Epidemiology and social
Molecular epidemiological tracing of HIV-1 outbreaks in Hainan island of southern China
Deng, Weia,b,*; Fu, Pengc,*; Bao, Linlina,b; Vidal, Nicoled; He, Qiyac; Qin, Chuana,b; Peeters, Martined; Delaporte, Ericd; Andrieu, Jean-Mariee; Lu, Weia,d
aSino-French Collaboration Center for AIDS Research, People's Republic of China
bCenter for Comparable Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tsinghua University, Beijing, People's Republic of China
cHainan Center for Disease Control and Prevention, Haikou, Hainan, People's Republic of China
dUMR145, Institut de Recherche pour le Développement and University of Montpellier 1, Montpellier, France
eInstitut de Recherche sur les Vaccins et l'Immunothérapie des Cancers et du SIDA at the Centre Biomédical des Saints-Pères, Université Paris Descartes, Paris, France.
* Wei Deng and Peng Fu contributed equally to the writing of this article.
Received 7 November, 2008
Revised 22 December, 2008
Accepted 7 January, 2009
Correspondence to Professor Wei Lu, MD, PhD, IRD-UMR145 China Branch at the Tropical Medicine Institute, 12 Jichang Road, Guangzhou, People's Republic of China. Tel: +86 20 3659 0091; fax: +86 20 3658 5321; e-mail: firstname.lastname@example.org
Objectives: To determine the prevalence of HIV in Hainan island and the molecular epidemiological linkages related to risk factors of viral transmission.
Methods: A governmental HIV/AIDS surveillance program, HIV serological study was conducted in volunteers from several high-risk groups between 1991 and 2006 in Hainan province. By phylogenetic reconstruction, we performed a molecular epidemiological tracing in a representative subset of the HIV-1-seropositive individuals diagnosed during this survey.
Results: Of 499 752 volunteers, 523 (0.1%) accumulated cases of HIV-1 infection (69.2% needle-sharing drug users, 19.3% heterosexually acquired adults, 3.3% receivers of blood transfusion, 0.8% children born from HIV-1-infected mothers, and 7.7% remained unknown) were diagnosed. Among 83 patient samples examined (70 were infected with HIV-1 subtypes CRF01_AE and eight, two, one, one, and one were B', C, CRF08_BC, B, and a new CRF01_AE/B' recombinant, respectively), 66 (79.5%) were segregated into one large cluster (59 sequences) (founder effect) and one small cluster (three sequences) of CRF01_AE, one small cluster (two sequences) of B', and one small cluster (two sequences) of C. Phylogenetic and epidemiological linkages confirmed four heterosexual transmission events and rejected two potential heterosexual transmission suggested by contact tracing. Only two cases of CRF01_AE showed mutations conferring resistance to nucleoside and nonnucleoside reverse transcriptase inhibitors.
Conclusion: The reconstruction of current HIV-1 outbreaks by molecular epidemiological tracing is helpful for identifying epidemic sources and for defining prevention strategies.
The first HIV-1 outbreak in China was reported among injecting drug users (IDUs) in Ruili city of western Yunnan's Dehong prefecture in 1989 [1,2]. Heroin use and needle sharing were the major risk factors, which led to an HIV-1 prevalence rate reaching as high as 89% among IDUs in Ruili with a provincial average of 25%. HIV-1 then spreads through drug trafficking to both neighboring and remote provinces among IDUs who then infected their sexual partners and children, leading to the exponential growth of nationwide (including all 31 provinces) outbreaks from 1998 to 2004 . Although most HIV-1-infected individuals in China are IDUs, the incidence of sexual transmission of HIV-1 increased steadily over time, with less than 5% of total HIV/AIDS cases in 1996 and up to 43.6% of HIV-1 infections by the end of 2005.
Hainan island, the far southern province of China, has 10 counties and eight cities with an estimated population of 8.3 million people. With an agreeable tropical season, Hainan is among the most popular destinations for tourists in China and has a total of 38 etnicities including Han (80%), Li (12%), and others (such as Miao, Hui, Zhuang, etc.) (8%). As being one of the major tourist destinations in China, Hainan has long been suspected to be a site of potential HIV-1 outbreaks with a dominant sexual transmission, as were the case in Thailand in the 1990s [4,5] and in Xishuangbanna of China's Yunnan province in recent years . A governmental surveillance program for conducting HIV serological studies in local volunteers with high-risk behaviors was initiated as from 1991. To assess the impact of injection drug use and sexual transmission on HIV outbreaks in Hainan, we first analyzed the serological and epidemiological data collected between 1991 and 2006 and then designed a molecular study to trace the phylogenetic and epidemiological linkages in a representative subset of HIV-1-infected individuals diagnosed during this survey.
Materials and methods
Participants and specimens
A total of 499 752 (the repeated tests over years performed on the same individuals were excluded) volunteers from several high-risk groups (including drug users, female sex workers, sexually transmitted diseases (STD) clinic attendees, long-distance truck drivers, pregnant women, men who have sex with men, clients of female sex workers, tuberculosis patients, former plasma donors, and children born from HIV-infected mothers) participated in the national HIV/AIDS surveillance system and sentinel surveillance program  in Hainan between 1 January 1991 and 31 December 2006. An enzyme immunoassay for screening HIV-1/HIV-2/O antibodies (Vironostika Uni-form II plus O kit; Organon Teknika BV, Turnhout, Belgium) in sera was performed in the Hainan Center for Disease Control and Prevention (CDC). The positive sera were confirmed by western blot (HIVBlot2.2 kit; Genelabs Diagnostics, Singapore). Participants completed standardized CDC-administered questionnaires describing risk factors, mode of transmission, ethics, sex, age, education, profession, geographic location, and potential sexual contact. No systematic storage of serum/plasma samples or peripheral blood mononuclear cells (PBMCs) was available during this survey.
Having obtained the geographic distribution of all diagnosed HIV-1-seropositive cases in Hainan, we decided to conduct a molecular epidemiological study in a representative subset of HIV-1-infected individuals. When the number of infected cases was more than 50 in a county or city, we planned to recruit randomly 20 patients; when the number of infected cases was between 20 and 50, we planned to recruit randomly 10 patients; and when the number of infected cases was less than 20, we planned to recruit up to five patients. The institutional ethics committees of both Peking Union Medical College (PUMC) and local CDC approved the study protocol. All participants signed informed consent for blood collection. To control the sampling variability, we took the blood samples of all patients within two single days (7 and 14 March 2007). Of 125 selected patients, a total of 88 HIV-1-infected individuals had finally donated their bloods for participating the study. A total of 15 ml of EDTA-treated whole blood was taken from each patient, and all samples were sent to our PUMC-based central laboratory in Beijing by flight within 12 h after sampling. The blood samples were then used immediately for routine blood count and CD4 T-cell count measurements as well as to separate plasma and PBMCs. Plasma samples were stored at −80°C while PBMC samples were resuspended in the storage buffer containing 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich Corporation, St Louis, Missouri, USA) and 50% fetus bovine serum (Invitrogen Corporation, Grand Island, New York, USA), kept at −80°C for 24 h, and then stored in liquid nitrogen.
Contact tracing was initiated before blood sampling for the molecular study and was conducted again in individuals who were found within clusters in order to confirm sexual contacts and to identify the potential source of infection. Sequence and epidemiological data were made anonymous by the assignment of irretrievable patient identifiers prior to analysis. However, clinicians and patients remained blind to the laboratory data and vice versa.
Viral load measurement
All plasma samples were thawed at the same time and were used for viral RNA measurement using the Food and Drug Administration (FDA)-approved Amplicor HIV-1 Monitor Test kit (version 1.5) (Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) according to the manufacturer's instructions.
PCR, subtyping and resistance mutations
Viral RNA was extracted from the patients' plasma using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, California, United States) according to the manufacturer's instructions. The viral RNA was then subjected to reverse transcription polymerase chain reaction (RT-PCR). On the basis of the reference sequences obtained from the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID)-funded HIV database, the genetic subtypes were identified in pol gene (1864 base pairs) spanning protease and reverse transcriptase regions and in the V3–V5 region of the env gene (660 base pairs) as previously described . The newly obtained sequences were aligned under ClustalX with reference sequences representing overall HIV-1 group M genetic variability. We included all pure subtypes, subsubtypes, Asian circulating recombinant forms (CRFs) references (01, 07, 08, 15, 33, 34), and also some other CRFs prevalent elsewhere but reported to circulate sometimes at low level in Asia, such as CRF02 and CRF13. Phylogenetic analysis was first conducted for each new sequence individually. A general tree was then made with all new sequences and drawn with the minimal number of references, that is, excluding those that were not represented in this study. The pol fragments, of sufficient length, were analyzed using Simplot version 3.5.1 software (http://sray.med.som.jhmi.edu/SCRoftware) to determine if the new sequences were recombinant. Genotypic data identified minor and major resistance mutations in protease and reverse transcriptase genes, based on the last updated (October 2007) guidelines from the International AIDS Society Resistance Testing – USA panel (http://www.iasusa.org/resistence_mutations/index.html). Three drug resistance interpretation algorithms (ANRSV2007.10, HIVDB, RegaV7.1.1) were used and compared in line on the Stanford Resistance Database tool: HIValg version 4.3.0 (http://hivdb.stanford.edu/pages/algs/HIValg.html). In addition and in accordance with the latest WHO recommendations, resistance mutations were also evaluated with the last updated list for surveillance of transmitted drug-resistant strains in untreated patients (http://cpr.stanford.edu/cpr/).
Phylogenetic inter-relationships among viral sequences were estimated using neighbor-joining trees and maximum likelihood methods by using PHYML 2.4.4 integrated molecular evolutionary genetics analysis software . The maximum likelihood analysis used the HKY85 model of nucleotide substitution, including gamma-distributed rate heterogeneity among sites. Maximum likelihood bootstrap values were obtained for 1000 replicates. The existence of transmission clusters was determined using the statistical robustness of the maximum likelihood topologies assessed by high bootstrap values (>98%) with 1000 resamplings and short branch lengths (genetic distances <0.015) of HIV-1 pol gene sequence .
Differences in the CD4 T-cell count and viral load among different subgroups of patients were determined using a Mann–Whitney nonparametric test.
HIV prevalence in Hainan
Of 499 752 volunteers participating in the government-supported HIV/AIDS serological surveillance program in Hainan between 1991 and 2006, 523 (0.1%) cases were confirmed to be infected with HIV-1. As many as 69.2% (362 out of 523) of HIV-1-infected cases acquired the virus by injection drug using while 19.3% (101 out of 523) of cases were infected by heterosexual transmission, 3.3% (17 out of 523) were infected by blood transfusion, 0.8% (4 out of 523) were children born from HIV-1-infected mothers, and 7.7% (40 out of 523) remained unknown. Individuals with HIV-1 infection identified during this survey were mainly male patients (85.7%), Han ethnics (53.9%), 20–39-year-olds (85%), and had low levels (below high school) of education (88%) (Table 1). HIV-1-seropositive cases were located in 14 out of the 18 counties/cities with a domination of south-east and north-east coast (Fig. 1a) with a nearly 10-fold increase of year-diagnosis rate as from 2002 (Fig. 1a).
Characteristics of participants for the molecular study
The blood samples were collected from a representative set of 88 HIV-1-infected individuals located in 13 out of the 18 counties/cities in Hainan. The epidemiological characteristics of the 88 participants (Table 2) were comparable with that of the overall 523 cases diagnosed in Hainan (Table 1). Of 88 participants, 10 were currently on antiretroviral combination therapy with nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) (#2, #4, #5, #11, #19, #24, #25, #29, #41 and #68). Apart from five cases (#1, #16, #17, #26 and #27) in which the PCRs were repeatedly negative in pol and env despite a sufficient viral load, the HIV-1 genotyping tests were successfully performed in the remaining 83 samples.
Identification of viral subtypes
Of 83 patients samples examined in pol, 70 (84.3%) were infected with HIV-1 subtypes CRF01_AE, eight (9.6%) with the B′ variant, two (2.4%) with C subtype, one (1.2%) with B subtype, one (1.2%) represented CRF08_BC, then one (1.2%) constituted a new CRF01_AE/B′ recombinant, respectively. No obvious geographic correlation was observed. The same subtypes were obtained in the pol and env regions, with one exception (#7) which was CRF01_AE/B′ recombinant in pol but clearly subtype B′ in env. Only two cases (#2 and #11) of CRF01_AE who were currently under treatment showed primary mutations conferring resistance to NRTIs and NNRTIs as analyzed with three different algorithms but remained sensitive to antiprotease inhibitors. The 73 patients that were not under ARV treatment were submitted to a PCR analysis (http://cpr.stanford.edu/cpr/) for the surveillance of transmitted drug resistance, as developed recently . No SDRM-2008 mutation was detected, only a few borderline/suspicious mutations have been detected in the reverse transcription for six patients (K223Q, n = 4; T69S, n = 1; V108I, n = 1). The peripheral blood CD4 T-cell counts (mean ± SD, 303 ± 230 cells/μl) have an inverse correlation (R2 = 0.271, P < 0.001) to the plasma viral loads (geometric mean ± SE, 37 810 ± 40 958 RNA copies/ml). The 36 (40.9%) cases with a CD4 T-cell count (cells/μl) less than 200 [eligible for antiretroviral therapy (ART)] received antiretroviral drugs thereafter (Table 3). No difference in the CD4 T-cell count or in the viral load among different subgroups of patients was observed.
Identification of transmission clusters
Of the 83 pol sequences analyzed, 66 (79.5%) segregated into four clusters (clusters 1–4) having sequence similarity based on the established criteria of high bootstrap values (>98%) and short genetic distances (<0.015), whereas the remaining 17 cases (20.5%) represented nonclustered sequences in the maximum likelihood pol tree. Of the four transmission clusters, cluster 1 was very large including 59 sequences of CRF01_AE, whereas clusters 2–4 were quite small with three, two, and two sequences of CRF01_1E, C, and B′, respectively (Fig. 2a). Of the four identified transmission clusters in the pol gene, only one (cluster 3) had a relatively low boot strap value (<90%), and the three others had a high bootstrap value (>98%) in the maximum likelihood env tree (Fig. 2b). Only one nonclustered transmission event (#25) defined by the maximum likelihood pol tree displayed a bootstrap value of more than 90% in the env tree.
Of six potential heterosexual transmission pairs (#3/#4, #6/#44, #7/#9, #22/#23, #24/#25, and #81/#82) initially presumed by contact tracing, four pairs (#3/#4, #6/#44, #22/#23, and #81/#82) were strongly supported by phylogenetic reconstruction. One pair (#24/#25) was rejected because the bootstrap value (94.8%) and the genetic distances (0.018) in pol were below the cut-off values, and also the two strains had a separate clustering in env (#25 was located inside the ‘cluster 1’ and #24 was not). One more pair (#7/#9) was rejected because the sequences did not cluster together in pol; however, they clustered together in env despite a low bootstrap value. As one of them (#7) was subtyped as CRF01_AE/B′ in pol, one cannot exclude the possibility that a superinfection with the CRF01_AE variant occurred in this patient, leading to recombination between CRF01_AE and B′ infecting strains and subsequently to the emergence of the recombinant strain as the major infecting virus. As the phylogenetic reconstruction is based on the major sequences analysis over the whole fragment, it rejected this presumed contact tracing relationship at first. To further elucidate the phylogenetic relationships between the two cases #7 and #9, we cut the pol alignment at the breakpoint in which #7 is no more CRF01_AE but B′ and took the alignment segment corresponding to the B′ subtype shared by the two samples over 1252 unambiguously aligned nucleotides. A maximum likelihood analysis under the criteria previously used in this study no more rejected the initially presumed contact tracing relation. The two samples were epidemiologically linked as the genetic distance was 0.005 nucleotides and the bootstrap value 993.
No additional sexual transmission pair was identified by phylogenetic reconstruction. One huge (53 sequences in cluster 1) and one small (three sequences in cluster 2) IDU transmission clusters were identified by phylogenetic reconstruction.
Since the first HIV-1 outbreak in 1989, the mainland China has today a national figure of over 650 thousands of HIV-1-infected people  with diverse subtypes including B, B′, C, CRF07_BC, CRF08_BC, and CRF01_AE [10–14]. Recent studies have reported that CRF01_AE became a dominant subtype in Yunnan province mainly through sexual transmission [6,15]. In present study, we demonstrated a dominant HIV-1 CRF01_AE outbreak (84%) mainly among the IDUs in Hainan, suggesting that this subtype may indeed surpass the previously documented major subtypes (B′, C, CRF07_BC, or CRF08_BC) and lead to a new epidemic in China.
Phylogenetic analysis of viral gene sequences has recently been used to construct direct or indirect epidemiological links in geographically defined populations with acute/primary or chronic HIV-1 infection [16–19]. These recent findings underscore the importance of tracing the molecular linkage of HIV transmission to better understand the population-based epidemic nature/trend and to define an efficient strategy to control the epidemics in the region. Phylogenetic reconstruction of HIV-1 pol gene allows us to identify nearly 80% (66 out of 83) epidemiological links in the representative samples of Hainan's HIV-1-seropositive individuals. In the striking contrast to our previous findings in Xishuangbanna where only small sizes of clusters (2–4) were observed [6,15], a very huge cluster (59) representing 89% of all clustered samples was identified. The finding of significant subclustering of these sequences within CRF01_AE clade both in pol and env raises issues unique to a geographically defined outbreak, such as that occurs on islands. The specific differentiation of this cluster from the other CRF01_AE sequences is most likely due to a founder effect. Founder effects of one particular HIV-1 variant have already been demonstrated in many areas in the world [20–26]. Evidence of a strong founder effect of one particular HIV-1 strain and variant among IDUs was also seen in the Netherlands (subtype B variant), Russia (CRF03_AB and subtype A), China (CRF07_BC and CRF08_BC), northern Vietnam (CRF01_AE variant), and Myanmar (B′ variant). Genetic distances were very low in the cluster 1, indicating a high homogeneity of the strains concomitant with a low evolutionary rate and a fast-spreading epidemic . In our case, of the 59 closely clustered cases, 53 (90%) contracted HIV-1 through IDU transmission as confirmed by contact tracing. Ten percent of the closely related cases were contracted through another mode of transmission thus suggesting that IDUs could potentially serve as a bridge between a high-risk group and a lower-risk population, such as heterosexually active adults that constitute the second HIV-infected group in Hainan. Given that the prevalence of HIV infection is still low (0.1%) in Hainan, an efficient management of local IDUs could be the key strategy for holding back the epidemic in this region.
Phylogenetic means are also widely used to infer or reject transmission links in suspected cases by contact tracing [28–31]. In this regard, our results, in keeping with our previous findings [6,15], strongly support that phylogenetic analysis is a powerful mean for providing molecular evidence to approve or reject close linkage in suspected transmission events, or eventually suggest new linkage of potential transmission. Although the potential sexual transmissions in two couples (#7/#9 and #24/#25) were suggested by contract tracing, phylogenetic analysis rejected such linkages by distinct genotyping and maximum likelihood trees. However, the epidemiological linkage between #7, which was classified as a recombinant, and #9 was subsequently demonstrated in the pol region constituted with the same subtype. It is thus important to carefully check the recombinant nature of the studied strains and to analyze a second genomic region that may bring useful information over the presumed linkage.
Taken together, our findings illustrate that HIV-1 CRF01_AE is becoming a major subtype accounting for current HIV outbreaks in China. Provided that nearly 80% of people with HIV-1 infection are undiagnosed and that there is an increasing mobility of population (vast majority are peasants) in China, a considerable large number of undiagnosed persons with HIV-1 infection may represent potential sources leading to new nationwide HIV outbreaks through sharing syringe needle, unprotected sex, particularly in poorly educated populations. The molecular epidemiological information provided by the present study is critical not only for the design of prevention policies but also for guiding the design of vaccine strategies in the targeting region .
All sequences analyzed in the present study are deposited in EMBL under the accession numbers FM251948-FM252030 and FM865453-FM865531 for the pol and env sequences, respectively.
We thank all participants for their enthusiastic cooperation. We acknowledge Ellène Gozard, Véronique Jagot, Alain Tiafvoon, Ju Yuan, and Liang Yu for technical assistance. This study was supported by Chinese Ministry of Science and Technology (grant 2005AA218020 to W.L.), Institut de Recherche pour le Développement, and Institut de Recherche sur les Vaccins et l'Immunothérapie des Cancers et du SIDA.
W.L. was responsible for the overall study design, organization, data analyses, and writing of the report. W.D., P.F., L.B., N.V., and Q.H. participated in design and implementation of the study. C.Q., M.P., E.D., and J.-M.A. contributed to discussions on study design and interpretation of the findings.
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