First Data on HIV-1 Resistance Mutations to Antiretroviral Drugs in Central African Republic

P revious studies have shown that there is a high genomic variability of HIV-1 in Central African Republic (CAR). ^{1 –5} Antiretroviral drugs (ARVs) were introduced in CAR in 2004. Failure to HAART is mainly associated with emergence of viral mutants resistant to the drugs; the resistance mutations are identified by sequencing of the viral genomic parts of interest, and genotypic international algorithms (e.g., ANRS and Stanford) have been proposed for interpretation. However, they are mainly based on subtype B and more data are still useful for non-B subtypes. ^{6 –8} We present here sequence data of non-B HIV-1 isolates obtained in 2008–2009 from Central African patients at failure of a d4T/AZT-3TC-NVP/EFV regimen, together with two patients at failure of a regimen including a protease inhibitor (PI) (indinavir: IDV). We present also polymorphism of protease from patients whose regimen did not contain a PI.

Patients at clinical and/or immunological failure of their HAART were proposed a genotypic assay for determination of viral resistance and their consent was obtained directly or via their family in the case of children; ages ranged from 5 to 63 years. Blood was collected in ethylenediaminetetracetic acid (EDTA) tubes and plasma was separated and stored at − 80°C. Plasma was thawed, homogenized, and applied to spots of Whatman 903 filter paper cards with five spots of 50 μl of plasma. The samples were allowed to dry at room temperature and were placed in plastic bags with desiccants. They were stored at 4°C before being carried at room temperature to Bordeaux, France during a 2–3 day journey. The spots were cut with scissors and eluted in 220 μl/spot of a buffer containing PBS, Tween 20, and fetal calf serum. The samples were then agitated at 4°C for 1 h before being vortexed. 140 μl of the final elution were used for RNA extraction using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The RNA was used in reverse transcription polymerase chain reaction (RT-PCR) of RT and Prot using two sets of primers in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) thermal cycler as described previously. ⁵ The obtained fragments were sequenced on both strands using the CEQ DTCS Quick Start kit on an automated sequencer Beckman CEQ 2000 DNA Analyser System in the Virology Department of the University Hospital of Bordeaux. The RT and Prot sequences were aligned on the different HIV-1 reference strains of the Los Alamos database, and phylogenetic trees were constructed and analyzed. The same sequences were introduced in the Smartgene database and the resistance mutations were listed according to the ANRS algorithm.

The sequences have been submitted to GenBank and the accession numbers are HM117932-HM117933 for the Prot sequences of the two patients at failure of IDV containing regimen, HM117936 to HM117946 for the Prot sequences of patients receiving a HAART without a PI and whose Prot sequences were studied for polymorphism, and HM117948 to HM 117959 for RT sequences of patients at failure of a HAART containing d4TAZT/3TC/NVP-EFV. The data are summarized in Table 1. The twelve RT sequences cluster with CRF11_cpx, CRF22_01A1, CRF06_cpx, CRF15_01B, and CRF25_cpx. Clearly, the resistance to d4T/AZT-3TC-NVP/EFV is associated with mutations that are the same as those recorded for subtype B : 184V to 3TC, TAMs and K65R to d4T/AZT and 103N, 181C, 190A/S, 101E, 106I, and 98G to NNRTIs.

The two Prot sequences from patients at failure of a regimen containing IDV cluster with CRF11_cpx and CRF13_cpx; one bears two major mutations 46L and 82A, while the other exhibits 90M associated with minor mutations.

The other eleven Prot sequences analyzed for polymorphism cluster with CRF11_cpx, B, CRF22_01A1, CRF02_AG, and CRF09_cpx. There is a high but not surprising polymorphism of Prot with particularly association of 36I + 69K +89M; in the ANRS algorithm, this pattern is associated with resistance to tipranavir but there is a need for phenotypic investigation. Although limited, these data confirm the high diversity of HIV-1 in CAR and, regarding the different non-B subtypes/CRFs and the different drugs used, do not provide evidence of new mutation profiles compared to subtype B.