Genomic profiling and characterization of ocular Chlamydia trachomatis reference strain B/HAR36.

The human pathogen Chlamydia trachomatis has multiple serovariants that have distinct organotropisms. We recently revised genomic sequence data linked to ocular reference strain, B/HAR36. Now linked to its correct genomic data in the European Nucleotide Archive, we describe its genomic features.

Unravelling Chlamydia trachomatis diversity in Amhara, Ethiopia: MLVA-ompA sequencing as a molecular typing tool for trachoma.

Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections, alongside improving facial cleanliness and environmental conditions to reduce transmission. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes could be useful. While many studies have used the Ct major outer membrane protein gene (ompA) for genotyping, it has limitations. Our study applies a typing system novel to trachoma, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing 3 variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control.

Genomic insights into local-scale evolution of ocular Chlamydia trachomatis strains within and between individuals in Gambian trachoma-endemic villages.

Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovars A-C, is the leading infectious cause of blindness worldwide. Africa bears the highest burden, accounting for over 86 % of global trachoma cases. We investigated Ct serovar A (SvA) and B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and the implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swabs across nine Gambian villages during a 6 month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted whole-genome sequencing (WGS) on a limited number (n=43) of Ct-positive samples with an omcB load ≥10 from four villages. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had highest identity to ompA from strain A/HAR13 (NC_007429) and 30 had highest identity to ompA from strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains, Ct A/HAR13 and Ct B/Jali20, indicated an single nucleotide polymorphism accumulation rate of 2.4×10-5 per site per year for the Gambian SvA and 1.3×10-5 per site per year for SvB variants (P<0.0001). Variant calling resulted in a total of 1371 single nucleotide variants (SNVs) with a frequency >25 % in SvA sequences, and 438 SNVs in SvB sequences. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP) and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB, hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.

When Bacteria and Viruses Collide: A Tale of Chlamydia trachomatis and Sexually Transmitted Viruses.

The global incidence of sexually transmitted infections (STIs) remains high, with the World Health Organization (WHO) estimating that over 1 million people acquire STIs daily. STIs can lead to infertility, pregnancy complications, and cancers. Co-infections with multiple pathogens are prevalent among individuals with an STI and can lead to heightened infectivity and more severe clinical manifestations. Chlamydia trachomatis (CT) is the most reported bacterial STI worldwide in both men and women, and several studies have demonstrated co-infection of CT with viral and other bacterial STIs. CT is a gram-negative bacterium with a unique biphasic developmental cycle including infectious extracellular elementary bodies (EBs) and metabolically active intracellular reticulate bodies (RBs). The intracellular form of this organism, RBs, has evolved mechanisms to persist for long periods within host epithelial cells in a viable but non-cultivable state. The co-infections of CT with the most frequently reported sexually transmitted viruses: human immunodeficiency virus (HIV), human papillomavirus (HPV), and herpes simplex virus (HSV) have been investigated through in vitro and in vivo studies. These research studies have made significant strides in unraveling the intricate interactions between CT, these viral STIs, and their eukaryotic host. In this review, we present an overview of the epidemiology of these co-infections, while specifically delineating the underlying mechanisms by which CT influences the transmission and infection dynamics of HIV and HSV. Furthermore, we explore the intricate relationship between CT and HPV infection, with a particular emphasis on the heightened risk of cervical cancer. By consolidating the current body of knowledge, we provide valuable insights into the complex dynamics and implications of co-infection involving CT and sexually transmitted viruses.

Comparison of genovars and Chlamydia trachomatis infection loads in ocular samples from children in two distinct cohorts in Sudan and Morocco.

Trachoma is a blinding disease caused by repeated conjunctival infection with different Chlamydia trachomatis (Ct) genovars. Ct B genovars have been associated with more severe trachoma symptoms. Here, we investigated associations between Ct genovars and bacterial loads in ocular samples from two distinct geographical locations in Africa, which are currently unclear. We tested ocular swabs from 77 Moroccan children (28 with trachomatous inflammation-follicular (TF) and 49 healthy controls), and 96 Sudanese children (54 with TF and 42 healthy controls) with a Ct-specific real-time polymerase chain reaction (PCR) assay. To estimate bacterial loads, Ct-positive samples were further processed by multiplex real-time qPCR to amplify the chromosomal outer membrane complex B and plasmid open reading frame 2 of Ct. Genotyping was performed by PCR-based amplification of the outer membrane protein A gene (~1120 base pairs) of Ct and Sanger sequencing. Ct-positivities among the Moroccan and Sudanese patient groups were 60·7% and 31·5%, respectively. Significantly more Sudanese patients than Moroccan patients were genovar A-positive. In contrast, B genovars were significantly more prevalent in Moroccan patients than in Sudanese patients. Significantly higher Ct loads were found in samples positive for B genovars (598596) than A genovar (51005). Geographical differences contributed to the distributions of different ocular Ct genovars. B genovars may induce a higher bacterial load than A genovars in trachoma patients. Our findings emphasize the importance of conducting broader studies to elucidate if the noted difference in multiplication abilities are genovar and/or endemicity level dependent.

Effects of iota-carrageenan on ocular Chlamydia trachomatis infection in vitro and in vivo.

Ocular chlamydial infections with the ocular serovars A, B, Ba, and C of Chlamydia trachomatis represent the world's leading cause of infectious blindness. Carrageenans are naturally occurring, sulfated polysaccharides generally considered safe for food and topical applications. Carrageenans can inhibit infection caused by a variety of viruses and bacteria. To investigate whether iota-carrageenan (I-C) isolated from the red alga Chondrus crispus could prevent ocular chlamydial infection, we assessed if targeted treatment of the conjunctival mucosa with I-C affects chlamydial attachment, entry, and replication in the host cell. Immortalized human conjunctival epithelial cells were treated with I-C prior to C. trachomatis infection and analyzed by flow cytometry and immunofluorescence microscopy. In vivo effects were evaluated in an ocular guinea pig inclusion conjunctivitis model. Ocular pathology was graded daily, and chlamydial clearance was investigated. Our study showed that I-C reduces the infectivity of C. trachomatis in vitro. In vivo results showed a slight reduced ocular pathology and significantly less shedding of infectious elementary bodies by infected animals. Our results indicate that I-C could be a promising agent to reduce the transmission of ocular chlamydial infection and opens perspectives to develop prophylactic approaches to block C. trachomatis entry into the host cell.

Detection of Chlamydiaceae and Chlamydia-like organisms on the ocular surface of children and adults from a trachoma-endemic region.

Trachoma, the leading infectious cause of blindness, is caused by Chlamydia trachomatis (Ct), a bacterium of the phylum Chlamydiae. Recent investigations revealed the existence of additional families within the phylum Chlamydiae, also termed Chlamydia-like organisms (CLOs). In this study, the frequency of Ct and CLOs was examined in the eyes of healthy Sudanese (control) participants and those with trachoma (case). We tested 96 children (54 cases and 42 controls) and 93 adults (51 cases and 42 controls) using broad-range Chlamydiae and Ct-specific (omcB) real-time PCR. Samples positive by broad-range Chlamydiae testing were subjected to DNA sequencing. Overall Chlamydiae prevalence was 36%. Sequences corresponded to unclassified and classified Chlamydiae. Ct infection rate was significantly higher in children (31.5%) compared to adults (0%) with trachoma (p < 0.0001). In general, 21.5% of adults and 4.2% of children tested positive for CLOs (p = 0.0003). Our findings are consistent with previous investigations describing the central role of Ct in trachoma among children. This is the first study examining human eyes for the presence of CLOs. We found an age-dependent distribution of CLO DNA in human eyes with significantly higher positivity in adults. Further studies are needed to understand the impact of CLOs in trachoma pathogenicity and/or protection.

The effect of infectious dose on humoral and cellular immune responses in Chlamydophila caviae primary ocular infection.

Following infection, the balance between protective immunity and immunopathology often depends on the initial infectious load. Several studies have investigated the effect of infectious dose; however, the mechanism by which infectious dose affects disease outcomes and the development of a protective immune response is not known. The aim of this study was to investigate how the infectious dose modulates the local and systemic humoral and the cellular immune responses during primary ocular chlamydial infection in the guinea pig animal model. Guinea pigs were infected by ocular instillation of a Chlamydophila caviae-containing eye solution in the conjunctival sac in three different doses: 1×102, 1×104, and 1×106 inclusion forming units (IFUs). Ocular pathology, chlamydial clearance, local and systemic C. caviae-specific humoral and cellular immune responses were assessed. All inocula of C. caviae significantly enhanced the local production of C. caviae-specific IgA in tears, but only guinea pigs infected with the higher doses showed significant changes in C. caviae-specific IgA levels in vaginal washes and serum. On complete resolution of infection, the low dose of C. caviae did not alter the ratio of CD4+ and CD8+ cells within guinea pigs' submandibular lymph node (SMLN) lymphocytes while the higher doses increased the percentages of CD4+ and CD8+ cells within the SMLN lymphocytes. A significant negative correlation between pathology intensity and the percentage of CD4+ and CD8+ cells within SMLN lymphocyte pool at selected time points post-infection was recorded for both 1×104, and 1×106 IFU infected guinea pigs. The relevance of the observed dose-dependent differences on the immune response should be further investigated in repeated ocular chlamydial infections.

Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA-Implications for Epithelial-Mesenchymal Transition.

Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.

PURPOSE: To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. METHODS: HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated. RESULTS: HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. CONCLUSIONS: We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.

Infectious dose and repeated infections are key factors influencing immune response characteristics in guinea pig ocular chlamydial infection.

The aim of this study was to determine whether infectious dose of Chlamydia caviae after repeated infections influences the immunological responses and subsequent clearance of pathogen at the ocular surface of guinea pigs. Animals were infected three times via the conjunctiva at six- and twelve-week intervals by applying either 1 × 10(4) or 1 × 10(6) inclusion-forming units (IFUs) of C. caviae. Ocular pathology, infection course, C. caviae-specific serum IgG levels and their capacity to bind and neutralize infection ex vivo were assessed. Animals infected with 1 × 10(4) IFUs had completely diminished ocular infection and pathology after the 2nd infection with increased levels of C. caviae-specific serum IgG and their effective capacity to bind and neutralize C. caviae. Only partial protection was observed in animals infected with 1 × 10(6) IFUs after the 2nd and 3rd infections. Our findings show that full protection was observed in animals repeatedly infected with the lower dose. The lower dose appeared not to compromise the host immune system, thereby enabling fast clearance of the pathogen and the establishment of competent neutralizing antibodies.

Evaluating the Effect of Copper Nanoparticles in Inhibiting Pseudomonas aeruginosa and Listeria monocytogenes Biofilm Formation.

BACKGROUND: Biofilm formation is a major virulence factor in different bacteria. Biofilms allow bacteria to resist treatment with antibacterial agents. The biofilm formation on glass and steel surfaces, which are extremely useful surfaces in food industries and medical devices, has always had an important role in the distribution and transmission of infectious diseases. OBJECTIVES: In this study, the effect of coating glass and steel surfaces by copper nanoparticles (CuNPs) in inhibiting the biofilm formation by Listeria monocytogenes and Pseudomonas aeruginosa was examined. MATERIALS AND METHODS: The minimal inhibitory concentrations (MICs) of synthesized CuNPs were measured against L. monocytogenes and P. aeruginosa by using the broth-dilution method. The cell-surface hydrophobicity of the selected bacteria was assessed using the bacterial adhesion to hydrocarbon (BATH) method. Also, the effect of the CuNP-coated surfaces on the biofilm formation of the selected bacteria was calculated via the surface assay. RESULTS: The MICs for the CuNPs according to the broth-dilution method were ≤ 16 mg/L for L. monocytogenes and ≤ 32 mg/L for P. aeruginosa. The hydrophobicity of P. aeruginosa and L. monocytogenes was calculated as 74% and 67%, respectively. The results for the surface assay showed a significant decrease in bacterial attachment and colonization on the CuNP-covered surfaces. CONCLUSIONS: Our data demonstrated that the CuNPs inhibited bacterial growth and that the CuNP-coated surfaces decreased the microbial count and the microbial biofilm formation. Such CuNP-coated surfaces can be used in medical devices and food industries, although further studies in order to measure their level of toxicity would be necessary.

Association between respiratory viruses and exacerbation of COPD: a case-control study.

BACKGROUND: Respiratory viral infection is the main cause of exacerbations of chronic obstructive pulmonary disease (COPD) in all age groups. The present study aimed to find out the association between viral infection in exacerbated and stable patients with COPD as well as evaluating the frequency of respiratory viruses in the Iranian exacerbated patients. METHODS: The study included 170 patients as the sample group with acute exacerbations and a control group consisting of 96 stable patients over a period of 3 years. Reverse transcription- nested polymerase chain reaction (RT-nested PCR) and nested PCR methods were used to diagnose the presence of 16 respiratory viruses. RESULTS: Viral infection was detected in 81 (47.6%) exacerbations and 24 (25%) stable patients (p < 0.05). Adenovirus was more frequent among the exacerbated patients than the stable patients (p < 0.05). Furthermore, influenza virus, respiratory syncytial virus, and enterovirus turned out to be the most common viruses in both groups. Moreover, respiratory viral co-infection has a possible role in exacerbation, severity, and longer hospitalization. Muscle pain and fever were found as significant symptoms in the infected patients with exacerbations. CONCLUSIONS: The current study investigated the probable roles of the respiratory viruses, and dual infections during acute exacerbations of COPD. Since climate-dependent respiratory viral incidence patterns in Iran are often dissimilar, preparing a comprehensive global model of respiratory infections with seasonal details in different geographical zones might decrease the morbidity and mortality rate in exacerbations of COPD.

The aetiological role of human papillomavirus in colorectal carcinoma: an Iranian population- based case control study.

BACKGROUND: Human papillomavirus (HPV) is one of the most common sexually transmitted infections worldwide and the association between HPV infection and genital cancers has been well established. This study concerned the possible role of HPV infection in colorectal carcinoma (CRC) in the Iranian population. MATERIALS AND METHODS: We examined 80 tissues obtained from patients with colorectal cancer consisting of 58 colon cancer samples and 22 rectal cancer samples and 80 tissues from patients with unremarkable pathologic changes as matched controls by sex, study center and anatomical sites. HPV infection and genotypes were detected using nested PCR and sequencing methods, respectively. RESULTS: HPV DNA was detected in 5/80 (6.25%) cases including 1 of 22 (4.54%) patients with rectum cancer and 4 of 58 (6.9%) patients with colon cancer and 1/80 (1.25%) of controls. Furthermore, HPV-18 was detected as the most frequent type and we found no significant correlation between prevalence of HPV infection and anatomical sub- sites. CONCLUSIONS: Although a causal relation between human papillomavirus and colorectal cancer was not found through this study, analysis of medical records pointed to a possible role for high- risk types of HPV in increasing the potential of aggressiveness in colorectal cancer. This study shows a particular frequency of HPV genotypes in patients with colorectal cancer in Iran. Since HPV vaccines are limited to a few types of virus, using cohort studies in different geographical zones to screen for patterns of HPV infection in different organs might increase the efficacy and optimization of the current vaccines.

Evaluation of human papillomavirus infections in prostatic disease: a cross-sectional study in Iran.

BACKGROUND: The role of inflammation in prostate diseases is suggested by the presence of inflammatory cells within the prostate in benign prostatic hyperplasia (BPH) and prostate cancer (PCa) patients. In addition, bacterial and viral infection may lead to chronic and recurrent inflammation of the prostate. The human papillomaviruses (HPVs) are a family of sexually transmitted viruses which have been implicated in the aetiology of cervical cancer and several other malignancies. This study evaluated the frequency of HPV infection in individuals with prostatic disease in Iran. MATERIALS AND METHODS: The study included formalin fixed paraffin- embedded tissue samples of 196 primary prostate cases, including 29 PCa and 167 BPH samples. HPV DNA was purified and amplified through MY09/MY11 and GP5+/GP6+ primers with nested PCR. All patients were interviewed using a questionnaire to collect demographic information. RESULTS: Nested PCR showed that HPV DNA was found in 17.2 percent of PCa samples and 4.8 percent of BPH samples (not significant). CONCLUSIONS: Our data do not support a significant role of HPV infection in prostatic disease in Iranian patients, but demographic data indicated a probable association between presence of HPV DNA and risk of inflammation in prostate tissue which might lead to prostate carcinoma. Further studies are required to elucidate any roles of HPV infection in prostatic disease.