Heping Zheng

Prospective, multi-centre clinic-based evaluation of four rapid diagnostic tests for syphilis

Objectives: To evaluate prospectively four rapid, point-of-care serological tests for syphilis in prenatal or high risk populations in four countries. Methods: Tests were performed on consecutive clinic attenders, using whole blood in the clinic, and whole blood and serum in the laboratory. The sensitivity and specificity of each test was evaluated, using a standard treponemal test (Treponema pallidum haemagglutination assay (TPHA) or fluorescent treponemal antibody, absorbed (FTA-ABS) as gold standard. Non-treponemal tests (rapid plasma reagin (RPR) or venereal diseases research laboratory (VDRL) tests) were also performed on all subjects at three sites. Results: The specificity of each rapid test was >95% at each site. Sensitivities varied from 64–100% and, in most cases, were lower when whole blood was used rather than serum. Conclusions: Rapid serological tests for syphilis are an acceptable alternative to conventional laboratory tests. Since they do not require equipment or electricity, they could increase coverage of syphilis screening, and enable treatment to be given at the first clinic visit.

Clinic-based evaluation of Clearview Chlamydia MF for detection of Chlamydia trachomatis in vaginal and cervical specimens from women at high risk in China

Objectives: To determine the performance of a rapid Chlamydia trachomatis (CT) test (Clearview Chlamydia MF) compared to the current “gold standard” (Roche Amplicor CT assay) test, and to assess acceptability of the tests to patients. Methods: A total of 1497 women at sexually transmitted diseases (STD) clinics or re-education centres in six urban cities (Shanghai, Nanjing, Shenzhen, Guangzhou, Chengdu and Fuzhou) in China participated in the study. Three vaginal and three cervical swabs were collected from each participant. Rapid CT tests were performed locally on the first vaginal and cervical swabs and the results were read independently by two staff members. The second and third swabs were randomised for performing the Roche CT assay at the National STD Reference Laboratory. Acceptability of the rapid tests to patients was determined by asking patients in clinics about their willingness to wait for the results. Results: The prevalence of CT was 13.2% (197/1497), as determined by the Roche assay with cervical specimens. CT was detected in 78 vaginal and 127 cervical specimens by the rapid test and the positive rates determined with cervical specimens were significantly higher than those with vaginal specimens (p<0.001). There was good agreement between the results read by two independent staff for either vaginal or cervical specimens (both κ = 0.98, p<0.001). Sensitivities for vaginal and cervical specimens were 32.8% and 49.7%, respectively, and specificities were 99.2% and 97.9%, respectively. The positive predictive value was 85.7% for vaginal and 78.4% for cervical specimens. The vast majority of the patients (99.1%) were willing to wait up to two hours for the results. Conclusion: Clearview Chlamydia MF, while yielding a rapid result and requiring minimal laboratory facilities, had unacceptably low sensitivity compared to a nucleic acid amplification test. Rapid tests yielding results within one hour are generally accepted by the clients.

Molecular Typing of treponema pallidum Causing Early Syphilis in China: A Cross-sectional Study

Background: There have been limited data on molecular epidemiology of syphilis in China. This study aimed to analyze strain type distribution of Treponema pallidum causing early syphilis across geographic areas in China using an enhanced method. Methods: Genital samples were collected from patients in East, South, and North China. Positive DNA of T. pallidum was analyzed by arp, tpr, and tp0548 genes. Results: Sufficient DNA for full molecular typing existed in 197 of 324 samples, and 27 strain types were identified. A range of 3 to 20 repeats (except 4, 11, and 19 repeats) and 25 repeats were found for the 60-bp tandem repeats of the arp gene. This was the first time the 9 and 25 repeats were detected. For the RFLP analysis of the tpr genes, patterns a, d, h, j, and l were identified. This was the first time the h, j, and l patterns were observed in China. For the sequence analysis of the tp0548 gene, sequences c, e, and f were identified. Strain type distribution was significantly different across geographic areas (&khgr;2 = 20.6, P = 0.006). Overall, 14d/f was most predominant (39% of fully typed samples, 95% CI = 32%–46%); 13d/f, 15d/f, and 16d/f were next most common (each 13% of fully typed samples, 95% CI = 9%–18%). Conclusions: There is substantial genetic diversity of T. pallidum in China. The broad and ununiform distribution of strain types may reflect differences in regional sexual network patterns. Predominance of few strain types may indicate a linked transmission.

A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis

We have developed a high-resolution melting analysis (HRMA) for the genotyping of Chlamydia trachomatis and applied it specifically to the 11 sexually transmitted infection-related genotypes: D through K and L1 through L3. The variable segment 2 (VS2) was selected as the target for HRMA genotype identification. Eleven C. trachomatis genotypes were amplified by a nested real-time polymerase chain reaction (PCR) in the presence of the LCGreen saturating dye and showed no cross-reaction with 10 pathogenic bacteria or commensals from urogenital tract. The detection limit of HRMA method was 100 elementary bodies (EB)/mL. All of the 11 genotypes can be distinguished from each other by following an HRMA workflow. Genotype F, G, H, I, J, K, L2, and L3 could be directly identified from each other, whereas D, E, and L1 could be distinguished from each other by a second analysis with fewer curves or by heteroduplex formation with a known reference strain. In the validation panel of 36 C. trachomatis-positive urogenital samples genotyped by VS1-VS2 sequencing, nested real-time VS2 PCR followed by HRMA was able to discriminate between all samples correctly. This assay requires no fluorescence-labeled probes or separate post-PCR analysis and provides a simple and rapid approach for genotyping the C. trachomatis strains that are the most commonly sexually transmitted.

Association of the in vitro susceptibility of clinical isolates of chlamydia trachomatis with serovar and duration of antibiotic exposure.

Background The presence of persistent Chlamydia trachomatis infection after treatment does not always correlate with in vitro susceptibility testing. Methods The in vitro minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of azithromycin, clarithromycin, roxithromycin, doxycycline, tetracycline, ofloxacin, and penicillin were tested against 61 clinical isolates of C. trachomatis on 6 serovars, and the MIC/MBC of azithromycin and ofloxacin at different points in time after antibiotic administration to infected cultures. Results Of the 7 antibiotics tested, clarithromycin showed the greatest activity against C. trachomatis isolates with MIC90 of 0.032 &mgr;g/mL and MBC90 of 0.064 &mgr;g/mL, followed by doxycycline with MIC90 0.064 &mgr;g/mL and MBC90 0.064 &mgr;g/mL, and azithromycin with MIC90 0.160 &mgr;g/mL and MBC90 0.320 &mgr;g/mL. Azithromycin had roughly the same MIC50 values (0.08 &mgr;g/mL) as the other serovars isolates tested, and other antibiotics showed a 2- to 4-fold difference in MICs50 between serovars. In addition, an increase in the azithromyin MIC was observed by 8 hours and the ofloxacin MIC by 16 hours. At 24 hours, the azithromycin MICs were greater than 40 &mgr;g/mL and ofloxacin MICs were greater than 64 &mgr;g/mL. Conclusions The current data demonstrated that the antimicrobial susceptibility of C. trachomatis was influenced by both the serovar type and the duration of exposure to antibiotics in infected cultures.

Susceptibility of Neisseria gonorrhoeae to azithromycin and ceftriaxone in China: A retrospective study of national surveillance data from 2013 to 2016

Background Gonorrhea remains one of the most common sexually transmitted diseases worldwide. Successful treatment has been hampered by emerging resistance to each of the antibiotics recommended as first-line therapies. We retrospectively analyzed the susceptibility of gonorrhea to azithromycin and ceftriaxone using data from the China Gonococcal Resistance Surveillance Programme (China-GRSP) in order to provide evidence for updating the treatment recommendations in China. Methods and findings In this study, we included 3,849 isolates collected from patients with a confirmed positive Neisseria gonorrhoeae (N. gonorrhoeae) culture at clinic visits during the period of 1 January 2013 through 31 December 2016 in 7 provinces. Antimicrobial susceptibility testing of gonorrhea isolates using agar dilution was conducted to determine minimum inhibitory concentration (MIC). Resistance to azithromycin (RTA) was defined as MIC ≥ 1.0 mg/l, and decreased susceptibility to ceftriaxone (DSC) was defined as MIC ≥ 0.125 mg/l. The prevalence of isolates with RTA was 18.6% (710/3,827; 95% CI 17.4%–19.8%). The percentage of patients with DSC fluctuated between 9.7% and 12.2% over this period. The overall prevalence of isolates with both RTA and DSC was 2.3% (87/3,827; 95% CI 1.9%–2.8%) and it increased from 1.9% in 2013 to 3.3% in 2016 (chi-squared test for trend, P = 0.03). Study limitations include the retrospective study design and potential biases in the sample, which may overrepresent men with symptomatic infection, coastal residents, and people reporting as heterosexual. Conclusions To our knowledge, this is the first national study on susceptibility of N. gonorrhoeae to azithromycin and ceftriaxone in China. Our findings indicate high rates of RTA and DSC from 2013 to 2016. Although dual therapy with azithromycin and ceftriaxone has been recommended by WHO and many countries to treat gonorrhea, reevaluation of this therapy is needed prior to its introduction in China.

Upregulated ataxia-telangiectasia group D complementing gene correlates with poor prognosis in patients with esophageal squamous cell carcinoma

The ataxia-telangiectasia group D complementing gene (ATDC) plays significant roles in various human cancers. However, the clinical significance of ATDC in esophageal squamous cell carcinoma (ESCC) has not been investigated. The ATDC messenger RNA level of 40 paired ESCC and nonneoplastic tissues were evaluated using quantitative real-time polymerase chain reaction, 10 pairs of which were also used for Western blot analysis. In addition, immunohistochemical staining was performed to detect the ATDC expression in 118 paraffin-embedded cancerous and matched nonneoplastic tissues, and the correlation of ATDC expression with the clinicopathological parameters and prognosis of the ESCC patients was analyzed. The quantitative real-time polymerase chain reaction, immunohistochemical staining, and Western blot results demonstrated that the expression level of ATDC was significantly higher in ESCC tissue than in matched noncancerous tissues. Both ATDC messenger RNA and protein expression in the ESCC tissue were significantly correlated with tumor differentiation, stage, and lymph node metastasis. However, there was no significant difference in ATDC expression based on patient age or gender. Moreover, the results of both univariate and multivariate analyses showed that increased ATDC expression was correlated with a shorter 5-year survival time for ESCC patients after surgery. We concluded that increased ATDC expression is associated with poor clinical outcomes and that this marker might be a useful indicator for prognosis and a promising target for therapy in ESCC patients.

Prevalence of Chlamydia trachomatis Genotypes in Men Who Have Sex with Men and Men Who Have Sex with Women Using Multilocus VNTR Analysis-ompA Typing in Guangzhou, China.

Background Chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infection in China. Although C. trachomatis genotypes can be discriminated by outer membrane protein gene (ompA) sequencing, currently available methods have limited resolutions. This study used a high-resolution genotyping method, namely, multilocus variable number tandem-repeat analysis with ompA sequencing (MLVA)-ompA, to investigate the local epidemiology of C. trachomatis infections among men who have sex with men (MSM) and men who have sex with women (MSW) attending a sexually transmitted diseases (STD) clinic in Guangzhou, China. Methods Rectal specimens from MSM and urethral specimens from MSW were collected between January 2013 and July 2014 at the Guangdong Provincial Center STD clinic. The specimens were sent to the laboratory for analyses. All specimens that were tested positive for C. trachomatis by the commercial nucleic acid amplification tests were genotyped by MLVA-ompA. Results Fifty-one rectal specimens from MSM and 96 urethral specimens from MSW were identified with C. trachomatis. One hundred and forty-four of the 147 specimens were fully genotyped by MLVA-ompA. Rectal specimens from MSM were divided into four ompA genotypes and urethral specimens from MSW into nine genotypes. No mixed infections were found among all specimens. The most frequent genotypes were D, G, J, E and F. All specimens were further divided into 46 types after ompA genotyping was combined with MLVA. Genotypes D-8.7.1 and G-3.4a.3 were the most frequent among MSM, whereas genotypes D-3.4a.4, E-8.5.1, F-8.5.1, and J-3.4a.2 were the most frequent subtypes among MSW. The discriminatory index D was 0.90 for MLVA, 0.85 for ompA, and 0.95 for MLVA-ompA. Conclusions The most prevalent MLVA-ompA genotypes were significantly different between MSM and MSW from Guangzhou, China. Moreover, MLVA-ompA represented a more favorable degree of discrimination than ompA and could be a reliable complement for ompA for the routine subtypes of C. trachomatis.

Generalisability of an online randomised controlled trial: an empirical analysis

Background Investigators increasingly use online methods to recruit participants for randomised controlled trials (RCTs). However, the extent to which participants recruited online represent populations of interest is unknown. We evaluated how generalisable an online RCT sample is to men who have sex with men in China. Methods Inverse probability of sampling weights (IPSW) and the G-formula were used to examine the generalisability of an online RCT using model-based approaches. Online RCT data and national cross-sectional study data from China were analysed to illustrate the process of quantitatively assessing generalisability. The RCT (identifier NCT02248558) randomly assigned participants to a crowdsourced or health marketing video for promotion of HIV testing. The primary outcome was self-reported HIV testing within 4 weeks, with a non-inferiority margin of −3%. Results In the original online RCT analysis, the estimated difference in proportions of HIV tested between the two arms (crowdsourcing and health marketing) was 2.1% (95% CI, −5.4% to 9.7%). The hypothesis that the crowdsourced video was not inferior to the health marketing video to promote HIV testing was not demonstrated. The IPSW and G-formula estimated differences were −2.6% (95% CI, −14.2 to 8.9) and 2.7% (95% CI, −10.7 to 16.2), with both approaches also not establishing non-inferiority. Conclusions Conducting generalisability analysis of an online RCT is feasible. Examining the generalisability of online RCTs is an important step before an intervention is scaled up. Trial registration number NCT02248558.

Human Papillomavirus Positivity in the Anal Canal in HIV-Infected and HIV-Uninfected Men Who Have Anal Sex with Men in Guangzhou, China: Implication for Anal Exams and Early Vaccination

Background. The epidemiology of HPV in men who have sex with men (MSM) in Guangzhou, China, had not been reported previously. Methods. HIV-infected and HIV-uninfected MSM were recruited from a Guangzhou-based MSM clinic in 2013. Sociodemographic characteristics and sexual behaviors were collected. An anal cytological sample was taken for HPV testing. Results. We recruited 79 HIV-infected and 85 HIV-uninfected MSM. The median age was 26 years in both groups. The positivities of anal HPV of any type (81.0% versus 48.2%), any high risk type (50.6% versus 27.1%), any low risk type (55.7% versus 31.8%), and any 9-valent vaccine type (74.7% versus 36.5%) were all significantly higher among HIV-infected compared to that among HIV-negative MSM (p for all < 0.05). The great majority of HPV-infected MSM were infected with 9-valent vaccine types (59 out of 64 HIV-infected and 31 out of 41 HIV-uninfected). Anal bacterial infections were associated with higher anal HPV positivity and greater number of anal HPV types. Conclusion. Sexually active MSM in Guangzhou, especially those infected with HIV, had high and multiple HPV detections. The majority of these cases were potentially preventable by HPV vaccine. Regular anal exams and early HPV vaccination are warranted in this population.