Evolutionary Dynamics and Complicated Genetic Transmission Network Patterns of HIV-1 CRF01_AE among MSM in Shanghai, China.

To explore the evolutionary dynamics and molecular transmission patterns of HIV-1 CRF01_AE in depth among men who have sex with men (MSM) in Shanghai, we constructed phylogenetic tree and genetic transmission networks based on 1, 152 pol sequences from MSM, 282 from other risk groups and 795 references. Phylogenetic analyses identified two distinct major CRF01_AE lineages and a Shanghai-based sub-lineage. The estimated tMRCAs for lineage 1 and 2 were 1996.0 (1992.9-1999.2) and 1997.8 (1994.3-2001.4), respectively. Of the 1, 152 MSM, 681 (59.1%) were identified as belonging to 241 separate networks. Of these 681 individuals in networks, 74.2% were linked to cases diagnosed in different years, 4.3% were linked to heterosexual women, and 0.7% were linked to persons who inject drugs. A total of 71 networks including 180 individuals diagnosed in Shanghai with the same domicile were found. Recent infection (P = 0.022) and sampling year after 2011 (P < 0.001) were significantly associated with potential transmission links among the networks. Besides, a significant transmission of viruses with drug resistant mutations at V179D/E were found in the networks. Given these findings, we propose that genetic transmission analysis is a useful tool in HIV intervention strategies to curb the spread of virus and promoting public health.

Design and implementation of a sexual health intervention for migrant construction workers situated in Shanghai, China.

BACKGROUND: China's growing population of internal migrants has exceeded 236 million. Driven by rapid development and urbanization, this extreme population mobility creates opportunities for transmission of HIV and sexually-transmitted infections (STI). Large numbers of rural migrants flock to megacities such as Shanghai in search of employment. Although migrants constitute a key population at heightened risk of acquiring HIV or an STI, there is a lack of easily accessible sexual health services available for them. In response, we designed a short, inexpensive sexual health intervention that sought to improve HIV and STI knowledge, while reducing stigma, risky sexual behaviour, and sexual transmission of HIV and STI among migrant construction workers (MCW) situated in Shanghai, China. RESULTS: We implemented a three-armed, community-randomized trial spread across three administrative districts of Shanghai. The low-intensity intervention included educational pamphlets. The medium-intensity intervention included pamphlets, posters, and videos. The high-intensity intervention added group and individual counselling sessions. Across 18 construction sites, 1871 MCW were allocated at baseline to receive one intervention condition. Among baseline participants, 1304 workers were retained at 3-months, and 1013 workers were retained at 6-months, representing a total of 579 person-years of follow-up. All workers, regardless of participation, had access to informational materials even if they did not participate in the evaluation. Overall outputs included: 2284 pamphlets distributed, 720 posters displayed, 672 h of video shown, 376 participants accessed group counselling, and 61 participants attended individual counselling sessions. A multivariable analysis of participation found that men (aOR = 2.2; 95 % CI 1.1, 4.1; p = 0.036), workers situated in Huangpu district (aOR = 5.0; 95 % CI 2.6, 9.5; p < 0.001), and those with a middle school education (aOR = 1.9; 95 % CI 1.2, 3.0; p = 0.01) were more likely to have participated in intervention activities. CONCLUSION: A brief educational intervention that prioritized ease of delivery to a highly mobile workforce was feasible and easily accessed by participants. Routine implementation of sexual health interventions in workplaces that employ migrant labour have the potential to make important contributions toward improving HIV and STI outcomes among migrant workers in China's largest cities.

HIV-1 Genetic Diversity and Its Impact on Baseline CD4+T Cells and Viral Loads among Recently Infected Men Who Have Sex with Men in Shanghai, China.

The HIV-1 epidemic among men who have sex with men (MSM) has been spreading throughout China. Shanghai, a central gathering place for MSM, is facing a continuously increasing incidence of HIV-1 infection. In order to better understand the dynamics of HIV-1 diversity and its influence on patient's immune status at baseline on diagnosis, 1265 newly HIV-1-infected MSM collected from January 2009 to December 2013 in Shanghai were retrospectively analyzed for genetic subtyping, CD4+T cell counts, and viral loads. HIV-1 phylogenetic analysis revealed a broad viral diversity including CRF01_AE (62.13%), CRF07_BC (24.51%), subtype B (8.06%), CRF55_01B (3.24%), CER67_01B (0.95%), CRF68_01B (0.4%), CRF08_BC (0.08%) and CRF59_01B (0.08%). Twenty-four unique recombination forms (URFs) (1.98%) were identified as well. Bayesian inference analysis indicated that the introduction of CRF01_AE strain (1997) was earlier than CRF07_BC strain (2001) into MSM population in Shanghai based on the time of the most recent common ancestor (tMRCA). Three epidemic clusters and five sub-clusters were found in CRF01_AE. Significantly lower CD4+T cell count was found in individuals infected with CRF01_AE than in those infected with CRF07_BC infection (P<0.01), whereas viral load was significantly higher those infected with CRF01_AE than with CRF07_BC (P<0.01). In addition, the patients with >45 years of age were found to have lower CD4+T cell counts and higher viral loads than the patients with <25 years of age (P<0.05). This study reveals the presence of HIV-1 subtype diversity in Shanghai and its remarkable influence on clinical outcome. A real-time surveillance of HIV-1 viral diversity and phylodynamics of epidemic cluster, patient's baseline CD4+T cell count and viral load would be of great value to monitoring of disease progression, intervention for transmission, improvement of antiretroviral therapy strategy and design of vaccines.

Impact evaluation of a community-based intervention to reduce risky sexual behaviour among female sex workers in Shanghai, China.

BACKGROUND: Female sex workers (FSWs) are at risk for sexually transmitted infections (STIs), including HIV. We implemented an HIV/STI preventive intervention among FSWs in Shanghai that aimed to increase condom use, improve HIV knowledge, and reduce STI and HIV incidence. METHODS: From six districts in Shanghai, 750 randomly selected venue-based FSWs were allocated to either a behavioural intervention or control group. In the intervention and control groups, 221 and 278 participants, respectively, had at least one follow-up at three or six months. In analysis, we randomly selected 57 lost to follow-up cases in the intervention group and imputed baseline values to equalize the arms at n = 278 (74.1% follow-up rate in each group). The impacts of the intervention on condom use, HIV/STI risk perception and knowledge, and STI incidence were assessed using either a logistic or linear model, adjusting for the baseline measure of the outcome and venue type. RESULTS: The intervention improved consistent condom use with any partner type in the previous month (AOR = 2.09, 95% CI, 1.43-3.04, p = 0.0001). Consistent condom use with clients in the three most recent sex acts increased in both arms, and with primary partners in the intervention arm, but there was no difference between groups after adjusting for baseline condom use and venue type. There were no differences in cumulative incidence of any STI (i.e., chlamydia, gonorrhoea, syphilis) between groups. HIV transmission knowledge (p = 0.0001), condom use skill (p = 0.0421), and self-efficacy for using condoms (p = 0.0071) were improved by the intervention. HIV-related stigma declined (p = 0.0119) and HIV and STI risk perception were improved (4.6 to 13.9%, and 9.4 to 20.0%, respectively). The intervention was associated with these improvements after adjusting for the baseline measure and venue type. CONCLUSION: Following a preventive intervention among Shanghai FSWs, our findings demonstrate that a simple, community-based educational intervention improved overall condom use, HIV and STI knowledge, and attitudes in relation to HIV/AIDS. The intervention should be implemented widely after tailoring educational materials regarding condom negotiation with different partner types (i.e., commercial sex clients and primary partners).

Evidence that HIV-1 CRF01_AE is associated with low CD4+T cell count and CXCR4 co-receptor usage in recently infected young men who have sex with men (MSM) in Shanghai, China.

Men who have sex with men (MSM) have recently accounted for an alarmingly increasing proportion of HIV-1 transmission in China. In order to investigate the immune status as a result of CRF01_AE infection and CXCR4 co-receptor usage in a young Shanghai-based HIV-1-infected MSM population in Shanghai, 364 HIV-1-infected MSM with average age of 22.7 years old, newly diagnosed between Jan 2009 and Jul 2013 were analyzed for CD4+T cell count, subtyping using phylogenetic analysis, and viral co-receptor tropism using Geno2pheno and webPSSM in combination. A total of 276 individuals were identified as recently infected. Subtype assignment were as follows: 176 (63.8%) CRF01_AE, 77 (27.9%) CRF07_BC, and 23 (8.3%) subtype B. Besides, 24 second-generation recombinant strains were identified. A lower CD4+T cell count at baseline survey was observed among CRF01_AE strain-infected individuals, compared to those who were infected with CRF07_BC (P<0.01). The frequency of baseline CD4+T cell count <200 was higher and the frequency of CD4 T counts >500 lower in CRF01_AE infection than CRF07_BC infection. It is worth noting that 32.4%-40.9% of CRF01_AE strain-infected individuals were predicted to carry CXCR4-tropic viruses whereas none of CRF07_BC and subtype B were found to be as CXCR4-tropic viruses (P<0.001). As could be expected CXCR4 tropism was associated with lower CD4 T counts. This study revealed that CRF01_AE strains with high frequency of CXCR4 tropism are prevailing in the young MSM population in China and could potentially cause a severe loss of CD4+T cell count and rapid disease progression. A regular surveillance of HIV-1 subtypes, CD4+T cell count and viral co-receptor usage would be greatly beneficial for effectively monitoring disease progression, improvement of antiretroviral therapy strategy and prompt intervention of transmission.

HIV drug resistance and its impact on antiretroviral therapy in Chinese HIV-infected patients.

BACKGROUND: Highly active antiretroviral therapy (HAART) has significantly decreased mortality among Chinese HIV patients. However, emerging HIV drug resistance (HIVDR) poses a growing threat to the long-term success and durability of HAART. METHODS: Three cross-sectional surveys were conducted across the country from 2004 to 2006, respectively. Patients completed a questionnaire and provided blood for CD4 cell count, HIV viral load (VL), and HIV resistance genotyping. Factors associated with HIVDR were identified by logistic regression. RESULTS: 3667 unique patients were included across the three surveys. Among 2826 treatment-experienced patients, median duration of treatment was 17.4 (IQR 8.6-28.4) months and HIVDR was identified in 543 (19.2%). Factors significantly associated with HIVDR included ART drug distribution location, CD4 cell count, initial HAART regimen, self-reported medication adherence, and province. CONCLUSIONS: Virologic failure increased over time on therapy but a significant proportion of patients in failure had no resistance mutations identified, suggesting that treatment adherence is suboptimal and must be emphasized. Due to the significantly higher risk of HIVDR in certain provinces, additional steps to reduce HIVDR should be taken.

[Clinical characteristics of 638 patients infected with hepatitis C virus by post blood transfusion, in Shanghai].

OBJECTIVE: To investigate the clinical-epidemiologic characteristics of patients with hepatitis C virus (HCV) infection by post blood transfusion. METHODS: Polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect HCV RNA and anti-HCV, respectively. Analysis was performed on patients' age distribution, cause of primary diseases, years of exposure, ingredient and amount of transfusion, incubation period, disorder on liver function and changes on abdominal ultrasound image, etc. RESULTS: HCV RNA levels were higher than 3.0 log(10) copy/ml in 90.8% infected patients with a median as 6.10 log(10) copy/ml. 19.2% of the patients showed viral load 3.0 to 4.0 log(10) copy/ml, and 66.1% of them showed 5.0 to 6.0 log(10) copy/ml. Only 14.7% of the infected persons had HCV RNA levels higher than 7.0 log(10) copy/ml. Eighty-one point five percent (44/54) of the infected persons were confirmed as HCV RNA positive by HCV RNA qualitative analysis with HCV genotype as primarily type 1. 99.8% (636/637) of the patients were detected as anti-HCV positive by serological test. The sensitivity of serological test was higher than both quantitative and qualitative HCV RNA assays (P = 0.000, P = 0.000, respectively). HCV infection post blood transfusion was more seen in common people at 40 to 60 years old. Most cases (85.7%) had their first exposure during 1990 to 1994. More than 10% of the cases had primary diseases as obstetrics, orthopedics or gastrointestinal tract hemorrhage. 79.9% of the patients received whole blood product transfusion. The mean interval between transfusion and clinical diagnosis was 8.5 ± 5.5 years. 90.1% of the infected patients had liver function damage, while most of them showed elevated alanine aminotransferase (ALT) no more than 5 upper limits of normal (ULN), whereas Serum total bilirubin (TBIL), ALT and aspartate aminotransferase (AST) ≥ 5 × ULN level were showing more clinical manifestations (P = 0.000, P = 0.001, P = 0.009, respectively). Abdominal ultrasound among 8.9% of the infected persons showed changes in cirrhosis, and most of them were older than 50 years of age. CONCLUSION: Most of the post transfusion HCV infected cases happened in adulthood, and were mainly exposed during 1990 to 1994. Infected patients usually had their liver function damaged with elevated ALT no more than 5 × ULN and with medium HCV RNA levels. HCV genotype was mainly for type 1. Patients who were of older age showed higher incidence of cirrhosis. If a patients' infection period was longer than 5 years, he/she would show higher incidence of cirrhosis.

Factors associated with HIV virologic failure among patients on HAART for one year at three sentinel surveillance sites in China.

BACKGROUND: Emerging HIV drug resistance (HIVDR) poses a growing threat to the long-term success and durability of highly active antiretroviral therapy (HAART). OBJECTIVE: To understand the incidence of HIVDR and estimate the proportion of potential HIVDR and its associated risk factors among the patients on HAART for one year. METHODS: Antiretroviral-naïve patients ≥ 18 years old were invited to participate in this one-year prospective study from seven clinics in Yunnan, Guangxi, and Xinjiang provinces. A questionnaire and blood draw were collected at baseline and 12 month follow-up. The protocol used was modified slightly from the WHO Protocol for Surveys of HIV Drug Resistance Emerging During Treatment and Related Program Factors in Sentinel ART Sites in Resource-limited Settings. RESULTS: 435 subjects were included in the final analysis, with median baseline CD4 cell count 139 cells/mm3. Of the total 417 patients who fall under WHO guidelines for 'Classification of outcomes based on endpoints' (on ART at 12 months, switch, lost to follow-up, and stop), 90 (21.6%) did not have any drug resistant mutations (potential HIVDR) and 17 (4.1%) did (HIVDR, Table 2). The remaining 310 (75.3%) had a viral load <1000 copies/ml (HIVDR prevention). Among 351 patients retained at 12 months, 41 (11.7%) had a viral load >1000 copies/ml.Patients who self-reported missing doses in the previous month were 6.2 fold (95% CI 2.5-15.7) more likely to fail than those who did not. and those from Xinjiang were 12.1 fold (95% CI 5.2-28.1) more likely to fail compared to those from Yunnan and Guangxi. Why Xinjiang was associated with virologic failure was not clear but may be related to the demographics of the participants from Xinjiang, being significantly more IDUs, poorer, and less adherent than those from Yunnan and Guangxi. CONCLUSIONS: Although successful virologic outcomes were seen in the vast majority (75.3%) of those treated at one year, virologic failure continues to be a problem particularly among those less adherent and from Xinjiang. Additional data are needed to understand the generalizability of these results, particularly those related to Xinjiang. For IDUs, enhancing adherence to HAART and considering the treatment of drug addiction as an integral part of the treatment for HIV infection should be considered. As China's National Free Antiretroviral Treament Program continues to mature and improve, ramping up treatment in these settings may be important considerations to the long-term success of the program.

The prevalence of transmitted antiretroviral drug resistance in treatment-naive HIV-infected individuals in China.

BACKGROUND: Transmission of HIV drug resistance (TDR) gives rise to reduced efficacy of initial antiretroviral treatment and has become a public health concern. METHODS: A nationwide survey on TDR was conducted in antiretroviral therapy-naive HIV-1-infected individuals from September 2004 to October 2005 in China. Drug resistance genotyping was performed on subjects' plasma samples. Drug resistance mutations were determined and scored by Stanford HIV Drug Resistance algorithm. RESULTS: Sequences were obtained from 676 individuals, of whom 61.2% were former plasma and blood donors, 17.3% were infected sexually, and 17.2% were intravenous drug users. Subtype B' HIV-1 strains were found in 73.5%, CRF01_AE in 13.9%, CRF07_BC in 6.2%, CRF08_BC in 2.7%, subtype C in 1.04%, subtype B in 0.9%, CRF02_AG in 0.4%, and B'/C intersubtype recombinant strains in 1.3% of the subjects. Twenty-six (3.8%) were found to harbor drug resistance strains. The rates of resistance to protease inhibitors, nucleoside reverse transcriptase inhibitors, and nonnucleoside reverse transcriptase inhibitors were 0.4%, 1.6%, and 2.1%, respectively. Though there was no significant difference in TDR rates between 2004 and 2005 (2.9% vs. 4.4%), an increased trend was observed in the rate of high-level drug resistance (0.8% in 2004 vs. 3.0% in 2005, P = 0.0634). CONCLUSIONS: The rate of TDR was relatively low in China, as compared with those in developed countries. Surveys among recently HIV-infected subjects should be performed continually to ensure the success of the scale-up antiretroviral treatment.

[Frequency of the CD4+CD25nt/hiCD127lo regulatory T lymphocytes from the peripheral blood in Chinese healthy individuals].

AIM: To determine the frequency of the CD4(+)CD25(nt/hi)CD127(lo) regulatory T lymphocytes from the peripheral blood in the Chinese healthy individuals and provide some useful evidence for clinical research of correlative diseases. METHODS: From the CD4(+)CD25(nt/hi)CD127(lo) regulatory T lymphocytes of peripheral blood in 312 Chinese healthy male and female individuals aged from 8 to 60(five age groups were collected) The expression of transcription factor Foxp3 was detected by triplex immuno fluorescence and the frequency of CD4(+)CD25(nt/hi)CD127(lo) regulatory T lymphocytes was determined by flow cytometry. RESULTS: The frequency of CD4(+)CD25(nt/hi)CD127(lo) regulatory T lymphocytes in Chinese healthy individuals was (6.55+/-0.11)%, and the frequency differed among age groups(P=0.015) and sex groups(P<0.05). CD4(+)CD25(nt/hi)CD127(lo) regulatory T lymphocytes specifically express transcription factor Foxp3. CONCLUSION: The frequency of the CD4(+)CD25(nt/hi)CD127(lo) regulatory T lymphocytes from the peripheral blood in the Chinese healthy individuals has been preliminarily determined which lays the foundation for further clinical research of regulatory T lymphocytes. As a specific cell surface marker, CD25(nt/hi)CD127(lo) can helpful obtain pure CD4(+)CD25(+) regulatory T lymphocytes and suppress the interference of other cells during cell separation.

Genetic diversity and drug resistance of human immunodeficiency virus type 1 (HIV-1) strains circulating in Shanghai.

The HIV-1 epidemic in Shanghai is rapidly increasing. To better understand the HIV-1 genetic diversity and the mutations associated with resistance to protease inhibitors (PIs) and reverse transcriptase inhibitors (RTIs), 95 antiretroviral (ARV)-treated and treatment-naive HIV-1-seropositive individuals living in Shanghai were investigated. The HIV-1 pol gene in 70 of the 95 plasma samples was successfully amplified and analyzed. The result showed that CRF01_AE predominated in Shanghai with 42.9%, followed by subtype B (10%), B' (12.9%), CRF07_BC (11.4%), CRF08_BC (10%), CRF02_AG (4.3%), G (2.9%), and K (1.4%). In addition, three new intersubtype and/or inter-CRF recombinants were detected including B'/CRF01_AE (1.4%), U/G (1.4%), and U/CRF01_AE (1.4%). The mutations conferring primary and secondary resistance to PIs were detected in 3 of 70 (4.3%) patients and the mutations conferring resistance to RTIs were identified in 12 of 70 (17.2%) patients, among whom 11 of 15 (73.3%) and 1 of 55 (1.8%) were ARV-treated and treatment-naive individuals, respectively (p < 0.01). This study reveals the emergence of genetic diversity of HIV-1 currently circulating in Shanghai. HIV-1 infection by heterosexual contact is still a major route for introduction of HIV-1 variants into this city in recent years. It is believed that this information may help to guide recommendations for diagnostic assays, vaccine design, and antiretroviral regimen strategies in China.

[Study on using the multiplier method in estimating the size of men who have sex with men population in Shanghai].

OBJECTIVE: To estimate the size of men who have sex with men (MSM) in Shanghai. METHODS: Multiplier method was used in the study and two popular Shanghai-based MSM websites were selected as targeted institutions. The number of MSM (r) who visited website during a given period was investigated by a web-based questionnaire. Meanwhile, a survey was conducted among MSM living in Shanghai to obtain the proportion of MSM who had visited the websites during the given period, the reciprocal number of this proportion was the multiplier (m), therefore, population size can be estimated by r multiplied by m. RESULTS: The MSM population was estimated as 398 433 when website A was selected as target institution and 370 755 was estimated when website B was selected. The estimated population of MSM accounted for 7.1% and 6.6% of male population aged 15 to 49 years old in Shanghai, respectively. CONCLUSION: It was feasible to use multiplier method which selecting MSM website as target institution to estimate the size of MSM population, however, the representativeness of this study sample should be considered seriously.

Normal values for CD4 and CD8 lymphocyte subsets in healthy Chinese adults from Shanghai.

The aim of this study was to establish reference ranges for lymphocyte subsets in Chinese adults. Venous blood specimens were obtained from 614 healthy, human immunodeficiency virus (HIV)-seronegative adults in Shanghai. Flow cytometry was used to determine percentages and absolute numbers of CD4 and CD8 T lymphocytes. Mean values for CD4 and CD8 lymphocytes were 727 and 540 cells/microl, respectively, yielding a CD4/CD8 ratio of 1.49. While CD8 lymphocyte values varied with age and gender, no significant differences in CD4 lymphocyte values were observed. Shanghai adults had approximately 100 fewer CD4 lymphocytes/microl on average than Caucasians, suggesting that lower CD4 lymphocyte cutoffs for classifying and monitoring HIV infection may be needed in China.

Identification and distribution of HIV type 1 genetic diversity and protease inhibitor resistance-associated mutations in Shanghai, P. R. China.

OBJECTIVE: To investigate the genetic diversity of the HIV-1 circulating in Shanghai and to analyze the mutations in the protease (PR) gene associated with resistance to protease inhibitors (PIs). DESIGN: The genetic diversity of HIV-1 and PI resistance-associated mutations was studied in 40 Shanghai HIV-1-seropositive treatment-naive residents. The patients studied were exposed to the infection mainly through contaminated blood products (hemophiliacs) (n = 17) and sexual contacts (n = 19). Samples from 2 injecting drug users (IDUs) and 2 children born to HIV-1 infected mothers were also analyzed. METHODS: HIV-1 partial gag, pol, and env genes in infected plasma samples were amplified by reverse transcriptase polymerase chain reaction, sequenced, and phylogenetically analyzed. Analysis of PI resistance-associated amino acid substitution in PR was performed. RESULTS: HIV-1 genes in 38 of the 40 plasma samples were successfully amplified and analyzed. Polymerase chain reaction amplification was successful for 16/17 hemophilia patients and 18/19 sexually infected individuals. While all the 16 hemophilia patients infected through contaminated blood products were infected with subtype B', the 18 patients infected through sexual contact were infected with several subtypes including subtype A (n = 2), B (n = 4), B' (n = 1), C (n = 2), CRF08_BC (n = 1), CRF01_AE (n = 7), and intersubtype recombinant CRF01_AE/B (n = 1). The 2 IDUs were infected with CRF08_BC and the 2 children born to HIV-1 infected mothers were infected with subtype B' and CRF01_AE. PI resistance-associated amino acid substitutions were found at 1 codon in primary and 7 codons in secondary regions of the PR gene. Amino acid substitutions were more frequently found in the B/B' sequences (69%) than in the non-B sequences (31%). Substitutions characteristic with the subtype B/B' sequences mainly among hemophiliacs included L63P (87%), A71V/T (27%), and V77I (93%) while those that characterized the non-B sequences mainly found among heterosexuals included M36I (69%) and K20R (19%). CONCLUSION: This study reveals the presence of multiple HIV-1 subtypes and recombinants infecting Shanghai residents. The broad HIV-1 diversity is being introduced into this city through heterosexual contacts. This study also reveals that viruses infecting these treatment-naive patients have acquired both primary or secondary mutations in their PR genes. These studies should provide the basis for further epidemiologic surveys of HIV-1 subtypes and set strategies for treatment intervention and vaccine programs.