Subtype-specific problems with quantification of plasma HIV-1 RNA.

OBJECTIVE: To determine whether two commercial assays for quantification of plasma HIV-1 RNA levels detect different genetic subtypes of HIV-1 with equal efficiency. DESIGN: Blind testing of stored plasma samples from 95 individuals infected with different genetic subtypes of HIV-1 (27 subtype A, 24 B, 18 C, 18 D, two E, two G, two H, and two J). The HIV-1 subtype had previously been determined by direct sequencing of the V3 domain of the env gene. METHODS: One plasma sample from each individual was tested once by the Roche HIV monitor assay and once by the Organon nucleic acid sequence-based amplification (NASBA) HIV-1 RNA quantitative assay, according to the manufacturers' recommendations. Information about CD4+ lymphocyte counts and antiretroviral treatment was available. RESULTS: The results from the two assays were strongly correlated with each other for subtypes B, C and D, but not for subtype A because many samples had RNA levels close to or below the lower detection limit of the assays. Thus, 15 out of 27 (56%) subtype A samples were negative by the HIV monitor assay and 12 (44%) were negative by the NASBA assay. These frequently occurring negative results among subtype-A-infected individuals were not due to better immunological status, more aggressive antiretroviral treatment, or differences in sample storage conditions. CONCLUSIONS: The HIV monitor assay and, possibly to slightly lesser degree, the NASBA assay appear unable to accurately quantify HIV-1 RNA levels in plasma samples from many subtype-A-infected individuals. These problems are likely to be due to primer mismatches and they limit the possibility of using these assays for routine monitoring of HIV-1-infected individuals in many parts of the world.

Strong correlation between the complement-mediated antibody-dependent enhancement of HIV-1 infection and plasma viral load.

OBJECTIVE: We have previously demonstrated that complement-mediated antibody-dependent enhancement (C-ADE) of HIV-1 infection correlates with accelerated immunosuppression and disease progression in HIV-1-infected individuals. In the present work the relationship between C-ADE and plasma HIV-1 RNA concentrations was studied to determine the effect of C-ADE on viral replication. METHODS: Three studies were performed: (a) C-ADE and HIV-1 RNA concentrations were determined in the serum and plasma aliquots taken at the same time from 98 HIV patients, mostly in the advanced stage of the disease; (b) the above two parameters as well as HIV enzyme-linked immunosorbent assay (ELISA)-reactive antibodies (Abbott HIV 1/2 test), and p24 antigen levels (Abbott antigen test; Abbott, Delkenheim, Germany) were determined in four seroconversion panels purchased from the Boston Biomedica firm; (c) changes of HIV-1 RNA concentration and C-ADE during a 17 month follow-up period were determined in 18 HIV-infected patients. C-ADE was measured by the method previously established in our laboratories. The results were expressed by an enhancement/neutralization index (E/NI). HIV-1 RNA levels were determined with the Amplicor monitor kit (Roche, Basel, Switzerland), and in some experiments with the nucleic acid sequence based amplification (Organon Teknika, Turnhout, Belgium) kits. RESULTS: (a) We found a highly significant (P<0.0001) positive correlation between E/NI values reflecting the extent of HIV-1 infection enhancement and plasma HIV-1 RNA levels. Both E/NI and HIV-1 RNA levels negatively correlated to the CD4 cell counts. (b) C-ADE was first detected just before, or concomitantly with, seroconversion in 4/4 seroconversion panels. (c) Both E/NI values and HIV-1 RNA levels significantly (P<0.001) increased during a 17 month observation period in 18 HIV-infected patients. CONCLUSION: We found strong association between the extent of the complement-mediated antibody-dependent enhancement of HIV-1 infection and the plasma viral load in HIV patients. On the basis of these findings, C-ADE correlates with HIV replication in vivo, and potentially contributes to the progression of HIV disease.

Comparative analysis of commercial assays for the detection and quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in plasma from patients infected with HIV-1 subtype C.

The commercial assays commonly used to quantify plasma human immunodeficiency virus type 1 (HIV-1) RNA in clinical settings were designed to assess HIV-1 subtype B. We compared the performance of 4 commercial assays (Amplicor versions 1.0 and 1.5 [Roche]; Quantiplex [Chiron]; and NASBA HIV-1 RNA QT [Organon Teknika]) in detecting and quantifying HIV-1 RNA in plasma from HIV-infected persons from Zambia, an area where HIV-1 subtype C is predominant. Each assay detected plasma HIV-1 RNA, but they do not all measure statistically similar quantities of plasma HIV-1 RNA.

Study on mutations and antiretroviral therapy (SMART): preliminary results.

Resistance to antiretroviral drugs is believed to be an important cause of treatment failure in human immunodeficiency virus (HIV)-infected patients, however, the role of susceptibility assays in the management of these individuals needs to be defined. SMART (study on mutations and antiretroviral therapy) is an ongoing study on mutations and antiretroviral therapy focused particularly on HIV-infected patients treated with two nucleoside analogue reverse transcriptase inhibitors (NRTIs). Plasma HIV-1 RNA was assessed by NASBA (nucleic acid sequence-based amplifications) (Organon Teknika, Boxtel, The Netherlands) with a detection limit of 80 copies/ml, whereas resistance was assessed by direct sequencing of the RT pol gene in patients with detectable viraemia, and by Antivirogram (Virco) in non-responder patients. The preliminary results of this study show that both genotypic and phenotypic assays identify mutated viral strains in the majority of patients failing a dual regimen. Furthermore, the data indicate a high rate of genotypic resistance to lamivudine in both responders and non-responders, a high rate of phenotypic resistance to lamivudine in non-responders, no genotypic resistance to didanosine and stavudine in responders, and a very low rate of both genotypic and phenotypic resistance to didanosine and stavudine in non-responders.

Comparison between a nucleic acid sequence-based amplification and branched DNA test for quantifying HIV RNA load in blood plasma.

HIV RNA was quantified in blood plasma from 209 patients and in control specimen comparing the NucliSens HIV-1 QT test (Organon Teknika), which is based on the nucleic acid sequence amplification procedure, and the Quantiplex 3.0 test (Bayer), which uses hybridization signal enhancement by branched DNA (bDNA) probes. A highly significant correlation (P=0.01) was found between the two methods with 88% of the samples showing similar results. In cases of discrepant findings, higher virus load was observed with either test (14xNASBA>bDNA; 12xbNDA>NASBA). Differences could neither be related to clinical features nor to divergent virus subtypes. Standard preparations containing 35000 and 222000 copies were quantified with intra-assay coefficients of variation of <20% using both methods. A preparation of 192 copies was measured with lower precision by both tests, yet was detected more reliably by the bDNA method.

Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods.

The efficacy of eight different methods for the extraction of HIV-1 RNA from plasma was compared. The RNA preparation method that gave the best results by RT-PCR was the one described by Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159). This method consists of a guanidine thiocyanate treatment followed by three phenol-chloroform-isoamylalcohol extractions and an ethanol precipitation. The disadvantage of this method is that it is time consuming and less suitable for the extraction of large series of samples. Moreover, due to the large number of procedural steps, there is a greater risk of sample mix-up or contamination. Of the single-step RNA purification methods, good results were obtained with the TRIzol method (Gibco Life Technologies, Paisley, UK) and with the extraction method offered by the NASBA kit (Organon Teknika, Turnhout, Belgium). The above single-step methods are recommended since both are sensitive enough to detect low copy numbers of HIV-RNA in the plasma of asymptomatic patients, and require only 2 h for completion. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extraction varied between 17.3 and 47.3%). Inter-laboratory reproducibility was evaluated only for the TRIzol-method and found to be low (mean variation coefficient 63.4).

Techniques for the evaluation of nucleic acid amplification technology performance with specimens containing interfering substances: efficacy of boom methodology for extraction of HIV-1 RNA.

Accurate HIV-1 RNA quantitation with nucleic acid amplification assays (NAAA) is partly dependent on overall assay design to ensure proper and reproducible functioning in the presence of endogenous interfering substances present in a clinical specimen, or exogenous interfering substances introduced as a result of specimen collection or handling. This study tested various methods of evaluating interfering substances that could potentially affect the outcome of HIV-1 RNA amplification in a NAAA. Clinical specimens from HIV-1 seronegative subjects containing various endogenous interferents were evaluated with and without an HIV-1 RNA spike to assess recovery and specificity, respectively, with a non-PCR NAAA (NASBA HIV-1 RNA QT, Organon Teknika) that incorporates Boom methodology for nucleic acid extraction. Additional specimens were prepared to simulate various circumstances that might occur during specimen preparation to result in the introduction of exogenous interferents. A retrovirus reverse transcriptase inhibitor, zidovudine (AZT), was added to plasma specimens prior to testing. NAAA results obtained with 127 total clinical specimens, 10 bacterially contaminated specimens, 5 platelet enriched specimens, 5 AZT specimens, and 30 anticoagulated specimens were consistent with the expected outcomes in the presence and absence of the HIV-1 RNA spike, giving an assay specificity of 100%. The spiked HIV-1 RNA copies in the clinical specimens reported by the assay were 99% of the copies reported for a positive index control (normal plasma plus HIV-1 RNA spike). Compared to the amplification levels of the three internal assay calibrators obtained for normal plasma controls, no differences in the amplification levels of the calibrators for each type of specimen were observed. This result indicated that the interferents examined did not affect adversely assay function. Addition of known PCR interferents (hemoglobin and heparin) and AZT to isolated HIV-1 RNA resulted in a substantial reduction of amplification and invalid results, whereas no inhibition was observed when these interferents were added to the test system prior to isolation; these results directly demonstrate the efficient removal of such interferents during the NASBA HIV-1 RNA QT isolation procedure. The several approaches to investigate interference described in this study may be utilized for the evaluation of other assays using nucleic acid amplification technology.

Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) irrespective of the stage of HIV-1 infection.

Using an experimental assay for isothermal amplification of HIV RNA in plasma (NASBA, Organon Teknika, Boxtel, The Netherlands), 70 samples from 59 HIV-1-infected persons and 29 samples from 28 uninfected volunteer blood donors were tested for the presence of HIV-1 RNA. The HIV-1-positive plasma samples were drawn from patients at various stages of infection: two samples from persons with signs of acute HIV infection (CDC stage I); 29 samples from 25 symptom-free persons (CDC stage II) and 39 samples from 32 persons with AIDS-related symptoms (CDC stage IV). All samples from HIV-1-infected persons had HIV-1 RNA, irrespective of the stage of infection (100% sensitivity). Testing the donor samples in duplicate, initially 54/58 samples (93%) tested negative for HIV-1 RNA. Repeated testing of the 4 samples with inconsistent test results showed all samples to be negative (specificity 100%). Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) promises to be a valuable alternative to the detection of HIV-1 RNA or DNA by the polymerase chain reaction.

Molecular-based methods for quantifying HIV viral load.

Viral load quantitation has become the major prognostic marker for disease prognosis and outcome of antiretroviral therapy in the treatment of HIV-infected individuals. The three major methodologies for viral load quantitation: the reverse transcriptase-polymerase chain reaction (RT-PCR; Amplicor HIV-1 Monitor Test, Roche Diagnostic Systems, Pleasanton, CA), the nucleic acid sequence-based amplification (NASBA; NucliSens HIV-1 QT Test, Organon Teknika, Bostel, The Netherlands); and a signal amplification methodology termed branchedchain DNA (bDNA) technique (Quantiplex HIV-1 RNA test, Bayer Diagnostics, Emeryville, CA) are briefly reviewed here.

Viral load comes of age.

AIDS: The Food and Drug Administration (FDA) granted the Roche Molecular Systems a license to market the Amplicor HIV-1 Monitor Test on June 3, 1996. How and when the test should be used, and to what extent the insurance establishment will pay for its use, remains to be determined. Three available viral load tests on the market--Roche's PCR assay, Chiron's bDNA test, and Organon Teknika's NASBA test--all measure viral load in different ways. The consensus is that viral load testing is a good prognostic indicator.^ieng

A population-based approach to determine the prevalence of transmitted drug-resistant HIV among recent versus established HIV infections: results from the Canadian HIV strain and drug resistance surveillance program.

OBJECTIVES: Published results on primary or transmitted HIV drug resistance may be biased because they have been largely derived from specific cohort studies or higher risk individuals who present symptomatically. Here, we present results from a representative population-based study of newly diagnosed cases of HIV in Canada and compare the prevalence of transmitted drug resistance between recent and established infections. METHODS: Available archived sera taken for the purpose of diagnostic HIV testing from all treatment-naive HIV-positive individuals who were newly diagnosed between 2000 and 2001 were tested for recency of infection, HIV-1 subtype, and mutations conferring reduced susceptibility to reverse transcriptase inhibitors and protease inhibitors (PIs). Recent infections were identified using the Organon Teknika Vironostika HIV-1-LS assay. After full-length sequencing of the pol gene, drug resistance mutations were identified using the 2004 International AIDS Society-USA mutations panel. Differences in drug resistance profiles between recent and prevalent infections were examined using the chi test and the Fisher exact test. The variables examined included gender, age at diagnosis, year of diagnosis, exposure category, ethnicity, and HIV-1 subtype. RESULTS: Among the study population, 8.1% had genotypic evidence of transmitted drug resistance: 4.1% against nucleoside reverse transcriptase inhibitors, 1.4% against nonnucleoside reverse transcriptase inhibitors, 1.5% against PIs, and 1% against > or =2 classes of drugs. A higher proportion of recent infections had genotypic evidence of transmitted drug resistance when compared with established infections (12.2% vs. 6.1%, respectively; P = 0.005). Transmitted drug resistance was identified mainly among recently infected Caucasian men who have sex with men but it was not limited to this group. Compared with the year 2000, a higher proportion of recently infected individuals with resistance-conferring mutations were diagnosed during the year 2001 (66.7% vs. 46.6%). CONCLUSIONS: In Canada, transmitted drug resistance is occurring within all 3 drug classes and across different population groups. The results suggest that the prevalence rates may be higher among recent versus established infections. Given the public health implications of transmitting drug-resistant HIV, it is important to continue population-based drug resistance surveillance to guide optimum prevention and treatment of HIV infection.

The first B/G intersubtype recombinant form of human immunodeficiency virus type 1 (HIV-1) identified in Germany was undetected or underquantitated by some commercial viral load assays.

The high level of genetic diversity of human immunodeficiency virus type 1 (HIV-1) and the continual emergence of recombinant forms have important implications not only for the global evolution of HIV but also for diagnosis, monitoring, and treatment strategies. The present study reports the first intersubtype B/G recombinant strain of HIV-1 in Germany. This strain is notable from a clinical perspective, since it was undetectable in the NucliSens HIV-1 QT assay (Organon Tecknika/bioMérieux) and was significantly underquantitated in the Monitor v1.5 test (Roche Molecular Systems) relative to the LCx HIV RNA Quantitative assay (Abbott Laboratories). Gag-encoded p24 (gag p24), pol-encoded integrase (pol IN), and env-encoded gp41 (env gp41) immunodominant region (IDR) sequences were characterized to establish group and subtype designation and to evaluate the degree of genetic diversity at primer and probe binding sites of the viral load assays. Phylogenetic analysis revealed that this virus is an intersubtype B/G recombinant strain. The gag p24 region is subtype G, env gp41 IDR is subtype B, and pol IN is a B/G chimera. Nucleotide mismatches within primer and probe-binding sites provided the molecular basis for differences in quantitation observed between viral load assays. Genetic diversity of HIV-1 continues to challenge the reliability of detection and quantitation by viral load assays.

HIV-1 viral load: comparative evaluation of three commercially available assays in Argentina.

Viral load (HIV-RNA copies per milliliter of plasma) has good correlation to prognosis considering progression to AIDS. The evaluation of commercial kits to measure viral load has become a need to find the most specific, sensitive and reproducible procedure to follow up HIV-infected patients. Hereby, a comparative analysis was done by using three different assays available in Argentina for quantitation of HIV-RNA in plasma. A plasma panel: 20 from HIV-1 infected individuals (9 asymptomatic and 11 symptomatic) and 9 from HIV-1 seronegative individuals was studied. Samples were run by Amplicor HIV-1 Monitor (Roche Diagnostic System, USA) Quantiplex HIV-1 RNA 2.0 Assay (Chiron Corporation, USA) and NASBA HIV-1 RNA QT (Organon Teknika, Holland). RNA was extracted from 0.2 ml of plasma for Amplicor, 0.1 ml and 1 ml of plasma for NASBA and, duplicates of 1 ml of plasma was centrifuged and pellet was used for bDNA assay no RNA extraction step. For a given specimen, a log difference of < 0.5 between assays was considered as concordant result. All seronegative samples were bellow the detection limit for all assays (Amplicor 200 c/ml, NASBA 400 c/ml and Quantiplex (bDNA) 500 c/ml). Two samples from asymptomatic patients were not detectable by NASBA (Sensitivity: 90%) Sensitivity was increased to 100% by using 1 ml of plasma. All samples were detectable by the other assays (sensitivity: 100%). For NASBA-bDNA, 74% samples were concordant, 35% for Amplicor-bDNA and 53% for NASBA-Amplicor. By using 1 ml of plasma from asymptomatic patients, concordance was 65% for NASBA-bDNA and 60% for NASBA Amplicor. Comparing samples from asymptomatic patients, only 22% was concordant in both cases. Reproducibility of NASBA was low (33% with differences lower than 0.5 Log) when 0.1 and 1 ml were used. Due to the levels of concordance of these results, it would be suggested to use always the same technique to follow up HIV-1 infection. The reproducibility of the assays should be tested by every laboratory and for every technician in charge of the assay in order to have confidence in the results specially to follow up HIV-infected patients or to monitor anti-viral therapies.

Accuracy of plasma HIV RNA quantification: a multicentre study of variability.

Inaccurate quantification of plasma HIV RNA concentration may be detrimental to patient care, yet little is known about how reproducible results are within and between laboratories. Each week between January and April 1998 a different laboratory represented at the Public Health Laboratory Service HIV Diagnosis Forum sent aliquots of the same anti-HIV positive plasma specimen by First Class Mail to the other 12 laboratories and to itself. Aliquots were frozen on receipt and examined in the next assay run. At the end of the 13 week period each laboratory reported their findings and provided further information about the specimen that they had dispatched. The correlation of results between laboratories and between the four different assay kits used was generally satisfactory. HIV RNA concentrations determined by the Roche Monitor and AcuGen kits were higher, and by the Chiron Quantiplex v 2.0 kit lower, than average. The Chiron Quantiplex gave the most reproducible concentrations. Nine 'below detection limit' results occurred, associated with three specimens. One specimen gave a below detection limit result in every one of six laboratories using the Organon Teknika Nuclisens HIV-1 QT kit, and was found to contain viral RNA of HIV-1 clade G. Another below detection limit result was probably due to technical error, and the remaining two to assay insensitivity. The findings suggested that an unsustained change in HIV RNA of

Underevaluation of HIV-1 plasma viral load by a commercially available assay in a cluster of patients infected with HIV-1 A/G circulating recombinant form (CRF02).

The authors studied the correlation and agreement of commercially available assays in detection and quantification of the HIV-1 intersubtype A/G circulating recombinant form CRF02. The assays under comparison were Bayer Versant HIV-1 RNA, version 3.0; Roche Amplicor HIV-1 Monitor, version 1.5 (standard procedure); and Organon Teknika NucliSens HIV-1 RNA QT. Plasma samples from 114 patients infected with CRF02 were tested by the three assays under standard conditions. Although correlation among the assays was high and statistically significant for subtype B and CRF02, in the latter instance, NucliSens measured average viral load values (3.29 +/- 0.71 log(10) copies/mL) about 4 and >8 times lower than those obtained by Versant (3.90 +/- 0.90 log(10) copies/mL) and Amplicor (4.22 +/- 1.05 log(10) copies/mL), respectively. Furthermore, in a statistically significant percentage of CRF02-harboring samples, NucliSens produced viral load values undetectable or 1 log(10) lower than those obtained in Versant and Amplicor assays. Altogether, these data underline a low performance of NucliSens in detecting and quantifying viremia in plasma samples harboring the CRF02. These results are potentially important as global distribution of new HIV-1 subtypes is expanding, and recombinant strains, particularly CRF02, are emerging and becoming highly prevalent.

Comparison of NucliSens and Roche Monitor assays for quantitation of levels of human immunodeficiency virus type 1 RNA in plasma.

We compared the performance of Organon Teknika's NucliSens and Roche Diagnostic Systems' Monitor quantitative human immunodeficiency type 1 RNA assays. Both had similar linearity and sensitivity over most of the dynamic range of the assays, although the Monitor assay was superior at the low range of RNA values while the NucliSens assay was more consistent at higher RNA values. NucliSens generally showed less interassay variability.

Comparison of three different commercial methods for measuring plasma viraemia in patients infected with non-B HIV-1 subtypes.

In order to determine whether commercial assays presently in use for the quantification of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels detect different genetic viral subtypes with the same reliability, a panel of 38 samples corresponding to 22 HIV-1-infected patients, representing non-B subtypes A-F, was examined. One to three plasma samples belonging to each individual were tested by the second-generation HIV-1 branched DNA (bDNA) assay (Chiron, Spain), the Nuclisens assay (Organon-Teknika, Spain), the Amplicor Monitor reverse transcriptase polymerase chain reaction assay (Roche, Spain), and the Ultradirect Monitor (Roche) using primers specifically designed to amplify non-B HIV-1 subtypes. Each of the different methods for measuring viral load showed a distinct sensitivity for non-B HIV-1 subtypes. Values higher than the assay detection limit were obtained in 22 (57.9%), 33 (86.8%), and 37 (93.3%) samples using the bDNA, Nuclisens, and Monitor assays, respectively. Significantly different values (>0.5 logs) were found in 55.3%, 81.6%, and 71.1% of specimens comparing results provided by bDNA and Nuclisens, bDNA and Monitor, and Nuclisens and Monitor, respectively. Quantitative values provided by the Ultradirect Monitor test using non-B primers were particularly discordant with the other tests. Overall, 44.7% of samples yielded higher viral load values with this assay than with the regular Monitor assay, reflecting its enhanced sensitivity for non-B subtypes; however, the reproducibility of this test was low. These results support the recommendation of always using the same assay when monitoring plasma viraemia.

Comparison of two amplification technologies for detection and quantitation of human immunodeficiency virus type 1 RNA in the female genital tract. Division of AIDS Treatment Research Initiative 009 Study Team.

Human immunodeficiency virus type 1 (HIV-1) RNA levels in female genital tract and peripheral blood samples were compared using two commercial amplification technologies: the Roche AMPLICOR HIV-1 MONITOR test and either the Organon Teknika nucleic acid sequence-based amplification (NASBA-QT) assay or the NucliSens assay. Estimates of HIV-1 RNA copy number were derived from internal kit standards and analyzed unadjusted and adjusted to a common set of external standards. We found a discordance rate of approximately 18% between the two technologies for the detection of HIV-1 in either the genital tract or peripheral blood samples. Detection discordance was not consistent among specimens or among women. There were no significant differences in adjusted or unadjusted estimates of HIV-1 RNA copy number in the genital tract samples using the AMPLICOR HIV-1 MONITOR test and either the NASBA-QT assay or the NucliSens assay. In addition, the estimated HIV-1 RNA copy number in peripheral blood samples did not differ when tested with the NucliSens assay and the AMPLICOR HIV-1 MONITOR test using kit standards. However, there was a significant difference in estimated RNA copy number between the NASBA-QT assay and the AMPLICOR HIV-1 MONITOR test for internal kit standards, which, as we have previously shown, was eliminated after adjustment with the external standards. Our results suggest that the Roche and Organon Teknika assays are equivalent for quantifying HIV-1 RNA in female genital tract specimens, although variation in detection does exist.

Development of multiplexed nucleic acid testing for human immunodeficiency virus type 1 and hepatitis C virus.

BACKGROUND AND OBJECTIVES: In most Western countries, blood donations are routinely screened for hepatitis C virus (HCV) RNA by polymerase chain reaction (PCR) or other nucleic acid tests. We describe the development of a multiplexed assay for human immunodeficiency virus type 1 (HIV-1) and HCV in an internally controlled PCR suitable for large-scale blood donor screening. MATERIALS AND METHODS: The HIV/HCV multiplexed PCR used primers from highly conserved regions in the long terminal repeat region. The National Institute for Biological Standards and Controls (NIBSC) International HIV-1 RNA standard, run control and HIV-1 subtype panel were used for assay evaluation. RESULTS: The HIV-1 PCR showed a sensitivity of 24 IU/ml for HIV-1 RNA (a dilution where 95% of replicate reactions were positive), which was at least five times more sensitive than the Roche Monitor version 1.5 (using the ultrasensitive extraction protocol) and Organon NASBA assays. The assay was capable of detecting all subtypes of HIV-1 (A to H), as well as the more divergent group N and O variants. The sensitivity of the PCR was unaffected by multiplexing with HCV primers and by the presence of a bovine viral diarrhoea virus (BVDV) internal control. CONCLUSION: We have developed a highly sensitive multiplexed PCR for HIV-1 and HCV RNA screening that can be introduced into current PCR-based blood donor screening at minimal cost and without significant operational changes.

Intra-assay performance characteristics of five assays for quantification of human immunodeficiency virus type 1 RNA in plasma.

Three kits (Roche AMPLICOR human immunodeficiency virus type 1 [HIV-1] Monitor, Chiron enhanced-sensitivity bDNA, and Organon Teknika NASBA HIV-1 QT) and two in-house assays (from National Genetics Institute and Baylor College of Medicine) were compared with a blinded panel. The results were evaluated as to intra-assay sensitivity, precision, and ability to detect differences in a dilution series.