Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus.

BACKGROUND: Epidemiologic data suggest that infection with herpes simplex virus type 2 (HSV-2) is associated with increased genital shedding of human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 transmissibility. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of HSV suppressive therapy with valacyclovir (at a dose of 500 mg twice daily) in Burkina Faso among women who were seropositive for HIV-1 and HSV-2; all were ineligible for highly active antiretroviral therapy. The patients were followed for 24 weeks (12 weeks before and 12 weeks after randomization). Regression models were used to assess the effect of valacyclovir on the presence and quantity of genital and plasma HIV-1 RNA and genital HSV-2 DNA during treatment, adjusting for baseline values, and to evaluate the effect over time. RESULTS: A total of 140 women were randomly assigned to treatment groups; 136 were included in the analyses. At enrollment, the median CD4 cell count was 446 cells per cubic millimeter, and the mean plasma viral load was 4.44 log10 copies per milliliter. With the use of summary-measures analysis, valacyclovir therapy was found to be associated with a significant decrease in the frequency of genital HIV-1 RNA (odds ratio, 0.41; 95% confidence interval [CI], 0.21 to 0.80) and in the mean quantity of the virus (log(10) copies per milliliter, -0.29; 95% CI, -0.44 to -0.15). However, there was no significant decrease in detection of HIV (risk ratio, 0.93; 95% CI, 0.81 to 1.07). HSV suppressive therapy also reduced the mean plasma HIV-1 RNA level by 0.53 log(10) copy per milliliter (95% CI, -0.72 to -0.35). Repeated-measures analysis showed that these effects became significantly stronger during the 3 months of follow-up. CONCLUSIONS: HSV suppressive therapy significantly reduces genital and plasma HIV-1 RNA levels in dually infected women. This finding may have important implications for HIV control. (ClinicalTrials.gov number, NCT00158509 [ClinicalTrials.gov].).

Evaluation of transmitted HIV drug resistance among recently-infected antenatal clinic attendees in four Central African countries.

BACKGROUND: The rapid expansion of antiretroviral treatment in resource-limited settings is raising concerns regarding the emergence and transmission of HIV drug resistance (HIVDR). We evaluated the extent of transmission of drug-resistant HIV strains in four Central African countries: the Republic of Congo, Central African Republic, Chad and Cameroon. METHODS: The World Health Organization (WHO) HIVDR threshold survey was implemented in major treatment areas in each country. Pregnant women who were aged <25 years, who were at first pregnancy and who were HIV type-1-positive were enrolled at each site in 2006-2007 for genotyping. HIVDR prevalence was categorized using the WHO threshold survey binomial sequential sampling method. RESULTS: The prevalence of HIVDR in Brazzaville and Bangui sites could not be classified because the eligible sample number was not reached. HIVDR prevalence was low (<5%) in N'Djamena for all drug classes. In Yaoundé, we found one individual with the D67D/N mutation and two with K103N. HIVDR prevalence was categorized as low (<5%) for protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), and moderate (> or =5-< or =15%) for non-NRTIs (NNRTIs). HIVDR prevalence in Douala was low for PIs and NNRTIs, and moderate for NRTIs as we identified one individual with M184V plus K101E plus G190A mutations and a second with D67D/N. CONCLUSIONS: The moderate HIVDR prevalence found in Yaoundé and Douala indicate that efforts should be made in Cameroon to prevent HIVDR; however, additional surveys are needed to confirm this trend. This study highlighted challenges presented by the WHO methodology, such as additional costs, workload, difficulties in acquiring even small sample numbers and the necessity for better quality assurance of HIV testing and record keeping at antenatal clinics.

Long-term safety, effectiveness and quality of a generic fixed-dose combination of nevirapine, stavudine and lamivudine.

We assessed the long-term safety, effectiveness and quality of a fixed-dose combination of nevirapine, stavudine and lamivudine (triomune). HIV-1-infected adults initially enrolled in a one-year, open-label, single-arm, multicentre trial in Cameroon were followed for 2 years. Our results support the safety and effectiveness of the triomune combination for first-line treatment of HIV infection. Virological effectiveness appeared to wane somewhat during the second year of treatment, however, and plasma nevirapine concentrations were relatively high.

Impact of suppressive herpes therapy on genital HIV-1 RNA among women taking antiretroviral therapy: a randomized controlled trial.

OBJECTIVE: To demonstrate a causal relationship between herpes simplex virus 2 (HSV-2) and increased genital HIV-1-RNA shedding in women on HAART. DESIGN: A randomized, double-blind, placebo-controlled trial of herpes-suppressive therapy (valacyclovir 500 mg twice a day) in HIV-1/HSV-2-infected women taking HAART in Burkina Faso. METHODS: Participants were followed for a total of 12 biweekly visits before and after randomization. The presence and frequency of genital and plasma HIV-1 RNA, and of genital HSV-2 were assessed using summary measures, adjusting for baseline values. Random effect linear regression models were used to assess the impact of treatment on genital and plasma viral loads among visits with detectable virus. RESULTS: Sixty women were enrolled into the trial. Their median CD4 lymphocyte count was 228 cells/mul, and 83% had undetectable plasma HIV-1 RNA at baseline. Valacyclovir reduced the proportion of visits with detectable genital HSV-2 DNA [odds ratio (OR) 0.37, 95% confidence interval (CI) 0.13, 1.05], but had no significant impact on the frequency (OR 0.90, 95% CI 0.31, 2.62) or quantity (reduction of 0.33 log copies/ml, 95% CI -0.81, 0.16) of genital HIV-1 RNA. However, according to pre-defined secondary analyses restricted to women who shed HIV-1 at least once in the baseline phase, valacyclovir reduced both the proportion of visits with detectable HIV-1 shedding (OR 0.27, 95% CI 0.07, 0.99) and the quantity of genital HIV-1 RNA during these visits (-0.71 log10 copies/ml, 95% CI -1.27, -0.14). CONCLUSION: HSV-2 facilitates residual genital HIV-1 replication among dually infected women taking HAART despite HIV-1 suppression at the systemic level.

HIV-1 drug-resistance mutations among newly diagnosed patients before scaling-up programmes in Burkina Faso and Cameroon.

We analysed whether mutations associated with resistance to antiretroviral (ARV) drugs circulate among treatment-naive HIV-1-infected individuals at a period when these drugs started to become more widely available in Africa. Overall, major resistance mutations in the pol gene, as defined by the International AIDS Society Resistance Testing-USA panel, were observed in 16 treatment-naive individuals. Eight of the 97 patients tested in Burkina Faso bore mutations conferring resistance to one drug class of ARV drugs: two to nucleoside reverse transcriptase inhibitors (NRTIs; M41L [n = 1], M41L+T69S [n = 1]), four to non-NRTIs (NNRTIs; V106A/V [n = 1] and V1081 [n = 3]) and two to protease inhibitors (PIs; L33F [n = 2]). In Cameroon, resistance mutations were identified in 8 of 102 patients: three to PIs (M461/L [n = 2], L33F [n = 1]), three to NRTIs (T69N/T [n = 1], M184V [n = 1], A62V [n = 1]) and two to NNRTIs (P236L [n = 1], V1081 [n = 1]). It is important to note that not all genotypic drug-resistance algorithms give similar interpretations to the observed mutations. Population surveillance for ARV drug resistance is required and should be included in all implementation programmes.

Natural polymorphism in protease and reverse transcriptase genes and in vitro antiretroviral drug susceptibilities of non-B HIV-1 strains from treatment-naive patients.

BACKGROUND: Most studies on antiretroviral (ARV) resistance of human immunodeficiency virus (HIV) have been done on subtype B which only represent a limited proportion of infections worldwide. OBJECTIVE: Understand baseline susceptibilities to ARVs in non-B strains. METHODS: To explore in greater detail possible intrinsic resistance to antiretroviral drugs in non-B subtypes, phenotypic resistance was tested in 35 non-B (A, D, F, G, J; CRF02, 06, 09, 11, 13) HIV-1 isolates obtained from ARV treatment naive patients. The panel includes strains with an increasing number of minor mutations in the protease gene and/or with atypical mutations at positions associated with resistance in protease and RT. RESULTS: We detected phenotypic resistance (fold-change values equal or superior to biological test cut-offs (BCO) in 14 of the 35 strains, 4 strains had decreased in vitro susceptibility to more than one drug. However, it is important to note that in most cases the increased fold-changes were close to the BCOs. Phenotypic resistance was observed against each of the three ARV drug classes: ritonavir (n=3), nelfinavir (n=2), saquinavir (n=2), zidovudine (n=2), stavudine (n=1), didanosine (n=1); delavirdine (n=6), efavirenz (n=1) and nevirapine (n=1). Some mutations could be associated with decreased in vitro susceptibility: 1 of 3 strains only with mutations M46I/L in protease, 1/2 A98S, K101N, V108I, V179I, and P236L in reverse transcriptase. Interestingly, the presence of an increasing number of minor mutations in the protease gene was not associated with decreased in vitro susceptibility to protease inhibitors. CONCLUSION: It is necessary to continue phenotypic studies on non-subtype B strains to identify the role of all polymorphisms present in protease and RT genes and to optimize interpretation algorithms. Data obtained from large, diverse populations of HIV-1 infected individuals is critical for defining and standardizing the quantification of resistance (phenotypic and genotypic testing).

Genotypic drug resistance interpretation algorithms display high levels of discordance when applied to non-B strains from HIV-1 naive and treated patients.

Genotypic drug resistance interpretation algorithms have been developed on patients infected with HIV-1 subtype B to interpret complex patterns of mutations. As non-B strains are characterised by the natural presence of several resistance-related mutations, we examined to what extent this might result in interalgorithm discordances in naive and treated patients. We compared the prediction by three algorithms (ANRS, Stanford and Rega) of drug susceptibilities to diverse HIV-1 strains from 272 naive and 156 treated patients. In naive patients, higher levels of interalgorithm discordance were observed for predictions of protease inhibitor (0.60-39%) than for predictions of reverse transcriptase inhibitor susceptibility (0-4%). The main reason for discordant protease inhibitor interpretation was the presence of resistance mutations that were natural protease polymorphisms. In contrast, in the treated patients, more interalgorithm discordances were observed for predictions of reverse transcriptase inhibitor (5-48%) than protease inhibitor susceptibilities (10-31%). Discordances were related to disagreement between the intermediate and susceptible scores, the intermediate and resistant scores and the interpretations of complex mutation patterns, related to cross-resistance and antagonistic interactions.

Low rate of genotypic HIV-1 drug-resistant strains in the Senegalese government initiative of access to antiretroviral therapy.

OBJECTIVE: To monitor the prevalence of antiretroviral (ARV)-resistant HIV-1 viruses, and the genotypic mutations in patients enrolled in the Senegalese initiative for access to antiretroviral treatment (ART). METHODS: A total of 80 patients with a virological follow-up of at least 6 months were selected, 68 were ART-naive and 12 ART-experienced. Genotypic resistance to ARV was studied at baseline for a random subset of patients and at each rebound in plasma viral load during ART, by sequencing the protease and reverse transcriptase genes. RESULTS: At baseline, 66 patients received highly active antiretroviral therapy (HAART) [2 nucleoside reverse transcriptase inhibitors (NRTIs) +1 protease inhibitor (PI) (n = 64) or 2 NRTIs + 1 non-nucleoside reverse transcriptase inhibitor (NNRTI) (n = 2)] and 14 patients (17.5%) started with a dual therapy because of ongoing antitubercular therapy or efficient previous bitherapy for the ART-experienced patients. The emergence of drug-resistant viruses (n = 13) during follow-up was more frequent in ART-experienced patients than in ART-naive patients, 41.7 versus 11.8%, resistant viruses emerged at comparable follow-up periods, a median of 17.8 and 18.3 months, respectively. In patients receiving zidovudine and lamivudine in their drug regimen, resistance to lamivudine was more frequent than to zidovudine. Two of the three patients, with viruses resistant to PIs, acquired mutations associated with cross-resistance. Strikingly, five (39%) of the 13 patients developed resistances to drugs that they had never received (n = 3) or that they received 18 or 36 months ago (n = 2). Didanosine/stavudine pressure had selected zidovudine-resistant viruses in four patients, and indinavir had selected a nelfinavir-resistant virus in one patient. CONCLUSION: In contrast to other reports from developing countries where patients had received ARVs in an uncontrolled manner, our study showed that implementation of HAART together with good clinical, biological and logistical monitoring can reduce the emergence of resistant strains in Africa.

The Senegalese government's highly active antiretroviral therapy initiative: an 18-month follow-up study.

OBJECTIVE: To study the feasibility, effectiveness, adherence, toxicity and viral resistance in an African government HAART initiative. METHODS: A prospective observational cohort study started in Dakar in August 1998. Initial treatment consisted of two nucleoside reverse transcriptase inhibitors and one protease inhibitor. The patients attended monthly medical examinations. Plasma HIV-1 RNA and CD4 cell counts were determined at baseline and every 6 months. Intention-to-treat analyses were performed. RESULTS: Fifty-eight treatment-naive patients, mostly infected by HIV-1 strain CRF02-AG, were enrolled. Most were at an advanced stage of HIV disease (86.2% had AIDS). Adherence was good in 87.9% of patients and treatment was effective in most of them. Thus, HIV-1 RNA was undetectable in 79.6, 71.2, 51.4 and 59.3% of patients at months 1, 6, 12 and 18, respectively and the median viral load reduction was approximately 2.5 log10 copies/ml. The CD4 cell count rose by a median of 82, 147 and 180 x 106 cells/l at months 6, 12 and 18, respectively. At the same time points, the cumulative probability of remaining alive or free of new AIDS-defining events was 94.8, 85.0 and 82.3%. Most adverse effects (80.8%) were mild or moderate and only two cases of drug resistance occurred. CONCLUSION: This study shows that HAART is feasible and well tolerated in African patients. Clinical and biological results were comparable to those seen in western cohorts, despite differences in the HIV-1 subtype distribution and an advanced disease stage when the treatment was initiated. Contrary to other recent studies in Africa, viral resistance rarely emerged.

Identification of a new circulating recombinant form of HIV type 1, CRF11-cpx, involving subtypes A, G, J, and CRF01-AE, in Central Africa.

In this study, we characterized three full-length genome sequences with a similar mosaic structure from epidemiologically unlinked individuals from Cameroon (97CM-MP818) and the Central African Republic (99CF-MP1298 and 99CF-MP1307). Phylogenetic and recombinant analysis confirmed that the three strains had a similar complex recombinant genome, which we can designate now as CRF11-cpx. This new CRF was composed of successive fragments of subtype A, G, J, and CRF01-AE. The previously reported GR17 virus from a Greek patient infected in the Democratic Republic of Congo (DRC) has a similar structure and should be considered as the prototype strain of CRF11-cpx. This new CRF circulates in Cameroon, Central African Republic, Gabon, and DRC, although the exact prevalences remain to be determined.

Low prevalence of HIV type 1 drug resistance mutations in untreated, recently infected patients from Burkina Faso, Côte d'Ivoire, Senegal, Thailand, and Vietnam: the ANRS 12134 study.

The frequency of transmitted HIV drug resistance (HIVDR) was evaluated in the context of rapid scale-up of antiretroviral treatment in Thailand, Vietnam, Burkina Faso, Côte d'Ivoire, and Senegal by using an adaptation of the WHO generic protocol of the HIV Drug Resistance Threshold Survey (HIVDR-TS) for sample collection and classification. Resistance-associated mutations were interpreted using the 2009 WHO list for epidemiological surveys. We included 266 subjects from the five study sites. Of the 266 RT and PR sequences analyzed, two from Vietnam harbored virus with major drug resistance mutations (G190A in RT for one individual and M46I in PR for the second individual). Phylogenetic analysis revealed that CRF01_AE predominates (>90%) in Thailand and Vietnam. CRF02 (>65%) cocirculates with other HIV-1 variants in Senegal and Côte d'Ivoire. The prevalence of HIVDRM is scored as low (< or = 5%) in all the five sites for the three drug classes analyzed. A continuous population survey for HIVDRM will provide a rational basis for maintaining or changing the current first line regimen in these countries.

Antiretroviral drug resistance and routine therapy, Cameroon.

Among 128 patients routinely receiving highly active antiretroviral therapy in an HIV/AIDS outpatient clinic in Cameroon, 16.4% had drug resistance after a median of 10 months. Of these, 12.5% had resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 10.2% to non-NRTIs, and 2.3% to protease inhibitors.

Long-term benefits of highly active antiretroviral therapy in Senegalese HIV-1-infected adults.

OBJECTIVES: To assess the long-term survival, as well as the immunologic and virologic effectiveness, adherence, and drug resistance, in HIV-infected patients receiving highly active antiretroviral therapy (HAART) in one of the oldest and best-documented African cohorts. METHODS: A prospective observational cohort study included the first 176 HIV-1-infected adults followed in the Senegalese government-sponsored antiretroviral therapy initiative launched in August 1998. Patients were followed for a median of 30 months (interquartile range, 21-36 months). HAART comprised 2 nucleoside reverse transcriptase inhibitors and either 1 protease inhibitor or 1 nonnucleoside reverse transcriptase inhibitor. RESULTS: At baseline, 92% of patients were antiretroviral naive and 82% had AIDS; the median CD4 count was 144 cells/mm, and median viral load was 202,368 copies/mL. The survival probability was high (0.81 at 3 years; 95% CI, 0.74-0.86) and was independently related to a baseline hemoglobin level <10 g/dL and a Karnofsky score <90%. Antiviral efficacy was consistently observed during the 3 years of treatment (-2.5 to -3.0 log10 copies/mL; 60-80% of patients with viral load <500 copies/mL) and the CD4 count increase reached a median of 225 cells/mm. Most patients reported good adherence (80-90%). The emergence of drug resistance was relatively rare (12.5%). CONCLUSION: This study shows that clinical and biologic results similar to those seen in Western countries can be achieved and sustained during the long term in Africa.

Comparison of drug resistance mutations and their interpretation in patients infected with non-B HIV-1 variants and matched patients infected with HIV-1 subtype B.

OBJECTIVE: To compare the prevalence of mutations associated with resistance to antiretroviral drugs and their interpretation in patients infected with non-B HIV-1 variants versus HIV-1subtype B-infected patients with similar treatment regimens. METHODS: The reverse transcriptase (RT) and protease genes of HIV-1 were sequenced, and subtypes were determined by phylogenetic analysis. Each sequence belonging to a non-B variant was matched with a sequence belonging to subtype B. Patterns of resistance mutations were interpreted in terms of drug resistance using the HIV db algorithm. RESULTS: RT mutations M41L, L210W, and, to a lesser extent, T215Y were less prevalent in patients infected with non-B variants. This lower prevalence was associated with subtypes A (A1/A2), C, F (F1/F2), and CRF06_cpx. A lower prevalence of high-level resistance to zidovudine was also observed in patients infected with these HIV-1 variants. In the protease gene, differences between patients infected with B or non-B strains were mainly observed for mutations playing a minor role in drug resistance and known to occur mainly as a natural polymorphism in non-B strains: K20R/M/I, M36I, L63P, A71V/T, and V77I. Interpretation of genotypes using the HIV db algorithm indicated that resistance to saquinavir, ritonavir, indinavir, and amprenavir was more frequently a high-level resistance for subtype B and an intermediate-level resistance for non-B variants, but this difference was only significant for amprenavir. CONCLUSION: Our results suggest that the genetic diversity of HIV-1 does not play a major role in the development of resistance to antiretroviral drugs.

CRF06-cpx: a new circulating recombinant form of HIV-1 in West Africa involving subtypes A, G, K, and J.

Phylogenetic analysis of numerous strains of HIV-1 isolated from diverse geographic origins has revealed three distinct groups of HIV-1: groups M, N, and O. Within group M, subtypes, sub-subtypes and circulating recombinant forms (CRFs) exist. Recently, two near-full-length genomes of similar complex mosaic viruses containing fragments of subtypes A, G, I, and J were described in patients from Burkina Faso (BFP-90) and Mali (95ML-84). Here, we report on the characterization of two additional full-length genome sequences with similar mosaic structure in epidemiologically unlinked individuals from Senegal (97SE-1078) and Mali (95ML-127). Phylogenetic and recombinant analysis confirmed that the previously described strains, BFP-90 and 95ML-84, were indeed a new CRF of HIV-1, which we can now designate as CRF06-cpx. This new CRF fits the complex (cpx) designation, because four different subtypes (A, G, K, and J) were involved in the mosaic genome structure. The fragment in the pol gene, which was initially characterized as unknown in the BFP-90 strain and subsequently as subtype I in the 95ML-84 strain, is now, with the recent description of the new K subtype, clearly identified as subtype K. CRF06-cpx circulates in Senegal, Mali, Burkina Faso, Ivory Coast, and Nigeria, although the exact prevalence remains to be determined. Importantly, this new variant has also been documented on other continents (Europe [France] and Australia), showing that these viruses are spreading not only locally but globally.

Resistance to antiretroviral treatment in Gabon: need for implementation of guidelines on antiretroviral therapy use and HIV-1 drug resistance monitoring in developing countries.

The protease and reverse transcriptase (RT) genes were studied in antiretroviral (ARV)-experienced and drug-naive HIV-1-infected individuals in Libreville, Gabon. We have shown, although on a limited number of samples that in 58% (11/19) of the patients, with a mean of 17.7 months of ARV drug experience, major mutations inevitably inducing resistances to ARV drugs were present. Resistance was mainly observed to the NRTIs (nucleoside analogue RT inhibitors). This high prevalence may reflect inappropriate ARV drug use. In order to avoid the rapid emergence of resistant viruses on a large scale in the developing world, it is important that the infrastructures necessary to monitor ARV treatment are also rapidly implemented in these countries and that clinicans are trained in the appropriate use of ARV drugs. A continuous surveillance of the circulation of ARV drug-resistant viruses must be organized to guide ARV treatment strategies and policies.

Biological and genetic characteristics of HIV infections in Cameroon reveals dual group M and O infections and a correlation between SI-inducing phenotype of the predominant CRF02_AG variant and disease stage.

In Yaounde, Cameroon, HIV-1 group-specific V3 serology on 1469 HIV-positive samples collected between 1996 and 2001 revealed that group O infections remained constant around 1% for 6 years. Only one group N sample was identified and 4.3% reacted with group M and O peptides. Although the sensitivity of the group-specific polymerase chain reaction (PCR) in two genomic regions was not optimal, we confirmed, in at least 6 of 49 (12.2%) dual O/M seropositive samples and in 1 of 9 group O samples, dual infection with group O and M viruses (n = 4) or with group O or M virus and an intergroup recombinant virus (n = 3). Partial env (V3-V5) sequences on a subset of 295 samples showed that at least eight subtypes and five circulating recombinant forms (CRFs) of HIV-1 group M co-circulate; more than 60% were CRF02_AG and 11% had discordant subtype/CRF designations between env and gag. Similarly as for subtype B, the proportion of syncytium-inducing strains increased when CD4 counts were low in CRF02_AG-infected patients. The V3-loop charge was significantly lower for non-syncytium-inducing strains than for syncytium-inducing strains but cannot be used as an individual marker to predict phenotype. The two predominant HIV-1 variants in Africa, CRF02_AG and subtype C, thus have different biological characteristics.

Development and evaluation of a DNA enzyme immunoassay method for env genotyping of subtypes A through G of human immunodeficiency virus type 1 group M, with discrimination of the circulating recombinant forms CRF01_AE and CRF02_AG.

The tools currently available for genetic subtyping of human immunodeficiency virus type 1 are laborious or can be used only for the analysis of a limited number of samples and/or subtypes. We developed and evaluated a molecular biology-based method using subtype-specific oligonucleotide probes for env genotyping of subtypes A through G, CRF01_AE, and CRF02_AG. DNA enzyme immunoassay (DEIA) genotyping is based on nested PCR amplification of the 5' end of the env gene (proviral DNA), followed by subtype-specific hybridization and immunoenzymatic detection on microplates. DEIA genotyping was validated with a large number of samples (n = 128) collected in Europe (France; n = 47), West-Central Africa (Cameroon; n = 36), and West Africa (Senegal; n = 45). Three different formats, depending on the distribution of subtypes in the three countries, were developed. The results were compared with those obtained by sequencing of the V3-V5 region and phylogenetic analysis or an env heteroduplex mobility assay. Additional sequencing and phylogenetic analyses of the DEIA region (the first codon of the env coding sequence to the middle of conserved region C1 of gp120) were performed to investigate the reasons for discrepancies. Intense and highly specific reactions between the oligonucleotide probes and the corresponding samples were observed. Overall, correct identification was achieved for 107 of 128 samples (83.6%). One sample was not amplified, 10 (8%) were nontypeable (NT), and 10 (8%) were misidentified. Six of the 10 discordant samples were further investigated by phylogenetic analysis, which indicated that these samples corresponded to recombinants involving the env 5' end and the V3 and V5 regions of the two parental clades. Sequencing of NT samples showed numerous differences between sample and probe sequences, resulting in a lack of hybridization, and revealed the limitations of the selected probes in terms of specificity and sensitivity. We demonstrated the feasibility of DEIA genotyping: six subtypes plus the two most prevalent circulating recombinant forms were discriminated by using the 5' end of the env gene. This method can be adapted to the local situation by including only probes that correspond to the prevalent strains.