattern of detection is however reversed with data supplied for panel member 5 (group O) whereby the Ampliprep reflects results at least 0.5log10/ml lower than the Abbott. Two versions of the Roche Taqman assays were used, v1 whereby samples are typically extracted via the manual high pure method and v2 in which samples are extracted via the automated Ampliprep system.
It is anticipated that whilst the extraction methods vary it is unlikely that this will contribute to the difference observed values but more likely due to the difference in probes and primers incorporated in the amplification step of each method V1 targets the single area gag area of the genome, whilst the v2 assay has a dual target of the LTR and gag regions, with the LTR region of the HIV genome known to be more conserved than gag it is likely that the primer/probe design is more able to detect across the range of subtypes.
Qualitative assays
The results from the qualitative assays, shaded in grey, are more variable. However, with the limited number of repeat assays, use of the dilution series to obtain PCR/detectable units is not expected to be precise, and the variability is not unexpected. Although as noted above, the assays from laboratory 12 were generally of low sensitivity, for those samples that were positive, expression relative to the concurrently tested HIV-1 IS produces results in line with other laboratories and assays, for example for panel member S03.
Table 4 also gives the overall means of laboratories using the two most common assay methods, the Abbott Real Time (ART) and the Roche COBAS Ampliprep/Taqman. For most panel members, expression of results relative to the concurrently tested HIV-1 IS has removed the differences between the results of these methods. The exceptions are panel member S05 (group O) where the Abbott assay is around 0.3 log higher, panel member S06 (group N) where the Abbott assay is around 1.0 log lower, and panel member S09 (subtype C) where the Abbott assay is around 0.3 log lower. For the other panel members, the differences are no longer statistically significant at 1% (p>0.01).
Table 7 shows the overall median of the estimated log10 IU/ml across all laboratories performing quantitative assays. The majority of panel members are between 3.5 and 4.0 log10 IU/ml. The exceptions are S05 (group O) and S06 (group N) which have medians of 3.32 and 2.74 log10 IU/ml respectively, as a result of systematic under-estimation of these subtypes by some assays.
In cases where laboratories reported data in qualitative format and the sample was positive at all dilutions tested, it was not possible to obtain an estimate. These are shown in table 8.
There is considerable variability between laboratories and assays. For example, for the 3rd HIV-1IS 10/152, estimates range between 1.4 and 5.0 log10 PCR detectable units/ml. It is clear that the assays vary in sensitivity, with the in-house assays from laboratory 12 having particularly low sensitivity.
It is clear that there are differences in efficiency of detection and quantitation by different assays for different subtypes. The effect is perhaps less marked than was the case in the first subtype study conducted in 2001 (Holmes et al.) as is illustrated in figures 11a and b whereby it can be seen that there has been a large overall improvement in the ability of all assays to detect a representative from group O. Overall, the data for panel members give a value around 3.5 – 4.0 log10 IU/ml when expressed relative to the concurrently tested IS, with the subtype O and N coming out lower. For samples S02 (G), S08 (AG-GH), S09 (C), S10 (F) the histograms show good agreement in the estimate of IU/ml for all quantitative assays.
However, this may reflect the assay performance rather than the actual content of these panel members. Despite good agreement between some methods, it is still considered that there is a disparity between some assays and subtypes and therefore it is not beneficial to assign values in IU to panel members.
Conclusions
This study has shown that the proposed replacement lyophilised panel performs well in all assays used within the study and is suitable for establishment as a WHO reference panel (?).
Proposal
We propose that the panel is established as the 2nd International Reference panel for HIV-1 NAT Subtypes. Due to variations in the assays ability to detect different subtypes with equal efficiency we propose that no unit can be assigned to any panel member.
Participant feedback comments
The report was circulated to all participants for comment. Two participants replied with minor typographical corrections. No further corrections were received.
References
Collet, D. 1991. Modelling binary data. Chapman Hall, London.
Davis, C., Heath, A., Best S., Hewlett, I., Lelie, N., Schuurman, R. and Holmes, H 2002.
Calibration of HIV NAT working reagents against the 1st international standard for HIV-1 RNA. Journal of Virological Methods (accepted for publication).
Holmes, H., Davis, C., Heath, A., Hewlett, I. And Lelie, N, 2001. An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques. Journal of Virological Methods 92, 141-150.
Holmes H, Davis C, Heath A
Development of the 1st International Reference Panel for HIV-1 RNA subtypes for use in nucleic acid-based techniques.J Virol Methods. 2008 Dec;154(1-2):86-91.
Holmes H, Berry N, Heath A, Morris C
Preparation and evaluation of the 1st international standard for the quantitation of HIV-2 RNA in plasma. J Virol Methods. 2011 Aug;175(2):246-52.
Morris C and Heath A
Evaluation of the 3rd HIV-1 NAT International Standard WHO report (waiting for details)
Plantier JC, Leoz M, Dickerson JE, De Oliveira F, Cordonnier F, Lemée V, Damond F, Robertson DL, Simon F (August 2009). "A new human immunodeficiency virus derived from gorillas". Nature Medicine 15 (8): 871–2
Table 1: Characteristics of the Subtype Reference Panel
Subtype /Group
Strain
Study Code
NIBSC
Code Origin Source
Full length
Sequence Accession Number
A 92UG037
7 11/150
Uganda UNAIDS Yes U51190
B 92TH014
4 11/152
Thailand UNAIDS Yes U86572
C 98TZ017
9 11/154
Tanzania UNAIDS Yes AF286235
D 94UG114
3 11/156
Uganda UNAIDS Yes U88824
AE 92TH001
1 11/158
Thailand UNAIDS Near full
length U86565
F 93BR020
10 11/160
Brazil UNAIDS Yes AF005494
G RU570
2 11/162
Russia CFAR
(Dr. A Bobkov) No U08368 AG-GH VI525
8 11/164
Gabon CFAR
(Dr. v.d.Groen) No
L11792 U09665 AJ277822
N YBF30
6 11/166
Cameroon CFAR
(Dr. F Simon) Yes AJ006022
O MVP5180
5 11/168
Cameroon CFAR
(Dr. L Gurtler) Yes L20571
Table 2: Fill characteristics 92UG037 (sample 7)
92TH014 (sample 4)
98TZ017 (sample 9)
94UG114 (sample 3)
92TH001 (sample 1)
No vials filled 2130 2131 2149 2121 2124
Mean residual oxygen
0.380 0.648 0.609 0.467 0.279
Mean residual moisture
0.31 0.36 0.31 0.66 0.74
93BR020 (sample 10)
RU570 (sample 2)
V1525 (sample 8)
YBF30 (sample 6)
MVP5180 (sample 5)
No vials filled 2150 2143 2146 2162 2143
Mean residual oxygen
0.657 0.575 0.974 1.04 0.94
Mean residual moisture
0.61 0.62 0.75 0.20 0.14
Table 3a: Accelerated Degradation data – 4 months
S01 S02 S03 S04 S05 S06 S07 S08 S09 S10
-20oC 3.66 4.57 3.73 3.70 3.36 3.42 3.52 4.07 3.87 3.94 +4oC 3.77 3.94 3.64 3.66 3.37 3.42 3.56 4.02 3.71 3.88 +20oC 3.62 4.00 3.66 3.63 3.30 3.51 3.61 3.93 3.95 3.84 +37oC 3.62 3.74 3.43 3.37 3.25 3.23 3.26 3.78 3.59 3.61 +45oC 3.29 3.66 3.30 3.31 3.15 2.95 3.11 3.60 3.38 3.65
Table 3b: Accelerated Degradation data – 8 months
S01 S02 S03 S04 S05 S06 S07 S08 S09 S10
-20oC 3.59 4.03 3.50 3.61 3.05 3.83 3.26 3.92 3.84 3.83 +4oC 3.69 3.90 3.50 3.64 3.02 3.86 3.55 3.75 3.84 3.86 +20oC 3.62 3.99 3.44 3.59 3.10 3.70 3.48 3.94 3.81 3.70 +37oC 3.41 3.72 3.08 3.27 2.99 3.40 3.16 3.47 3.58 3.40 +45oC 2.34 2.94 2.46 2.54 2.57 2.32 2.49 2.88 2.75 3.62
Table 4: Samples Scored Positive or Negative
Type Method Lab S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11
Quantitative
RT
1B P P P P P P - P P P P
2B P P P P P P P P P P P
8 P P P P P P P P P P P
12 P P P P P P/N P P P P P
13 P P P P P N P P P P P
15 P P P P P P P P P P P
16A P P P P P N P P P P P
CAP
1A P P P P P P P P P P P
2A P P P P P P P P P P P
9 P P P P P P P P P P -
11 P P P P P P P P P P P
20 P P P P P P P P P P P
RHP 17 P P P P P P P P P P P
IH
3 P P P P P P P P P P P
4A P P P P P P P P P P P
4B P P P P P P P P P P P
6 P P P P N P P P P P P
16B P P P P N P P P P P P
Qualitative
ART 16A N 1/10 N 1/10 P P P N 1/10 P P P P P
PUP 18 P P P P P P P P P P P
VSP 19 P P P P P P P P P P P
IH
5 P P P P P N P P P P P
7 P P P P P P P P P P P
12A N N P/N N N N P/N P/N N N P
12B P/N N P/N N N N P/N N P/N P P
14 P P P P N N P P P P/N P
16B P P P P ? P P P P P P
P - Positive
N - Negative or < LLD
P/N - Both Positive and Negative replicates or assays N 1/10 - Not tested Neat but Negative at 1 in 10
? Negative at 1 in 10 – Not tested neat (correct?)
Assay Codes:
ART Abbott Real Time
CAP Roche cobas Ampliprep/cobas Taqman RHP Roche HighPure System/cobas Taqman PUP Procleix Ultrio Plus Assay
VSP Virus Screening PCR Kit (manufacturer GFE Blut mbH) IH In-House
Table 5: Estimated Copies or IU /ml (log10) from Quantitative Assays
Units Method Lab S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11
Copies
ART
1B 3.39 3.79 3.24 3.49 3.48 2.27 nt 4.01 3.36 3.60 5.15 2B 3.17 3.53 3.16 3.33 3.29 2.08 3.32 3.76 3.25 3.35 4.86 8 3.15 3.57 3.22 3.23 3.25 1.98 3.30 3.60 3.07 3.34 4.89 12 3.29 3.67 3.27 3.39 3.34 2.24 3.38 3.78 3.30 3.50 4.94 15 3.26 3.74 3.09 3.36 3.32 2.18 3.41 3.90 3.33 3.50 5.05 16A 3.07 3.29 2.90 3.12 2.95 neg 2.99 3.50 3.07 3.39 4.77 Mean 3.22 3.60 3.15 3.32 3.27 2.15 3.28 3.76 3.23 3.45 4.94
CAP
1A 3.53 4.06 3.54 3.54 3.16 3.29 3.48 3.90 3.80 3.74 5.19 2A 3.60 3.90 3.49 3.60 3.09 3.31 3.52 3.87 3.76 3.73 5.11 9 3.77 4.00 3.70 3.74 3.23 3.49 3.76 4.00 3.93 3.87 nt 11 3.76 4.04 3.71 3.85 3.41 3.59 3.72 3.97 3.94 3.89 5.41 20 3.53 3.84 3.50 3.58 3.01 3.40 3.56 3.76 3.75 3.71 5.14 Mean 3.64 3.97 3.59 3.66 3.18 3.42 3.61 3.90 3.84 3.79 5.21
RHP 17 2.93 3.34 3.02 2.99 1.89 2.20 2.93 2.93 3.20 2.97 4.73
IH 6 3.07 3.22 3.13 3.18 neg 3.26 3.22 3.57 3.20 3.37 4.87 16B 3.31 4.00 3.49 2.90 neg 3.20 3.01 3.94 3.29 3.45 4.97
IU
ART 13 3.46 3.82 3.56 3.66 3.33 neg 3.65 4.05 3.49 3.72 5.27
IH
3 3.42 4.00 3.60 3.01 4.41 3.54 3.39 3.87 3.23 3.72 5.02 4A 1.92 3.55 2.31 2.42 2.36 2.33 2.57 3.72 3.38 3.20 4.86 4B 2.09 4.07 2.47 2.62 3.43 2.57 2.92 4.20 3.76 3.72 5.39
neg – Negative or < LLD nt – Not Tested
Table 6: Estimated PCR detectable units (log10) from Qualitative Assays
Method Lab S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 ART 16A ? ? 2.06 3.08 2.06 ? 3.08 2.06 2.06 2.06 3.08
PUP 18 3.13 3.03 2.56 2.67 3.51 2.50 3.13 3.51 2.74 2.97 pos VSP 19 1.85 2.96 2.30 2.30 pos 3.30 2.55 3.65 2.61 2.96 pos
IH
5 1.81 1.40 1.81 2.40 1.40 neg 2.22 2.35 1.81 1.81 4.37 7 1.98 3.08 3.02 2.48 3.29 2.88 3.27 3.47 2.78 3.27 5.02 12A neg neg 0.40 neg neg neg 0.40 0.40 neg neg 1.42 12B 0.74 neg 0.40 neg neg neg 0.40 neg 0.74 0.88 2.54 14 0.55 1.82 1.55 1.82 neg neg 0.72 1.07 1.82 <0 3.08 16B 2.36 2.61 2.44 2.12 ? 2.16 2.38 2.88 2.61 2.78 4.08
neg – Negative at neat
? – Negative at 1 in 10 – Not tested neat
pos – Positive at all dilutions tested, so no estimate of PCR/detectable units possible.
Table 7: Estimated IU/ml (log10) from Quantitative Assays
Method Lab S01 S02 S03 S04 S05 S06 S07 S08 S09 S10
ART
1B 3.51 3.91 3.36 3.60 3.60 2.39 nt 4.13 3.47 3.72 2B 3.58 3.95 3.57 3.74 3.70 2.50 3.73 4.17 3.66 3.77 8 3.53 3.94 3.60 3.61 3.63 2.35 3.67 3.97 3.45 3.72 12 3.62 4.00 3.60 3.72 3.67 2.57 3.71 4.11 3.63 3.83 13 3.45 3.81 3.56 3.66 3.33 neg 3.65 4.04 3.48 3.72 15 3.49 3.96 3.32 3.59 3.54 2.41 3.63 4.12 3.55 3.73 16A 3.57 3.79 3.40 3.62 3.45 neg 3.49 4.00 3.57 3.89 Mean 3.54 3.91 3.49 3.65 3.56 2.44 3.65 4.08 3.54 3.77
CAP
1A 3.61 4.13 3.62 3.62 3.24 3.37 3.56 3.98 3.88 3.82 2A 3.76 4.07 3.65 3.76 3.25 3.47 3.69 4.03 3.93 3.89
9 - - - -
11 3.62 3.90 3.57 3.71 3.27 3.45 3.58 3.83 3.80 3.75 20 3.65 3.97 3.63 3.71 3.14 3.53 3.69 3.88 3.88 3.84 Mean 3.66 4.02 3.62 3.70 3.23 3.46 3.63 3.93 3.87 3.83 RHP 17 3.47 3.87 3.56 3.53 2.43 2.74 3.47 3.47 3.74 3.50
IH
3 3.67 4.25 3.85 3.26 4.66 3.79 3.64 4.12 3.48 3.97 4A 2.33 3.96 2.72 2.83 2.78 2.74 2.98 4.13 3.79 3.61 4B 1.97 3.95 2.35 2.50 3.32 2.46 2.80 4.09 3.65 3.60 6 3.47 3.62 3.53 3.58 neg 3.65 3.62 3.96 3.60 3.76 16B 3.61 4.30 3.79 3.19 neg 3.50 3.31 4.24 3.59 3.75 Overall Median 3.57 3.95 3.57 3.61 3.32 2.74 3.63 4.04 3.63 3.75
neg – Negative or < LLD nt – Not Tested
Lab 9 did not test Sample 11, the IS, and so results could not be expressed in IU.
Table 8: Estimated IU/ml (log10) from Qualitative Assays
Method Lab S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 ART 16A ? ? 4.25 5.27 4.26 ? 5.27 4.26 4.26 4.26
PUP 18 - - - -
VSP 19 - - - -
IH
5 2.71 2.30 2.71 3.30 2.30 neg 3.12 3.25 2.71 2.71 7 2.24 3.33 3.27 2.73 3.55 3.14 3.52 3.72 3.03 3.52 12A neg neg 4.25 neg neg neg 4.25 4.25 neg neg 12B 3.47 neg 3.13 neg neg neg 3.13 neg 3.47 3.61
14 2.74 4.02 3.75 4.02 neg neg 2.92 3.27 4.02 1.89 16B 3.54 3.79 3.63 3.31 ? 3.35 3.57 4.06 3.79 3.96
Neg – Negative at neat
? – Negative at 1 in 10 – Not tested neat
pos – Positive at all dilutions tested, so no estimate of PCR/detectable units possible.
Figure 1 - Panel Member 1 - 11/158 Subtype AE
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
12A 16A 04B04A
07 05 14
01A 01B 02B 06 08 12 12B
13 15 16A 16B 16B 17
02A 03 11 20
IH ART IH IH
IH IH IH
CAP ART ART IH ART ART IH ART ART ART IH IH RHP
CAP IH CAP CAP
Quantitative Qualitative
Figure 2 -Panel Member 2 - 11/162 Subtype G
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
12A 12B
16A 05 07 06 13
16A 16B 17
01B 02A 02B 04A 04B 08 11 12 14 15 20
01A 03 16B
IH IH
ART IH IH IH ART
ART IH RHP
ART CAP ART IH IH ART CAP ART IH ART CAP
CAP IH IH
Quantitative Qualitative
Figure 3 - Panel Member 3 - 11/156 Subtype D
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
04B 04A
05 01B
07 12B
15
01A 02B 06 08 11 12 13 16A
17
02A 03 14 16B 16B 20
12A 16A
IH IH
IH ART
IH IH ART
CAP ART IH ART CAP ART ART ART RHP
CAP IH IH IH IH CAP
IH ART
Quantitative Qualitative
Figure 4 - Panel Member 4 - 11/152 Subtype B
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
12A 12B
04B04A 07
03 05 16B 16B
01A 01B 06 08 15 16A
17
02A 02B 11 12 13 20
14 16A
IH IH
IH IH IH
IH IH IH IH
CAP ART IH ART ART ART RHP
CAP ART CAP ART ART CAP
IH ART
Quantitative Qualitative
Figure 5 - Panel Member 5 - 11/168 Subtype O
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
06 12A 12B 14 16B
16B 05 17 04A 01A
02A 04B 11 13 20
01B 07 15 16A
02B 08 12
16A 03
IH IH IH IH IH
IH IH RHP IH CAP
CAP IH CAP ART CAP
ART IH ART ART
ART ART ART
ART IH
Quantitative Qualitative
Figure 6 - Panel Member 6 - 11/166 Subtype N
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
05 12A 12B 13 14 16A
16A 08 01B
02B 04B 12 15
04A 17
01A 07 16B
02A 11 16B
20
03 06
IH IH IH ART
IH ART
ART ARTART
ART IH ART ART
IH RHP
CAP IH IH
CAP CAP IH CAP
IH IH
Quantitative Qualitative
Figure 7 - Panel Member 7 - 11/150 Subtype A
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
04B04A 05 14
12B 16B
01A 06 07 11 16A 16B 17
02A 02B 03 08 12 13 15 20
12A 16A
IH IH IH IH
IH IH
CAP IH IH CAP ART IH RHP
CAP ART IH ART ART ART ART CAP
IH ART
Quantitative Qualitative
Figure 8 - Panel Member 8 - 11/164 Subtype AG-GH
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
12B 05
14 17 07
11 01A 02A 03 04B
06 08 12 13 15 16A 16B 20
01B 02B 04A 12A 16A 16B
IH IH
IH RHP IH
CAP CAP CAP IH IH IH ART ART ART ART ART IH CAP
ART ART IH IH ART
IH
Quantitative Qualitative
Figure 9 - Panel Member 9 - 11/154 Subtype C
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
12A 05 07 01B
03 06 08 12B
13 15 16A 16B
02B 04A 04B 11 12 16B
17
01A 02A 14 20
16A
IH IH IH ART
IH IH ART
IH ART ART ART IH
ART IH IH CAP ART IH RHP
CAP CAP IH CAP
ART
Quantitative Qualitative
Figure 10 - Panel Member 10 - 11/160 Subtype F
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
IU/ml (log10)
Neg Neg? 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
12A 14 05 04A
04B 07 12B
17
01A 01B 02B 06 08 11 12 13 15 16B
20
02A 03 16A 16B
16A
IH IH IH IH
IH IH IH RHP
CAP ART ART IH ART CAP ART ART ART IH CAP
CAP IH ART
IH
ART
Quantitative Qualitative
Appendix 1 – Study Protocol
Appendix 2: Panel IFU
Appendix 3: Participants
Véronique Michel-Treil Genomics Department
Covance Central Laboratory Services SA Ru Moise Marcinhes 7
1217 Meyrin Geneva
SWITZERLAND
Pavel Hložek GeneProof a.s.
Viničnί 235 615 00 Brno
CZECH REPUBLIC
Therese Boström Cepheid AB Cirkusgrönd 4 17263 Sundbyberg SWEDEN
Angela Light
Genomics Department
Covance Central Laboratory Services 8211 SciCor Dr.
Indianapolis.
IN 46214 USA Maria Susana Vitali
UNC – Hemoderivados Av Valparaiso
s/n Ciudad Univeritaria
X5000HRA Córdoba – Argentina ARGENTINA
Mashashi Tatsumi
AIDS Research Centre, National Institute of Infectious Disease
1-23-1 Toyama Shinjuku
Tokyo 162-8640 JAPAN
Cristina Alemany Biomat S.A
c/ llevant n°11, Pol. Llevant 08150- Parets del Vallès Barcelona
SPAIN
Dr DF York
Molecular Diagnostic Services (Pty) Ltd 6 Ribston Place
Westville 3630 SOUTH AFRICA
Bryan Cobb Roche
4300 hacienda Ave Pleasanton
CA 94609 USA
Erin Wigglesworth Division of Retrovirology NIBSC
Potters Bar EN6 3QG UK Yiming Shao
National Center for AIDS/STD Control and Prevention
Chinese Center for Disease Control and Prevention
155 Changbai Road Changping District Beijing 102206 CHINA
Micha Nuebling Julia Kress
Paul Ehrlich Institute Paul Ehrlich Str 51-59 63225 Langen
GERMANY
Dr. Agnieszka Dryla Baxter AG
Plasma Analytics Industriestrasse 20 1221 Vienna
Indira Hewlett FDA
9000 Wisconsin Ave Bethesda
MD 20892
AUSTRIA USA Dominique Baudon
Amfumbom Kfutwah HIV Laboratory
Centre Pasteur du Cameroun BP 12 74
Yaoundé
REPUBLIC OF CAMEROON
Christine Defer EFS Nord De France U.R.B.M
10-12 Bd de Belfort 59000 Lille
FRANCE Polona Nograsek
Blood Transfusion Centre of Slovenia Slajmerjeva 6
1000 Ljubljana SLOVENIA
Lutz Pichl
German Red Cross Blood Transfusion Service Nat Laboratory
Feithstrasse 180-186 D-58097 Hagen GERMANY Bharathi Anekella
SeraCare Life Sciences 217 Perry Parkway Gaithersburg MD 20877 USA
Almoustapha Maiga
HIV/TB Research & Training Centre “SEREFO”
Faculty of Medicine, Pharmacy and Dentistry University of Bamako
BP: 1805 Bamako MALI
