The current mainstream antiretroviral therapy (ART) in developed countries is a combination of reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). Since its establishment in the late 1990s, ART has benefited many HIV-1-infected patients1,2 and has recently become prevalent even in developing countries. On the other hand, the emergence of mutants resistant to these antiviral agents has become a serious concern. In countries with sufficient treatment modalities, the prevalence of drug-resistant variants has ranged from 10% to 20% among drug-naive patients,3-10 whereas in developing countries, resistance has rarely been reported. Recently, the World Health Organization's (WHO) "3 by 5" policy has promoted ART coverage in low- and middle-income countries. As of June 2005, about 500,000 people were receiving ART in sub-Saharan Africa, although the regional coverage rate was still only 11% of the estimated need. In countries with rapidly scaled-up ART provision such as Kenya, Uganda, and Cameroon, the threat of resistance now exists (3 by 5 progress report, June 2005, WHO/UNAIDS). In Kenya, for example, where ART has been provided for 12% to 17% of the estimated need, the prevalence of resistant strains among drug-naive patients has recently risen from 1% (2002) to 11% (2003) (personal communication). In Botswana, the prevalence of primary mutations against PIs was found to be 4% among drug-naive patients.11
In Cameroon, where all representative major groups and subtypes of HIV-1 cocirculate,12-18 there have as yet been no reports suggesting the emergence of ART resistance-associated primary mutations among drug-naive, HIV-1-infected patients. According to WHO/UNAIDS, as of October 2004, the prevalence of HIV-1 infection in Cameroon was estimated to be 4.8% to 9.8% in adults; the estimated number of people needing treatment was about 95,000; and 12,896 people were reported to have received ART. With a firm political leadership, Cameroon set a national target of providing ART based on WHO guidelines [ie, a combination of 2 nucleotide analog, reverse transcriptase inhibitors (NRTIs) and 1 nonnucleotide reverse transcriptase inhibitor (NNRTI)] to 36,000 people by the end of 2005, which means that the ART-treated population would nearly triple in 1 year (Summary country profile for HIV/AIDS treatment scale-up, WHO/UNAIDS). With such a rapid introduction of ART, this and other developing countries might suffer a phase of drug resistance in the near future.
In the current study, we evaluated the prevalence of drug-resistant strains in previously untreated HIV-1-infected patients at the dawn of the ART era in rural western Cameroon. It has been reported that conventional genotyping tests can underestimate the overall prevalence of resistant strains,19,20 and the importance of minor resistant mutants has been discussed recently.21,22 Therefore, we also investigated the rate of drug-resistant strains found as minor populations.
Fifty-four HIV-1-infected individuals (24 men and 30 women; mean age ± SD, 32.9 ± 10.1 years) attending antenatal/STD clinics [Infectious Disease Wards of the Maternal and Child Health Clinic (Nkwen) and the Azire Integrated Health Center (Kumbo), both in the northwestern province of Cameroon] were enrolled in this study (Table 1). With thorough ethical clearance and informed consent, blood samples were collected from the individuals in February 2004, when none had been treated with ART. The presence of plasma anti-HIV-1 antibody was screened with an immunochromatography assay kit (Determine HIV 1/2; Abbott, Tokyo, Japan) and confirmed with a microparticle enzyme immunoassay kit (AxSYM HIV1/2; Abbott). Peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation. Genomic DNA was extracted from the PBMCs using a DNA extraction kit (Qiagen, Hilden, Germany).
Polymerase Chain Reaction, Cloning, and Sequencing
A region of the HIV-1 group M pol gene including the protease sequence (Pol-PR; corresponding to nt 2265-2555 in HIV-1HXB2) was amplified by nested polymerase chain reaction (PCR) with primers NYUPOL7 (5′-GGGAATTTTCTTCAGAGCAG-3′) and NYUPOL8 (5′-TCTTCTGTCAATGGCCATTGT-3′) in the first round, and NYUPOL9 (5′-TCCTTAACTTCCCTCAAATCACT-3′) and NYUPOL10 (5′-CTGGCACGGTTTCAATAGGACT-3′) in the second round.23 A region of the HIV-1 pol gene including the reverse transcriptase sequence (Pol-RT; corresponding to nt 2513-3209 in HIV-1HXB2) was amplified by nested PCR with primers RT18 (5′-GGAAACCAAAAATGATAGGGGGAATTGGAGG-3′) and K104 (5′-TGACTTGCCCAATTTAGTTTTCCCACTAA-3′) in the first round, and K101 (5′-GTAGGACCTACACCTGTTCAACATAATTGGAAG-3′) and K102 (5′-CCCATCCAAAGAAATGGAGGAGGTTCTTTCTGATG-3′) in the second round. A region of the HIV-1 pol gene including the integrase sequence (Pol-IN; corresponding to nt 4493-4780 in HIV-1HXB2) was amplified with primers unipol5 (5′-TGGGTACCAGCACACAAAGGAATAGGAGGAAA-3′) and unipol6 (5′-CCACAGCTGATCTCTGCCTTCTCTGTAATAGACC-3′) in the first round, and unipol1 (5′-AGTGGATTCATAGAAGCAGAAGT-3′) and unipol2 (5′-CCCCTATTCCTTCCCCTTCTTTTAAAA-3′) in the second round.24 A region of the HIV-1 env gene including the C2V3 sequence (corresponding to nt 6975-7520 in HIV-1HXB2) was amplified with primers M5 (5′-CCAATTCCCATACATTATTGTGCCCCAGCTGG-3′) and M10 (5′-CCAATTGTCCCTCATATCTCCTCCTCCAGG-3′) in the first round, and M3 (5′-GTCAGCACAGTACAATGCACACATGG-3′) and M8 (5′-TCCTTGGATGGGAGGGGCATACATTGC-3′) in the second round.24
Nested PCR was performed using the AmpliTaq Gold PCR kit (Perkin-Elmer, Foster City, CA) according to the manufacturer's instructions. Amplification was done with 1 cycle of 95°C for 10 minutes and 35 cycles of 95°C for 30 seconds, 45°C to 55°C for 30 seconds, and 72°C for 1 minute, with a final extension of 72°C for 10 minutes. PCR amplification was confirmed by ethidium bromide staining of samples electrophoresed on an agarose gel. The PCR products were cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced as described previously (Applied Biosystems, Foster City, CA).
Phylogenetic Analysis and Subtyping
The samples were aligned with subtype reference sequences from the Los Alamos database by CLUSTAL W (version 1.81) with minor manual adjustments. Phylogenetic trees were constructed and visualized as described previously.25,26 To improve the accuracy, we used the genotyping tool (http://www.ncbi.nih.gov/projects/genotyping/formpage.cgi), the RIP 2.0 system (http://www.hiv.lanl.gov/content/hiv-db/RIPPER/RIP.html), and the REGA subtyping tool (http://dbpartners.stanford.edu/RegaSubtyping/) as needed.
PI and RTI Resistance-Associated Mutations
The PR and RT sequences (297 bp and 697 bp, respectively) were translated into the corresponding amino acids (99 and 232 amino acids, respectively) and analyzed for previously reported drug resistance-associated mutations in subtype B strains.27 For each sample, at least 4 clones were obtained and genotyped to evaluate minor populations.
Nucleotide Sequence Accession Numbers
GenBank accession numbers of the sequences reported in this study are as follows: DQ461820 to DQ461873 for Pol-PR, DQ461874 to DQ461925 for Pol-RT, DQ461926 to DQ461973 for Pol-IN, and DQ464287 to DQ 464330 for Env-C2V3.
All 54 samples could be analyzed in the PR region. In the RT, IN, and Env regions, 51, 48, and 44 samples were analyzed, respectively. The subtype of each sample was identified. Of the 54 HIV-1 strains, 45 (83.3%) had a concordant subtype or circulating recombinant form (CRF) designation: 40 (74.1%) CRF02_AG, 2 (3.7%) subtype A1, 2 (3.7%) subtype G, and 1 (1.8%) subtype F2. The remaining 9 (16.7%) strains had a discordant subtype or CRF: 6 (11.1%) subtype A1/CRF02_AG, 2 (3.7%) subtype D/CRF02, and 1 subtype (1.8%) G/CRF02. Thus, these 9 HIV-1 strains were considered to be recombinant; and in all cases, CRF02_AG was involved in the recombination.
PI Resistance-Associated Mutations
The overall prevalence of primary PI resistance-associated mutations was 7.4% (4 of 54 cases) (Table 1), although the prevalence of full-clone primary mutations was only 1.9% (1 of 54 cases). There were 3 cases that had strains with primary mutations as minor populations (1 of 4, 1 of 4, and 1 of 5 clones). One patient carried a strain with primary mutation M46L in full clones, and its determined subtype was A1. A mutation at codon 82 (V82I) was detected in full clones from 2 patients, but V82I confers minimal resistance to available PIs. These 2 cases were subtype G and CRF02_AG, in which V82I is regarded as a natural polymorphism.28,29
As minor populations, V82A, one of the most important "cleft" mutations, was detected in 1 case (1 of 5 clones); and the "flap" mutations M46L and M46I29 were observed in 1 case each (both 1 of 4 clones).
As for secondary mutations, most were detected at polymorphic sites such as codons 10, 20, 36, 63, and 77 (Table 2).29 Of the 54 cases, 51 (94.4%) had the M36I mutation, 1 had M36V, and 1 had M36N. Thus, all except 1 case had a mutation at this site. K20I appeared with a frequency of 85.2% (46 of 54 cases); and together with K20R and K20N, 87.0% of cases had a mutation at this site. These findings are consistent with previous reports showing mutation of codon 36 in more than 80% of non-B subtypes and mutation of codon 20 in more than 50% of subtype A and in more than 80% of subtype G and CRF02_AG.28,30 Other secondary mutations at frequent polymorphic sites were L10V/I (7 cases, 13.0%), L63P (3 cases, 5.6%), and V77I (3 cases, 5.6%). G73S was detected in 1 case (1.9%).
RTI Resistance-Associated Mutations
Of the 51 cases analyzed, 5 (9.8%) had strains with primary RTI resistance mutations as minor populations (Table 1). No case was found to have primary mutations in full clones.
The prevalence of primary mutations associated with NRTI resistance was 3.9% (2 of 51 cases). M184V, a potent inducer of resistance to lamivudine and emtricitabine with less potency against multiple drugs,29 was detected in 1 patient (1 of 4 clones), and V75I was detected in 1 case as a minor population (1 of 4 clones).
The prevalence of primary mutations associated with NNRTI resistance was 5.9% (3 of 51 cases). Y188C, which causes intermediate-to-high resistance to nevirapine,29 was detected in 2 cases (04CM058 and 04CM095) (1 of 4 and 1 of 7 clones, respectively). L100I was detected as a minor population (1 of 4 clones) in 1 case. HIV-1 subtype/CRF of the 2 strains with Y188C were different as shown in Table 1 and Figures 1A, B, suggesting that these 2 persons were epidemiologically unrelated.
As for secondary mutations, E44D, which is associated with multi-NRTI resistance,29 was detected in 1 case with full clones; yet there were no coexistent primary mutations.
Secondary mutations as minor populations were omitted from the figure because they were numerous, and their importance is likely much less than that of the other mutations.
In the current study, we found PI and RTI resistance-associated primary mutations in 7.4% and 9.8%, respectively, of drug-naive HIV-1-infected individuals in Cameroon when drug-resistant minor populations were included. This is one of the first reports of the emergence of primary ART resistance-associated mutations among drug-naive, non-B subtype HIV-1-infected individuals in Cameroon.
The current study was designed to evaluate the prevalence of potential resistant strains as minor populations, so we used proviral DNA extracted from PBMCs and analyzed several clones per sample. There is no doubt that direct sequencing of plasma RNA is the gold standard for drug resistance surveillance.31 Recently, however, it has been reported that conventional genotype testing may overlook minor virus populations if their frequencies are less than 25%.19,20 Minor resistant strains that eventually overgrow and affect the clinical course of disease can emerge from levels of virus that are undetectable by conventional assays.32,33 Therefore, we focused on the baseline frequency of the dormant hazard in a newly ART-promoting country.
Unlike developed countries where PI-including regimens are frequently administered, the current first line of ART in Cameroon is the combination of 2 NRTIs and 1 NNRTI, which means that PI use is limited compared with that of industrialized countries. Konings et al23 reported that only secondary mutations associated with PI resistance were detected among drug-naive HIV-1 patients in Cameroon during 2000 to 2002. Nonetheless, in our study, primary mutations of PI resistance were detected in drug-naive patients at the rate of 7.4% as of February 2004. This is consistent with findings by Ndembi et al suggesting the emergence of the drug resistance primary mutations during the last few years (personal communication). The HIV-1 strains with primary mutations were found to be CRF02_AG, subtypes A1 and F2, which circulate in west central Africa. It would, therefore, be natural to explain this phenomenon by simple transmission of the resistant strains from patients treated with PIs in Cameroon, although PI use has been limited in this country. However, the possibility that genetic diversity gave birth to resistant strains in the drug-naive individuals cannot be excluded. In addition, the possibility of laboratory artifact that was induced by PCR error cannot be fully excluded either.
As for RTI resistance, previous reports have not detected primary mutations in sub-Saharan Africa during 1999 to 2003,34,35 including the report by Konings et al36 that was based on samples collected in Cameroon between 2000 and 2002. In our study, samples were collected in western Cameroon in February 2004, and 5 cases (9.8%) yielded HIV-1 strains with primary mutations, although all were found as minor populations. Recently, there have been several reports noting the importance of drug-resistant strains detected as minor populations. Minor drug-resistant HIV-1 populations have been detected both in the early phase of treatment failure32,33 and during successful structured treatment interruption.21 Minor drug-resistant populations undetectable by conventional assays can eventually overgrow and affect the clinical course. They also have been found to persist longer than previously expected in untreated patients, a favorable condition for wild-type virus to overgrow,37-39 which also indicates the risk of resistance transmission even from minor strains. Thus, careful follow-up studies should be conducted to assess whether the drug-resistant mutants found in our study as minor populations might impact future ART.
Most of the published data focusing on minor populations of resistant strains are based on the analysis of subtype B strains using quantitative real-time PCR with specific primers for V82A, L90M, or M184V of RT.21,40 In our study, the entire regions of PR and RT genes were amplified, cloned, and genetically analyzed to identify mutation sites other than these 3. This is particularly important in the context of analyzing non-B subtype HIV-1 strains, which often contain polymorphisms. As a result, we detected some other important mutations as minor populations, such as V82A in the PR gene and V75I/Y188C in the RT gene.
In our study, several non-B subtype-specific polymorphisms were confirmed in the protease gene. First, we detected K20I/N and M36I/N at relatively higher frequencies than previous reports. Because Holguin et al reported that K20I and M36I were detected in all cases of subtype G strains,30 the higher rate can be explained by the fact that most of our samples were CRF02_AG, whose Pol-PR is subtype G. Other secondary mutation sites that we detected, such as L10, L63, and V77, are consistent with previous reports that consider these mutations non-B subtype natural polymorphisms. Secondly, V82I was detected in 2 cases, one of which belonged to subtype G; and the amino acid change could be regarded as a subtype-specific natural polymorphism.28 In this study, there were no other subtype-specific polymorphisms that could cause considerable natural drug resistance.
The subtype distribution of HIV-1 circulating in western Cameroon was found to be slightly different from those of other districts.16-18,23,36 The predominance of CRF02_AG was much more conspicuous in the western district (85%) than in other districts (about 60%), although the prevalences of other constituents such as A1, G, F2, and D were almost identical to those in other reports. As expected, independent recombinants were detected between CRF02_AG and subtype A1, D, or G, although the rate of recombination in this area was slightly lower (16.7%) than in other areas of Cameroon (about 21%).18 Still, the independent recombinants carry the potential hazard of developing natural resistance.
In conclusion, as of February 2004, primary mutations of the HIV-1 protease exist in drug-naive patients in rural western Cameroon, despite little use of protease inhibitors. As the scheduled rapid provision of ART can give rise to resistant strains in developing countries, we should be careful not to overlook the emergence of resistant strains. More importantly, we also observed the emergence of minor populations of both RTI- and PI-resistant strains, which could have been underestimated by conventional surveillance. Thus, careful drug regimen design is needed to prevent the rise of overt resistant strains from minor populations.
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