Cocirculation of subtype B and CRF01_AE in Southeast Asia has led to the establishment of new recombinant forms. In our previous study, we found five samples suspected of being recombinants between subtype B and CRF01_AE, and here, we analyzed near full-length sequences of two samples and compared them to known CRFs_01B, subtype B, and CRF01_AE. Five overlapped segments were amplified with nested PCR from PBMC DNA, sequenced, and analyzed for genome mosaicism. The two Indonesian samples, 07IDJKT189 and 07IDJKT194, showed genome-mosaic patterns similar to CRF33_01B references from Malaysia, with one short segment in the 3' end of the p31 integrase-coding region, which was rather more similar to subtype B than CRF01_AE, consisting of unclassified sequences. These results suggest gene-specific continuous diversification and spread of the CRF33_01B genomes in Southeast Asia.
HIV infection is a major problem in Indonesia. The number of people living with HIV has been increasing from year to year, especially among injecting drug users (IDUs). Since there were only limited data about molecular epidemiology profiles of HIV/AIDS in Indonesia, a cross-sectional study involving 208 HIV-1-seropositive individuals was conducted in 2007 in Jakarta. The majority of participants were 16-30 years of age (64.9%) and 74.5% were male. The most frequent risk factor was injecting drug use (IDU) (45.7%) followed by heterosexual transmission (34.1%). Phylogenetic analysis of gag (p17 and p6) and env C2V3 regions showed 200 (96.2%) of 208 DNA samples were CRF01_AE and only 3 (1.4%) were subtype B. Five samples (2.4%) indicated discordant subtypes between the three aforementioned regions: three of them showed unique CRF01_AE/B recombination patterns in 2.3-kbp nucleotide sequences (from p17 to part of RT), including one sample showing similarity to CRF33_01B, reported previously in Malaysia. This study shows the current predominant subtype is CRF01_AE in every risk group, with a decreasing number of pure subtype B, and the first identification of CRF01_AE/B recombinant forms among HIV-1-seropositive Indonesians.
A new HIV-1 circulating recombinant form (CRF), CRF33_01B, has been identified in Malaysia. Concurrently we found a unique recombinant form (URF), that is, the HIV-1 isolate 06MYKLD46, in Kuala Lumpur, Malaysia. It is composed of B or a Thai variant of the B subtype (B') and CRF01_AE. Here, we determined the near full-length genome of the isolate 06MYKLD46 and performed detailed phylogenetic and bootscanning analyses to characterize its mosaic composition and to further confirm the subtype assignments. Although the majority of the 06MYKLD46 genome is CRF01_AE, we found three short fragments of B or B' subtype inserted along the genome. These B or B' subtype regions were 716 and 335 bp, respectively, in the protease-reverse transcriptase (PR-RT) region, similar to those found in CRF33_01B, as well as an extra 590 bp in the env gene region. Thus we suggest that 06MYKLD46 is a possible second-generation HIV-1 recombinant derived from CRF33_01B.
To assess the prevalence of mutations associated with drug resistance in antiretroviral-naive patients in Kuala Lumpur, Malaysia, genotypic resistance testing was conducted among drug-naive HIV-1 patients attending the University Malaya Medical Center (UMMC) between July 2003 and June 2004. Reverse transcriptase (RT) and protease genes of plasma virions were sequenced from 100 individuals. The majority of the patients were recently diagnosed. Codons 20-255 of the RT and 1-96 of the protease gene were examined for major and minor mutations associated with antiretroviral resistance reported by the International AIDS Society- USA (IAS-USA) Drug Resistance Mutations Group. The prevalence of patients with at least one major mutation conferring drug resistance was 1%, with only one patient having a Y181C amino acid substitution in the RT gene that confers high-level resistance to nevirapine and delavirdine. Minor mutations were detected in high prevalence in the protease gene. Amino acid substitutions I13V, E35D, and M36I were associated with CRF01_AE while L63P, V77I, and I93L were associated with subtype B. Baseline prevalence of major mutations associated with resistance to antiretroviral drugs was low among antiretroviral-naive HIV-1 patients, suggesting that routine drug resistance testing may be unnecessary for all individuals newly diagnosed with HIV or all patients beginning antiretroviral therapy.
Many HIV-infected individuals do not enter health care until late in the infection course. Despite encouraging earlier testing, this situation has continued for several years. We investigated the prevalence of late presenters and factors associated with late presentation among HIV-infected patients in an Asian regional cohort. This cohort study included HIV-infected patients with their first positive HIV test during 2003-2012 and CD4 count and clinical status data within 3 months of that test. Factors associated with late presentation into care (CD4 count <200 cells/μl or an AIDS-defining event within ±3 months of first positive HIV test) were analyzed in a random effects logistic regression model. Among 3,744 patients, 2,681 (72%) were late presenters. In the multivariable model, older patients were more likely to be late presenters than younger (≤30 years) patients [31-40, 41-50, and ≥51 years: odds ratio (OR) = 1.57, 95% confidence interval (CI) 1.31-1.88; OR = 2.01, 95% CI 1.58-2.56; and OR = 1.69, 95% CI 1.23-2.31, respectively; all p ≤ 0.001]. Injecting drug users (IDU) were more likely (OR = 2.15, 95% CI 1.42-3.27, p < 0.001) and those with homosexual HIV exposure were less likely (OR = 0.45, 95% CI 0.35-0.58, p < 0.001) to be late presenters compared to those with heterosexual HIV exposure. Females were less likely to be late presenters (OR = 0.44, 95% CI 0.36-0.53, p < 0.001). The year of first positive HIV test was not associated with late presentation. Efforts to reduce the patients who first seek HIV care at the late stage are needed. The identified risk factors associated with late presentation should be utilized in formulating targeted public health intervention to improve earlier entry into HIV care.
We investigated the molecular epidemiology of HIV-1 subtypes in Malaysia among injecting drug users (IDUs) and sexual transmission risk groups, using serologic and genetic techniques. Frozen sera collected at a general hospital, a blood bank, several drug treatment centers, and an STD clinic in Kuala Lumpur, between 1992 and 1996, were investigated retrospectively. V3 peptide serotyping and monomeric gp120 capture serotyping were used to study 89 known HIV-1-infected subjects. The methods differentiate subtypes B, E, and C. V3 peptide and gp120 capture results were comparable. No subtype C-specific reactive sera were found; one specimen was dually reactive for subtypes C and B, using the V3 peptide ELISA; and four were durally reactive for subtypes E and C using this assay. Genotypic analysis of HIV-1 gag RNA in serum was done on a subset of subjects and confirmed serologic findings. HIV-1 subtypes differed significantly by risk category: of 53 IDUs, 29 (55%) were infected with subtype B and 19 (36%) were infected with subtype E, 3 (6%) were dually reactive, and 2 (4%) were not typable. Of 36 persons with heterosexual risks, 29 (81%) were infected with subtype E, 5 (14%) were infected with subtype B, and 2 (5%) were not typable. Persons with IDU risks were significantly more likely to be infected with subtype B than were those with sexual risks (OR 5.89; 95% CI, 1.94-18.54; p < 0.001). Subtypes B and E of HIV-1 appear to predominate in Malaysia; subtype B was more prevalent among IDUs; subtype E was more prevalent among all other groups. These results may have important HIV-1 vaccine implications.
Zidovudine (ZDV) is converted to its active triphosphate (ZDVTP) by intracellular kinases. The intermediate ZDV monophosphate (ZDVMP) is believed to play a major role in ZDV toxicity. Manipulation of ZDV phosphorylation is a possible therapeutic strategy for altering the risk-benefit ratio. Here we investigate whether combining RBV with ZDV is able to modulate efficacy and toxicity of ZDV. We have measured the intracellular activation of ZDV (0.3 microM) in the absence and presence of ribavirin (RBV; 2 and 20 microM) in Molt 4 and U937 cells. MTT cytotoxicity of ZDV (10-1000 microM) was also measured with and without RBV (2 microM) in Molt 4 and U937 cells. Measurement of endogenous deoxythymidine triphosphate (dTTP) allowed investigation of the dTTP/ZDVTP ratio. The antiviral efficacy of ZDV in combination with RBV (2 microM) was assessed by HIV p24 antigen measurements. In the presence of RBV (2 and 20 microM) a decrease in total ZDV phosphates was observed, owing mainly to an effect primarily on ZDVMP rather than the active ZDVTP. RBV also increased endogenous dTTP pools in both cell types, resulting in an increase in the dTTP/ZDVTP ratio. ZDV alone significantly reduced p24 antigen production, with an IC50 of 0.34 microM. Addition of RBV increased the IC50 approximately fivefold (1.52 microM). However, at higher concentrations of ZDV (10 and 100 microM) the antagonistic effect of RBV (2 microM) on ZDV was lost. The RBV-mediated decrease in ZDVMP may explain the reduction in ZDV toxicity when combined with RBV (2 microM). Cytotoxicity of ZDV was reduced in the presence of RBV (2 microM) at all concentrations in both cell lines, probably owing to saturation of ZDVTP formation. The interaction of ZDV and RBV is concentration dependent.
Global surveillance of HIV-1 subtypes for genetic characterization is hampered by the biohazard of processing and the difficulties of shipping whole blood or cells from many developing country regions. We developed a technique for the direct automated sequencing of viral DNA from dried blood spot (DBS) specimens collected on absorbent paper, which can be mailed unrefrigerated in sturdy paper envelopes with low biohazard risk. DBS were collected nonrandomly from HIV-1-infected, mostly asymptomatic, patients in five Asian countries in 1991, and shipped via airmail or hand carried without refrigeration to Bangkok, and then transshipped to North America for processing. After more than 2 years of storage, including 6 months at ambient temperatures, proviral DNA in the DBS was amplified by nested PCR, and a 389-nucleotide segment of the C2-V3 env gene region was sequenced, from which 287 base pairs were aligned and subtyped by phylogenetic analysis with neighbor-joining and other methods. From southern India, there were 25 infections with subtype C and 2 with subtype A. From Myanmar (Burma), we identified the first subtype E infection, as well as six subtype BB, a distinct cluster within subtype B that was first discovered in Thailand and that has now appeared in China, Malaysia, and Japan. From southwest China, one BB was identified, while a "classical" B typical of North American and European strains was found in Indonesia. From Thailand, five DBS of ambiguous serotype were identified as three B, one BB, and one E. A blinded control serotype E specimen was correctly identified, but a serotype BB control was not tested. Most HIV-1 in southern India appears to be env subtype C, with rare A, as others have reported in western and northern India. The subtypes BB and E in Myanmar, and the BB in China, suggest epidemiological linkage with these subtypes in neighboring Thailand. DBS are a practical, economical technique for conducting large-scale molecular epidemiological surveillance to track the global distribution and spread of HIV-1 variants.
Earlier studies in the 1990s indicate that human immunodeficiency virus type 1 (HIV-1) subtype B has been the predominant subtype among injecting drug users (IDUs) in Malaysia. More recent studies performed between 2003 and 2004, however, show a high prevalence of unique CRF01_AE/B intersubtype recombinants among IDUs. To determine the subtype distribution among IDUs in Kuala Lumpur prior to the emergence of CRF01_AE/B intersubtype recombinants, the gag-pol or the reverse transcriptase gene was sequenced from IDUs who were diagnosed as HIV positive between 1993 and 2002. Subtype B was present at 50.0% followed by CRF01_AE/B recombinant at 41.7%, with more CRF01_AE/B recombinants detected between 2000 and 2002. All CRF01_AE/B recombinants shared similar recombination patterns. Interestingly, we found that this potential new candidate of circulating recombinant form (CRF) could have emerged as early as the mid-1990s. The results showed evidence of changing HIV-1 molecular epidemiology toward the predominance of CRF01_AE/B intersubtype recombinants among IDUs in Kuala Lumpur.
We investigate mutations and correlates according to HIV-1 subtype after virological failure (VF) of standard first-line antiretroviral therapy (ART) (non-nucleoside/nucleotide reverse transcriptase inhibitor [NNRTI] +2 nucleoside/nucleotide reverse transcriptase inhibitor [N(t)RTI]). SECOND-LINE study participants were assessed at baseline for HIV-1 subtype, demographics, HIV-1 history, ART exposure, viral load (VL), CD4(+) count, and genotypic ART resistance. We used backward stepwise multivariate regression (MVR) to assess associations between baseline variables and presence of ≥3 N(t)RTI mutations, ≥1 NNRTI mutation, ≥3 thymidine analog-N(t)RTI [ta-N(t)RTI] mutations (TAMs), the K65/K70 mutation, and predicted etravirine (ETV)/rilpivirine (RPV) activity. The inclusion p-value for MVR was p .05. Of 541 participants, 491 (91%) had successfully characterized baseline viral isolates. Subtype distribution: B (n = 123, 25%), C (n = 202, 41%), CRF01_AE (n = 109, 22%), G (n = 25, 5%), and CRF02_AG (n = 27, 5%). Baseline CD4(+) 200-394 cells/mm(3) were associated with <3 N(t)RTI mutations (OR = 0.47; 95% CI 0.29-0.77; p = .003), absence of the K65/K70 mutation (OR = 0.43; 95% CI 0.26-0.73; p = .002), and higher ETV sensitivity (OR = 0.52; 95% CI 0.35-0.78; p = .002). Recent tenofovir (TDF) use was associated with K65/K70 mutations (OR = 8.91; 95% CI 5.00-15.85; p
To date, there are 16 types of CRF01_AE/B circulating recombinant forms identified, and most of them are distributed in Asian countries such as China, Malaysia, and Singapore. Previous HIV molecular epidemiological surveys showed that CRF01_AE (27.6%) and B (9.6%) subtypes are predominant strains in mainland of China. At the same time, the HIV-1 virus spreads faster in the men who have sex with men (MSM) population than in other risk groups. In Shanghai district, ∼66.0% of newly reported cases were infected through homosexual transmission. In this study, we report a novel recombinant strain of CRF01_AE/B. The near full-length genome phylogenetic tree showed that the strain clustered with the CRF01_AE reference sequence and placed in the peripheral position within the branch of the CRF01_AE strain. Subregional evolutionary results indicated that the CRF01_AE subtype was derived from cluster 4 of CRF01_AE, which is mainly distributed in northern China. The subtype B was correlated with the U.S./Europe B, which are widely prevalent in the Chinese MSM population. In recent years, a large number of recombinant forms between CRF01_AE and B strains are continuously emerging in China. Therefore, understanding the current epidemic recombinant forms will have significant implications for prevention and treatment of HIV/AIDS.
We sought to assess the impact of routine HIV viral load (VL) monitoring on the incidence of switching from a first- to a second-line antiretroviral therapy (ART) regimen, and to describe factors associated with switch. Data from a regional cohort of 16 clinical programs in six Asian countries were analyzed. Second-line switch was defined as a change from a non-nucleoside reverse transcriptase inhibitor (NNRTI) to a protease inhibitor (PI) or vice versa, and ≥1 of the following: (1) reported treatment failure by local criteria, (2) switch of ≥1 additional drug, or (3) a preceding HIV VL ≥1,000 copies/ml. Routine VL was having ≥1 test after ≥24 weeks of ART and ≥1 time/year thereafter. Factors associated with time to switch were evaluated with death and loss to follow-up as competing risks. A total of 2,398 children were included in this analysis. At ART initiation, the median (interquartile range) age was 6.0 (3.3-8.9) years, more than half had WHO stage 3 or 4, the median CD4 was 189 (47-456) cells/mm3, 93% were on NNRTI-based first-line ART, and 34% had routine VL monitoring. Treatment switch occurred in 17.6% of patients, at a median of 35 (22-49) months. After adjusting for country, sex, first ART regimen, and CD4% at ART initiation, children with routine VL monitoring were 1.46 (95% confidence interval 1.11-1.93) times more likely to be switched (p = .007). Scale-up of VL testing will lead to earlier identification of treatment failure, and it can help guide earlier switches to prevent resistance.