*AIDS Prevention and Research Center, National Yang-Ming University, Taipei, Taiwan
†Institute of Public Health, National Yang-Ming University, Taipei, Taiwan
‡Department of Urology, Mackay Memorial Hospital, Taipei, Taiwan
§Mackay Medicine, Nursing and Management College, New Taipei City, Taiwan
‖Institute of Population Health Sciences, National Health Research Institutes, Miaoli, Taiwan
¶Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
#Department of Infectious Diseases, Taipei Veterans General Hospital, Taipei, Taiwan
**Department of Infectious Diseases, Taichung Veterans General Hospital, Taichung, Taiwan
††Department of Infectious Diseases, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
‡‡Department of Infectious Diseases, China Medical University Hospital, Taichung, Taiwan
§§Department of Microbiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
Correspondence to: Yi-Ming Arthur Chen, PhD, AIDS Prevention and Research Center, National Yang-Ming University, Li-Nong Street, Section 2, Taipei, Taiwan 112 (e-mail: email@example.com).
Supported in part by two grants from the Centers for Disease Control of the Department of Health of the Republic of China (DOH95-DC-1109 and DOH97-DC-1202) and a grant from Ministry of Education of the Republic of China (Genomic Research Center of the National Yang-Ming University, Top University and Center Grant).
Parts of this paper have been presented at the 15th International Bioinformatics Workshop on Virus Evolution and Molecular Epidemiology held in Rotterdam, the Netherlands in 2009.
The authors have no conflicts of interest to disclose.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.jaids.com).
Received September 9, 2011
Accepted December 6, 2011
The epidemiological patterns of HIV-1 infection vary widely across different Asian countries. In mainland China, where the epidemic was driven by injected drug use previously, heterosexual transmission has become the predominant mode of HIV transmission recently.1 The patterns of HIV transmission in Thailand have changed over time, with virus spreading increasingly to persons considered to be at lower risk.2 In 2007, it was estimated that female infections accounted for 29% of adults HIV infections in Asia.3 The majority of women were probably infected with HIV by their husbands or partners who had been infected either during unsafe paid sex or through injected drug use.2
According to the Taiwan Centers for Disease Control, the epidemiology of HIV-1 infection in 2004 started to increase exponentially and the major transmission route shifted from unprotected sexual intercourse to sharing syringes and diluted heroine solution.4 Due to the implementation of harm-reduction program in 2005 and 2006, the HIV-1 epidemic among injection drug users (IDUs) started to subside in 2007.5 Unfortunately, a resurgence of HIV-1 epidemic among men who have sex with men (MSM) has been observed since 2005, and the trend remained the same in the past few years (Fig. 1).
Concerning the molecular epidemiology, previous surveys reported that HIV-1 subtype B had been the major subtype from the beginning of the epidemic till 2003.6–9 Since 2004, almost all new reported cases who were IDUs had CRF07_BC which shares about 97% nucleotide sequence homology with the isolates from mainland China.8,10 We hypothesized that CRF07_BC may have been transmitted from IDUs to other risk groups through unprotected sexual intercourse and CRF07_BC/subtype B dual infection may have emerged in different risk populations. Besides, we would like to find out whether other HIV-1 strains besides subtype B have transmitted to Taiwanese MSM and caused the recent epidemic in this population? Therefore, we conducted a molecular epidemiological study on the HIV-1 subtypes among different risk groups in Taiwan from 2005 to 2008.
MATERIALS AND METHODS
From 2005 to 2008, a total of 1133 subjects were recruited from the following settings: 48 persons from Xindian drug abuse treatment center, 71 female inmates from Taoyuan Woman's prison (northern region), 137 inmates from Taichung prison, 6 persons from Nantou detention center (central region), 127 from Yunlin second prison, 8 from Chiayi detention center and 118 from Kaohsiung prison (southern region), 537 patients from the AIDS clinics of Taipei City Hospital (northern region) and 64 from China Medical University Hospital (central region), and 16 cases referred from nongovernmental organizations (see Figure S1, Supplemental Digital Content 1, http://links.lww.com/QAI/A293). Demographic data, knowledge of AIDS, drug abuse history, risk factors associated with HIV-1 transmission, and year (date) of HIV-1 diagnosis were obtained via a self-administered questionnaire. For participants who were illiterate, well-trained interviewers were provided to assist them to complete the questionnaires. Blood samples were collected for HIV-1 subtyping. Informed consent was obtained from all the participants. The study was approved by all the institutes providing the settings and the Institutional Review Board of the National Yang-Ming University, Taiwan.
HIV-1 Subtyping, Cloning, and Sequencing
DNA was extracted from peripheral blood mononuclear cells using QIAmp blood extraction kits (QIAgen,Valencia, CA). The HIV-1 subtypes were determined using the following 2 methods: (1) nested multiplex polymerase chain reaction (PCR) with specific primers for subtypes B, C, 01_AE, and 07_BC; and (2) phylogenetic analysis using nucleotide sequences of HIV-1 env (C2-V5) or pol region obtained through direct sequencing of the PCR products using a DNA analyzer (ABI 3730, Applied Biosystems, Foster City, CA). Detailed procedures of the methods mentioned above have been described previously.10
Confirmation of Dual Infection
To confirm the effectiveness of nested multiplex PCR in detecting mixed infection, 12 patients (subject No. 4V491, 4V514, 4V739, 4V765, 4V894, D733, D734, D735, D775, D966, D970, and D1007) were randomly selected from those 49 patients who had been identified to have dual infection using nested multiplex PCR. The following 2 methods were used for confirmation: (1) Gene subcloning/sequencing/phylogenetic analysis; and (2) Heteroduplex mobility assay (HMA). HIV-1 viral RNA was extracted from the plasma samples using QIAamp RNA extraction kit (Qiagen, Hilden, Germany). To generate cDNA, random primers (Promega, Sunnyvale, CA) were used for the reverse transcription. The PCR products of the env region were subcloned into pGEM-T easy vectors (Promega), and 20 colonies were selected randomly for further sequencing and phylogenetic analysis. The nucleotide sequences of the gag region of the patients were obtained from direct sequencing of the PCR products from the nested multiplex PCR using a DNA analyzer.
Heteroduplex Mobility Assay
HMA was used to confirm the existence of dual infection (see Figure S2, Supplemental Digital Content 2, http://links.lww.com/QAI/A294). The PCR products of the env gene of these 12 patients were subjected to HMA. Detailed procedures of HMA have been described previously.6
The nucleotide sequences of the patients were aligned with different reference sequences obtained from Los Alamos HIV database (http://hiv-web.lanl.gov) using the BioEdit.11 MEGA program (version 5.05)12 was used to find the best-fit nucleotide substitution model and to construct phylogenetic trees. Besides, neighbor-joining (NJ) method was also used in this study.13 To construct NJ tree, the substitution model TN93 + G model was used to calculate the evolutionary distance and followed by bootstrap value analysis with 1000 replicates.13 At least 4 strains from each HIV-1 subtypes were included for the phylogenetic analysis. SIVcpz was used as the out-group in the rooted trees. To trace the origin of the CRF01_AE or CRF07_BC stains, sequences representing isolates from different regions in the world, with special emphasis on the neighboring Asian countries, were used as reference sequences in the analysis. Bootstrap values >70 were used as an indicator of significance of the sequences clustered.
Hepatitis C Virus Antibody Test and Genotyping
In this study, 8 individuals who denied having intravenous drug use were found to be infected with CRF07_BC. To rule out whether they actually were IDUs, both anti-hepatitis C virus (HCV) antibody test (Murex 3rd, Abbott Laboratories, North Chicago, IL) and genotyping were employed. Nested multiplex PCR was used for HCV genotyping. Detailed methods have been described by our laboratory previously.14
The Pearson χ2 test and Fisher exact test were performed in univariate analysis to investigate the differences in demographic data and behavior patterns in various Taiwanese risk populations. Student t test and 1-way Analysis of Variance test were conducted to examine age differences. Multivariate logistic regression analysis was performed to identify risk factors associated with dual infection.
Nucleotide Sequence Accession Numbers
The HIV-1 env and pol sequences obtained in this study have been deposited in GenBank with accession numbers GQ231554 to GQ231593.
To understand the distribution of various HIV-1 subtypes among different risk groups after the outbreak of CRF07_BC infection in Taiwan 2002–2004, 1133 HIV-1–infected subjects were recruited from various settings from 2005 to 2008. As shown in Supplemental Digital Content 3 (see Table S1, http://links.lww.com/QAI/A295) the male to female ratio of the study population was approximately 8:1. Their mean age was 33.4 ± 7.9 years, and the male heterosexuals were significantly older than other risk groups except male IDUs (P < 0.001). Most subjects (60.8%) had received education up to senior high school, whereas male IDUs and female patients had much lower education levels (P < 0.001). About half of the subjects had experiences of traveling to foreign countries, especially in Southeast Asia. The prevalence of sexually transmitted diseases in the MSM was significantly higher than the other groups (P < 0.001). About 10% (79 of 789) of subjects had sexual contact with sex workers. The percentage of IDUs who were receiving antiretroviral therapy was significantly lower than other groups (P < 0.001).
The distribution of HIV-1 subtypes and CRFs among different high-risk groups is shown in Table 1. Subtype B was predominant in all the male risk groups except IDUs, whereas infections among Taiwanese female heterosexuals were mainly non–B subtypes (66.7% CRF01_AE, 25% subtype B, and 8.3% CRF07_BC). There was 1 male heterosexual infected with CRF08_BC and 49 (4.3%) individuals had subtype B and CRF07_BC dual infection. Most of the dual infections were seen in IDUs (45/49, 91.8%). In addition, there were 8 subjects including 4 male heterosexuals, 2 MSM and 2 female heterosexuals who denied of using intravenous drugs had CRF07_BC infection (Table 1).
Twelve of 49 specimens with dual infection had been randomly selected to confirm the effectiveness of nested multiplex PCR in detecting mixed infection. Using reverse transcriptase—polymerase chain reaction, gene subcloning and sequencing, we found that 9 specimens (4V491, 4V514, 4V739, 4V765, 4V894, D733, D734, D735, and D966) contained sequences from both subtype B and CRF07_BC (data not shown). Besides, HMA showed 10 samples (4V491, 4V514, 4V894, D733, D734, D735, D775, D966, D970, and D1007) had both subtype B and CRF07_BC signals (see Figure S2, Supplemental Digital Content 2, http://links.lww.com/QAI/A294). Altogether, all 12 cases were confirmed to have dual infection by using methods mentioned above.
Concerning risk factors for dual infection, we divided the single infection into 2 groups—CRF07_BC and subtype B, and compared the risk factors with patients dually infected with subtype B and CRF07_BC separately (Table 2; see Table S2, Supplemental Digital Content 4, http://links.lww.com/QAI/A296). Compared with male IDUs infected with CRF07_BC, those male IDUs had dual infection seems to be younger, higher educated (P = 0.012), having higher rate of traveling to mainland China (14.3% vs. 8.8%, P = 0.002), longer median years of sharing syringes, higher median frequencies of sharing syringes per month (5 vs. 2, P = 0.027), and lower rate of sharing both syringes and dissolved heroine solution (37.5% vs. 58.3%, p = 0.037) (Table 2). No risk factors were identified for dual infection when we compared the data with IDUs infected with subtype B infection. It is important to note that 15.4% (6 of 39) of IDUs with dual infection had sexual contact with female sex workers, whereas only 8.7% (36 of 415) of IDUs with single infection had such experience (Table 2; see Table S2, Supplemental Digital Content 4, http://links.lww.com/QAI/A296). Multivariate logistic regression was used to elucidate independent risk factors associated with dual infection, and the results showed that neither younger age nor higher educational level were associated with dual infection. Only sharing syringes more than 5 times per month remained to be a risk factor for dual infection and the odds ratio was 4.7 (95% confidence interval: 1.3 to 17.7) (Table 3).
In this study, we identified eight adults who denied using intravenous drugs had CRF07_BC infection. Their demographic and clinical data are summarized in Supplemental Digital Content 5 (see Table S3, http://links.lww.com/QAI/A297). In brief, 2 were married (subject No. 4V739 and C001F) and 3 (4V491, 4V845 and D682) were divorced. In terms of potential risk factors, a heterosexual male (No. 4V845) admitted to having sexual contact with sex workers and a heterosexual female admitted that her husband was an IDU (No. C001F). Because previous study showed that almost 100% of HIV-1–infected IDUs has HCV infection and many of them infected with HCV genotype 6, we analyzed their anti-HCV antibody and genotypes.10,15 The results showed that 3 cases had anti-HCV antibody and their genotypes were 6a, 1a, and 1b, respectively.
Phylogenetic trees were constructed using either maximum likelihood or NJ method with nucleotide sequences from env (C2-V5) or pol (Pro-reverse transcriptase). As shown in Figure 2A, there were 2 major groups of CRF07_BC strains in the NJ tree using nucleotide sequences from env gene. Besides, 3 CRF07_BC strains (D850, D970 and D734) had significant bootstrap value in the CRF07_BC clade, suggesting they may originate from different source. Group 1 consists of 16 strains which were diagnosed from 2002 to 2005, and group 2 consists of the remaining strains diagnosed from 2003 to 2007. Group 1 includes 3 referenced strains (D76, D118 and D120) from IDUs from Tainan DC (southern region of Taiwan), and these 3 patients have been postulated as the earliest CRF07_BC cases in Taiwan.8 These 3 cases were clustered with other 4 strains diagnosed in 2003, and their bootstrap value was 67. Besides, the following 9 strains were also found in group 1: 1 reference strain (subject D126) from IDU from Tainan DC, 5 IDUs from Taipei City Hospital, 2 from Taichung DC and Prison, and 1 reference strain (subject D338) from Yunlin second Prison. Most of the CRF07_BC strains from other regions of Taiwan were belonged to Group 2. Notably, a reference strain (subject D79) from IDU from Tainan DC was found in group 2. It is important to note that 5 cases (4V739, 4V514, 4V894, 4V491, and C001) denied having intravenous drug use behavior but had CRF07_BC infection were clustered in the group 2.
Concerning CRF01_AE infection, there were 2 groups found in this study. Group 1 consists of strains from 1992 to 2005, whereas group 2 consists of strains from 13 IDUs (9 from Yunlin second Prison and 4 from Taipei DC and Prison) with bootstrap value of 100. Group 2 were all diagnosed in 2005. Besides, there were 2 CRF01_AE from IDUs were found in group 1 (Fig. 2A).
In terms of phylogenetic analysis using nucleotide sequences from pol gene, there were 2 small clusters found in CRF07_BC strains (Fig. 2B). Besides, 1 CRF01_AE strain from IDUs (D728) were clustered with other CRF01_AE strains from heterosexual population in central region of Taiwan with bootstrap value of 89, suggesting that CRF01_AE strains may have spread to Taiwanese IDUs through unprotected sexual intercourse. In addition, a CRF08_BC strain isolated from a male heterosexual adult (4V858) clustered with other CRF08_BC strains from mainland China in both NJ trees using nucleotide sequences from either env or pol genes (Figs. 2A, B). Finally, the results of phylogenetic analysis using maximum likelihood method were similar to those using NJ method (see Figure S3, Supplemental Digital Content 6, http://links.lww.com/QAI/A298).
In this study, we conducted a molecular epidemiological survey to examine the distribution of different HIV-1 subtypes/CRFs in various risk groups in Taiwan from 2005 to 2008. Subtype B has been the major subtype of HIV-1 infection since 1988; however, CRF07_BC has become the most prevalent HIV-1 strain among Taiwanese IDUs since 2004. Furthermore, this investigation revealed that CRF07_BC may have been transmitted from IDUs to other risk groups through unprotected sexual intercourse because there were 8 adults who were not IDUs had contracted the infection. In addition, people involved in riskier behaviors may be at a higher risk of becoming dual-infection carriers. Such information has not been reported previously.
In this molecular epidemiological study, we used nested multiplex PCR with subtype-specific primers to identify mixed infections. The effectiveness of this method for identifying dual infection has been proved by 2 methods.
In this study, 60.8% of HIV-1–infected subjects had received education up to senior high school; however, male IDUs and women were less educated (see Table S1, Supplemental Digital Content 3, http://links.lww.com/QAI/A295). Previously, we reported that in Taiwan, most IDUs injected illegal drugs when they were young, which is when they are less likely to practice self-protection. Therefore, even though the period of syringe sharing among IDUs was not long, the cumulative numbers of exposures were sufficient for them to be infected with HIV.10 Overall, 11.5% (34 of 295) of IDUs had had sexual contact with sex workers (see Table S1, Supplemental Digital Content 3, http://links.lww.com/QAI/A295); however, only 62.8% (228 of 363) of subjects having sexual intercourse used condoms more than 3 of 10 times, which explained why CRF07_BC was able to spread to the other risk groups through unprotected sex.
In Taiwan, the harm-reduction programs began operating in November 2005, resulting in a decline in the numbers of newly infected HIV cases who were IDUs.4,5,16 Unfortunately, a resurgence of HIV-1 epidemic among MSM occurred in the past few years (Fig. 1). According to our phylogenetic analysis, the main subtype circulating among Taiwanese MSM was still subtype B. Therefore, further studies are needed to identify psychosocial and behavioral factors related to the HIV-1 transmission among MSM in Taiwan.
In this study, we found that men with dual infection had a longer period and greater frequently of syringe sharing than those with single infection. After adjustment, the number of times syringe sharing took place per month was associated with dual infection (Table 3).
Previously, we reported that subtype B is predominant in homosexual/heterosexual/bisexual males, while CRF07_BC is predominant among IDUs in Taiwan.8–10 In addition, the earliest group of IDUs infected with CRF07_BC was found in Taiwan in 2002.10 In this study, we found that 49 cases had CRF07_BC/subtype B dual infection. According to the questionnaires, all of them were diagnosed to have HIV-1 infection after 2003 except 1 heterosexual male (4V894) who was diagnosed to have HIV-1 infection in 2001 (see Table S3, Supplemental Digital Content 5, http://links.lww.com/QAI/A297). Therefore, we speculate that he had been infected with subtype B before he contacted CRF07_BC infection. It is interesting to note that among 10 plasmid subclones that we isolated from 4V894 patient's reverse transcriptase—polymerase chain reaction product, 3 clones contained subtype B signals and 7 clones were CRF07_BC. The dual infection may have important impact to the patients' disease progression and clinical management.
In this study, we found that there were 8 individuals who denied of any intravenous drug use history had CRF07_BC infection (see Table S3, Supplemental Digital Content 5, http://links.lww.com/QAI/A297). We used HCV genotyping to further prove that they were not IDUs because general population in Taiwan were mainly infected by genotypes 1 to 3, although genotype 6 is mainly found among IDUs.15,17 The results showed that 5 of 8 individuals mentioned above did not have anti-HCV antibody. Furthermore, among those 3 cases who had HCV infection, only 1 case (D682) had genotypes associated with IDUs—genotype 6a. Therefore, CRF07_BC infection has spread to other risk groups in Taiwan as we hypothesized. However, compared with the outbreak of HIV-1 infection among IDUs and sex workers in Thailand in 1987–1988 and its subsequent transmission to other risk groups,18–20 the scale of CRF07_BC transmission to heterosexual population in Taiwan is much smaller than we expected.
In the early 1990s, CRF01_AE was a major subtype of HIV-1 among heterosexual populations in Southeast Asia and it was believed to originate from central Africa.6,21 Subsequently, studies have indicated that there were several waves of CRF01_AE that were epidemic among IDUs in Thailand and mainland China.22,23 Previously, we have reported 10 IDUs infected with CRF01_AE who did not cluster with other CRF01_AE strains from either the heterosexual populations in Taiwan or IDU populations from mainland China or Thailand.10 However, it is worthy to note that in this study, several CRF01_AE strains from IDUs clustered with other CRF01_AE strains from Taiwanese heterosexual population (Fig. 2). Because there was a report of isolation of a unique recombinant form AE/07_BC from an IDU in Guangxi Province, mainland China,24 studies are needed to determine whether there is any such recombinant form in Taiwan.
There are several limitations to this study. The questionnaires collected in this study may be subject to recall bias. More cases of single infection than dual infections were excluded from statistical analysis due to missing data. In addition, we could not determine whether cases with dual infection were infected with subtype B or CRF07_BC first based on the date of diagnosis with HIV-1 infection. Despite these limitations, this study was able to determine that CRF07_BC may have been transmitted from IDUs to other risk groups through unprotected sexual intercourse. This study also showed the presence of dual infections in Taiwanese risk groups and that people having riskier behavior were at a higher risk of becoming dual-infection carriers. Based on these findings, which have never been reported before, health authorities in Taiwan are urged to confront these health issues immediately.
The authors wish to thank all subjects who participated in this study; peer educators and social workers for their helps in collecting the questionnaires; and staffs from the AIDS Prevention and Research Center, Genomic Research Center of the National Yang-Ming University, and from the health clinics of prisons and detention centers for their technical assistance and administrative supports.
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