Saeng-Aroon, Siriphan MSc*; Wichukchinda, Nuanjun MSc*; Myint, Lay MD, PhD†; Pathipvanich, Panita MD‡; Ariyoshi, Koya MD, PhD*†; Rojanawiwat, Archawin MD*; Matsuda, Masakazu†; Sawanpanyalert, Pathom MD, PhD*; Sugiura, Wataru MD, PhD†; Auwanit, Wattana PhD*
HIV-1 has tremendous ability to mutate swiftly and to develop resistance to almost all clinically used antiretroviral drugs. Reduced sensitivity to nucleoside reverse transcriptase inhibitors (NRTIs), non-NRTI (NNRTIs), and protease inhibitors has been studied extensively and is linked to specific point mutations in either the reverse transcriptase or the protease gene. 1 Most current knowledge for interpreting these genotypic changes has been derived from studies on HIV-1 subtype B viruses. Worldwide, however, the majority of HIV-1–infected people live in developing countries and most of them are infected with non-B subtypes. Non-B subtypes differ from subtype B in pol gene by 10–15%. 2 We and other groups have published data showing some discrete differences in the patterns of drug resistance mutations between subtypes. 3–5 With a growing demand for access to antiretroviral therapy in resource-limited countries, the resistance patterns of non-B subtype viruses to antiretroviral drugs are becoming an important issue.
In Thailand, with a population of approximately 62 million, it was estimated that 695,000 people were living with HIV-1 infection. Of these, the majority are infected with CRF01_AE (previously known as subtype E), and 55,000 people had AIDS in 2000. 6 In the past, only a small minority of HIV-1–infected patients could afford antiretroviral drugs due to the high monthly price; thus most were either not treated or were treated with suboptimal antiretroviral regimens, mostly dual therapy. 7 When patients are treated suboptimally, HIV-1 acquires resistance to drugs more quickly.
Furthermore, the prevention of mother-to-child HIV-1 transmission (PMTCT) program, which uses a short-course zidovudine (AZT) regimen, was expanded first in northern Thailand in 1997 and later throughout the country. 8 This expanded program could also trigger the emergence of AZT drug resistance. Studies of HIV-1–infected individuals with primary HIV infection have shown that drug-resistant HIV strains can be transmitted from one adult to another and occasionally transmitted vertically from mother to child. 9–12 To control the spread of drug-resistant HIV-1, a monitoring system of anti-retroviral drug-resistant HIV-1 in an epidemiologic scale is urgently needed.
The pol gene has been commonly sequenced for testing drug resistance in many HIV laboratories of developed countries. However, access to the sequencing test in developing countries is limited due to the relatively high costs of reagents and unavailability of expensive equipment such as an automated sequencer. Polymerase chain reaction (PCR)-based assays are an alternative method of detecting point mutations, having the advantage of increased sensitivity and low cost. Allele-specific primer extension assays have been applied to detect drug-resistant HIV-1; however, they have not been adequately specific for widespread application. 13,14 Conversely, mutagenically separated PCR (MS-PCR) is a PCR-based point mutation assay that overcomes this specificity limitation and has been successfully applied to detect drug-resistant HIV-1 of non-B subtypes. 15–17 Previous papers evaluated the performance of MS-PCR in developed countries, but it has not yet been used in resource-limited settings.
We conducted this study with the following objectives: to evaluate the performance of MS-PCR specific for M41L and K70R mutations in detecting AZT-resistant HIV-1 strains in Thailand; to apply the MS-PCR to the screening of AZT-resistant HIV-1 among drug-naive HIV-1–infected Thais; and to investigate the patterns and prevalence of drug-resistant genotypes among HIV-1–infected Thai individuals who had been treated with suboptimal antiretroviral regimens by sequencing the pol gene.
PATIENTS AND METHODS
We used samples obtained from HIV-1–infected individuals who attended the Day Care Center clinic at the Lampang Hospital from July 6, 2000, to July 15, 2001 and gave a written informed consent. The Lampang HIV study was approved by the Thai government ethics committee. The Lampang Hospital is a government referral hospital with approximately 800 beds, situated in the center of Lampang province, which is 100 km south of Chiang Mai in northern Thailand. Plasma samples were collected from these individuals and stored at −80°C until their use. Viral load measurement was conducted using a commercial kit (Amplicor HIV-1 Monitor Test, version 1.5; Roche Diagnostics, Branchburg, NJ). CD4+ cell count was measured by flow cytometory (FACScan; Becton-Dickinson, Franklin Lakes, NJ).
After viral load measurement, the residual RNA was used for drug resistance genotyping by sequencing pol gene as previously described. 17 Briefly, an 888-basepair (bp) reverse transcriptase fragment (base number of nucleotide: 2485–3372) was amplified by PCR after a reverse transcription (RT) reaction from the RNA by an RNA-PCR kit (AMV One Step RNA PCR Kit; Takara, Osaka, Japan). Primary PCR products were further amplified with a high-fidelity DNA polymerase (KOD DNA polymerase; Toyobo, Osaka, Japan). Sequencing was performed using an autosequencer ABI-3100 (Applied Biosystems, Foster City, CA) with dye terminators (BigDye Terminator Cycle Sequencing Ready Reaction Kit; Applied Biosystems). The sequence results were assembled and aligned on the reference HIV sequence of HIV-1 HXB2 (Gen-Bank accession number M38432) by ABI Prism SeqScape Software (Applied Biosystems). They were submitted to Stanford HIV RT and Protease Sequence Database (http://hivdb.stanford.edu/) for drug resistance genotyping.
M41L and K70R MS-PCR
On measuring the viral load, we used the residual RNA for the MS-PCR experiments. The method of MS-PCR for detecting M41L and K70R AZT resistance mutations in CRF01_AE has been described in a previously published paper. 17 Briefly, the first-round RT-PCR was conducted to amplify a 370-bp RT region, which spans codon 41 and 70. The second- and third-round PCR were conducted with 1 common forward primer and 2 reverse primers, which are allele specific. The size of the wild-type specific primer was designed about 20 nucleotide bases longer than the mutant type-specific primer so that the wild-type PCR product could be easily differentiated from the mutant-type PCR product by electrophoresis with a 3% agarose gel. We used clinical samples, of which drug-resistant genotypes were confirmed by the sequencing method used for positive controls, and included them for every experiment. When an MS-PCR result showed double bands, of which one was faint, we retested the sample.
We compared proportions by the χ2 test. Means of continuous variables were compared by a nonparametric test, the Kruskal-Wallis one-way analysis of variance. The data management and statistical analysis were conducted using Epi Info version 6.04.
A total of 489 HIV-1–infected individuals attended the clinic during the observation period. History of antiretroviral drug therapy was available from 487 infected individuals, in whom 336 were drug naive and 151 were drug experienced; 22 individuals were exposed to a single NRTI, 114 to two NRTIs, 14 to three or more antiretroviral drugs including protease inhibitors, and one did not have regimen information.
Patterns and Prevalence of Drug Resistance Mutations Among Suboptimally Treated Individuals
Out of the 114 individuals who had been exposed to 2 NRTIs, we first analyzed 112 individuals who had received AZT/didanosine (ddI) or AZT/zalcitabine (ddC) dual therapy for the sequencing of RT region; the other 2 individuals had received either d4T/ddC or AZT/lamivudine (3TC). A total of 108 individuals (96.4%) were infected via the heterosexual route. The median (range) duration of dual therapy was 621 (28–1600) days; 76 individuals were on therapy and 36 patients were off therapy at the time of sampling; 28 individuals were exposed to AZT/ddI, 76 to AZT/ddC, and 8 to both AZT/ddI and AZT/ddC. Sequence data were obtained from 99 individuals, of whom 57 (57.6%) had mutations that are known to be associated with AZT, ddI, or ddC resistance. We could not obtain sequence data from 13 individuals, mainly because of their low viral load; 10 had a viral load under the detectable level (<400 copies/mL). Assuming that the individuals without sequence data did not have any drug-resistant virus, the prevalence of drug-resistant viruses detected by the sequencing method was 57/112 (50.9%) among those who had received dual therapy. If confined to the 76 individuals on therapy, 48 (63.2%) had drug-resistant viruses. Figure 1 summarizes amino acid variations at the sites, which are known to be associated with AZT, ddI, or ddC resistance mutations in subtype B infection. The most common drug resistance mutations were D67N, followed by K70R and T215Y/F. Other mutations were also commonly seen at codon 41, 210, and 219. No mutation of Q151M was found. We also analyzed associations among these specific mutations. The presence of D67N was strongly associated with K70R and less significantly with M41L; 31 (86.1%) out of 36 individuals with D67N mutation had either a K70R or M41L mutation (Table 1). The presence of T215Y/F was strongly associated with M41L but not with K70R; 22 (71%) of 31 individuals with T215Y/F mutation had K70R or M41L mutation (Table 1).
Drug Resistance Mutations in Relation to the Duration of Antiretroviral Therapy
The prevalence of drug resistance mutations correlated with the duration of dual therapy among 76 individuals on therapy. Among those with the duration of therapy for <180 days, 180–365 days, and >365 days, drug-resistant viruses were found to be predominant in 3 (30%), 8 (62%), and 33 (69%) individuals, respectively, with the median numbers of drug resistance mutations of 0, 2, and 2, respectively (Fig. 2). The number of drug resistance mutations was significantly associated with the median level of viral load: the median (inter-quartile range, IQR) viral load of individuals with no mutation, with 1–4 mutations, and with ≧5 mutations was 7412 (<400–62,432); 37,871 (7866–105,105); and 156,989 (32,682–184,767) copies/mL, respectively (P = 0.018 by Kruskal Wallis one-way analysis of variance).
Drug Resistance Mutations After Stopping Antiretroviral Drug Therapy
The interval between stopping dual therapy and the time of sampling was also associated with the presence of drug resistance mutations. Twenty-six individuals who had been substantially exposed to dual therapy for >180 days were off therapy at the time of sampling. Seven individuals (27%) were off therapy for >180 days and 19 (73%) were off therapy for <180 days. Drug-resistant viruses were detected in only 1 individual (14%) in the former group but 7 individuals (37%) in the latter group.
Evaluation of M41L and K70R MS-PCR in Detecting AZT-Resistant Strains
We then compared direct sequence methods with M41L and K70R MS-PCR in 96 antiretroviral drug–experienced individuals for whom both sequence and MS-PCR results were available (Table 2). Overall concordant rate for codon 41 was 96.9% (93/96) where M41I was regarded as a mutant type and concordant rate for codon 70 was 92.7% (89/96). Discordant results were seen mainly in the samples that were determined as mutant type by the MS-PCR and as wild type by the sequencing method.
To study the sensitivity of M41L and K70R MS-PCR as a screening strategy in detecting AZT-resistant strains, we defined the AZT-resistant strains as viruses with at least one AZT resistance mutation, which was detected by the sequencing method. Out of the 96 plasma samples that were tested by both the sequencing and the MS-PCR methods, 52 samples had no AZT resistance mutation and 44 samples had at least one AZT resistance mutation and were regarded as containing AZT-resistant viruses. Of the 44 samples with AZT-resistant viruses, the M41L and K70R MS-PCR detected either M41L or K70R mutation in 38 samples, resulting in the sensitivity of the M41L and K70R MS-PCR in detecting the AZT-resistant viruses at 86.4%. The number of AZT resistance mutations related to the detection rate of AZT resistance mutations by the MS-PCR (Table 3). When the viruses had multiple mutations, the sensitivity of the M41L and K70R MS-PCR was considerably higher. Of 39 samples containing HIV-1 with more than one AZT resistance mutation, 37 samples (94.5%) were diagnosed as having resistant viruses by the M41L and K70R MS-PCR.
Screening AZT-Resistant Viruses Among Antiretroviral Drug–Naive Individuals in Northern Thailand
We applied the M41L and K70R MS-PCR to the screening of 292 antiretroviral drug–naive HIV-1–infected individuals attending the Lampang Hospital for the existence of AZT drug-resistant viruses. There were 271 individuals (92.8%) who were known to be infected with HIV-1 via the heterosexual route. We found 2 patients (0.7%) who carried mutant viruses: one had M41L and the other had K70R mutation. Later it was noted that these 2 patients, as well as their spouses, had never received any antiretroviral drugs but both had participated in clinical trials of herbal medicine in the past.
Our observation showed that AZT, ddI, or ddC resistance mutations were found in >50% of individuals who had received dual therapy. The prevalence of drug-resistant viruses was higher among individuals who had received the drugs for a longer period, as previously reported. 18,19 We attribute the high prevalence of resistant viruses to the fact that the dual therapy was suboptimal. Clinicians working in government hospitals, however, did not have other options because the more efficient antiretroviral therapy such as triple or quadruple therapy was not affordable for most patients when this study was conducted. 7 Recently, access to multiple antiretroviral drugs has been dramatically improved, because the Government Pharmaceutical Organization (GPO) started the production of generic antiretroviral drugs known as “GPOvir,” which is a combined tablet of stavudine, lamivudine, and nevirapine. We nevertheless anticipate that individuals who had already had viruses resistant to NRTI dual therapy may not gain as much benefit from the generic medicine as antiretroviral drug–naive individuals do.
The most common mutations observed in this study were D67N, K70R, and T215Y/F, and we found few mutations at codons 65, 74, 108, 151, and 184. Such patterns of NRTI resistance mutations are similar to the patterns in CRF01_AE infection as well as in subtype B infections that have been reported in our previous report. 4 M184V mutation was often found in our previous study but not in the current study. We think that this difference reflects on the rare use of 3TC in Thailand when this study was conducted. Our current study, though a cross-sectional observation, showed several associations among resistance mutations such as D67N and M41L or K70R, T215Y/F and M41L in Thai strains as known in subtype B infection. 20,21
We found a high concordance rate of MS-PCR with the sequencing method in detecting M41L and K70R point mutations. The finding is compatible with previous papers. 16,17 Discordant results between the MS-PCR and sequencing method were seen in some samples, most of which showed mutant type by the MS-PCR but wild type by the sequencing method. We think that such discordances are due to the greater sensitivity of MS-PCR for detecting a minor virus population than the sequencing method. However, a high sensitivity and specificity of detecting 2 particular point mutations do not specifically justify the application of M41L and K70R MS-PCR for the screening of AZT-resistant viruses in the field. D67N and T215Y/F mutations are very common but it is technically difficult to establish MS-PCR specific for these mutations due to a higher degree of polymorphism around the mutation sites. Our data showed that these mutations were frequently accompanied by M41L and/or K70R as previously reported in subtype B. 22 Furthermore, we evaluated how efficiently the M41L and K70R MS-PCR could detect AZT-resistant viruses that were detected by the sequencing. The overall sensitivity was reasonably high particularly among the viruses with multiple drug resistance mutations.
This is the first report that addressed the transmission of drug-resistant HIV-1 using a large number of samples in Thai-land. We found that the prevalence of HIV-1 strains with either M41L or K70R mutation was as low as 0.7% among our drug-naive population. Considering that the overall sensitivity of the MS-PCR for detecting HIV-1 with any AZT resistance mutation was 86.4%, the prevalence of AZT-resistant HIV-1 was estimated to be 0.8%, which is still very low. There is still the concern that the low prevalence of resistant virus could be a consequence of the fact that the resistance to AZT in the drug-naive population was often associated with mutations at codon 60 or 215. To exclude this possibility, we further tested 60 samples, which were randomly selected from the drug-naive samples and confirmed that none had drug resistance mutations at these sites. The majority (127/292) of drug-naive individuals (43.5%) were initially diagnosed as HIV infected in 1997 or before, when the PMTCT program started in the region, and many were likely to have been infected several years prior to their first diagnosis of HIV infection. Thus, our result may not show an effect, which could have been triggered by the PMTCT program. A report from the United Kingdom suggests that transmission of drug-resistant HIV-1 is increasing. 23 We believe that our report is important in providing the baseline information on AZT-resistant HIV-1.
There has not been a consensus on the strategy of monitoring the transmission of drug-resistant HIV-1 in developing countries. Detecting individuals with primary viremia is ideal but not practical. In our study, we surveyed a drug-naive population for the presence of drug-resistant viruses. One concern with this approach is that drug-resistant viruses, which are generally less fit, might have been overwhelmed by the wild-type viruses in the absence of antiretroviral drug pressure because drug-resistant viruses among drug-treated individuals disappear following the interruption of antiretroviral therapy. 24 However, a recently published paper showed 2 cases of transmission of drug-resistant HIV-1 in which the resistant genotypes remained as a dominant population for a prolonged period in the absence of antiretroviral therapy. 25 Another way to monitor the spread of antiretroviral drug–resistant viruses is to screen infected individuals shortly after they receive antiretroviral therapy, which selects a minor population of insidious resistant viruses, before de novo resistance mutations occur. Further studies are needed.
This study demonstrates that it is feasible to apply MS-PCR techniques for screening a large number of field samples for the presence of AZT-resistant viruses in Thailand. Taking into account the enormous benefits of MS-PCR such as much lower cost, ease of use, no requirement of automated sequencers, and higher sensitivity of detecting a minor virus population, we think that the M41L and K70R MS-PCR is a useful technique for the screening of AZT-resistant HIV-1 in epidemiologic surveys in developing countries. Recently, GPOvir has become widely available in Thailand. As the patterns of drug-resistant mutations against 3TC and nevirapine are relatively simple, we propose that MS-PCR technique should be considered for monitoring viruses resistant to this combination of antiretroviral drugs.
We thank Ms. Suthira Kasemusuk, Ms. Sriprai Seneewong-na-ayoottaya, Ms. Anongnard Ariyakrua, Ms. Nutira Boonna, and Mr. Praphan Wrongramnong for their field works; Ms. Kanha Lor-yont, Mr. Wicharn Khaewkarnka, Mr. Sithichai Umnajsirisuk, Mr. Somchai Ni-yom-thai, and Ms. Jirawan Srisawat for their laboratory assistance. Finally, we would like to thank the HIV-infected individuals who participated in this study.
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