HIV-1 subtype C drug-resistance background among ARV-naive adults in Botswana.

Current HIV-1 antiretroviral (ARV) drug resistance knowledge is limited to HIV-1 subtype B (HIV-1B). We addressed whether unique genetic and phenotypic properties of HIV-1 subtype C (HIV-1C), southern Africa's most prevalent subtype, may foment earlier and/or distinct resistance mutations. Population-level HIV-1C genotypes were evaluated with respect to drug resistance prevalence before Botswana's public ARV treatment programme began. Viruses were genotyped from 11 representative districts of northern and southern Botswana, and consensus sequences from these 71 individuals and 51 previously reported sequences from HIV-positive blood donors were constructed. Phylogenetic analysis classified all 71 sequences but one, which exhibited pol gene mosaicism, as HIV-1C. The protease and reverse transcriptase coding region had no detectable known primary mutations associated with HIV-1B protease inhibitor (PI) drug resistance. Secondary mutations associated with PI drug resistance were found in all sequences. Several HIV-1C-specific polymorphic sites were found across the pol gene. Northern and southern Botswana viral sequences showed no significant differences from each other. Population genotyping shows that, without countrywide ARV treatment, HIV-1C-infected Batswana harbour virtually no primary mutations known to confer resistance to the three major HIV-1B ARV drug classes. Some secondary PI mutations and polymorphic sites in the protease enzyme necessitate continuous population monitoring, particularly after introduction of countrywide ARV treatment in Botswana. Although its PI resistance development rate and kinetics are not known, our data may suggest increased susceptibility and readiness of HIV-1C to develop resistance under drug pressure when the PI class of drugs is used.

Mutations and polymorphisms associated with antiretroviral drugs in HIV-1C-infected African patients.

To detect and characterize polymerase gene (pol) polymorphisms and mutation patterns in HIV-1C-infected Batswana patients treated with reverse transcriptase inhibitors, samples from AIDS patients treated with highly active antiretroviral therapy (HAART) were sequenced for the region encompassing the entire HIV-1 protease (PR) and the first 335 amino acids of reverse transcriptase (RT). Amongst the 16 patients treated with antiretroviral (ARV) drugs, eight started HAART regimens containing didanosine, stavudine and nevirapine (ddI/d4T/NVP) or efavirenz (EFV) (arm A) while the others started with zidovudine (AZT) and lamivudine (3TC) given together as combivir (CBV) with either NVP or EFV as arm B. Arm B is the first line regimen currently provided by the Botswana ARV national programme. Greater efficacy, in terms of treatment duration, was observed in patients in arm B (14 months) as compared with patients in arm A (9 months); P<0.05, n=8. Appearance of the M184V mutation in the arm B patients coincided with a rebound of viral load (VL) (4.3 +/-0.1 log10 RNA copies/ml) and a significantly improved immunological parameter (deltaCD4=207.0+/-48.1 cells/microl; P<0.05). Interestingly, patients developing the M184V mutation preferentially harboured polymorphisms Q174K and/or I178L located in close proximity to pol position 184. The M184V mutation occurred following a clear clinical benefit consisting of increased CD4 cell counts and lower plasma viral loads. Primary mutations known to be associated with NNRTI and NRTI resistance for HIV-1B were observed in 10 of the 16 treated patients.

Gag drug resistance mutations in HIV-1 subtype C patients, failing a protease inhibitor inclusive treatment regimen, with detectable lopinavir levels.

The development of antiretroviral (ARV) drugs and their use in human immunodeficiency virus type 1 (HIV-1) has led to the effective control of HIV replication in infected patients. However the emergence of resistant HIV-1 strains still remains a problem. Literature has shown that mutations may accumulate in the protease (PR) and gag regions of HIV-1 patients who fail therapy with protease inhibitor (PI) drugs (1, 2). Gag mutations have also been found to play an important role in the evolution of PI resistance (2). Despite this, the standard genotypic drug-resistance test examines mutations in the reverse transcriptase (RT) and PR region of HIV-1 and not gag (3). This study investigated the frequency of gag drug resistance mutations in the absence of major PI mutations in HIV-1 subtype C patients, failing a PI inclusive treatment regimen. Sixty-eight samples were retrieved from patients that were classified as second line treatment failures as they had a viral load greater than 1000 copies\mL, as well as detectable lopinavir (LPV) levels. The gag and protease region of these patients were genotyped. Mutations in the gag and protease region were assessed using the REga Db sequencing tool and the CPR programme on the Stanford University HIV drug resistance database. The mean LPV level of these samples was 11.66 µg/mL. 69.11% (n=46) of the patients have no major PI mutations in protease. The following mutations that are associated with PI exposure were present in the data set: G62R (n=6), H219Q (n=11), S737T (n=8), I389T (n=8) and Q474L (n=7). Predictably, mutations that are associated with PI resistance were found, which are generally located in the p7/p1 and p1/p6 cleavage site. These mutations are K436R (n=4), I437V (n=1), L449P (n=5), R452K (n=4) and P453L\T (n=9). These results contribute to the knowledge of resistance mutations in gag and their impact on PI resistance.

Comparison of a clinical prediction rule and a LAM antigen-detection assay for the rapid diagnosis of TBM in a high HIV prevalence setting.

BACKGROUND/OBJECTIVE: The diagnosis of tuberculous meningitis (TBM) in resource poor TB endemic environments is challenging. The accuracy of current tools for the rapid diagnosis of TBM is suboptimal. We sought to develop a clinical-prediction rule for the diagnosis of TBM in a high HIV prevalence setting, and to compare performance outcomes to conventional diagnostic modalities and a novel lipoarabinomannan (LAM) antigen detection test (Clearview-TB®) using cerebrospinal fluid (CSF). METHODS: Patients with suspected TBM were classified as definite-TBM (CSF culture or PCR positive), probable-TBM and non-TBM. RESULTS: Of the 150 patients, 84% were HIV-infected (median [IQR] CD4 count = 132 [54; 241] cells/µl). There were 39, 55 and 54 patients in the definite, probable and non-TBM groups, respectively. The LAM sensitivity and specificity (95%CI) was 31% (17;48) and 94% (85;99), respectively (cut-point ≥ 0.18). By contrast, smear-microscopy was 100% specific but detected none of the definite-TBM cases. LAM positivity was associated with HIV co-infection and low CD4 T cell count (CD4<200 vs. >200 cells/µl; p = 0.03). The sensitivity and specificity in those with a CD4<100 cells/µl was 50% (27;73) and 95% (74;99), respectively. A clinical-prediction rule ≥ 6 derived from multivariate analysis had a sensitivity and specificity (95%CI) of 47% (31;64) and 98% (90;100), respectively. When LAM was combined with the clinical-prediction-rule, the sensitivity increased significantly (p<0.001) to 63% (47;68) and specificity remained high at 93% (82;98). CONCLUSIONS: Despite its modest sensitivity the LAM ELISA is an accurate rapid rule-in test for TBM that has incremental value over smear-microscopy. The rule-in value of LAM can be further increased by combination with a clinical-prediction rule, thus enhancing the rapid diagnosis of TBM in HIV-infected persons with advanced immunosuppression.