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OBJECTIVES: Invasive lumbar puncture is the conventional method for diagnosing neurosyphilis (NS). We investigated a non-invasive alternative method to detect serum Treponema pallidum-specific antibodies against highly immunogenic antigens TP0171 (TP15), TP0435 (TP17), and TP0574 (TP47) by using luciferase immunosorbent assay. METHODS: A total of 816 HIV-negative patients suspected of NS from the Beijing and Guangzhou cohorts were retrospectively selected and tested for serum anti-TP15, TP17, and TP47 IgG antibodies. Two diagnostic prediction models were developed using stepwise logistic regression in the Beijing cohort, and evaluated in the Guangzhou cohort for external validation. RESULTS: Serum antibodies against TP15, TP17, and TP47 showed moderate capability for NS diagnosis in the Beijing cohort and the corresponding area under the receiver operating characteristic curves (AUCs) were 0.722 [95% confidence interval (CI): 0.680-0.762)], 0.780 (95% CI: 0.741-0.817), and 0.774 (95% CI: 0.734-0.811), respectively. An expanded NS prediction model integrated with anti-TP17 and anti-TP47 antibodies showed better performance than the base NS diagnostic model without anti-TP17 and anti-TP47 antibodies with the AUC of 0.874 (95% CI: 0.841-0.906) vs. 0.845 (95% CI: 0.809-0.881) (p = 0.007) in the development cohort, and 0.934 (95% CI: 0.909-0.960) vs. 0.877 (95% CI: 0.840-0.914) (p < 0.001) in validation cohort, respectively. Decision curve analysis revealed that the net benefit of the expanded model exceeded that of the base model when the threshold probability was between 0.10 and 0.95 in both the development and external validation cohorts. DISCUSSION: Serum antibodies against TP17 and TP47 exhibited promising diagnostic capability for NS and significantly enhanced the predictive accuracy of model for NS diagnosis. Our study highlights the potential of serum treponemal antibody detection as a non-invasive method for NS diagnosis to substitute invasive lumbar puncture in NS diagnosis.
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To assess the clinical applicability of a semi-quantitative luciferase immunosorbent assay (LISA) for detecting antibodies against Treponema pallidum antigens TP0171 (TP15), TP0435 (TP17), and TP0574 (TP47) in diagnosing and monitoring syphilis. LISA for detection of anti-TP15, TP17, and TP47 antibodies were developed and evaluated for syphilis diagnosis using 261 serum samples (161 syphilis, 100 non-syphilis). Ninety serial serum samples from 6 syphilis rabbit models (3 treated, 3 untreated) and 110 paired serum samples from 55 syphilis patients were used to assess treatment effects by utilizing TRUST as a reference. Compared to TPPA, LISA-TP15, LISA-TP17, and LISA-TP47 showed a sensitivity of 91.9%, 96.9%, and 98.8%, specificity of 99%, 99%, and 98%, and AUC of 0.971, 0.992, and 0.995, respectively, in diagnosing syphilis. Strong correlations (rs = 0.89-0.93) with TPPA were observed. In serial serum samples from rabbit models, significant differences in the relative light unit (RLU) were observed between the treatment and control group for LISA-TP17 (days 31-51) and LISA-TP47 (day 41). In paired serum samples from syphilis patients, TRUST titres and the RLU of LISA-TP15, LISA-TP17, and LISA-TP47 decreased post-treatment (P < .001). When TRUST titres decreased by 0, 2, 4, or ≥8-folds, the RLU decreased by 17.53%, 31.34%, 48.62%, and 72.79% for LISA-TP15; 8.84%, 17.00%, 28.37%, and 50.57% for LISA-TP17; 22.25%, 29.79%, 51.75%, and 70.28% for LISA-TP47, respectively. Semi-quantitative LISA performs well for syphilis diagnosis while LISA-TP17 is more effective for monitoring syphilis treatment in rabbit models and clinical patients.
Asunto(s)
Anticuerpos Antibacterianos , Antígenos Bacterianos , Sensibilidad y Especificidad , Sífilis , Treponema pallidum , Sífilis/diagnóstico , Sífilis/microbiología , Sífilis/sangre , Treponema pallidum/inmunología , Animales , Humanos , Conejos , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Masculino , Femenino , Adulto , Luciferasas/genética , Serodiagnóstico de la Sífilis/métodos , Persona de Mediana Edad , Modelos Animales de Enfermedad , Adulto JovenRESUMEN
Precise genotyping is necessary to understand epidemiology and clinical manifestations of Chlamydia trachomatis infection with different genotypes. Next-generation high-throughput sequencing (NGHTS) has opened new frontiers in microbial genotyping, but has been clinically characterized in only a few settings. This study aimed to determine C. trachomatis genotypes in particular mixed-genotype infections and their association with clinical manifestations and to characterize the sensitivity and accuracy of NGHTS. Cervical specimens were collected from 8,087 subjects from physical examination center (PEC), assisted reproductive technology center (ART) and gynecology clinics (GC) of Chenzhou Hospital of China. The overall prevalence of C. trachomatis was 3.8% (311/8087) whereas a prevalence of 2.8, 3.7 and 4.8% was found in PEC, ART and GC, respectively. The most frequent three C. trachomatis genotypes were E (27.4%, 83/303), F (21.5%, 65/303) and J (18.2%, 55/303). Moreover, NGHTS identified 20 (6.6%, 20/303) mixed-genotype infections of C. trachomatis. Genotype G was more often observed in the subjects with pelvic inflammatory disease than genotype E (adjusted OR = 3.61, 95%CI, 1.02-12.8, p = 0.046). Mixed-genotype infection was associated with severe vaginal cleanliness (degree IV) with an adjusted OR of 5.17 (95%CI 1.03-25.9, p = 0.046) whereas mixed-genotype infection with large proportion of minor genotypes was associated with cervical squamous intraepithelial lesion (SIL) with an adjusted OR of 5.51 (95%CI 1.17-26.01, p = 0.031). Our results indicated that NGHTS is a feasible tool to identity C. trachomatis mixed-genotype infections, which may be associated with worse vaginal cleanliness and cervical SIL.
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A longitudinal serological study to investigate the seropositive frequency, incidence, and antibody dynamics of Chlamydia trachomatis infection in the general population of China is urgently needed in order to optimize the strategies for surveillance and precise prevention of C. trachomatis infection. This longitudinal study enrolled 744 subjects aged 18-65 years from Jidong Community of Northern China from 2014 to 2018. Seropositive frequency, incidence, and reinfection of C. trachomatis were determined by detecting antibody against C. trachomatis Pgp3 using "in-house" luciferase immunosorbent assay (LISA). The dynamic of anti-Pgp3 antibody was analyzed using the Generalized Estimating Equation (GEE) model. The overall Pgp3 seropositive frequency among the 18-65-year-old population was 28.1% (95% CI 24.9-31.5), and significantly increased from 12.0% in those aged 18-29 years to 48.6% in the 60-65 years old. The seropositive frequency was slightly higher in women than in men (31.3% vs. 25.4%) without statistical significance. The C. trachomatis incidence and reinfection rate were 11 and 14 per 1,000 person-years, respectively, and showed no significant difference with respect to age, gender, ethnicity, marital status, and education levels. Furthermore, anti-Pgp3 antibody remained detectable in 93.3% (195/209) of the seropositive subjects during the 5 years of follow-up. The overall decay rate for anti-Pgp3 antibody for CT-infected persons was -0.123 Log2 RLU/year, which was dramatically slower than in CT new infection (-3.34 Log2 RLU/year) or reinfection (-1.1 Log2 RLU/year). In conclusion, at least one quarter of the people aged 18-65 years have been infected with C. trachomatis over their lifetime while all age groups are susceptible to C. trachomatis infection in the community of Northern China. Therefore, comprehensive prevention strategies are urgently needed.
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Human immunodeficiency virus type one (HIV-1) infection is one of the major public health problems worldwide. Effective control of HIV-1 epidemic relies on early diagnosis of HIV-1 infection by using simple, rapid point-of-care test (POCT). An integrated assay was developed and evaluated in this study to combine a real-time isothermal reverse-transcription recombinase-aided amplification (rRT-RAA) and CRISPR Cas12a-mediated detection for HIV-1. The testing results could be directly observed with naked eye using a blue light imager, making it a suitable on-site testing assay. Our preliminary data indicated that the assay was capable of detecting 20 copies of purified HIV-1 DNA or RNA per reaction or as low as 123 copies/ml of HIV-1 viral load in clinical samples. When screening 155 clinical samples with or without HIV-1 infection, the sensitivity and specificity of the rRT-RAA assay were 98.95% (94/95) and 100% (60/60), respectively. The coefficient value was 0.986 when compared with the Chinese FDA approved HIV-1 RT-qPCR assay. Furthermore, the newly developed HIV-1 rRT-RAA assay could detect the major HIV-1 genotypes CRF01_AE, CRF07_BC, CRF08_BC, CRF08_BC and subtype B in China. Our preliminary results indicated that the rRT-RAA assay or its combination with CRISPR Cas12a-mediated detection could serve as a rapid, convenient, and robust assay for HIV-1 detection.