Journal of Virological Methods 154 (2008) 86–91

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Journal of Virological Methods

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Development of the 1st International Reference Panel for HIV-1 RNA genotypesfor use in nucleic acid-based techniques

Harvey Holmesa,∗, Clare Davisa, Alan Heathb

a Division of Retrovirology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UKb Division of BioStatistics, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK

Article history:Received 3 June 2008Received in revised form 20 August 2008Accepted 21 August 2008Available online 2 October 2008

Keywords:HIV-1RNAGenotypesNAT

a b s t r a c t

Twenty-eight laboratories from 16 countries participated in a collaborative study to evaluate an HIV-1 RNAGenotype Reference Panel for use with nucleic acid-based tests (NAT). The Reference Panel consisted of 11coded samples representing different HIV-1 genotypes (subtypes A–D, AE, F, G, AA-GH, groups N and O) aswell as a negative diluent control. Each laboratory assayed the eleven panel members concurrently withthe 1st International Standard for HIV-1 RNA (NIBSC Code 97/656) on at least three separate occasionsand the data collated and analysed at NIBSC. Twenty-nine sets of data from NAT were received, 19 fromquantitative and 10 from qualitative assays, with six different commercial assays and five “in-house”assays represented.

The results showed that viruses from subtypes A–D and recombinant virus AE [CRF01 AE] were detectedconsistently, but that some assays had difficulty with the detection and quantification of viruses fromsubtypes F and G, a mixed recombinant virus AA-GH and a representative of group N. Furthermore, mostassays failed to detect the group O representative. The study illustrated the limitations of some molecular

assays particularly in detection of certain non-B genotypes which are important viruses in the globalAIDS pandemic and illustrated the value of a well-characterised genotype panel. The panel has beenestablished by the World Health Organisation’s Expert Committee on Biological Standardisation as the

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. Introduction

The HIV/AIDS pandemic remains the most serious of infectiousisease challenges to public health in the early 21st century. Recentgures from the United Nations Programme on HIV/AIDS (UNAIDS,008) show that during 2007 some 33 million individuals were liv-

ng with HIV, 2 million had died from an AIDS-related illness andhere were 2.7 million new infections. HIV-1 exhibits a high muta-ion and recombination rate resulting in a highly variable virus thatxhibits substantial genetic diversity combined with rapid evolu-ion (WHO Network, 1994; Tatt et al., 2001). As a result, three mainenetic groups exist, a major group (group M) mainly responsi-le for the global pandemic and consisting of nine subtypes, A–D,–H, J and K, and a more diverse collection of outliers mainly con-

ned to Central Africa that have been referred to as groups N and ORobertson et al., 2000). Genetic analysis of virus isolates includingull-length genome sequencing has established that inter-subtypeecombination is common, presumably as a result of individu-

∗ Corresponding author. Tel.: +44 1707 641218; fax: +44 1707 641060.E-mail address: hholmes@nibsc.ac.uk (H. Holmes).

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166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2008.08.014

el HIV-1 RNA Genotypes (code 01/466).© 2008 Elsevier B.V. All rights reserved.

ls being concurrently infected by more than one strain of HIV-1Robertson et al., 1995). Some of these recombinant viruses aremportant epidemic viruses circulating widely in certain regionsf the world and have been referred to as circulation recombinantorms (CRFs) (Leitner et al., 2005). An example is the CRF01 AE,riginally classified as subtype E, which is a common cause of HIVnfection and AIDS in South-East Asia (WHO Network, 1994). It hasecently been shown that some 50% of all infections worldwide areue to subtype C, subtypes A, B, D and G account for another 31%nd recombinant forms were responsible for 18% (Hemelaar et al.,006).

Although HIV diagnosis is based on sensitive antibody-detectionssays, the use of nucleic acid-based molecular assays (NAT) haveecome more widespread in the field of blood safety, as HIV-1NA can be identified some 6–11 days before antibody becomesetectable (AuBuchon et al., 1997; Busch et al., 1995). It is alsoidely used in the monitoring of HIV-infected individuals and

n assessing the outcome of HIV vaccine trials. There has beensubstantial range of NAT developed over the years, includingany commercially available assays, some based on target ampli-

cation technologies and some on signal amplification (Peter andevall, 2004). The former include thermocyclic methods involving

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everse transcription followed by polymerase chain reaction (PCR)r ligase chain reaction (LCR) as well as isothermal methods suchs nucleic acid sequence-based amplification (NASBA) and tran-cription mediated amplification (TMA); an example of a signalmplification method is the branched DNA (dDNA) assay.

Many of the early NAT had a rather narrow band of specificityargeted primarily at subtype B viruses, as these predominated inhe developed world (Alaeus et al., 1997; Coste et al., 1996). Greaterwareness of HIV genetic diversity, the desire to detect as broadrange of HIV strains as possible and the recognition that early

ssays would not detect certain of the non-subtype B field isolatesed to a number of improvements in assay design. However, it haseen recognised that some assays may have difficulties in detectingertain subtypes/groups, occasionally giving sub-optimal or evenegative results for samples that are clearly positive in other assaysCoste et al., 1996; Holguin et al., 1999; Nolte et al., 1998; Parekht al., 1999; Kim et al., 2007; Geelen et al., 2003; Truchsess et al.,006; Wang et al., 2008). It is therefore desirable to have well char-cterised standards such as reference panels containing a range ofIV-1 subtypes and groups available for evaluating the sensitivitynd analytical performance of such assays (Lee et al., 2006).

Representatives of the WHO Collaborating Centres involved inhe Working Group on Reference Preparations for testing HBsAg,CV and HIV Diagnostic Kits as well as the WHO Internationalorking Group on Standardisation of Gene Amplification Tech-

iques for the Virological Safety Testing of Blood and Blood ProductsSoGAT) agreed that there was a need for a well-characterised ref-rence panel of different HIV-1 subtypes/groups. Such a referenceanel would be of particular use in regions of the world where non-genotypes of HIV-1 predominate or are frequently encountered

y laboratories involved in molecular (NAT) diagnosis and patientonitoring as well as by kit manufacturers. NIBSC agreed to formu-

ate a candidate reference panel and evaluate it in an Internationalollaborative Study.

. Materials and methods

.1. Selection of viruses

A representative of each of the main HIV-1 group M subtypes–D, F and G as well as the outlier groups N and O was included in

he panel. Where possible, non-recombinant viral isolates with anown full-length genomic sequence were selected for use. Excep-ions were a CRF01 AE (AE) recombinant, as no pure subtype Eirus has been isolated to date, and a mixed recombinant virus (AA-H), as an example of the more complex recombinant viruses thatre increasingly being encountered in the field. All viruses wererovided as low passage isolates by the Programme EVA Centreor AIDS Reagents, NIBSC, UK. Isolates from subtypes A (92UGO37ARP177.8]), B (92TH014 [ARP180.8]), C (98TZ017 [ARP1032.17]), D94UG114 [ARP177.30]), AE (92TH001 [ARP180.1]) and F (93BR020ARP179.25]) were donated by the WHO-UNAIDS Network for HIVharacterisation (Dr. S. Osmanov, WHO, Switzerland). A subtype G

solate (RU570 [ARP174]) was donated by Dr. A. Bobkov, Ukraine, theA-GH isolate (VI525 [ARP176]) by Dr. G. van der Groen, Institute ofropical Medicine, Belgium, the group N isolate (YBF30 [ARP190])y Dr. F. Simon, Institute Pasteur, Paris, and the group O isolateMVP5180 [EVA167]) from Dr. L. Gürtler, Max von Pettenkofer Insi-ute, Germany.

.2. Preparation of panel

All viruses were propagated on phytohaemagglutinin-timulated human peripheral blood mononuclear cells that

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al Methods 154 (2008) 86–91 87

ere maintained in RPMI 1640 medium supplemented with5% foetal calf serum, antibiotics and interleukin-2. Virus wasarvested when p24 antigen levels were shown to be rising and,fter clarification by low speed centrifugation, a stock of cell-freeulture supernatant was stored under liquid nitrogen vapour asml aliquots.

Prior to formulating the Reference Panel, an estimate of theNA concentration of the undiluted viral stocks was obtained using

our diverse NAT systems, Roche Amplicor HIV-1 Monitor Ver-ion 1.5 (Monitor), Biomerieux Nuclisens HIV-1 QT (Nuclisens),ayer Versant bDNA V3 (bDNA) and an “in-house” assay basedn the LTR region of the viral genome (Cleland et al., 2001). Welso investigated quantification of the virus stocks using confocalicroscopy counts and electron microscope counts as described in

hattacharya et al. (2004). Based on these results, dilutions werestimated for each viral stock to give a final concentration for eachanel member in the region of 2000–10,000 copies/ml. In order torepare the reference panel, viral stocks were spun at 10,000 × g for0 min to remove cellular debris and diluted by the pre-determinedmount in defibrinated plasma (Base Matrix, Boston Biomedica Inc.,SA) which had been pre-screened for HBsAg, Anti-HCV and Anti-IV-1/2 and found to be negative. A total of 550 ml of each panelember was produced and these were distributed into 500 aliquots

f 1.1 ml. Each panel member was prepared on a separate occa-ion to minimise the risk of cross-contamination. A virus-negativeiluent control was included. A total of 500 Genotype Referenceanels (NIBSC Code 01/466) were prepared each containing 11anel members. For the collaborative study, each member of 100 ofhe Reference Panels was assigned a unique code. All the aliquotsere batch frozen and stored below −70 ◦C. The 1st International

tandard for HIV-1 RNA (NIBSC Code 97/656) was included in thetudy (Holmes et al., 2001).

.3. Organisation of the Collaborative Study

Twenty-nine laboratories participated in the collaborative studynd were each assigned a code number for the duration of the study.ach participant was sent 3 sets of the Genotype Reference PanelNIBSC Code 01/466) containing the 10 coded HIV-1 RNA geno-ypes (subtypes A–D, AE, F, G, AA-GH, groups N and O) and negativeontrol and three vials of the WHO 1st International Standard forIV-1 RNA (NIBSC code 97/656). All samples were shipped on dry

ce and stored by the participants at −70 ◦C to −85 ◦C until required.hawing and re-freezing of the vials was not recommended.

Participants were asked to thaw the liquid preparations (geno-ype panel) at 37 ◦C in a water bath. The International Standards a lyophilised preparation and participants were instructed toeconstitute the vial by removing the tear-off crimp, loosening theubber seal and adding 1.0 ml of de-ionised RNAse-free water. Theial was gently agitated for 20 min to fully dissolve the contentsefore use.

Participating laboratories were requested to assay the elevenanel samples concurrently with the International Standard on at

east three separate occasions. Laboratories performing quantita-ive assays were requested to assay each panel member and thenternational Standard undiluted. Laboratories performing qual-tative assays were requested to perform an initial assay using.0 log10 dilution steps followed by at least two further assays usingdilution series of 0.5 log10 steps selected to cover the anticipatednd-point.

.4. Stability

Samples of the Genotype Reference Panel (NIBSC Code 01/466 -ubtypes A–D, AE, F, G, AA-GH and group N) were assayed period-

88 H. Holmes et al. / Journal of Virological Methods 154 (2008) 86–91

Table 1Laboratory mean estimates RNA copies/ml or PCR detectable units (log10/ml)

Lab Assaya IS A B C D AE F G AA GH N O

Quantitative45 LCx 4.51 3.57 3.40 3.44 3.71 3.64 3.53 3.34 3.62 2.48 2.1814 bDNA 4.51 3.05 3.17 3.04 3.50 3.27 3.41 3.23 3.57 2.58 –58 bDNA 4.65 3.07 3.23 3.11 3.48 3.39 3.53 3.28 3.58 2.65 –05 CMon 4.97 3.19 3.05 3.38 3.38 3.48 3.50 3.79 4.27 3.67 –09 CMon 4.65 3.39 3.19 3.30 3.61 3.62 3.63 3.73 3.84 3.40 –57 CMon 5.14 3.57 3.40 3.61 3.67 3.97 3.81 3.89 4.26 3.75 –69 CMon 4.94 3.26 3.27 3.54 3.76 3.62 3.77 3.78 4.10 3.73 –27 CMonb 4.66 3.18 2.99 3.25 3.22 3.47 3.48 3.61 3.95 3.53 –17 Mon 4.85 3.62 3.04 3.62 3.66 3.68 3.74 3.89 4.27 3.73 –22 Mon 4.81 3.42 3.19 3.35 3.44 3.49 3.54 3.61 4.12 3.62 –56 Mon 5.07 3.52 3.26 3.54 3.41 3.71 3.70 3.87 4.26 3.81 –60 Mon 4.75 3.29 2.89 3.40 3.45 3.59 3.84 3.51 4.11 3.87 –61 Mon 4.93 3.23 3.06 3.33 3.18 3.58 3.46 3.73 4.06 3.24 –63 Monc 4.99 3.62 3.37 3.37 3.68 3.80 3.92 4.01 4.20 3.46 –72 Mon 4.57 3.32 3.11 3.46 3.57 3.54 3.60 3.76 3.92 3.47 –04 Nuc 4.93 3.43 3.24 3.27 3.47 3.46 – – 2.06 – –48 Nuc 5.26 3.68 3.64 3.78 3.98 3.69 2.12 – 2.34 – –64 Nuc 4.95 3.49 3.54 3.65 3.73 3.50 2.54 – 2.14 – –68 Nuc 5.26 3.61 3.68 3.65 4.06 3.49 – – 2.18 – –

Qualitative65 AmS 4.74 3.38 2.54 3.61 3.21 3.51 2.77 3.11 3.21 3.38 –66 AmS 4.64 3.06 3.23 3.82 3.27 3.61 3.51 3.75 4.05 3.7919 CAmS 4.10 3.21 2.69 3.21 3.08 3.13 3.84 3.68 3.42 3.42 –30 CAmS 4.47 3.11 2.66 2.90 2.95 3.52 3.98 3.17 3.57 3.17 –17 TMA 4.52 3.16 3.04 3.52 3.33 3.00 3.80 3.07 3.97 2.78 1.6412 IH-ne 3.26 2.39 1.99 1.75 1.83 1.65 1.90 1.34 2.88 0.75 –52 IH-sg 4.24 2.68 2.57 3.15 3.21 2.68 1.47 3.68 2.77 – –54 IH-sg 4.30 2.00 3.13 2.42 2.99 2.58 2.42 2.81 3.00 – –59 IH-np 4.34 3.21 2.72 3.39 3.45 3.21 3.21 3.45 3.21 – –67 IH-np 4.53 2.20 2.61 2.47 2.47 1.99 2.59 2.25 2.46 2.10 1.08

a LCx = Abbott LCx HIV RNA quantitative; bDNA = Bayer Versant HIV 3.0 RNA bDNA; CMon = Roche COBAS Amplicor Monitor v1.5; Mon = Roche Amplicor Monitor v1.5;N S = RoI ag pri

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cally using the COBAS Amplicor HIV-1 Monitor test V1.5 assay inrder to determine the real time stability of the frozen panel.

.5. Statistical methods

.5.1. Quantitative assaysWhere laboratories returned quantitative results, a single esti-

ate was calculated for each sample as the geometric mean overll assays performed by the individual laboratory (arithmetic mean

f log10 estimates).

.5.2. Qualitative assaysWhere laboratories returned results from qualitative assays, a

ingle estimate was calculated for each laboratory using methods

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able 2stimated mean RNA concentration for quantitative (RNA copies/ml [log10]) and qualitati

ssay type No. IS A B C D

uantitative NATLCx 1 4.51 3.57 3.40 3.44 3.7bDNA 2 4.58 3.06 3.20 3.07 3.4Monitor 12 4.86 3.38 3.15 3.43 3.5Nuclisens 4 5.10 3.55 3.53 3.59 3.8Overall mean 19 4.86 3.39 3.25 3.43 3.5

ualitative NATAmpliscreen 4 4.49 3.19 2.78 3.38 3.1TMA 1 4.52 3.16 3.04 3.52 3.3In-House 5 4.14 2.50 2.60 2.64 2.7Overall mean 10 4.32 2.84 2.72 3.02 2.9

a Based of positive results only.

che COBAS Ampliscreen HIV-1 v1.5; TMA = transcription mediated amplification;mers; np = nested PCR with pol primers).

ased on the Poisson model, as described in the report of the col-aborative study to establish the International Standard (Holmes etl., 2001).

. Results

Data were received from 28 laboratories performing NAT ofhich one laboratory reported data from two different assays.

he data from NAT came from 19 (66%) quantitative assays and 10

34%) qualitative assays. A variety of assay methods were used inhe collaborative study. Of the 19 sets of results from quantitativessays, 12 were from versions of the Roche Amplicor Monitor assay1.5, including data from both the semi-automated COBAS systemnd the manual system and from standard and Ultrasensitive

ve (NAT detectable units/ml [log10]) assays

AE F G AA GH N O

1 3.64 3.53 3.34 3.62 2.48 2.189 3.33 3.47 3.25 3.58 2.62 –0 3.63 3.67 3.76 4.11 3.61 –1 3.53 2.33a – 2.18 – –8 3.58 3.36 3.66a 3.62 3.40a 2.18a

3 3.44 3.53 3.43 3.56 3.44 –3 3.00 3.80 3.07 3.97 2.78 1.649 2.42 2.32 2.71 2.87 1.43a 1.08a

8 2.89 2.95 3.03 3.26 2.77a 1.36a

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echniques, 4 were from the Biomerieux NucliSens HIV-1 QT assay,were from the Bayer Versant bDNA v3, and 1 was from the

bbott LCx HIV quantitative assay. Of the 10 sets of results fromualitative assays, 4 were from the Roche Ampliscreen HIV-1 v1.5ssay, 2 of which used the semi-automated COBAS system, 1 wasrom a transcription mediated amplification (TMA) system and 5ere from “in-house” PCR methods. Of the latter, 1 was a nested

CR assay with HIV-1 env gp41 primers, 2 were single PCR assaysith gag primers and 2 were nested PCR assays with pol primers.

Estimates of RNA concentration expressed as log10 copies/mlr log10 detectable units/ml were obtained for each sample andaboratory (Table 1) using the methods described above and the

ean RNA concentration determined for each assay type (Table 2).nalysis of the data shows that estimates from laboratories usingualitative assays were generally lower by approximately 0.5 log10han those from quantitative assays. Furthermore, it can be seenhat most assays detect subtypes A–D and CRF01 AE satisfactorily,ut that some difficulties were experienced by certain assays inhe detection of subtypes F and G, the mixed recombinant virusA-GH and groups N and O. The negative control sample contain-

ng diluent was found to be negative by all laboratories and assayypes.

Most laboratories satisfactorily detected the 1st Internationaltandard for HIV-1 RNA (code 97/656) and the results are in lineith that obtained in previous studies (Holmes et al., 2001; Davis

t al., 2002). Of the quantitative assays, the laboratories using theDNA and the LCx assays obtained estimates that were slightly

ower than those laboratories that used Monitor or Nuclisensssays. Of the qualitative assays, one laboratory using an “in-house”ssay (lab code 12) gave an estimate that was around 1.0–1.5 log10ower than the other laboratories.

There was good overall agreement in the detection of the fieldsolates from subtypes A–D and CRF01 AE between the majorityf laboratories using quantitative assays and the performance ofhe laboratories and assays for these genotypes was similar tohe performance of the International Standard. Laboratories usingDNA assays obtained estimates that were slightly lower than lab-ratories using other assays for the subtype A and C samples.hilst commercial qualitative assays generally gave values that

ell broadly within the range given by quantitative assays, the “inouse” qualitative assays showed somewhat lower values.

Detection of viral isolates from subtypes F and G, the mixedecombinant AA-GH and group N was less consistent. Of the quan-itative assays, the Nuclisens assay either detected the virusesub-optimally compared to the other assays (F and AA-GH) or failedo detect virus (F, G and N), whilst the Monitor assays gave valuespproximately 0.5–1.0 log10 higher than the LCx and bDNA assaysor viruses from G, AA-GH and N.

Of the qualitative assays, the commercial Ampliscreen and TMAssays gave values that were broadly similar to the quantitativessays for subtypes F and G, the mixed recombinant AA-GH androup N. However, the “in-house” qualitative assays gave more vari-ble results with three laboratories failing to detect the group Nirus and others giving sub-optimal values for subtypes F and G.hilst two laboratories (12 and 67) mostly gave values at the lower

nd of the range, one laboratory (59) gave values that in generallustered with the commercial quantitative and qualitative assays,lthough it failed to detect the group N virus.

The outlier group O virus was found to be negative or below theimit of detection by all but three laboratories. The exceptions were

he qualitative TMA assay, a qualitative “in-house” assay and thebbott LCx assay.

Tests on the panel showed that the panel retained its full activityfter 4 years storage at colder than −70 ◦C and that there was novidence of deterioration (data not shown).

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al Methods 154 (2008) 86–91 89

. Discussion

A total of 28 laboratories participated in the collaborative studyubmitting data sets from 19 quantitative and 10 qualitative assays.espite the extensive nature of the study, it is important to exerciseaution in making general comments regarding the comparativeerformance of the different assay methods, as methods other thanhe Roche Monitor assay had limited representation in the study.owever, from the tables it is clear that the results from laborato-

ies using qualitative assays, particularly the “in-house” versions,ere generally lower than those from quantitative assays. This was

o be expected, as the data from qualitative assays were treated asilution series and used to provide single estimates of detectablenits per ml in the undiluted samples using the method of max-

mum likelihood that takes account of the Poisson distributionCollet, 1991). This method assumes that the presence of at leastne ‘detectable unit’ in the sample volume tested will lead to a pos-tive result and makes no correction for assay inefficiencies, such asysis and extraction effects and those that occur during reverse tran-cription. The estimated ‘detectable units per ml’ cannot thereforee directly equated to an estimate of RNA copies per ml.

There was good agreement between all laboratories using theonitor assay, both in the semi-automated COBAS or manual for-at. The results from the bDNA assays were generally slightly lower

han those from the Monitor for the majority of the liquid sam-les, as well as for the freeze-dried International Standard. Thereas a difference of around 0.2–0.5 log10 between the overall means

Table 2) in most cases. The exceptions were subtypes B & D whereo difference was apparent and group N, where the bDNA was.0 log10 lower than Monitor.

The results from the Nuclisens assay appeared less consistentcross samples than the other assay kits. Subtypes G and group Nere not detected by the Nuclisens assay, and subtype F was eitherot detected or gave much lower estimates than other assays types.he Nuclisens assay also had much lower estimates for the recom-inant mixture subtype AA-GH than other assay types. For the otheramples, HIV-1 subtypes A–D and CRF01 AE, the results from theuclisens assay were similar to or slightly higher than those from

he other quantitative assays. Other groups have reported sub-ptimal values using the Nuclisens assay. Swanson et al. (2005)creened panels of HIV-1 seropositive plasma samples includingepresentatives of subtypes A–G and CRF01 AE and reported thathe Nuclisens gave lower detection of subtypes A and C comparedo the LCx, bDNA and Monitor assays and completely failed tomplify the subtype G sample, similar to that found in this study.ottesmann et al. (2004) examined a panel of HIV-1 subtype B and Clasma samples and found that the Nuclisens assay underestimateshe viral load results in subtype C patients. The Nuclisens HIV-1 QTssay has recently been superceded by the NucliSens Easy-Q, a real-ime NASBA assay with molecular beacon probe detection systemhat is claimed to detect a wide range of non-clade B HIV-1 isolatesdeMendoza et al., 2005).

The mixed recombinant virus AA-GH was originally reported asGH recombinant (Janssens et al., 1994), but later work showed

hat it was in fact a mixture of two HIV-1 strains, one an envG/gagHecombinant and the other an envA/gagA virus (Beirnaert et al.,000). The participants of the collaborative study described abovead not been informed that this sample was a mixed virus pop-lation. However, one of the collaborative study participants (J.ackett, personal communication), whilst confirming the sequence

esignation of the other panel members, noted that this sampleppeared to be subtype G over a 481 nt region of pol integrase and a98 nt region of env gp41, but that when the whole of pol integraseas amplified, the result was suggestive of a mixed population.

urther analysis of gag p24 showed a sequence that grouped with

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ubtype A. These findings were in accordance with those reportedy Beirnaert et al. (2000).

The Abbott LCx, a pol-based PCR assay, was the only quantitativessay to detect the representative of the outlier group O, a resultimilar to that reported by Swanson et al. (2005). This sample wasound negative by all other assays except the qualitative TMA andne of the qualitative “in-house” methods.

The performance of the “in-house” methods was more vari-ble than the commercial assays, with some methods giving muchower estimates than other qualitative assays. One of the two “in-ouse” assays based on nested PCR with pol primers (laboratory9) gave results that were consistent with many of the commercialssay results, although it failed to detect group N and O, whereashe other assay (laboratory 67), whilst giving lower PCR detectablenits, nevertheless was able to detect both groups N and O.

This study has identified the shortcomings of a number of com-ercial and “in-house” assays with regard to their ability to detect

nd, in the case of quantitative assays, to quantify HIV-1 RNA, par-icularly some of the less frequently encountered non-subtype BIV-1 genotypes. It underlines the need to use NAT that combineigh sensitivity with breadth of detection for blood screening andiagnostic applications. The 1st International Standard for HIV-1NA was included in the study and showed good correlation withhe results of two previous studies (Holmes et al., 2001; Davis etl., 2002). However, the sub-optimal detection of some genotypesith some assays makes the determination of potencies relative to

he International Standard inappropriate for these genotypes andherefore no attempt was made to calibrate the Genotype Referenceanel members in terms of international units.

A report on the collaborative study (WHO/BS/03.1961) was sub-itted to the World Health Organisation Expert Committee on

iological Standardisation who agreed that such a reference panelould aid the evaluation and application of NAT-based assays and,

n the basis of the results obtained, the Expert Committee estab-ished the reference panel of 10 individual genotypes, coded 01/466,s the 1st International Reference Panel for HIV-1 RNA Genotypes,ut did not assign unitages to its components. It was agreed that theIV-1 Reference Panel would provide a valuable set of well charac-

erised reagents for HIV-1 NAT for use in regions of the world whereon-B subtypes of HIV-1 predominate or are frequently encoun-ered by laboratories involved in molecular (NAT) diagnosis andatient monitoring as well as by kit manufacturers.

cknowledgements

We are grateful to all the participants in the collaborative studyithout whom this work could not have been undertaken. We wish

o thank the representatives of the WHO Collaborating Centresnvolved in the Working Group on Reference Preparations for test-ng HBsAg, HCV and HIV Diagnostic Kits and the WHO International

orking Group on the Standardisation of Genomic Amplificationechnologies for the Virological Testing of Blood and Blood ProductsSoGAT) for their support and encouragement. We thank Dr. Johnackett, Abbott laboratories, USA, for his independent genetic anal-sis of the panel and for bringing to our attention the mixed viralature of sample AA-GH. We acknowledge the help provided by theU Programme EVA Centre for AIDS Reagents, NIBSC, UK, in provid-ng the virus isolates and for distributing the panels. This work wasupported in part by a WHO Technical Services Agreement (Ref BCT

14/181/28) (WHO Technical Officer: Dr. Ana Padilla).

ppendix A. List of participants

Dr. C. Aberham/Dr. P. Gross, Baxter AG, Vienna, Austria

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al Methods 154 (2008) 86–91

Dr. H. Brummer-Korvenkontio, National Public Health Institute(KTL), Helsinki, FinlandDr. M. Cardoso, German Red Cross, Blood Transfusion Service ofBaden-Wurttemberg, Ulm, GermanyDr. M. Chudy/Dr. M. Nubling, Paul-Ehrlich-Institut, Langen, Ger-manyDr. T. Cuypers/Dr. N. Lelie, CLB-Sanguin, Amsterdam, The Nether-landsMs. C. Davis/Dr. H. Holmes, NIBSC, Potters Bar, UKDr. C. Defer, Etablissement de Transfusion Sanguine, Lille, FranceDr. R. Downing, CDC/Uganda Virus Research Institute, Entebbe,UgandaDr. K. Fransen, Institute of Tropical Medicine, Antwerp, BelgiumDr. T. Gierman, Bayer Corporation, Clayton, NC 27520, USADr. I. Hewlett/Dr. S. Lee, FDA/CBER, Rockville, MD 20852-1448, USADr. M. Imai, Kanagawa Prefectural Institute of Health, Yokohama241-0815, JapanDr. D. Jardine, National Serology Reference Laboratory, Fitzroy, Vic-toria, AustraliaDr. M. Jose, Instituto Grifols SA, Barcelona, SpainDr. C. Kücherer, Robert Koch Institut, Berlin, GermanyDr. E. Mathys/Dr. I. Thomas, Scientific Institute of Public Health,Brussels, BelgiumDr. M. Morgado, Fundation Oswaldo Cruz, Rio de Janeiro 21045-900, BrazilDr. K. Nakajima, The Japanese Red Cross Blood Centre, Tokyo 150-0012, JapanDr. J. Nkengasong, Project Retro-CI, CDC/HIV, Abidjan, Ivory Coast,West AfricaDr. M. Olsson/Dr. A. Friis, Plasma R & D, Biovitrum, Stockholm,SwedenDr. S. Rice, Windeyer Institute, RFUCMS, London, UK.Dr. J. Robinson/Dr. J. Hackett, Molecular diagnostics, Abbott Labo-ratories, IL 60064-6014, USADr. J. Turczyn, Bayer Corporation, Raleigh, NC 27610Dr. C. Wadey, CSL Bioplasma, Broadmeadows Victoria, Australia3047Dr. T. Weimer, Aventis Behring GmbH, Marburg GermanyDr. D. York, MDS, Westville 3630, South AfricaDr. A. Yoshikawa, Japanese Red Cross Saitama Blood Center,Saitama 350-1213, JapanDr. G. Zanganberg, Roche Molecular Systems, 82327 Tutzing, Ger-many

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