The current study reveals a high level of drug resistance among patients receiving HAART and drug-naïve individuals in Cameroon, demonstrated by 46% of the study individuals who were on HAART for a median period of 12 months harbored genotypic drug-resistance mutations. This finding is similar to that reported in other sub-Saharan African countries including Gabon (58%) [Vergne et al., 2002
], Cote d'Ivoire (57%) [Adje et al., 2001
], and Uganda (52%) [Richard et al., 2004
]. In particular, 39% of the patients receiving HAART in this study were predicted to have resistance to an NRTI, and 46% to an NNRTI. Of note is that the 13 out of 28 patients receiving HAART with drug-resistance mutations were on treatment for a median of 14 months, while the remaining 15 individuals whose viruses did not harbor drug-resistance mutations had been on treatment for a median of only 9 months, a finding which is consistent with what is known about prolonged ARV pressure on HIV-1 and the frequency of drug-resistance mutations. This therefore suggests that the individuals who do not show evidence of drug-resistance mutations are likely to develop resistance within another year. Therefore, monitoring of such patients for the emergence of drug resistance is a necessity to better manage their disease course.
Studies conducted in Yaoundé, Cameroon from 2002 to 2004 [Laurent et al., 2006
], found a lower level of prevalence of ARV resistance, whereby 16.4% of the participants had genotypic drug-resistance mutations after a median of 10 months, including 12.5% resistance to NRTIs, 10.2% to NNRTIs, and 2.3% to PIs [Laurent et al., 2006
], even though treatment and laboratory expenses were paid by the patient—an obstacle to drug adherence that often leads to a higher rate of drug-resistance mutations. In both studies conducted in Cameroon, the median of months from the start of HAART prior to sampling was comparable (10 months vs. 12 months). However, important differences in treatment were observed, including the administration of PIs, and a lower number of individuals on suboptimal ART regimens in the Yaoundé study (2% vs. 10% dual-therapy; 0% vs. 3% mono-therapy). Several factors could account for the differences in the rate of mutations including prescription practices, drug availability, support programs, and the training of physicians in both Yaoundé and in the less urban settings of Cameroon.
The resistance mutation observed most frequently among the patients receiving HAART in this study was M184V/I, present in all the patients with NRTI-resistance mutations, conferring a high-level resistance to 3TC, and was the drug taken by 96% of the study individuals. The M184V/I mutation also confers resistance to emtricitabine (FTC), which potentially abrogates the use of this drug for salvage therapy. The remaining NRTI-resistance mutations among patients receiving HAART in the current study include K65R, Q151M, and T215Y representing resistance to all the remaining NRTIs currently approved by the FDA. In fact, 77% of the individuals in this study with drug-resistance mutations are infected with viruses that are resistant to two or more NRTIs. The mean viral load of 10,997 copies/ml for these individuals reflects the great number of drug-resistance mutations present and suggests the need for salvage therapy. Of note, three individuals, 06ARC007, 06ARC009, and 07BDHS040 had undetectable viral loads at the time of collection. Individuals 06ARC007 and 06ARC009 were previously taking Triomune for 3 months and 4 months, respectively, then switched therapy 5 months prior to sample collection.
In the present study among patients receiving HAART, the majority of mutations conferred NNRTI resistance; however, no single mutation predominated, in contrast to the NRTI-resistance mutations. The most predominant NNRTI-resistance mutations were Y181C (38%), which alone confers high-level resistance to NVP, DLV, and ETR, and mutation K103NS identified in 31% (4/13) individuals with drug-resistance mutations. Irrespective of NVP resistance, 25% of all patients receiving HAART and 9.5% of all drug-naïve individuals had mutations that confer high-level resistance to at least one of the remaining NNRTIs approved by the FDA, including DLV, EFV, and ETR. Therefore, the mutations identified here serve as clear markers for the emergence of drug-resistant variants in this population and point to potential alternative second-line treatment options with drugs that would be effective in controlling disease progression in such individuals.
Subtype analysis of the RT gene revealed a predominance of CRF02_AG viruses (73%; 36/49) among the study individuals with 86% (24/28) of the patients receiving HAART harboring this variant. A comparison of the drug-resistance mutations among the CRF02_AG and non-CRF02_AG variants revealed subtle differences in the frequency of specific mutations suggesting that there could be subtype-specific differences, which may confer resistance to different medications. For example, among NRTI-resistance mutations, V75I and F77L confer resistance to ABC, DDI, D4T, and ZDV and were present in the non-CRF02_AG strains but were absent in the CRF02_AG strains. Differences in mutational patterns that cause resistance to NNRTIs were also identified among the CRF02_AG and non-CRF02_AG strains. Though the number of non-CRF02_AG strains studied is small, others have reported similar findings. Soares et al. 
, reported subtype differences in drug-resistance mutations among subtype B and C sequences of Brazilian HIV-1-infected patients on HAART. It was also striking to note differences in the mutational patterns between the Cameroon CRF02_AG and those available in the Stanford University HIV drug-resistance database, whereby the K103NS mutation that confers resistance to NNRTIs was present in 29% of the sequences from the database compared to 17% present among the Cameroonian sequences. Taken together, studies that examine subtype differences and the development drug-resistance mutations are urgently needed in the Central African region in particular, a region of the world that harbors the highest HIV-1 viral diversity and where first-line HAART use is increasing. Such studies are underway and will be reported elsewhere.
Data regarding the transmission of drug-resistance mutations in the current study found five (24%) drug-naïve individuals who harbored viruses with genotypic drug-resistance mutations to NRTIs and/or NNRTIs. Earlier studies conducted in Cameroon found the rate of drug-resistance mutation transmission among drug-naïve individuals ranging between 4.9% and 9.8% [Koizumi et al., 2006
; Vergne et al., 2006
; Vessiere et al., 2006
; Ndembi et al., 2008
]. The present analysis reveals an increase in the rate of transmission of viral variants with drug-resistance mutations, which could be due in part to the increasing availability of ARV therapy in the community and/or poor adherence to treatment. In addition, studies conducted in other African countries have found a lower frequency of RT drug-resistance mutations among drug-naive individuals including Mali (11.5%) [Derache et al., 2008
], South Africa (3.6%) [Jacobs et al., 2008
], and Tanzania (3% NRTI, 4% NNRTI) [Nyombi et al., 2008
]. Taken together, this study reveals a high prevalence of drug-resistance mutations among patients receiving HAART (46%) and drug-naïve (24%) individuals in Cameroon. From a public health standpoint, it is critical to improve efforts to monitor the emergence of such mutations, to initiate a second-line HAART that includes a PI, and to monitor drug-naïve individuals to prevent the spread of resistant variants.
Nucleotide Sequence Accession Numbers
The DNA sequences of the HIV-1 RT region of Pol that were determined in this study were submitted to GenBank under the following accession numbers: GQ344955–GQ345003.