Development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus type 1 subtype C and C' infections in Ethiopia

A gag-based molecular beacon assay utilizing real-time nucleic acid sequence-based amplification technology has been developed to differentiate between the two genetic subclusters of human immunodeficiency virus type 1 (HIV-1) subtype C (C and C') circulating in Ethiopia. Of 41 samples, 36 could be classified as C or C' by sequencing of the gag gene. All 36 isolates were correctly identified by the gag beacon test. Three isolates with genomes that were recombinant in gag were unambiguously typed as belonging to the C' subcluster. Further analysis revealed that these contained the most sequence homology with a reference subcluster C' sequence in the target region of the beacon and hence were correct for the analyzed region. For one sample, sequencing and gag molecular beacon results did not match, while another isolate could not be detected at all by the beacon assay. Overall, high levels of sensitivity and specificity were achieved for both beacons (90.5% sensitivity and 100% specificity for the C beacon and 100% sensitivity and 95.2% specificity for the C' beacon). The availability of a diagnostic test which can quickly and reliably discriminate between C and C' HIV-1 infections in Ethiopia is an important first step toward studying their respective biological characteristics. As the assay is specific to the Ethiopian HIV-1 subtype C epidemic, it will contribute to characterizing the circulating viruses in this population, thereby generating the information necessary for the development of a potential efficacious HIV-1 vaccine appropriate for the Ethiopian context.     

One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires interventions that can prevent transmission of numerous HIV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay that identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequence of the genome of all currently known HIV-1 subtypes and can identify and distinguish among the targeted subtypes as the reaction proceeds, because of the addition of subtype-specific molecular beacons with multiple fluorophores. The combination of isothermal nucleic acid sequence-based amplification and molecular beacons is a new approach in the design of real-time assays. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay on a panel of the culture supernatant of 34 viruses encompassing all HIV-1 subtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a group N isolate. Assay sensitivity on this panel was 92%, with 89% correct subtype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1 RNA per reaction. In 38% of 50 serum samples from HIV-1-infected individuals with a detectable amount of virus, we could identify subtype sequences with a specificity of 94% by using sequencing and phylogenetic analysis as the "gold standard." In conclusion, we showed the feasibility of the approach of using multiple molecular beacons labeled with different fluorophores in combination with isothermal amplification to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would not be suited for clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.     

HIV-1 subtype C in commerical sex workers in Addis Ababa, Ethiopia

In this study, we have investigated the diversity of the current HIV-1 strains circulating in Addis Ababa, Ethiopia; in addition, we have evaluated the applicability of peptide enzyme-linked immunosorbent assay (ELISA) and heteroduplex mobility assay (HMA) for HIV-1 subtyping. Previous studies have indicated that HIV-1 subtype C is the major subtype present in HIV-positive samples collected from various risk groups between 1988 and 1995 in Addis Ababa. To assess the possible influx of new HIV-1 subtypes, 150 commercial sex workers (CSW) reporting in 1997 to two Health Centers in Addis Ababa were enrolled in an unlinked anonymous cross-sectional study. Subtyping was performed according to the World Health Organization algorithm of peptide ELISA, followed by HMA and DNA sequencing. As a result, the HIV-1 prevalence among these CSWs was found to be 45% (67 of 150). Of the 67 samples, 66 contained HIV-1 of subtype C and only one was of subtype D. This confirms the persistent overall presence of HIV-1 subtype C in Addis Ababa and a low influx of other subtypes into this location.     

High genetic diversity of HIV-1 strains in Chad,West Central Africa

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.     

Measurement of HIV RNA in patients infected by subtype C by assays optimized for subtype B results in an underestimation of the viral load

Quantitation assays of HIV-1 RNA used currently were designed and optimized for subtype B viruses. However, infection with non-B HIV viruses has become more common worldwide. Unfortunately, little information is available regarding the suitability of these assays for measurement of viral load in specific non-B subtypes. The performance of two commercial HIV-1 RNA quantitation assays was evaluated in 82 HIV subtype C-infected patients and in 43 HIV-1 subtype B-infected patients. Blood samples were tested by the Amplicor HIV-1 Monitor Assay, Version 1.5, and by the nucleic acid sequence-based amplification HIV-1 assay (NucliSens). The results were compared by using a paired, two-tailed Student's t-test; the difference between the assays was found to be significant only for subtype C. Discordant results (>0.5 log difference) between the two assays were detected in 39% of subtype C samples, compared to 23.2% of subtype B samples. In all cases in which a discordant result was detected, the lower results were obtained by the NucliSens assay. Discordant results between CD4 and viral load (CD4 < 200 cells/ml with a viral load <5,000 copies/ml) were observed in eight of the subtype C-infected patients when a viral load was measured by NucliSens (9.7%), compared to three patients (3.6%) when measured by the Amplicor assay. In conclusion, in patients with HIV subtype C infection, measurement of HIV RNA by the NucliSens assay resulted in a significant underestimation of the viral load as compared to the Amplicor assay. As a consequence, such an underestimation may result in sub-optimal care of patients infected with HIV subtype C.     

Evaluation of a prototype Amplicor PCR assay for detection of human immunodeficiency virus type 1 DNA in blood samples from Tanzanian adults infected with HIV-1 subtypes A, C and D.

BACKGROUND: In previous evaluations, the standard Amplicor HIV-1 DNA PCR test (Roche Diagnostic Systems) has been reported to have low sensitivity for the detection of some non-B HIV-1 subtypes. It has therefore become necessary to determine the performance of commercially available as well as prototype HIV-1 PCR assays for HIV-1 DNA detection in samples from various geographical settings, in order to assess their ability to detect the different HIV-1 genotypes. OBJECTIVES: To determine the performance of the prototype Roche Amplicor version 1.5 PCR test in comparison to that of the standard Roche Amplicor PCR test for the detection of HIV-1 DNA in blood samples from HIV-1 seropositive pregnant Tanzanian women infected with various HIV-1 subtypes. STUDY DESIGN: This was a cross-sectional study done on 161 blood samples collected from 106 HIV-1 seropositive and 55 seronegative asymptomatic pregnant women attending antenatal clinic in Dar es Salaam, Tanzania. METHODS: Cell pellets for PCR were prepared from EDTA blood by the Amplicor whole blood PCR sample preparation method. Plasma was used for HIV serology by enzyme linked immunosorbent assays. Subtyping was done by the heteroduplex mobility assay (HMA) using cell pellets and/or plasma. RESULTS: The sensitivities of the prototype PCR and the standard assays were 99.1% (105/106) and 97% (99/102), respectively. All samples from 55 HIV-1 seronegative women were negative by both PCR assays. Among the 101 samples subtyped by HMA, 48 (47%) were subtype A, 30 (30%) subtype C, 20 (20%) subtype D and 3 (3%) were indeterminate. In the standard DNA PCR assay, a statistically significantly higher proportion of subtype A samples had a low level of reactivity as measured as optical density compared with the subtypes C and D samples while in the prototype assay all three subtypes showed a high level of reactivity. CONCLUSIONS: The Amplicor version 1.5 DNA PCR test has a high sensitivity for the detection of HIV-1 DNA in blood samples from Tanzanian adults. Since performance of this assay does not appear to be influenced by differences in HIV-1 subtypes A, C and D, it has the potential for use in the detection of HIV-1 DNA in samples from geographic areas where these subtypes are prevalent.     

Performance of ViroSeq HIV-1 Genotyping System in routine practice at a Belgian clinical laboratory

Since there are indications of an increasing amount of non-B subtypes in Western Europe it was decided to assess the performance of the ViroSeq HIV-1 Genotyping System on a set of samples from the AIDS Reference Laboratory at the University Hospitals Leuven, a hospital with an increasing number of patients infected with non-B subtypes. The set consisted of 383 samples comprising 12 different subtypes and the genotyping kit was assessed for its amplification capabilities as well as its sequencing capabilities. Amplification failed in 32 samples (8.4%) and there was a tendency of a lower performance of the kit when it concerned the amplification of non-B subtypes. Regarding the sequencing performance of the HIV-1 Genotyping System, three different results could be considered. The performance of the entire set of primers (A, B, C, F, G and H) on the different subtypes showed a significant decrease of positive results for subtypes A, G and the recombinants whereas a tendency to less positive results could be detected for subtypes CRF12_BF, D, H and J. When looking at the performance of the individual primers for the different subtypes, only one result differed significantly: there were less positive results by applying primer F on subtype A. A tendency to less positive results was found for other combinations of primer and subtype, most of which comprised combinations with primers B, C, F and H. A final result was obtained by comparing the overall sequencing results of a certain primer on all the non-B subtypes with the results of the same primer on subtype B. Primer F showed significant less positive results and a tendency to less positive results was found for primer H. The other primers showed comparable results. All of the above results regarding the sequencing primers did not include primer D since this is a back-up primer for primer A. Analysis of the results for primer D showed that less positive results were found for all the non-B subtypes, most of which were significant. The overall performance of primer D on all non-B subtypes was only 15.7%. The use of primer D as a back-up primer was also investigated: it generated a positive result in only 17.3% of the cases where primer A failed. Most of these positive results were subtype B (74%). As a result of sequencing problems 65 out of 351 (18.5%) samples had to be processed with "in-house" procedures.     

Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay

We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.     

Detection of intersubtype recombinants with respect to env and nef genes of HIV-1 among female sex workers in Calcutta, India

HIV-1 detected among female sex workers in Calcutta, India was characterized in respect to env and nef genes. A total of 39 HIV-1 seropositive samples were used in the study. Phylogenetic analysis of the nucleotide sequences of respective regions showed that 22 out of 39 samples (56.4%) were infected with subtype C with respect to both env and nef genes; however, 17 samples (43.6%) showed distinct subtype discordance. Simplot analysis of these samples showed the presence of intersubtype recombination between subtypes C and B. Both env C/nef B and env B/nef C recombinants were found to be present; 16 samples were found to be env C/nef B and 1 sample was detected as env B/nef C. This result indicates the emergence of intersubtype recombinants of HIV-1 for the first time in this eastern part of India.     

实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用

目的建立丙型副伤寒沙门菌和猪霍乱沙门菌的实时荧光PCR快速检测方法,用于沙门菌属内的分型鉴定。方法根据GenBank公布的丙型副伤寒沙门菌和猪霍乱沙门菌的保守序列,设计引物和改良分子信标探针,建立实时PCR检测方法。结果检测体系灵敏度高,纯DNA和菌液的最低检出限分别可达10fg和20CFU/反应体系;特异性好,对71株细菌的检测符合率达100%。20株沙门菌采取盲号模拟血培养标本进行血培养检测及鉴定,检出5株丙型副伤寒沙门菌和4株猪霍乱沙门菌,与试验的菌株相符。70份食品中用实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌均为阴性,而用传统方法分离培养未检出。结论建立的实时PCR检测方法可以快速、特异、灵敏地检测出丙型副伤寒沙门菌和猪霍乱沙门菌。     

检测亚甲基四氢叶酸还原酶基因C677T突变的分子灯塔技术

目的 研究分子灯塔技术检测N^5、N^10-亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase，MYHFR)基因C677T突变的可行性。方法 针对野生型和突变型MTHFR基因分别设计分子灯塔(荧光-淬灭修饰探针)，检测228例标本N^5，N^10-亚甲基四氢叶酸还原酶基因C677T。突变情况。结果 发现MTHFR基因型有3种，即纯合子突变(TT型)、杂合子突变(CT型)和野生型(CC型)，其基因型频率分别为18．0％、49．5％和32．5％。每例标本均可明确鉴定其基因型。结论 分子灯塔技术是一种能够高度特异性、高通量、高度自动化检测MTHFR基因突变的技术，而且这种技术还可运用于其他基因类似突变的检测，是一种检测已知基因突变的先进技术。     

Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O

Because human immunodeficiency virus type 1 (HIV-1) subtypes and circulating recombinant forms (CRFs) are spreading rapidly worldwide and are becoming less confined to a geographical area, RNA assays that can detect and quantify all HIV-1 isolates reliably are in demand. We have developed a fast, real-time monitored RNA assay based on an isothermal nucleic acid sequence-based amplification technology that amplifies a part of the long terminal repeat region of the HIV-1 genome. Real-time detection was possible due to the addition of molecular beacons to the amplification reaction that was monitored in a fluorimeter with a thermostat. The lower level of detection of the assay was 10 HIV-1 RNA molecules per reaction, and the lower level of quantification was 100 copies of HIV-1 RNA with a dynamic range of linear quantification between 10(2) and 10(7) RNA molecules. All HIV-1 groups, subtypes, and CRFs could be detected and quantified with equal efficiency, including the group N isolate YBF30 and the group O isolate ANT70. To test the clinical utility of the assay, a series of 62 serum samples containing viruses that encompassed subtypes A through G and CRFs AE and AG of HIV-1 group M were analyzed, and these results were compared to the results of a commercially available assay. This comparison showed that the quantification results correlated highly (R(2) = 0.735) for those subtypes that could be well quantified by both assays (subtypes B, C, D, and F), whereas improved quantification was obtained for subtypes A and G and CRFs AE and AG. A retrospective study with six individuals infected with either a subtype A, B, C, or D or an AG isolate of HIV-1 group M, who were treated with highly active antiretroviral therapy, revealed that the assay was well suited to the monitoring of therapy effects. In conclusion, the newly developed real-time monitored HIV-1 assay is a fast and sensitive assay with a large dynamic range of quantification and is suitable for quantification of most if not all subtypes and groups of HIV-1.     

Genotypic antiretroviral resistance testing for human immunodeficiency virus type 1 integrase inhibitors by use of the TruGene sequencing system

A sequencing assay for detection of mutations conferring resistance to human immunodeficiency virus type 1 (HIV-1) integrase inhibitors raltegravir and elvitegravir was developed using the automated TruGene sequencing system. The assay returned clear sequencing results for samples with ≥500 RNA copies/ml for mutation detection and HIV-1 subtyping across a spectrum of HIV-1 subtypes.     

Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1

We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification (NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant recombinant viruses and all group M HIV-1 subtypes.     

Field evaluation of the gag-based heteroduplex mobility assay for genetic subtyping of circulating recombinant forms of human immunodeficiency virus type 1 in Abidjan,Côte d'Ivoire

The gag-based heteroduplex mobility assay (gag-HMA) was evaluated for its ease and reliability in subtyping circulating recombinant forms (CRFs) of human immunodeficiency virus type 1 (HIV-1) in C?te d'Ivoire. One hundred thirty-two plasma samples were analyzed blindly for HIV-1 subtypes by sequencing the pol gene and by gag-HMA. DNA sequencing was used as the "gold standard." Of the 132 samples sequenced, 108 (82%) were CRF02_AG, 14 (11%) were pure subtype A, 5 (4%) were subtype G, 3 (2%) were subtype D, 1 was CRF01_AE, and 1 was subtype H. The gag-HMA correctly classified 126 (95.5%) of the samples. Of the 108 samples that were classified as CRF02_AG by DNA sequencing, 107 (99%) were correctly identified by gag-HMA, resulting in a positive predictive value of 96.4%. The gag-HMA seems to be a valuable tool for understanding the molecular epidemiology of HIV-1 CRF02_AG in C?te d'Ivoire and West Africa, which could be important for developing and evaluating AIDS vaccines, although DNA sequencing remains necessary for accurate molecular epidemiology.     

Infection with HIV type 1 group M non-B subtypes in individuals living in New York City

OBJECTIVE: To document infection with HIV type 1 (HIV-1) group M non-B subtypes in individuals living in New York City. DESIGN: From October 1999 through April 2003, HIV-1-seropositive individuals were selected from 3 clinics in New York City based on having risk factors for infection with HIV-1 non-B subtypes. METHODS: HIV-1 RNA was extracted from plasma samples, and partial gag, pol, or env genes were amplified by PCR analysis. The infecting HIV-1 group M subtype was determined based on results of either heteroduplex mobility assay or sequencing and phylogenetic analysis. RESULTS: Ninety-seven subjects were enrolled in the study. Of the 97 subjects, 91 (94%) were selected based on having emigrated from a non-European country, while 6 (6%) were native United States citizens. Subtypes were successfully determined in 53 (55%) of the 97 plasma samples tested. The subtypes in 2 plasma samples were unclassifiable. HIV-1 infections were classified as those due to the following group M subtypes: A (n = 4; 7%), B (n = 12; 22%), C (n = 8; 15%), F (n = 2; 4%), CRF01_AE-like (n = 7; 13%), CRF02_AG-like (n = 19; 34%), an intersubtype recombinant form G/A (n = 1; 2%), and unclassifiable viruses (n = 2; 4%). CONCLUSION: This study reveals infection with a broad variety of HIV-1 group M subtypes mostly in the immigrant population of New York City as well as how several non-B subtypes are being introduced into the United States.     

Performance evaluation of the two protease sequencing primers of the Trugene HIV-1 genotyping kit

This article describes the performance of the two protease sequencing modules available in the Trugene HIV-1 genotyping Kit on a sample population with a high prevalence of HIV-1 non-B subtypes (n=110). The relevance of the algorithm recommended by the kit was also evaluated. The results indicated a high sequencing failure rate of the PR module (34%). Forty-five percent of the failed sequences derived from non-B subtype viruses. Furthermore, no PR sequence could be obtained from any of the HIV-1 subtype A and C infected samples that were tested. In contrast, a sequence could be obtained from the entire panel using the P2 module. The data indicated that the high rate of sequencing failures of the PR module was related to both the HIV-1 non-B subtypes as well as lower levels of RNA viral load. In six out of the 73 samples for which both protease modules were successful, discrepancies between the two protease sequences were observed, which led to discordant resistance reports in two cases. The data highlight the problems and the clinical implications that may occur during resistance genotyping of clinical samples with a high prevalence of HIV-1 non-B subtypes.     

Genotyping of hepatitis C virus-comparison of three assays.

BACKGROUND: Genotyping of hepatitis C virus (HCV) is clinically relevant to epidemiology, prognosis, and therapeutical management of HCV infection. OBJECTIVES: Accuracy and specificity of three assays for HCV genotyping/subtyping were determined. The TruGene HCV 5'NC Genotyping Kit (TruGene), which is a direct sequencing test and two assays based on reversed hybridization, Inno-LiPA HCV II assay and ViennaLab HCV Strip Assay, were compared. Amplification products generated by the Cobas Amplicor HCV Test were used. STUDY DESIGN: A total of 100 consecutive HCV RNA positive samples derived from patients with chronic hepatitis C were examined for their genotypes/subtypes by the three assays. RESULTS: Identification of genotypes and subtypes by the TruGene assay as reference test for the Inno-LiPA HCV II assay and the ViennaLab HCV Strip Assay or Inno-LiPA HCV II assay as reference test for the TruGene and the ViennaLab HCV Strip Assay showed similar results for overall accuracies (TruGene as reference test for Inno-LiPA HCV II and ViennaLab HCV Strip Assay, genotypes/subtypes: 100%/95.5% and 97%/92%; Inno-LiPA HCV II as reference test for TruGene and ViennaLab HCV Strip Assay, genotypes/subtypes: 99%/85.9% and 97%/87.9%) and specificities (TruGene as reference test for Inno-LiPA HCV II and ViennaLab HCV Strip Assay, genotypes/subtypes: 100%/97.8% and 99%/97.7%; Inno-LiPA HCV II as reference test for TruGene and ViennaLab HCV Strip Assay, genotypes/subtypes: 100%/99.4% and 99.7%/98%). CONCLUSIONS: The three assays were found to be reliable for the detection and discrimination of all HCV genotypes common in Europe and in North America and to be suitable for the routine diagnostic laboratory.     

High specificity of V3 serotyping among human immunodeficiency virus type-1 subtype C infected patients with varying disease status and viral phenotype

V3 serotyping is a technique for determining HIV-1 genetic subtype based on the binding of antibodies from patient sera or plasma to synthetic V3 peptides derived from subtype consensus sequences. Variation in the performance of this assay has been attributed to V3 sequence heterogeneity, the degree of which varies with patient disease progression, virus co-receptor usage, and genetic subtype. This study assessed the performance of a competitive peptide enzyme immunoassay (cPEIA) in samples from HIV-1 subtype C infected patients with varying disease profiles, including those with syncytium (SI) and non-syncytium-inducing (NSI) viruses. Out of 90 sera tested, 94.4% reacted strongly against the subtype C peptide. There was no significant difference in assay sensitivity among samples from advanced AIDS patients in which humoral immune response may be lower, nor among SI viruses which carry changes in the V3 sequence. Four samples were found to be cross-reactive with other subtypes and one acutely infected patient sample was non-reactive due to low anti-gp120 antibody titers. A significantly higher number of samples showed secondary reactivity to subtype A, compared to other subtypes (P < 0.005). In conclusion, the assay was able to identify HIV-1 subtype C infection with a high level of sensitivity (94%) irrespective of the stage of disease and therefore provides a valuable resource for the large-scale epidemiological monitoring of the spread of HIV-1 subtypes in South Africa.