Chlamydia trachomatis antimicrobial susceptibility in colorectal and endocervical cells.

Background: Rectal Chlamydia trachomatis infections represent one of the most common sexually transmitted infections in the MSM population. Although current treatment guidelines suggest the use of either azithromycin or doxycycline, several clinical studies reported on azithromycin treatment failures in the case of rectal C. trachomatis localizations. In this context, the biological reasons behind the lack of azithromycin efficacy for C. trachomatis infections at the rectal level are still poorly understood. Objectives: To evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, mimicking the urogenital localization and the rectal site of infection. Methods: The susceptibility to macrolides (i.e. azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G), both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines. Results: For all the C. trachomatis strains, regardless of the genovar, macrolides showed higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Moreover, azithromycin MICs were significantly higher with increasing levels of C. trachomatis elementary bodies on Caco-2 cells. Conclusions: The higher azithromycin MICs observed in colorectal cells, together with the positive correlation between MICs and C. trachomatis loads found, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site.

Prevalence and antimicrobial resistance of genital Mollicutes in Italy over a two-year period.

Knowledge of the prevalence and antimicrobial susceptibility of genital Mollicutes is crucial to offer guidelines for empirical treatments. The aim of this study was to investigate the prevalence and the resistance profile of Mycoplasma hominis (MH) and Ureaplasma urealyticum/Ureaplasma parvum (UU/UP) in genital samples over a two-year period in Bologna, Italy. From January 2015 to December 2016, data on all the subjects providing uro-genital specimens for Mollicutes detection by culture were analyzed. A total of 4660 subjects (84.4% females) were enrolled and an overall Mollicutes prevalence of 30.9% was found. Women turned positive for Mollicutes infection twice as often as men (33.3% vs 17.8%) and the detection rate progressively decreased with increasing age. Ureaplasmas represented the commonest species isolated (overall prevalence: 24.2%), whereas mixed infections (6.5%) and MH single infections (3.9%) were far less common. Ureaplasma species showed significant levels of quinolone resistance, especially to ciprofloxacin (77%), whereas MH strains were non-susceptible to azithromycin and roxithromycin in about 90% of cases. Mollicutes co-infections showed a more severe resistance pattern than single infections. Over time, the resistance rate for azithromycin and roxithromycin increased significantly. Globally, our results revealed that minocycline and doxycycline can still be first-line drugs for Mollicutes treatment.

Distribution of genital Mollicutes in the vaginal ecosystem of women with different clinical conditions.

Ureaplasma urealyticum (UU), Ureaplasma parvum (UP), Mycoplasma hominis (MH) and Mycoplasma genitalium (MG) are the most common Mollicutes of the female genital tract. Although many studies have addressed their possible role in the vaginal ecosystem, many aspects remain to be elucidated. The aim of this study was to evaluate the vaginal presence of ureaplasmas/mycoplasmas in women with different clinical conditions. By means of quantitative PCR assays, the prevalence and load of each Mollicute were assessed in different groups of pre-menopausal women: 'healthy' (n=29), women with bacterial vaginosis (BV) (n=21), patients with Chlamydia trachomatis (CT) infection (n=25) and subjects with vulvo-vaginal candidiasis (VVC) (n=23). Globally, UP was the most prevalent Mollicutes in the vagina (67.3%), followed by MH (14.3%), UU (9.2%) and MG (3.1%). The presence of UU and UP was almost never associated. MH showed a significantly higher prevalence and higher bacterial loads in BV-positive women (P<0.05), whereas patients with CT and VVC were characterized by a Mollicutes pattern similar to healthy women. Mollicutes can be frequently found in the vaginal ecosystem, even in asymptomatic 'healthy' women. Although its presence is not a strict requirement, MH displays a significant role in the pathogenesis of BV.

Seroprevalence of a "new" bacterium, Simkania negevensis, in renal transplant recipients and in hemodialysis patients.

BACKGROUND: Simkania negevensis is an obligate intracellular bacterium belonging to the family Simkaniaceae in the Chlamydiales order. It is considered an ubiquitous microorganism and aquatic environments may be involved as a source of infection for humans. It was just isolated in samples from domestic water supplies and from mains water supplies, like spa water or swimming pool water, confirming its ability to resist to the common chlorination treatments. Evidence indicates a possible role of the microorganism in respiratory tract infections, in gastroenteric disorders and in the pathogenesis of cardiovascular disease, furthermore it has hypothesized that it could play a role in lung transplant rejection. Prevalence and possible effects in nephrology are unknown. METHODS: We examined the occurrence of Simkania negevensis in two differents populations, both characterized by a high susceptibility to infectious complications: 105 hemodialysis patients, 105 renal transplant recipients and 105 healthy subjects through the IgG and IgA response to Simkania negevensis in their sera. Serum antibodies to Simkania negevensis were detected by a homemade ELISA performed according to the Kahane's protocol. Furthermore water samples from hemodialytic circuit were collected, to evaluate Simkania negevensis resistance to usual treatment of disinfection. RESULTS: Our results were unexpected, showing a higher seroprevalence of antibodies against Simkania negevensis in the hemodialysis patients, compared to renal transplant patients (IgG 22% vs 9% - IgA 9% vs 3%). S. negevensis was isolated in all water samples analyzed. CONCLUSIONS: Our study detected for the first time the occurrence of S. negevensis in hemodialysis and in renal transplant patients. Our findings suggest that water used in hemodialysis could be one of the possible sources of S. negevensis infection, without clinical involvement risk for patients.

In vitro activity of a partially purified and characterized bark extract of Castanea sativa Mill. (ENC®) against Chlamydia spp.

Castanea sativa Mill (ENC®), containing tannins against 33 Chlamydia strains, was compared to SMAP-29 with inhibitory effect against C. trachomatis and C. pneumoniae. The ENC® activity against Chlamydia spp. was evaluated determining the lowest concentration to achieve more than half reduction of intact chlamydial inclusions versus controls. ENC® reduced all Chlamydia strains tested at 1 µg/mL, while SMAP-29 induced reductions of C. trachomatis and C. pneumoniae infectivity at 10 µg/mL. A great reduction of C. trachomatis, C. pneumoniae, and C. abortus infectivity was achieved with a 10 µg/mL ENC® concentration, whereas their infectivity was almost inhibited at 100 µg/mL ENC® concentration.

A Case of Reactive Arthritis Associated With Lymphogranuloma Venereum Infection in a Woman.

We report the first case of reactive arthritis associated with lymphogranuloma venereum (LGV) in an Italian human immunodeficiency virus-negative woman with urogenital and rectal Chlamydia trachomatis L2 serovar infection. The LGV-associated arthritis has to be considered even when classic symptoms of arthritis are missing and in case of asymptomatic or cryptic LGV localizations.

Evaluation of a new protocol for retrospective diagnosis of congenital toxoplasmosis by use of Guthrie cards.

The aim of this study was to assess the diagnostic value of IgM Western blotting (WB), IgA enzyme immunoassay (EIA), and DNA amplification by real-time PCR on Guthrie cards to retrospectively establish the diagnosis of congenital toxoplasmosis (CT). To this purpose, Guthrie cards were collected from 18 infants born to mothers with primary Toxoplasma gondii infection during pregnancy. Moreover, the analytical sensitivity of T. gondii PCR was assessed by testing mock dried blood specimens set up with several known DNA dilutions. IgM WB was demonstrated to be the most sensitive method. When the results of T. gondii DNA detection and specific IgM recovery were combined, retrospective CT diagnosis by using Guthrie cards was established in 3 out of 6 infected infants (sensitivity, 50%; 95% confidence interval, 26.8% to 73.2%). No positive PCR or serologic results were found in the group of 12 uninfected infants, demonstrating the excellent specificity of the three methods (95% confidence interval, 78.1% to 99.5%). The findings of the present study suggest that, in cases of missed diagnosis of CT at birth, analysis of Guthrie cards for children with compatible clinical findings after the perinatal period, in particular the combination of recovery of specific IgM antibodies and T. gondii DNA amplification, could be helpful. Nevertheless, since suboptimal conditions of storage of dried blood specimens can seriously affect sensitivity, negative results cannot rule out CT diagnosis. In contrast, because of the excellent specificity shown by IgM serologic testing and T. gondii DNA amplification on Guthrie cards, positive results obtained by either of the two methods should be considered diagnostic.

Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines.

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4(+)/IFN-γ(+) T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4(+)/IFN-γ(+)-inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4(+) T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

IgG and IgA response to Simkania negevensis in sera of patients with respiratory and gastrointestinal symptoms.

The presence of IgG and IgA antibodies to Simkania negevensis in adult Italian patients with respiratory or gastrointestinal symptoms was investigated by the microimmunofluorescence test. In patients with respiratory infections, IgG (50%) and IgA (13%) seropositivity was consistent with previous data. In patients with gastrointestinal disorders, IgG (68%) and IgA (18%) seroprevalence was significantly higher than in healthy controls. These results, in association with the previously described detection of S. negevensis in water sources, could suggest an oral route of infection other than droplets or close contact, and a possible association of S. negevensis with gastrointestinal infections.

Detection of Simkania negevensis in cell culture by using a monoclonal antibody.

In the present study, a monoclonal antibody (mAb), D5-14, raised in our laboratory against Chlamydia trachomatis LGV2 serotype, stained Simkania negevensis inclusions in S. negevensis-infected cells by using the immunofluorescence test. D5-14 mAb, reacting in immunoblot with an approximately 64-66-kDa protein of C. trachomatis LGV2 serotype, recognized a protein with the same molecular mass when tested with S. negevensis elementary bodies.

Dual-color bioluminescent assay using infected HepG2 cells sheds new light on Chlamydia pneumoniae and human cytomegalovirus effects on human cholesterol 7α-hydroxylase (CYP7A1) transcription.

Chlamydia pneumoniae and human cytomegalovirus (HCMV) are intracellular pathogens able to infect hepatocytes, causing an increase in serum triglycerides and cholesterol levels due to the production of inflammatory cytokines. We investigated whether these pathogens could interfere with cholesterol metabolism by affecting activity of hepatic cholesterol 7α-hydroxylase (CYP7A1) promoter. CYP7A1 is the rate-limiting enzyme responsible for conversion of cholesterol to bile acids, which represents the main route of cholesterol catabolism. A straightforward dual-reporter bioluminescent assay was developed to simultaneously monitor CYP7A1 transcriptional regulation and cell viability in infected human hepatoblastoma HepG2 cells. C. pneumoniae and HCMV infection significantly decreased CYP7A1 promoter activity in a dose-dependent manner, with maximal inhibitions of 33±10% and 32±4%, respectively, at a multiplicity of infection of 1. To support in vitro experiments, serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose levels were also measured in Balb/c mice infected with C. pneumoniae. Serum cholesterol and triglycerides also increased in infected mice compared with controls. Although further investigation is required, this work presents the first experimental evidence that C. pneumoniae and HCMV inhibit CYP7A1 gene transcription in the cultured human hepatoblastoma cell line.

Evaluation of the new test VERSANT CT/GC DNA 1.0 assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) are the two most common sexually transmitted bacterial infections in developed countries. The purpose of the present study was evaluating a new system for CT/GC detection in urine specimens. A total of 700 urine specimens were obtained from patients attending the STD Outpatients Clinic of St. Orsola University Hospital, Bologna, Italy. Samples were tested by VERSANT® CT/GC DNA 1.0 Assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY), a multiplex Real-Time PCR assay, for simultaneous CT/GC detection. Results obtained by VERSANT assay were compared with those obtained by culturing genital secretions of the same patients. Moreover, urine specimens testing positive in VERSANT assay were retested by in-house PCR assays, used as confirmatory tests. VERSANT® CT/GC DNA 1.0 Assay performed with 99.4% and 99.2% of specificity for GC and CT detection, respectively, whereas sensitivity was 100% both for CT and GC. Culture methods were 100% specific, but far less sensitive than VERSANT assay. VERSANT® CT/GC DNA 1.0 Assay demonstrated to be a highly sensitive and specific technique for CT/GC detection.

Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy.

We studied the prevalence of Chlamydia trachomatis (CT) urogenital infection and the distribution of different genotypes in a non-selected STD population of 1625 patients, evaluating presence of coinfections with other sexually transmitted diseases. Each patient was bled to perform serological tests for syphilis and HIV, then urethral or endocervical swabs were obtained for the detection of CT and Neisseria gonorrhoeae by culture. DNA extracted from remnant positive swabs was amplified by omp1 Nested PCR and products were sequenced. Total prevalence of CT infection was 6.3% (103/1625), with strong differences between men and women (11.4% vs 3.9%, P<0.01). Clinical symptoms and coinfections were much more frequent in men than in women (P<0.01). The most common serovar was E (prevalence of 38.8%), followed by G (23.3%), F (13.5%) D/Da (11.6%) and J (4.8%). Serovars distribution was statistically different between men and women (P=0.042) and among patients with or without coinfection (P=0.035); patients infected by serovar D/Da showed the highest coinfection rate. This study can be considered a contribution in increasing knowledge on CT serovar distribution in Italy. Further studies are needed to better define molecular epidemiology of CT infection and to investigate its correlation with other STDs.

In vitro activities of several antimicrobial agents against recently isolated and genotyped Chlamydia trachomatis urogenital serovars D through K.

A systematic evaluation of the susceptibility of all Chlamydia trachomatis urogenital serovars (D through K) to levofloxacin, erythromycin, doxycycline, clarithromycin, and azithromycin was performed. All C. trachomatis serovars had comparable susceptibilities with respect to the various antimicrobials tested, thus confirming the homogeneous data so far obtained regarding the susceptibility of C. trachomatis to antimicrobial agents.

Chlamydophila felis: plasmid detection in Italian isolates.

Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

CT043, a protective antigen that induces a CD4+ Th1 response during Chlamydia trachomatis infection in mice and humans.

Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4(+) Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4(+) Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.

Serological response to pgp3 protein in animal and human chlamydial infections.

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.

Laboratory diagnosis of syphilis with automated immunoassays.

The serological detection of specific antibodies to Treponema pallidum is of particular importance in the diagnosis of syphilis. The purpose of this study was to evaluate diagnostic performances of automated immunoassays in comparison with T. pallidum hemagglutination test (TPHA) and Western Blot (WB). The retrospective study was performed with different panels of sera: 244 clinical and serological characterized syphilitic sera and 203 potentially interfering samples. All the sera were tested by Enzygnost Syphilis, ARCHITECT Syphilis TP, TPHA, and homemade WB. The diagnostic performances of the two assays were very similar: both Enzygnost Syphilis and ARCHITECT Syphilis TP performed with a sensitivity of 99.2%, whereas the specificity was 98.5 and 98.4%, respectively. Considering the suitability for automation, both immunoassays may represent a good choice as a screening test. However, the use of a confirmatory test, such as TPHA or WB, remains a must in order to avoid false-positive results.

Insights Into Vaginal Bacterial Communities and Metabolic Profiles of Chlamydia trachomatis Infection: Positioning Between Eubiosis and Dysbiosis.

The vaginal microbiota plays a crucial role in maintaining the health and functioning of the female genital tract, preventing the colonization of urogenital pathogens and sexually transmitted infections. In this study, we characterized the vaginal bacterial communities and the metabolome associated to Chlamydia trachomatis infection (CT: 20 women), compared to healthy condition (H: 22 women) and bacterial vaginosis (BV: 19 women). A microarray-based tool (VaginArray), implemented with a real-time PCR for Gardnerella vaginalis, was used to determine the vaginal bacterial composition, whereas the metabolic profiles were assessed by a proton-based nuclear magnetic resonance (1H-NMR) spectroscopy. CT infection was characterized by bacterial and metabolic signatures similar to healthy condition, even though higher amounts of Lactobacillus iners, as well as depletion of some amino acids, biogenic amines, and succinate marked CT infection. Moreover, the frequency of Lactobacillus crispatus was higher in asymptomatic CT-positive patients than in women with CT-correlated symptoms. We also confirmed the marked differences in the microbiome and metabolome between healthy and BV-affected women. In conclusion, we highlighted microbial and metabolic peculiarities of the vaginal ecosystem in the case of CT infection, even though further studies are needed to understand if the observed alterations precede the infection onset or if the pathogen itself perturbs the vaginal environment.