Molecular Evidence of Chlamydia trachomatis Infection and Its Relation to Miscarriage.

BACKGROUND: Chlamydia trachomatis (CT) infection is the most common sexually transmitted disease in the world that can persist and also ascend in the genital tract. This intracellular and silent infection is related to some adverse pregnancy outcomes, such as miscarriage. The aims of this study were to explore the best CT screening tests using blood and vaginal samples and to investigate the correlation between CT infection and the incidence of miscarriage. MATERIALS AND METHODS: This case-control study was done in October 2013 through June 2014, using purposive sampling from 157 female participants with or without a history of miscarriage. The samples were taken after each participant had signed a letter of consent and had completed a questionnaire. To achieve the objectives of this study, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests were performed on vaginal swabs and blood samples, respectively. RESULTS: PCR results showed a significantly higher CT infection rate in the miscarriage group compared to the control group (11.3 vs. 0%, P=0.007). Anti-CT IgG and IgA antibodies were found in 4.2 and 2.1% of cases in the miscarriage group, and in 1.7 and 6.7% of cases in the control group, respectively (P>0.05). Despite lower humoral responses in this study, positive samples were detected only by one of the following techniques; PCR, ELISA IgA and ELISA IgG. It also should be noted that PCR worked best in terms of detection. CONCLUSION: Based on the obtained data, there is a strong association between molecular evidence of CT infection and miscarriage. A higher rate of CT detection in molecular tests compared to serological assays suggests that PCR could be used as the first-choice assay for detection of C. trachomatis. However, the importance of serological tests in detecting potential past CT infection or upper genital infection not amenable to sampling is undeniable.

Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is the most prevalent cause of bacterial sexually transmitted infections (STI) recognized throughout the world. The aim of this study is to determine different genotypes of genital C. trachomatis and the association between the serological markers of inflammation and genotypes of C. trachomatis in sexually active women (n=80) attending Shahid Beheshti Hospital in Isfahan, Iran. MATERIALS AND METHODS: In this descriptive study, endocervical swabs were collected from 80 women. There were 17 endocervical samples that showed positivity for C. trachomatis by plasmid polymerase chain reaction (PCR) using KL1 and KL2 primers. The omp1 gene was directly amplified in 17 plasmid PCR positive samples and was used to differentiate the clinical genotypes by omp1 gene PCR-restriction fragment length polymorphism (PCR-RFLP). The levels of IgG and IgA specific to C. trachmatis and C-reactive protein (CRP) were evaluated. RESULTS: Based on restriction-digestion patterns, four genotypes were identified. Genotypes E (35.3%) and F (35.3%) were the most prevalent, followed by D/Da (23.5%) and K (5.9%). There was no significant association between genotypes and the presence of IgG and CRP. Patients infected with genotype E showed a serological marker of chronic inflammation, i.e. IgA seropositivity, significantly more than patients infected with other genotypes (p=0.042). CONCLUSION: Nested PCR could increase the sensitivity of omp1 amplification. Based on the presence of IgA, chronic C. trachomatis infections were observed more frequently among genotype E-infected patients in our population.

Comparison of three methods of DNA extraction in endocervical specimens for Chlamydia trachomatis infection by spectrophotometry, agarose gel, and PCR.

Chlamydia trachomatis is the major cause of sexually transmitted disease in the world. The aim of this study was to determine the best method of DNA extraction for detecting C. trachomatis by polymerase chain reaction (PCR) in sexually active women (n = 80) attending Shahid Beheshti Hospital in Isfahan, Iran. Endocervical swabs were collected from 80 women, 22 of whom were asymptomatic and 58 symptomatic. Three different DNA extraction methods were used in this study (phenol-chlorophorm, proteinase K, and boiling). DNA yield was evaluated by spectrophotometry, agarose gel, and PCR. The internal control was assayed by beta-globin primers (PCO4, GH20). The DNA cryptic plasmid was selected as the target for C. trachomatis and samples were examined by PCR using specific KL1 and KL2 primers. It was shown that DNA extraction by boiling was the most sensitive with the highest yield of DNA. Of the 80 samples, 17 (21.25%) showed positivity for C. trachomatis by PCR. The highest rate of C. trachomatis infection was found in the group aged between 35 and 45 years old and those who used withdrawal or an intrauterine device as methods of contraception. It was demonstrated that DNA extraction by boiling was the least expensive and a very rapid method that gave the highest DNA yield. The infection rate in the sexually active women, including symptomatic and asymptomatic, was 21.25%, with a presumably high prevalence compared with other studies done in this field.

Detection of cloned strR, an antibiotic regulatory gene, using RFLP and nested PCR.

The genetics of streptomycin production is well characterized in Streptomyces griseus. More than 25 clustered genes encode proteins involved in biosynthesis, regulation and transport functions. StrR, the pathway specific transcriptional activator or regulator that located in this cluster, then induces transcription of other streptomycin production genes by binding multiple sites in the gene cluster. We aim to put strR gene in to different multicopy and integrated expression vector specifically designed for Streptomyces. To start with, the isolated strR gene was ligated into pBluescript (pBs) vector and transformed into different strains of Escherichia coli. The correct structure of the recombinant plasmid, isolated from transformed E. coli, was confirmed using gel electrophoresis, PCR and double digested with restriction enzymes BamHI and EcoRI. Finally the plasmid map, named pFDstrR. This unique vector has a much expanded Multiple Cloning Site (MCS), which makes it suitable for different purposes of gene cloning and also site directed mutagenesis or gene targeting. This gene will be lifted up and transfer into different varieties of Streptomyces specific vectors in order to make different transgenic or genetically manipulated Streptomyces.