Seroprevalence of group B Coxsackieviruses: Retrospective study in an Italian population.

Group B Coxsackieviruses (CVB) include six serotypes (B1-6) responsible for a wide range of clinical diseases. Since no recent seroepidemiologic data are available in Italy, the study aim was to investigate CVB seroprevalence in a wide Italian population. The study retrospectively included 2459 subjects referring to a large academic hospital in Rome (Italy) in the period 2004-2016. Seroprevalence rates and neutralizing antibodies (nAb) titers were evaluated in relation to years of observation and subjects' characteristics. Positivity for at least one serotype was detected in 69.1% of individuals. Overall, the prevalent serotype was B4, followed by B3 (33.3%), B5 (26.2%), B1 (12.7%), B2 (11.0%), and B6 (1.7%). For B2, a significant decrease in seroprevalence over years was observed. Positivity to at least one virus was 25.2% in children aged 0 to 2 years, but significantly increased in preschool (3-5 years) (50.3%) and school (6-10 years) children (70.4%). Higher nAb responses for B3 and B4 were observed in children aged 3 to 5 years. A high overall CVB prevalence was found. Type-specific variations in prevalence over time probably reflect the fluctuations in circulation typical of Enteroviruses. Children are at greater risk for CVB infection given the high number of seronegative subjects aged 0 to 10 years.

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria.

BACKGROUND: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells. RESULTS: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R2), ranging from 0,92 to 0,99. CONCLUSIONS: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.

Selected Immunological Mediators and Cervical Microbial Signatures in Women with Chlamydia trachomatis Infection.

In the female genital ecosystem, the complex interplay between the host immune system and the resident microflora protects against urogenital pathogens, like Chlamydia trachomatis C. trachomatis is responsible for urethritis and cervicitis; however, most chlamydial infections are asymptomatic and, thus, not treated, potentially leading to severe reproductive sequelae. Here we investigated the interaction between the levels of selected immune mediators and the community state types of the cervical microbiota in C. trachomatis-infected women. Cervical samples from 42 C. trachomatis-positive women and 103 matched healthy controls were analyzed through the metagenomic analysis of the hypervariable region v4 of the 16S rRNA gene and the determination of lactoferrin, interleukin 1α (IL-1α), IL-6, alpha interferon (IFN-α), IFN-ß, and IFN-γ by ELISA. Overall, C. trachomatis infection was significantly associated with a microbiota dominated by anaerobic bacteria (P = 0.000002). In addition, a network of Gardnerella vaginalis, Prevotella amnii, Prevotella buccalis, Prevotella timonensis, Aerococcus christensenii, and Variovorax guangxiensis has been identified as a potential biomarker of C. trachomatis infection through multiple statistical approaches. Again, chlamydial infection was significantly correlated with an increased production of lactoferrin, IL-6, IL-1α, IFN-α, and IFN-ß (P < 0.05), whereas very low levels of IFN-γ were observed in C. trachomatis-infected women, levels similar to those detected in healthy women. Our findings show a distinctive signature of C. trachomatis genital infection, characterized by a specific bacterial network, constituted by anaerobes, as well as by increased levels of lactoferrin and proinflammatory cytokines (IL-1α, IL-6, IFN-α, and IFN-ß), accompanied by low levels of IFN-γ.IMPORTANCE To our knowledge, this is the first study that investigated the association of C. trachomatis with the cervical levels of lactoferrin and selected inflammatory mediators and their correlation with the different community state types characterizing the female genital ecosystem. C. trachomatis, known as the leading cause of bacterial sexually transmitted diseases, continues to be an important public health problem worldwide for its increasing incidence and the risk of developing severe reproductive sequelae, like pelvic inflammatory disease and infertility. Specifically, C. trachomatis tend to persist in the female genital tract, leading to a chronic inflammatory state characterized by increased production of immune mediators responsible for tissue damage. Therefore, our study may help to broaden the knowledge on the complex interplay between the female genital microbiota and the host immune system in response to C. trachomatis infection.

Biofilm in Genital Ecosystem: A Potential Risk Factor for Chlamydia trachomatis Infection.

In healthy women, the cervicovaginal microbiota is mostly populated by Lactobacillus spp., the main host defense factor of the female genital tract. In addition to Lactobacilli, other microorganisms populate the cervicovaginal microbiota, like Candida spp. and Gardnerella vaginalis. The overgrowth of Candida spp. or G. vaginalis, known as biofilm-producing microorganisms in the genital ecosystem, may lead to microbial dysbiosis that increases the risk of acquiring sexually transmitted infections, like Chlamydia trachomatis. C. trachomatis, the leading cause of bacterial sexually transmitted diseases, is still considered an important public health problem worldwide because of the impact of asymptomatic infections on long-term reproductive sequelae, including pelvic inflammatory disease and infertility. The aim of our study was to investigate the interaction between C. trachomatis and the biofilm produced by Candida albicans or Gardnerella vaginalis, evaluating whether the biofilm can harbor C. trachomatis and influence its survival as well as its infectious properties. In order to do so, we developed an in vitro coculture transwell-based biofilm model. Our findings proved, for the first time, that C. trachomatis, an intracellular obligate pathogen, survived, for up to 72 hours after exposure, inside the biofilm produced by C. albicans or G. vaginalis, retaining its infectious properties, as evidenced by the typical chlamydial inclusions observed in the cell monolayer (chlamydial inclusion-forming units at 72 h: 9255 ± 1139 and 9873 ± 1015, respectively). In conclusion, our results suggest that the biofilm related to Candida or Gardnerella genital infections may act as a reservoir of C. trachomatis and, thus, contribute to the transmission of the infection in the population as well as to its dissemination into the upper genital tract, increasing the risk of developing severe reproductive sequelae.

TLR9 is expressed in human papillomavirus-positive cervical cells and is overexpressed in persistent infections.

Control of human papillomavirus (HPV) infection involves the activation of Toll-like receptors (TLRs), key components of the mucosal antiviral response. Available studies on TLR expression in HPV-positive cervical cells are limited and reported conflicting results. This study quantified TLR 2, 3, 4, 7 and 9 transcripts in low-risk (LR) and high-risk (HR) HPV-positive and HPV-negative cervical samples from 154 women attending a gynaecological clinic. Expression levels of TLR 2, 3, 4 and 7 did not differ among samples, whereas TLR9 levels were quite significantly higher in LR and marginally significant in HR HPV-positive samples, with respect to the HPV-negative samples. Interestingly, in a subgroup of women with documented previous HPV-infection, TLR9 levels were extremely higher in patients persistently positive to the same HPV genotype for more than 1 year, with respect to women who cleared HPV infection and to those re-infected with a different genotype. These findings implicate TLR9 in the response to LR and HR HPVs, including HPV 16 known to interfere with TLR9 transcription in cell lines. Elevated TLR9 levels without HPV clearance in persistently infected women could drive inflammation thereby contributing to cervical cancer risk.