Prevalence of viral load suppression and acquired drug resistance among people living with HIV in Nepal: a nationally representative surveillance study.

OBJECTIVES: This is the first nationally representative study to estimate the prevalence of viral load (VL) suppression and acquired HIV drug resistance (ADR) among people living with HIV (PLHIV) in Nepal. METHODS: A cross-sectional study recruited 1418 PLHIV from 20 ART centres in Nepal, using a two-stage cluster design. Participants were eligible if they were HIV-positive individuals on ART for 9-15 months or at least 48 months. Plasma specimens were collected and tested for the quantification of HIV-1 RNA. Specimens with a VL ≥1000 copies/mL were further processed for sequencing of PR and RT genes of HIV-1. The sequences were then analysed to detect mutations causing HIV drug resistance. RESULTS: The prevalence of ADR was 3.7% (95% confidence interval [CI]: 1.8-7.6) and 3.0% (95% CI: 1.8-5.2) among PLHIV who received ART for 9-15 months and 48 months or more, respectively. The prevalence of VL suppression was 95.3% (95% CI: 91.7-97.4) among those on ART for 9-15 months, and 96.5% (95% CI: 94.7-97.7) among those on ART for at least 48 months. The prevalence of any detectable acquired resistance to antiretroviral drugs was 80.7% (95% CI: 58.6-92.5) among those on ART for 9-15 months with VL ≥1000 copies/mL and 81.6% (95% CI: 55.4-94.0) among those on ART for at least 48 months with VL ≥1000 copies/mL. CONCLUSION: This study suggests that improved accessibility to VL monitoring and timely assessment of drug resistance in routine HIV programs are crucial in Nepal to ensure access to HIV treatment for all in need.

First Round of External Quality Assessment Scheme for SARS-CoV-2 Laboratories During the COVID-19 Pandemic in Thailand.

The emergence of SARS-CoV-2 necessitated the rapid deployment of tests to diagnose COVID-19. To monitor the accuracy of testing across the COVID-19 laboratory network in Thailand, the Department of Medical Sciences under the Ministry of Public Health launched a national external quality assessment (EQA) scheme using samples containing inactivated SARS-CoV-2 culture supernatant from a predominant strain in the early phase of the Thailand outbreak. All 197 laboratories in the network participated; 93% (n=183) of which reported correct results for all 6 EQA samples. Ten laboratories reported false-negative results, mostly for samples with low viral concentrations, and 5 laboratories reported false-positive results (1 laboratory reported false positives and false negatives). An intralaboratory investigation of 14 laboratories reporting incorrect results revealed 2 main causes of error: (1) RNA contamination of the rRT-PCR reaction and (2) poor-quality RNA extraction. Specific reagent combinations were significantly associated with false-negative reports. Thailand's approach to national EQA for SARS-CoV-2 can serve as a roadmap for other countries interested in implementing a national EQA program to ensure laboratories provide accurate testing results, which is crucial in diagnosis, prevention, and control strategies. A national EQA program can be less costly and thus more sustainable than commercial EQA programs. National EQA is recommended to detect and correct testing errors and provide postmarket surveillance for diagnostic test performance.

Adapting the geno2pheno[coreceptor] tool to HIV-1 subtype CRF01_AE by phenotypic validation using clinical isolates from South-East Asia.

OBJECTIVES: Geno2pheno[coreceptor] is a widely used tool for the prediction of coreceptor usage (viral tropism) of HIV-1 samples. For HIV-1 CRF01_AE, a significant overcalling of X4-tropism is observed when using the standard settings of Geno2pheno[coreceptor]. The aim of this study was to provide the experimental backing for adaptations to the geno2pheno[coreceptor] algorithm in order to improve coreceptor usage predictions of clinical HIV-1 CRF01_AE isolates STUDY DESIGN: V3-sequences of 20 clinical HIV-1 subtype CRF01_AE samples were sequenced and analyzed by geno2pheno[coreceptor]. In parallel, coreceptor usage was determined for these samples by replicative phenotyping in human cells in the presence of specific X4- or R5-inhibitors. RESULTS: The sole introduction of the CRF01_AE V3 region into a full-length otherwise subtype B provirus failed to produce replication-competent viral progeny. A successive genome-replacement strategy revealed that also CRF01_AE derived gag and pol sequences are necessary to generate HIV genomes with sufficient replication competence. Subsequent phenotypic analysis confirmed overcalling of X4-tropism for CRF01_AE viruses using the current version and the standard cut-off at 10% false positive rate (FPR) of geno2pheno[coreceptor]. Lowering the FPR cut-off to 2.5% reduced the X4-overcalling in our sample collection, while still allowing a safe administration of Maraviroc (MCV). CONCLUSION: This study demonstrates the successful adjustment of geno2pheno[coreceptor] rules for subtype CRF01_AE. It also supports the unique strength of combining complementing methods, namely phenotyping and genotyping, for validating new bioinformatics tools prior to application in diagnostics.

External Quality Assessment Scheme for HIV-1 Drug-Resistance Genotyping in Thailand.

The efficacy of antiretroviral (ARV) therapy can be compromised by the emergence and transmission of HIV-1 drug-resistant strains. HIV-1 drug-resistance (DR) genotypic testing thus plays an important role in the selection of optimal treatment regimens for HIV-infected individuals. Given the complexities of the testing procedures and the variety of approaches used, there is considerable potential for results to vary between laboratories. In Thailand, the national External Quality Assessment (EQA) scheme assesses the DR genotype testing performance of laboratories. Here, we evaluated the performance of laboratories in nucleotide sequencing and compared drug-resistance-associated mutations (DRMs) in the HIV-1 protease (PR) and reverse transcriptase (RT) genes during 2010-2015. The EQA samples in the 12 panels showed predominance for the CRF01_AE (85%) and subtype B (15%). Fourteen laboratory datasets were generated: eight using TruGene (TG), two using ViroSeq (VS), and four using in-house (IH) assays. All IH and VS laboratories had penalty scores <7, whereas five of the eight TG laboratories had fluctuating penalty scores. Moreover, seven and six TG laboratories could not amplify the two identical samples, 10B and 10E samples, or the CRF01_AE. Our findings demonstrate the requirement for laboratory participation in the ongoing EQA program and the optimization of kit assays using CRF01_AE samples. Our results also indicate that one advantage of participation is that the laboratories can monitor and investigate the source of laboratory errors.

Naturally Occurring Mutations in HIV-1 CRF01_AE Capsid Affect Viral Sensitivity to Restriction Factors.

TRIM5α and MxB are known as restriction factors that inhibit the early step of intracellular HIV-1 replication cycle. Both factors are believed to interact with the incoming virus core to suppress HIV-1 infection. The extreme diversity of HIV-1 is thought to be a consequence of its propensity to mutate to escape immune responses and host restriction factors. We recently determined the capsid sequences for 144 HIV-1 CRF01_AE viruses obtained in Thailand from 2005 to 2011. In this study, we further analyzed the amino acid variations among the capsid sequences of 204 HIV-1 CRF01_AE obtained in Thailand and China, including 84 of the aforementioned 144 viruses, to detect mutations permitting escape from restriction by host factors. We found a characteristic combination of E79D, V83T, and H87Q in sequences from Chinese viruses and subsequently showed that this combination conferred partial resistance to MxB. Interestingly, this combination conferred resistance to human TRIM5α as well. The H87Q mutation alone conferred resistance to MxB in the CRF01_AE background, but not in subtype B virus. In contrast, the H87Q mutation alone conferred resistance to human TRIM5α in both the CFR01_AE and subtype B backgrounds. BLAST analysis revealed the presence of the E79D, V83T, and H87Q combination in CRF01_AE viruses isolated not only in China but also in many other countries. Although the mechanistic details as well as precise role of MxB antiviral activity in infected individuals remain to be clarified, our data suggest an interaction between MxB and the HIV-1 capsid in vivo.

HIV Drug Resistance among Pre-treatment Cases in Thailand: Four Rounds of Surveys during 2006-2013.

In Thailand, antiretroviral therapy (ART) was initiated to treat human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) cases using the empirical regimen with no prior genotypic test to determine drug resistance. In order to assess prevalence rate of HIV drug resistance (HIVDR) among pre-treatment cases, four rounds of survey were carried out in ART clinics, including six, eight, 33 and four ART clinics in each round during 2006-2013. For which, HIVDR testing results were available in 310, 350, 797, and 413 cases in four rounds. It was revealed that HIVDR rates among naive cases were 2.0%, 2.8%, 4.0% and 4.8%, while in experienced cases, the rates were 0, 3.3%, 11.4% and 13.9%. The rates among all cases were 1.9%, 2.9%, 4.4% and 5.6%. Resistant drugs with the highest rates among all cases in the survey round 4 were nevirapine (3.6%) and efavirenz (3.1%). The results indicated the need to continue surveillance for pre-treatment HIVDR, and posed challenges to implement activities for protecting efficacy and prolong the use of empirical first-line regimen. A strategy to apply genotyping test, in a cost-effective approach, should be considered to prepare for situation when HIVDR increases beyond a critical level.

Circulation of HIV-1 Multiple Complexity Recombinant Forms Among Female Sex Workers Recently Infected with HIV-1 in Thailand.

The circulating subtype distribution of HIV-1 has not been well characterized in female sex worker (FSW) populations in Thailand. To understand the mechanisms and interrelationships of epidemics involving FSWs in Thailand, we performed a large molecular epidemiological study of FSWs aged 25 years with recently acquired HIV-1 infections. The samples were collected in 2005, 2007, 2009, and 2011 in 38 provinces, representing every region of Thailand. After gag (p24), pol (pro-RT), and env (C2/V3) were sequenced, comprehensive genome analysis was performed. Genetic subtypes were determined in 159 plasma samples. The percentage of circulating recombinant forms (CRFs) CRF01_AE (90.6%) predominated, while subtype B (1.3%), other CRFs (1.9%), and unique recombinant forms (URFs) (6.2%) were identified as minor populations. Interestingly, the unique recombinant nature of these HIV-1 strains was verified in 10 specimens, indicating the presence of new forms of HIV-1 intersubtypes G/A, C/B, AE/B/C, and AE/B with different recombination breakpoints. Subtype B has contributed to these new generations of unique CRF01/B recombinants, especially in the pol (RT) gene, in which the template switching of the RT genomes occurred during reverse transcription. These results imply that the several unique recombinant viruses circulating in Thailand were probably generated in the population or introduced from neighboring countries. Our study helps clarify the patterns of viral transmission and define transmission pathways in Thailand.

Drug-resistant mutation patterns in CRF01_AE cases that failed d4T+3TC+nevirapine fixed-dosed, combination treatment: Follow-up study from the Lampang cohort.

HIV/AIDS patients are treated in Thailand's national antiretroviral treatment (ART) program with a generic combination tablet of stavudine, lamivudine, and nevirapine (GPOvir). To determine GPOvir-resistant mutations, HIV-1 sequences of 59 GPOvir-failure cases from the Lampang cohort were compared with sequences from 76 randomly selected ART-naïve cases. The GPOvir-failure cases had not only known stavudine-, lamivudine- and nevirapine-resistant mutations, but also V118I, G196E, and H221Y. Among the 59 GPOvir-failure cases, 29 were ART-naïve prior to GPOvir (naïve group), and 30 had previous ART (exposed group). To clarify the effect of previous ART in drug-resistant acquisition pathways, naïve and exposed groups were compared. The exposed group had predominantly thymidine analogue-related mutations, whereas the naïve group had a higher prevalence of Q151M and K103N mutations. M184V lamivudine resistance was most frequent in both naïve and exposed groups. To identify which mutations in CRF01_AE pol were polymorphisms, the connection and RNase domains were also analyzed. CRF01_AE-specific polymorphisms were found in 19 residues, and GPOvir-failure cases had significantly higher frequency of N348I, E399D, P537S, and I542M. Our results expand identification of mutations in CRF01_AE pol that are polymorphisms by also analyzing the connection and RNase H domains.

Demographic, socio-economic, behavioral and clinical factors predicting virologic failure with generic fixed-dose combination antiretroviral therapy before universal health insurance coverage in northern Thailand.

We conducted a 2-year prospective cohort study to investigate multiple aspects of factors predicting the outcome of fixed-dose combination antiretroviral (ARV) therapy with lamivudine, stavudine, and nevirapine (GPOvir) at a government referral hospital in northern Thailand. At 6 and 24 months after the initiation of GPOvir, viral load (VL) was measured to determine virologic failure (>400 RNA copies/ml) and demographic, socio-economic, behavioral and clinical data were collected. From 10 April 2002 to 31 January 2004, 409 patients participated in this study: 64/364 (17.0%) at 6 months and 55/345 (15%) at 24 months virologically failed treatment. On univariate analysis, besides ARV experience [odds ratio (OR), 3.08, 95% confidence interval (CI), 1.71 -5.57] and the frequency of delayed doses (OR, 2.97; 95% CI, 1.47-6.00), we identified one socioeconomic factor significantly associated with virologic failure: "not having child" (OR, 1.85; 95% CI, 1.03 - 3.34). Although the association with "not having child" became marginal on multivariate analysis, results of in-depth interviews and group discussions indicated that having a child was a strong motivating factor for good treatment compliance. We suggest that patients without children may need more attention. Further investigation of socio-economic factors is warranted.

An efficient tool for surveying CRF01_AE HIV type 1 resistance in Thailand to combined stavudine-lamivudine-nevirapine treatment: mutagenically separated PCR targeting M184I/V.

Under programs organized by the government of Thailand, HIV-1-infected patients have been treated since 2002 with several regimens, including a tablet known as GPOvir, which contains lamivudine, stavudine, and nevirapine. The aim of this study was to establish an effective assay, based on mutagenically separated PCR (MS-PCR), with the goal of surveying GPOvir-resistant HIV-1 cases. To determine the target mutation point for the assay, we analyzed the patterns of acquired drug resistance in plasma samples from GPOvir-failed cases. Of 428 HIV-1-infected individuals treated with GPOvir at Lampang Hospital in northern Thailand from 2002 to 2004, 66 had detectable viral loads after 3 months of treatment. The HIV-1 sequences of these 66 GPOvir-failed cases and 55 pre-GPOvir baseline samples were analyzed. The most prevalent drug resistance mutation among the samples was the lamivudine resistance M184I/V mutation. Based on this finding, we developed a new MS-PCR assay to detect the M184I/V mutation, and evaluated the assay performance for detecting GPOvir-resistant CRF01_AE cases. Comparing the results of M184I/V MS-PCR and sequence analyses, we found a concordance rate of 95% and an overall sensitivity of the M184I/V MS-PCR for detecting GPOvir-resistant cases of 79%. Considering the relatively low price of the assay, approximately $12.50 per sample, M184I/V MS-PCR may be a candidate for monitoring a large number of GPOvir-treated patients, particularly in developing nations.

Atypical avian influenza (H5N1).

We report the first case of avian influenza in a patient with fever and diarrhea but no respiratory symptoms. Avian influenza should be included in the differential diagnosis for patients with predominantly gastrointestinal symptoms, particularly if they have a history of exposure to poultry.

Study of antiretroviral drug-resistant HIV-1 genotypes in northern Thailand: role of mutagenically separated polymerase chain reaction as a tool for monitoring zidovudine-resistant HIV-1 in resource-limited settings.

As the number of HIV-1-infected individuals receiving antiretroviral drugs has been rapidly increasing in developing countries, there is an urgent need for drug resistance genotype information of non-B subtype HIV-1 and for the establishment of a practical system of monitoring drug-resistant viruses. This study first sequenced the reverse transcriptase region of HIV-1 in 112 infected individuals who had been treated with zidovudine (AZT)/didanosine or AZT/zalcitabine as dual therapy at a government hospital in northern Thailand and then compared the above sequence method with mutagenically separated polymerase chain reaction (MS-PCR) for detecting M41L and K70R mutations. Concordant rates of detecting M41L and K70R mutations by the 2 methods were 96.9% (93/96) and 92.7% (89/96), respectively. The M41L and K70R MS-PCR could detect 86.4% of AZT-resistant strains with any resistance mutation, which was determined by the sequencing method. Then 292 drug-naive individuals were screened for the presence of drug-resistant HIV-1 by the MS-PCR assay and it was found that 2 individuals (0.7%) carried viruses with either the M41L or K70R mutation. It is feasible to test a large number of samples with MS-PCR, which is sensitive, cheap, and easy to perform and does not require sophisticated equipment. The M41L and K70R MS-PCR is potentially a useful tool to monitor the spread of AZT-resistant HIV-1 in resource-limited countries.