Male migrant workers
From May to October 2015 we conducted a survey on the characteristics and prevalence of various infections among male migrant workers in Shanghai, China (hereinafter referred to as male migrants). The included migrants were men aged 18 years or older, had lived in the community for more than 3 months and were able to provide a written informed consent. One thousand male migrants were invited and 983 of them fully complied with study requirements (median age = 27 years; range: 18-66). They were interviewed in private by trained staff in Mandarin language using an anonymized questionnaire that included information on socio-demographic characteristics and sexual behaviours. Venous blood was collected using sterilized needles and tubes, and transferred to laboratory within 2 h after collection while maintaining a cold chain. Plasma samples were stored at −80 °C until serological testing.
Plasma samples were tested by two monoclonal immunofluorescence assays (mIFAs) that target KSHV latent and lytic antigens . Briefly, two serology tests were performed: first, BC-3 cells (KSHV positive and Epstein-Barr virus negative B cell line, American Type Culture Collection, Manassas, VA), stimulated by tetradecanoyl phorbol acetate (TPA), were fixed and permeabilized and used for an enhanced mIFA. Second, Spodoptera frugiperda clone 9 expressing 3 KSHV proteins, ORF73, ORF65 and ORF-K8.1, was used. Sera taken from two KS patients and a normal person were as positive and negative controls, respectively, in each assay. The two assays were then compared, and only samples which were positive for both BC-3 and Sf9 assay at a standard serum dilution of 1:40 were considered KSHV-seropositive. The testing protocol used in our present study have been validated and shown to have sensitivity of 93.9% and specificity of 96.3% . To guarantee the quality, all slides were monitored for every batch and were read independently by two experienced laboratory workers.
A rapid plasma reagent test (Span Diagnostics Ltd., Surat, India), was used and confirmed by the Treponema pallidum hemagglutination test (TPHA, Syphagen TPHA, Biokit, Spain).
HSV-2 IgG antibodies were tested using an ELISA assay (HerpeSelect 2 ELISA IgG Kit, Focus Technologies, CA, USA). Equivocal samples were retested using another ELISA kit (HerpeSelect 2 ELISA IgG Kit, Euroimmun, Lübeck, Germany).
Anti-HCV immunoglobulin G (IgG) antibodies were tested by third-generation ELISA (Wantai Biomedical, Beijing, China).
HIV antibodies were assessed using ELISA (Vironostika HIV Uni-Form II plus O ELISA Kit, Biomerieux, Netherland) and confirmed by a western blot assay (Genelabs Diagnostic, Singapore).
The data from the male migrants study were pooled with those from five additional subpopulations that we had studied between 2010 and 2015 using similar questionnaires and the same serological assays described above. Briefly, we included 600 female migrant workers (age:29.34 ± 8.44), referred to as female migrants ; 600 female sex workers (FSW) (age: 26.47 ± 6.84) ; 1336 men attending sexually transmitted infection clinics, referred to as STI men (age: 37.39 ± 14.13) ; 439 intravenous drug users (IVDU) (age:45.39 ± 88.41) ; and 226 men having sex with men (MSM) (age: 27.79 ± 7.01) . Thus, a total of 3201 HIV negative individuals in addition to the aforementioned 983 HIV negative male migrants were finally included.
All studies had been approved by the Institutional Review Board (IRB) of Fudan University, Shanghai. Informed consent was obtained from all subjects, all study protocols and procedures were in accordance with the Declaration of Helsinki.
The prevalence of KSHV, syphilis, and when available, HSV2, was examined separately in the six available subpopulations and eventually combined into three groups: 1) heterosexuals, i.e., migrants of both sexes, FSW, and STI men; 2) IVDU; and 3) MSM (including 27 MSM who were formerly included in the STI survey ). On account of the limitations and inconsistencies of sexual history in study questionnaires, the prevalence of syphilis and HSV2 was preferred to number of sexual partners as proxies of sexual activity. Ninety-five percent confidence intervals (95% CI) of prevalence were computed according to the normal approximation to the binomial distribution. Prevalence ratios (PR) and 95% CI for KSHV positivity were estimated by the Poisson regression models with robust variance. PRs were adjusted by age group or by age group and subpopulation as indicated. Risk trends were assessed by considering categories as continuous variables. All statistical analyses were carried out using the SAS System for Windows (Cary, NC, USA), version 8.0.