Potential diagnostic value of miRNAs in sexually transmitted infections.

MiRNAs are small endogenous non-coding RNAs that have been demonstrated to be involved in post-transcriptional gene silencing, regulating a number of metabolic functions in the human body, including immune response, cellular physiology, organ development, angiogenesis, signaling, and other aspects. As popular molecules that have been studied in previous years, given their extensive regulatory functions, miRNAs hold considerable promise as non-invasive biomarkers. Sexually transmitted infections(STIs) are still widespread and have an adverse effect on individuals, communities, and society worldwide. miRNAs in the regulatory networks are generally involved in their molecular processes of formation and development. In this review, we discuss the value of miRNAs for the diagnosis of STIs.

Functional interferon-epsilon gene polymorphisms and sexually transmitted infections of the endometrium.

PROBLEM: Interferon-epsilon (IFNε) is the only type I IFN constitutively expressed in the female reproductive tract and fluctuates across the menstrual cycle in humans. Mouse models show that IFNε protects against Chlamydia trachomatis, Herpes Simplex Virus, HIV, and Zika in mice, but human studies are limited. Bacterial sexually transmitted infections (STI) can ascend to the upper genital tract and cause pelvic inflammatory disease (PID) and subsequent infertility. However, the host immunological mechanisms that play a role in the ascension and infection of the endometrium in individuals with clinically suspected PID are not elucidated. METHOD OF STUDY: This pilot investigation determined if IFNε gene variants are associated with bacterial vaginosis (BV) and endometrial infection with C. trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium using biospecimens from 154 self-report Black individuals who participated in the PID Evaluation and Clinical Health (PEACH) study. RESULTS: The T allele for rs2039381 was associated with endometrial STI infection (OR 2.7, 95% CI: 1.0-7.1) and the C allele for rs1125488 was inversely associated with BV (OR: .2, 95% CI: .05-.8). CONCLUSIONS: Few studies have examined IFNε gene variants, our study raises the possibility that IFNε gene variants may be a potential host contributor to STI pathogenesis.

STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences.

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5' tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis infections in pregnant women by multiplex recombinase polymerase amplification.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) are the main pathogenic microorganisms causing sexually transmitted infections. In this study, a multiplex thermostable recombinase polymerase amplification-lateral flow detection (RPA-LFD) assay was established, and the reaction conditions such as the ratio of primer concentration, magnesium ion concentration, amplification time and template DNA concentration in the multiplex RPA reaction were optimized. The optimized multiplex RPA-LFD method was used to detect both CT and NG positive control plasmids, and it was found that the LFD could be used to obtain visible results when the plasmid copy number was only 200. The sensitivity of the multiplex RPA-LFD method used for clinical samples was 85.62 (95% CI at 53.66-97.29) for NG detection and 90.90 (95% CI at 57.12-99.52) for CT detection.

Single-Cell Sequencing in Human Genital Infections.

Human genital infections are one of the most concerning issues worldwide and can be categorized into sexually transmitted, urinary tract and vaginal infections. These infections, if left untreated, can disseminate to the other parts of the body and cause more complicated illnesses such as pelvic inflammatory disease, urethritis, and anogenital cancers. The effective treatment against these infections is further complicated by the emergence of antimicrobial resistance in the genital infection causing pathogens. Furthermore, the development and applications of single-cell sequencing technologies have open new possibilities to study the drug resistant clones, cell to cell variations, the discovery of acquired drug resistance mutations, transcriptional diversity of a pathogen across different infection stages, to identify rare cell types and investigate different cellular states of genital infection causing pathogens, and to develop novel therapeutical strategies. In this chapter, I will provide a complete review of the applications of single-cell sequencing in human genital infections before discussing their limitations and challenges.

Sexual network and detection of anogenital human papillomavirus in a community cohort of men who have sex with men in Taiwan.

OBJECTIVES: We examined the association between anogenital human papillomavirus (HPV) infection and sexual networks in men who have sex with men (MSM). METHODS: A total of 253 MSM, 20 years of age and older, were recruited from the community in Southern Taiwan in 2015-2016. At baseline and at each follow-up visit, MSM were screened for HPV to identify 37 HPV genotypes. At the six-month follow-up, MSM were asked to fill out an egocentric network assessment and to report the last five persons with whom they had sex regarding the characteristics of sexual behavior with each network member. RESULTS: A total of 182 participants (71.9%) returned for the follow-up and one third had at least one HPV type detected. A higher level of bridging network position calculated by the level of constraints in the network was significantly less likely to have HPV detection at the anal site. A high level of concurrency was associated with penile HPV detection (AOR = 3.16, 95% CI = 1.01-9.86). CONCLUSIONS: Identifying network-related characteristics can advance our understanding of high-risk populations and for prioritizing HPV vaccine recommendations.

First report of sexually transmitted multi-drug resistant Shigella sonnei infections in Switzerland, investigated by whole genome sequencing.

Shigella sonnei causes foodborne infections, but has recently also been described as a sexually transmitted infection (STI), with increased levels of antimicrobial resistance. We describe three cases of sexually acquired Shigella sonnei infection - the first report of this emerging infection in Switzerland. We used in-house whole genome sequencing (WGS) to investigate possible transmission routes and epidemiological correlations between the three cases. The genomic analysis demonstrated that two of three case isolates were very closely related, with only two single nucleotide polymorphism differences between them, despite being isolated from two unrelated patients at time-points six months apart, and the infections having been acquired at different geographic locations within Europe. All three isolates were found to fall within two of the clusters (1 and 7) defined within UK men who have sex with men (MSM) isolate populations, but with higher divergence, suggesting a more diverse pool circulating within Europe. Phenotypic testing confirmed the genotypic findings, with all three isolates azithromycin resistant, and two out of three resistant to quinolones. This report underlines the importance of the implementation of new sequencing technologies in the investigation of epidemiological aspects of this STI circulating in the population of MSM. In such cases, therapy should always be guided by antimicrobial susceptibility testing owing to increasing resistances. Greater awareness of this emerging sexually transmitted infection is needed.

Nonpaternity and Half-Siblingships as Objective Measures of Extramarital Sex: Mathematical Modeling and Simulations.

BACKGROUND: Understanding the epidemiology of HIV and other sexually transmitted infections (STIs) requires knowledge of sexual behavior, but self-reported behavior has limitations. We explored the reliability and validity of nonpaternity and half-siblings ratios as biomarkers of current and past extramarital sex. METHODS: An individual-based Monte Carlo simulation model was constructed to describe partnering and conception in human populations with a focus on Sub-Saharan Africa (SSA). The model was parameterized with representative biological, behavioral, and demographic data. RESULTS: Nonpaternity and half-siblings ratios were strongly correlated with extramarital sex, with Pearson correlation coefficients (PCC) of 0.79 (95% CI: 0.71-0.86) and 0.77 (0.68-0.84), respectively. Age-specific nonpaternity ratios correlated with past extramarital sex at time of conception for different scenarios: for example, PCC, after smoothing by moving averages, was 0.75 (0.52-0.89) in a scenario of steadily decreasing nonmarital sex and 0.39 (0.01-0.73) in a scenario of transient drops in nonmarital sex. Simulations assuming self-reported levels of extramarital sex from Kenya yielded nonpaternity levels lower than global nonpaternity data, suggesting sizable underreporting of extramarital sex. CONCLUSIONS: Nonpaternity and half-siblings ratios are useful objective measures of extramarital sex that avoid limitations in self-reported sexual behavior.

Association of interleukin-8 gene polymorphisms in HIV patients with opportunistic infections in Limpopo Province, South Africa.

Opportunistic infections (OIs) are common among human immunodeficiency virus (HIV) patients; however, genetic susceptibility to these infections has not been studied. Recent studies have shown that interleukin-8 (IL-8) A/T genotype carriers are more susceptible to a variety of diseases. In this study, we showed the effects of IL-8 gene polymorphisms on OIs and symptoms such as sexually transmitted diseases (STDs), tuberculosis (TB), diarrhea, shortness of breath, weight loss, and viral load, in HIV and acquired immunodeficiency syndrome patients. Genomic DNA was purified from mouthwash samples collected from patients attending HIV centers in the Vhembe district. The IL-8 (-251) A/T locus was genotyped using allele-specific polymerase chain reaction followed by agarose gel electrophoresis. The results showed a weak association between the IL-8 AA genotype and OIs such as STDs (P = 0.143), diarrhea (P = 0.906), and TB (P = 0.762). Significant associations were found between the IL-8 AT genotype and weight loss (P = 0.019), shortness of breath (P = 0.043), and skin problems (P = 0.003). Low viral load was also found to be significantly associated with IL-8 AA genotype (P = 0.009). The present study suggests that different IL-8 genotypes are associated with resistance to various OIs. However, further studies using larger samples sizes are needed to confirm this hypothesis.

Increase in Sexually Transmitted Infections among Men Who Have Sex with Men, England, 2014.

Surveillance data from sexual health clinics indicate recent increases in sexually transmitted infections, particularly among men who have sex with men. The largest annual increase in syphilis diagnoses in a decade was reported in 2014. Less condom use may be the primary reason for these increases.

RNA Biosignatures in Adolescent Patients in a Pediatric Emergency Department With Pelvic Inflammatory Disease.

BACKGROUND: Adolescents are at high risk for pelvic inflammatory disease (PID). Because accurate diagnosis of PID is difficult, and complications of untreated PID are significant, novel methods to improve diagnosis are essential. OBJECTIVES: To determine if patients with PID have unique RNA expression patterns compared to controls. METHODS: Peripheral blood was collected from adolescent females with PID in the emergency department, and from control patients in the operating room. RNA was isolated, and microarray analysis was performed. Initial analysis involved a training set of 18 patients (9 PID patients with either Neisseria gonorrhoeae or Chlamydia trachomatis infection and 9 control patients). Supervised and unsupervised cluster analyses were performed, followed by network analysis. The training set was used to classify a set of 15 additional PID patients and 2 controls. RESULTS: Supervised cluster analysis of the training set revealed 170 genes which were differentially expressed in PID patients versus controls. Network analysis indicated that several differentially expressed genes are involved in immune activation. Analysis of additional PID patients based on the training set findings revealed that patients with positive testing for Trichomonas vaginalis partitioned with the PID group, whereas patients with no organism identified partitioned with both groups. CONCLUSIONS: RNA sample collection from adolescents in the emergency department is feasible. Genes were identified which were differentially expressed in PID patients versus controls, many of which are involved in inflammation. Future studies should confirm the training set findings on a larger sample and may lead to improved accuracy of PID diagnosis.

Interruption of CXCL13-CXCR5 axis increases upper genital tract pathology and activation of NKT cells following chlamydial genital infection.

BACKGROUND: Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection. METHODOLOGY AND PRINCIPAL FINDINGS: Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5-/- mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts) after Chlamydia muridarum genital infection (GT). Further studies in Cxcr5-/- mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα) in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d-/- mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922). CONCLUSIONS/SIGNIFICANCE: These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in human tubal pathology following C. trachomatis infection.

The molecular diagnosis of sexually transmitted genital ulcer disease.

Highly sensitive and specific nucleic acid amplification tests (NAATs) have emerged as the gold standard diagnostic tests for many infectious diseases. Real-time PCR has further refined the technology of nucleic acid amplification with detection in a closed system and enabled multiplexing to simultaneously detect multiple pathogens. It is a versatile, fast, and high-throughput system for pathogen detection that has reduced the risk of PCR contamination, eliminated post-PCR manipulations, and improved the cost-effectiveness of testing. In addition, real-time PCR can be applied to self-collected noninvasive specimens. Here, we describe an in-house developed TaqMan-based real-time multiplex PCR (M-PCR) assay for the diagnosis of sexually transmitted genital ulcer disease (GUD) and discuss briefly on issues associated with validation of assay performance.

Protocol for analyzing human leukocyte antigen variants and sexually transmitted infections: from genotyping to immunoassays.

This chapter describes experimental and analytical procedures that can be used to decipher the specific role of human leukocyte antigen (HLA) variants in infectious diseases. The techniques are distilled from more than one decade of active immunogenetics research, primarily on sexually transmitted infections (STIs) caused by viral and bacterial pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and Chlamydia trachomatis. The specific approaches cover (1) sequence-specific oligonucleotide (SSO) probe hybridization for low-resolution genotyping, (2) sequencing-based typing (SBT) for high-resolution, (3) statistical methods for testing associations between HLA variants and phenotypic traits, and (4) enzyme-linked immunospot (ELISpot) assay for enumerating HLA-restricted and epitope-specific T-lymphocyte responses. Proper application of these mature and robust techniques should help establish the importance of individual HLA alleles, haplotypes, and supertypes to host-pathogen interactions.

Influence of host genetic and ecological factors in complex concomitant infections - relevance to sexually transmitted infections.

While there is evidence that host genetics plays a role in susceptibility and subsequent sequelae of sexually transmitted infections (STIs), association findings have been inconsistent in deciphering the causal genes or biological pathways involved in the different life cycle and pathogenesis of infectious microbes. The lack of replication and validation studies from genome-wide association studies in general and specifically with infectious diseases, including STIs, is a continuing problem that limits the utility of these studies. Cohort heterogeneity, sample size, and confounding by population stratification due to differences in genetic polymorphisms in different ethnic groups are the usual explanations. However, in the context of genetic epidemiology studies of infectious disease, apart from the involvement of at least two genomes (the host and the pathogen), local environmental factors in the host shared by concomitant infections are often not examined. Different infectious microbes contribute to the shared local microenvironment, and the immune response can be favorable or unfavorable to different microbes individually and concomitantly at various levels. The balance of each infection relative to the other concomitant infections is a major confounder that has been under-studied. Thus, host genetic studies examining only one pathogen can yield inconsistent associations. This warrants a new paradigm that uses an ecological network-based study design and analysis tools. Defining the role of genetics in concomitant infection is likely to provide insight into pathogenic and protective mechanisms and to identify interdependent molecular targets for prophylactic and therapeutic interventions to multiple co-infections.

Transmitted HIV-1 drug resistance among young men of color who have sex with men: a multicenter cohort analysis.

BACKGROUND: Given the elevated potential for primary or transmitted drug resistance (TDR) among newly HIV-infected individuals, there is a need for a deeper understanding of the baseline resistance patterns present in young men of color who have sex with men. METHODS: Genotypic data were collected for participants aged 13-24 who were enrolled from seven sites. Univariate and bivariate methods were used to describe the prevalence of TDR and characteristics associated with TDR. RESULTS: Of the 296 individuals participating in the substudy, 145 (49%) had baseline genotypes. The majority of the individuals were African American (65%) and gay-identified (70%). There was significant variation in genotype availability by site (p < .001). Major surveillance drug resistance mutations were present in 28 subjects (19.3%); the majority were non-nucleoside reverse transcriptase inhibitor mutations (12.4%). Subjects with TDR were less likely to have used alcohol on 1 or more days in the prior 2 weeks. Location was not associated with acquisition of TDR. CONCLUSIONS: There was a high rate of TDR in a geographically and racially diverse sample of HIV-infected young men of color who have sex with men. This represents a serious public health concern given the young age of this sample and the potential need for long-term antiretroviral therapy. These findings underscore the critical roles of both early case identification and secondary prevention.