Molecular monitoring of HIV-1 drug resistance in Ifakara HIV-1 Cohort, Tanzania

HIV-1 resistance is one of the problems affecting success of antiretroviral therapy programmes worldwide. Many studies on efficacy of ART programmes, specifically on drug resistance, have been conducted in developed countries but not in developing countries due to lack of resources. So many tests have been optimized for HIV-1 subtype B which is prevalent in the developed countries but not for non-subtype B which is the main HIV-1 subtype in developing world.
Some few studies have been conducted in Tanzania to evaluate the efficacy of the ART regimens and programmes. These studies have evaluated either the virological efficacy and genotypic resistance in patients receiving ART (to evaluate development of acquired resistance) or genotypic resistance in drug naïve patients (to assess primary resistance). These few studies have been conducted in cities or urban areas with referral hospitals and have indeed shown some degree of resistance to ART and have provided some insights into degree of resistance in Tanzania, particularly in the urban areas. Most studies have not reached in the rural areas to investigate the pattern of resistance in these parts of the country.
This study was conducted to monitor HIV-1 drug resistance in Ifakara HIV/AIDS cohort, a rural located cohort that was established in 2004 as one of the first sites chosen for ART rollout in Tanzania. By October, 2011 the cumulative number of patients enrolled in this cohort was 5748 and a total of 3940 had been started on ART. So the first question in this study was to set to answer as to what extent is there a problem of drug resistance in this cohort and if there is a trend over time to this resistance. The other aim was to genotype samples from all patients with suspected clinical resistance. The third component of the study was other was to develop and validate a microarray tool for detection of resistance mutations to reverse transcriptase inhibitors which are the main drugs used as first line drugs in the Ifakara HIV-1 cohort.
To answer the first question, 187 and 200 randomly collected samples in 2005-2007 and 2009 respectively, from drug naïve HIV patients were processed in Ifakara, Tanzania, and CD4 assay was performed. The remaining aliquot were sent to Basel, Switzerland for viral load assay and molecular genotyping to identify HIV-1 subtypes and resistance mutations known to affect reverse transcriptase inhibitors and protease inhibitors which are the only official class of ART drugs used in Tanzania and Ifakara cohort.
Major drug resistance mutations were detected in 8.4% and 3.3% of analyzed samples in RT gene in 2005-2007 and 2009 respectively. The observed difference in resistance mutations in 2005-7 and 2009 was not statistically significant. The subtypes identified in 2005-7 were A (28.0 %), C (37.3%), D (24.0%) and CRF01_AE (10.7%). The subtype % frequencies in 2009 were as follows: A (24.2), C (45), D (17.5), CRF01_AE (7.5), B (5) and F (0.8). The pattern of HIV-1 subtypes observed in 2005-7 and 2009 were almost similar with the exception for the occurrence of five isolates of HIV-1 subtype B and one isolate of HIV-1 subtype F in 2009. In other studies conducted previously HIV-1 subtypes C, A and D were also observed as main HIV-1 subtypes in Tanzania. However, as with the other studies, local subtype prevalence variation was also observed.
A total of 16 samples collected from patients who had started ART for an average duration of 11 months were successfully genotyped and major drug resistance mutation to reverse transcriptase inhibitors were detected in two patients (12.5%). In the last analysis from 17 suspected clinical resistance cases 6 patient samples (35.3%) were idenfied to harbour major resistance mutations to reverse transcriptase inhibitors. No major drug resistance mutations to protease inhibitors were detected in all the samples analyzed, but only minor protease resistance mutations were observed. Resistance mutations were present in only one third of patients with suspected resistance, suggesting other factors may have played a role in the observed lower ART response in these patients. Though in general the ART response was good in the cohort, the low level of drug resistance observed likely could affect the ART program in the area.
The microarray was developed and optimized using 4 HIV-1 cloned RT fragments from Swiss HIV cohort patients and 102 samples from Ifakara cohort HIV patients. Overall the microarray had a sensitivity of 92 percent. One main challenge was a high level of failure to produce signals which could be due to either primer mismatch leading to failure in primer extension reaction or due to failure in the hybridization step. The disadvantages of this microarray are that it is only optimized for Ifakara HIV sequences and it can only test the selected mutations on the array and may not therefore be used as a diagnostic tool. The advantage of this microarray is that it is relatively cheap and it takes less time to get results.