Diversity of HIV Type 1 and Drug Resistance Mutations Among Injecting Drug Users in Kenya

A lthough the majority of HIV-1 transmissions are believed to be through heterosexual contact, ¹ injecting drug use (IDU) is gaining significant proportions as a mode of HIV-1 acquisition due to changing lifestyles. ² It has contributed substantially to HIV acquisition and associated morbidity and mortality. ^{3 –6} Injecting drug users (IDUs) are a high-risk group for HIV infection due to their tendency to share needles/syringes as well as engage in unprotected sex, facilitating the spread of HIV to the general population. In East Africa, drug injection occurs in most towns of Kenya and Tanzania, with HIV prevalence among IDUs at 52.5% and 15% in Nairobi and Mombasa, respectively. ⁷ Drug use leads to unprotected sex and more than 90% of women IDUs have been reported to trade sex for money and/or drugs. ⁸ Whereas data on the extent of heterosexual spread of HIV within the general population are easier to obtain, the estimation of the same among IDUs is hard because of the difficulties of selecting representative samples. ⁹

The first HIV case in Kenya was identified in 1984 and by the end of 2010, HIV prevalence was 6.3%. ¹⁰ To improve the life of patients and reduce the HIV/AIDS-related morbidity and mortality, the Kenyan government has significantly increased access to antiretroviral therapy (ART) since 2003 (http://nascop.or.ke/library/3d/Official_KAIS_Report). With this increase, the danger of increased drug-resistant strains among drug-naive patients became real. Furthermore, stigma among IDUs affects access to ART and compliance, resulting in an accelerated emergence of drug-resistant mutants, which are a potential source of transmitted drug resistance. ⁹ Antiretroviral drugs are limited in Kenya and only those meeting some criteria can receive treatment. Since IDUs are difficult to reach, not all those who are HIV infected may have access to available treatment. Therefore antiretroviral drug resistance among those on treatment could easily spread to those acquiring new infections and lead to early treatment failure among those on ART. The aim of this study was to establish the prevalence of drug resistance mutations among IDUs seeking rehabilitation and treatment at an established government facility in Mombasa, Kenya. These data would assist service providers to develop HIV prevention initiatives and service delivery among IDUs.

This was a cross-sectional pilot study involving 58 HIV-1-positive IDUs from Mombasa, Kenya. The study subjects were adults of 18 years or more and were seeking treatment from a government health facility. No ART history was available at the time of contact with the study subjects. After informed consent and ethical clearance from the Kenya Medical Research Institute ethical committee, blood samples were collected and sent to the laboratory for analysis. CD4⁺ T cell counts of peripheral blood were determined on site using the FACSCOUNT (Becton-Dickinson, Germany).

HIV-1 RNA was extracted from plasma using SMITEST EX-R and D (Genome Science Laboratories, Fukushima, Japan) according to the manufacturer's instructions. The HIV-1 reverse transcriptase (RT) gene was amplified by both one step RT and nested polymerase chain reaction (PCR) as previously described. ^{11 –13} The pol-RT nucleotide sequences were translated into the corresponding amino acids and analyzed for previously reported drug resistance-associated mutations. ¹⁴ Generated sequences were aligned using Clustal W, phylogenetic analysis was done by the neighbor-joining method, and trees were viewed using NJ plot (http://pbil.univ-lyon1.fr/software/njplot.html).

Of the 58 IDUs, 24 were males and 35 females. Their mean age was 31.5 years. Females were younger (mean, 30.6 years vs. 32.8 years) and had higher mean CD4⁺ T cell counts (367 vs. 342) than males, but this was not statistically significant. HIV-1 genotyping showed that all the individuals were infected with non-subtype B HIV-1. The most prevalent subtypes were A1 (52 of 58), D (5 of 58), and C (2 of 58). Figure 1 shows the phylogenetic tree of the 58 sequences and their clustering with reference sequences obtained from the Los Alamos HIV database.

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FIG. 1.

Phylogenetic tree of the HIV-1 pol-RT sequences from injecting drug users in Mombasa, Kenya. Patient samples were aligned and compared with reference sequences from the Los Alamos HIV database. The tree was constructed using the neighbor-joining method and rooted with SIVcpzGAB. The bootstrap values of 1000 replicates are indicated next to the node.

Drug resistance mutations were determined for all subjects in order to identify emerging mutations. Samples from eight individuals (13.8%) showed the presence of drug-resistant HIV-1 (Table 1). The mutations observed included M184V (1), K219Q (3), T215F (5), and K103N (1). One patient had multiple nucleoside reverse transcriptase inhibitor (NRTI) mutations (M184V and T215F).

In this pilot study, we evaluated drug resistance mutations among 58 IDUs from Mombasa, Kenya. Eight (13.8%) patient samples showed the presence of mutations that conferred resistance to available first-line regimens. One patient had a nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation, K103N. The prevalence of drug resistance in this group is higher than what has been reported in Kenya, albeit among drug-naive patients seeking treatment. ¹¹ This is because of the uniqueness of this population in seeking healthcare services. In nine samples, the T215S mutation was detected. It is a transition mutation between wild type and T215F/Y. The presence of the T215S mutation among drug-naive patients has been reported to be a risk for early virologic failure ¹⁴ and therefore an indication that T215F/Y mutations exist, possibly as minor populations. It was difficult to conclude with certainty whether the mutations observed among these people were transmitted.

Most IDUs in this study were infected with HIV-1 subtype A1. Although consistent with other findings in which subtype A1 has been reported to be the major subtype in circulation, the percentage of subtype A1 in this population was higher than expected. From our observation it is evident that most IDUs with HIV-1 subtype A1 might have had a single source of infection (Fig. 1). This is typical of IDUs. However, development of drug resistance may be independent, an indication that they were not transmitted but may have been acquired through exposure to ART. On the other hand, subtype D may have been acquired independently, i.e., sexual or other modes of transmission. Only three subtypes were detected among this population (A1, C, and D). However, the possibility of recombination with other subtypes if larger fragments are sequenced cannot be ruled out.

As in other cross-sectional studies, this study had limitations. One, the samples were anonymously collected with no baseline information regarding ART history or viral load data. Two, only positive samples were analyzed for subtypes and drug resistance; site prevalence analysis of HIV in the studied population was not possible. Three, the sample size was too small. However, this may be a representative of prevalent subtypes in this region although no recombination analyses were done and no protease sequencing was done to similarly check for resistance. From our observation, it is likely that some patients had a history of ART. Furthermore, the population sequencing methodology that we employed might have left out mutations that occur as minor populations.

Drug use in Kenya has been associated with the country's strategic position along drug trafficking routes within the Indian Ocean and therefore has a historical context. Since it has been shown that drug users are more likely to share syringes, this may become a source of new HIV infections and more so infection with drug-resistant virus. ¹⁵ Efforts to bring about structural changes in national policies should scale-up HIV prevention and proper management of already infected IDUs. This study is the first to document HIV-1 drug resistance among IDUs in Kenya and forms the basis upon which service providers in HIV prevention initiatives should increase ART management among IDUs and prevent further transmission of drug-resistant variants.