Major Outer Membrane Protein Gene Research Articles

A novel method for the preparation of reproducible, stable, and non-infectious quality control materials for Chlamydia trachomatis nucleic acid detection.

Chlamydia trachomatis (CT) is a significant sexually transmitted pathogen known to evoke severe complications, including infertility. Nucleic acid amplification tests (NAATs) are recommended by the World Health Organization to detect CT infection. Furthermore, the establishment of methods, performance validation, internal quality control, and external quality assessment for CT NAATs necessitate the utilization of quality control materials (QCs). QCs are specimens or solutions that are analyzed for quality control purposes in a test system. In this study, we established a novel cell line that stably integrates CT amplification target sequences for producing QCs for CT NAATs. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, we integrated the CT plasmid-mediated sequence (comprising the full length of the cryptic plasmid and the major outer membrane protein gene, 9,136 bp) into the MUC4 gene of HEK293T cells. Positive clones were screened through flow cytometric sorting, single-cell culture, and PCR-based identification, followed by the establishment of stable cell lines. These cells were then processed using optimized cell preservation procedures to prepare QCs. The sequence insertion copy number was confirmed by real-time quantitative PCR. This novel CT QCs demonstrate excellent clinical applicability, non-infectiousness, quantifiability, and stability. With an integrated sequence exceeding 9 kb in length, it offers exceptional flexibility for adapting to new kit developments. Furthermore, maintaining a well-defined copy number and stable shelf life, the QCs closely aligns with the quality control requirements of CT NAATs. This study presents an innovative method for preparing QCs for CT nucleic acid detection, making a valuable contribution to improving the performance of CT NAATs.IMPORTANCEUntreated CT infections impose significant burdens on individuals and communities, underscoring the importance of early and accurate testing via CT NAATs for disease control. QCs are instrumental in identifying testing process issues. Hence, we developed a cell line integrating CT-amplified target sequences as readily accessible non-infectious QCs. These QCs boast several advantages: the integration of over 9 kb of CT sequence allows for broad applicability, allowing flexible adaptation to the development of new kits. Confirming the CT sequence copy number provides a reliable basis for QC concentration preparation and kit detection limit evaluation. Optimized preservation protocol enhances QC stability during storage, facilitating convenient shipment to clinical laboratories at ambient temperatures. In summary, our novel CT QCs offer a powerful tool for improving CT NAAT performance and present a fresh perspective on QC preparation for detecting nucleic acids from intracellular parasitic pathogens.

Vaginitis with purulent vaginal discharge caused by artificial insemination using frozen Histophilus somni-contaminated semen

In April 2020, two cows in Japan, developed reproductive disorders accompanied by vaginitis with purulent discharge within 3 days of artificial insemination (AI) with the same lot of frozen semen. Histophilus somni was isolated from the vaginal swabs of both cows as well as from the same lot of frozen semen used for the AI. This incident marks the first reported case of H. somni infection in cattle through AI. The major outer membrane protein gene sequences and pulsed-field gel electrophoresis profiles of the isolates were identical. Moreover, we investigated the antimicrobial activity of 12 frozen semen straws against an H. somni isolate using a disk diffusion test. These straws were sourced from five AI centers and included the same lot of semen used for the AI. Although the composition of semen diluents from individual AI centers is not publicly available, both the same lot of frozen semen used in the AI and other lots produced by the same manufacturer showed lower antimicrobial activity than semen from other manufacturers. These results strongly suggest that the two vaginitis were caused by AI using H. somni-contaminated frozen semen because of insufficient antimicrobial activity to inhibit bacterial growth. The minimum inhibitory concentrations of the six antimicrobials recommended for addition to frozen semen in isolates were below the recommended concentrations, suggesting that proper addition could have prevented this incident. This highlights the importance of conducting periodical checks on the antibacterial activity of frozen semen to prevent the transmission of pathogens via AI.

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.

Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles

The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

Correction for Zhi et al., "Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis".

Correction for Zhi et al., "Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis".

A molecular survey of Chlamydia spp. infection in commercial poultry and detection of Chlamydia pneumoniae in a commercial turkey flock in Iran.

Chlamydiaceae are a group of gram-negative intracellular bacteria which can infect a wide variety of hosts. Some chlamydial agents are capable of crossing the host barrier and though they are potentially a risk to very different species. They also pose a zoonotic risk for human and different chlamydial agents are linked to several medical maladies. In this study, the presence of chlamydial agents in different commercial poultry flocks in Iran was investigated. Swab and tissue samples were collected from 435 birds in 24 different commercial poultry flocks. These samples were examined using a Chlamydiaceae-specific real-time PCR assay targeting 23S rRNA gene. Positive samples then were subjected to intergenic spacer rRNA (IGS) gene and major outer membrane protein gene (ompA) PCRs. Finally, positive PCR products were sequenced and analysed. Only one flock of commercial turkey became positive. Partial DNA sequencing of IGS gene revealed that all positive samples from the infected flock were Chlamydia pneumoniae and were identical to previously studied isolates from koala (LPCoLN) and frog (DC9). Further investigations showed slight dissimilarity in ompA gene of C. pneumoniae from different hosts. The detected turkey isolates were located in a different clade of phylogenetic tree, close to Western barred bandicoot and koala isolates. C. pneumoniae has passed the cross-species barrier in the past and therefor it could potentially be zoonotic. To the best of authors' knowledge, this is the first report of C. pneumoniae infection in commercial turkey.

Expression of recombinant Omp18 and MOMP of Campylobacter jejuni and the determination of their suitability as antigens for serological diagnosis of campylobacteriosis in animals.

Campylobacteriosis causes gastrointestinal tract lesions in adults and children and may result in severe complications. The primary sources of infection are infected animals and animal products. Immunochemical methods effectively diagnose intestinal infections but require highly specific antigens to detect their antibodies. This study aimed to obtain two recombinant immunogenic antigens of Campylobacter jejuni, an outer membrane protein with a molecular weight of 18 kDa (Omp18) and the major outer membrane protein (MOMP) with a molecular weight of 45 kDa, and evaluate their suitability for the serological diagnosis of campylobacteriosis using immunochromatographic assay (ICA). The C. jejuni Omp18 and MOMP gene sequences were synthesized de novo (Macrogen, Korea) and cloned into the pET32 expression plasmid. Using these genetic constructs, electrocompetent cells of the Escherichia coli BL21 strain were transformed and cultured under various conditions. Antigens were purified and refolded using metal affinity chromatography. The properties of the purified proteins were studied by western blotting, liquid chromatography with tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). We developed two recombinant E. coli BL21 cells producing rOmp18 and Recombinant MOMP (rMOMP) antigens with molecular weights of 36 and 64 kDa, respectively. Amino acid sequence analysis of the obtained antigens showed complete homology with the reference sequences in the PubMed NCBI database. Western blotting using positive-control sera demonstrated the specificity of the recombinant antigens. The results of ELISA with 94 bovine sera showed the interaction of recombinant antigens with specific antibodies. The obtained rOmp18 and rMOMP antigens can detect antibodies in the serum of infected or recovered animals and can be used to develop ICA.

Evaluation of 2 Commercial Assays for the Detection of Lymphogranuloma Venereum in Rectal Samples

The early identification of the Chlamydia trachomatis variants that cause lymphogranuloma venereum (LGV) is very important to establish an adequate antibiotic treatment. This identification should be made with molecular techniques that are easy to perform and accessible to most microbiology laboratories. The objective of this study was to evaluate 2 real-time polymerase chain reaction (PCR)-based assay (VIASURE Haemophilus ducreyi + C. trachomatis (LGV) real-time PCR detection kit and the Allplex Genital ulcer Assay) for the detection of LGV in rectal samples. Prospective study on positive rectal samples for C. trachomatis. All samples were processed in parallel by both tests. As a molecular reference method and to solve possible discrepancies between both assays, a PCR-based restriction fragment length polymorphism analysis of the major outer membrane protein gene (omp1) was used. In total, we detected 157 positive rectal samples for C. trachomatis, of which 36 were identified as LGV by PCR-based restriction fragment length polymorphism analysis. The positive percent agreement, negative percent agreement, and overall percent agreement were 88.9%, 100%, and 97.3%, respectively, for the Allplex Genital ulcer assay and 91.6%, 100%, and 97.1%, respectively, for the VIASURE assay. In the direct comparison between the Seegene assay and the VIASURE assay, we obtained a kappa concordance index of 0.98 between both tests. According to the results obtained, both tests could be used for the detection of LGV in rectal samples.

Genetic and Serological Diversity of Chlamydia trachomatis Strains Detected from Japanese Infants

Background: Classical fifteen serovars and variants of Chlamydia trachomatis were originally classified by the micro-immunofluorescence test which principally distinguished epitopes on the major outer-membrane protein (MOMP). The sequences of MOMP gene including four variable domains (VDs) have been determined for all classic 15 serovars. The PCR assay was used to amplify a large part of the MOMP gene (ompA or omp1) including four VDs and then cataloged restriction fragment length polymorphism (RFLP) was used to identify the serovars from genotypes of C. trachomatis as reported previously. Materials and Methods: Nasopharyngeal specimens were collected from 15 Japanese infants with pneumonia, from 1 month to 1 year old, during the period May 1990 to September 2013. Conjunctival swabs were also collected from 12 Japanese neonates under 1 month old with inclusion conjunctivitis during the same period. The specimens were collected and placed into Abbott LCR proprietary medium and then tested by PCR for typing. The sequencing strategy used was the double-stranded DNA cycle sequencing method. Results: Applied to 15 reference strains, this PCR-RFLP procedure allowed us to determine 13 of the 15 serovars of C. trachomatis. Thirteen of 15 isolates of nasopharyngeal origin and 11 of 12 isolates of conjunctival origin could be un-equivocally assigned to one of the 15 reference serovars with a restriction endonuclease profile identical to that of the prototype. The typing of 15 nasopharyngeal isolates gave the following results: 11 as E, 2 as H, and 2 as unclassified serovars. The typing of 12 conjunctival isolates gave the following results: 5 as D, 4 as E, 1 as F, 1 as H and 1 as unclassified serovar. Among the unclassified serovars, one was B-like and the rest was D-like. Discussion: The sequences of MOMP gene for all classic 15 serovars allowed the construction of restriction endonuclease cleavage-site maps that confirm the fragment-size patterns observed by electrophoresis. Sequencing the entire MOMP gene and cataloging the sequences of VDs of all serovars has confirmed the molecular basis of the serotyping procedure and provided a method for determining serovars by PCR-RFLP. Although geno-variants have also been reported for most serovars the PCR-RFLP method for typing allows quick and objective identification of serovars of C. trachomatis. Antigenic variations of C. trachomatis were also considered among the strains from nasopharyngeal and conjunctival origins.

A Predominant Clonal Thromboembolic Meningoencephalitis Group of Histophilus somni Assigned by Major Outer Membrane Protein Gene Sequencing and Pulsed-Field Gel Electrophoresis.

Histophilus somni, a member of the family Pasteurellaceae, causes a variety of diseases, including thromboembolic meningoencephalitis (TEME) and respiratory diseases, which result in considerable economic losses to the cattle and sheep industries. In this study, 132 chronologically diverse isolates from cattle in Japan and 68 isolates from other countries comprising 49 from cattle and 19 from sheep were characterized using major outer membrane protein (MOMP) gene sequence and pulsed-field gel electrophoresis (PFGE) analyses. The H. somni isolates formed nine MOMP genetic clades (clade Ia, Ib, and II–VIII) and 10 PFGE clusters (HS1–HS10). Except for two (1.0%), all isolates fell into one of the nine MOMP genetic clades, while 62 (31.0%) isolates belonged to no PFGE cluster. MOMP genetic clade Ia and PFGE cluster HS1 were the major groups, and all HS1 isolates possessed the clade Ia MOMP gene. Isolates from TEME cases were significantly associated with these major groups (chi-square test, p < 0.0001), as 88.2% of the TEME isolates belonged to MOMP genetic clade Ia and PFGE cluster HS1, which formed the most predominant clonal group. After an inactivated vaccine using an HS1 strain with the clade Ia MOMP gene was introduced in Japan in late 1989, the number of TEME cases and isolates assigned into the clonal group decreased simultaneously. However, the proportions of clade Ia and cluster HS1 isolates from TEME cases remained high after 1990. These results suggest a close association of TEME with PFGE cluster HS1 and MOMP genetic clade Ia, and imply the presence of factors or characteristics commonly possessed by those strains that contribute to the development of TEME.

Investigation on Ehrlichia Infection in Small Mammals and Ticks from Tengchong, Yunnan Province, Southern China.

Rare investigation on tick-borne pathogens was carried out in Yunnan, China. In this study, we did a survey on Ehrlichia infection in small mammals and ticks. A total of 40 small mammals and 49 ticks were collected from Tengchong, Yunnan province. PCR targeting 16S ribosomal RNA (rRNA), citrate synthase, GroEL heat-shock protein operon, and major outer membrane protein genes was performed and positive amplicons were sequenced. The 40 small mammals were identified as 10 species, 2 (5.0%) of which were infected with Ehrlichia, 4 (10.0%) were infected with Anaplasma phagocytophilum and another 2 (5.0%) were infected with Candidatus Neoehrlichia mikurensis. Six (12.2%) ticks were positive for Ehrlichia and another two (4.1%) were infected with A. phagocytophilum. Neither small mammals nor ticks had coinfection. The detected Ehrlichia was named as Ehrlichia sp. YN04, which was in the same clade of Ehrlichia sp. 360 by phylogenetic analysis. The sequences of the pathogen recovered from small mammals and ticks were identical with each other. The study reports one Ehrlichia species first detected from small mammals and ticks in mainland China. As Yunnan is a famous "Global Biodiversity Hotspot" in the world, we may expect much more tick-borne infectious pathogens existing and declare more public health attention in this region.

Erratum to: Molecular characteristics of the ompA gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students.

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene (ompA) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in GenBank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The ompA gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information (NCBI) BLAST search and homology analysis. The entire ompA gene sequence was compared with the corresponding gene sequences of serotype B strains available in GenBank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening (average age, (9.09±1.63) years; sex ratio, 1.0), accounting for 57.78% (26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half (13/26) of C. trachomatis-positive students had a bacterial copy number of >105. The compliance rate of the ompA gene sequences with the C. trachomatis serotype B strains in GenBank was up to 99%. Two novel genetic mutations were found when the ompA gene was compared with those of the 11 serotype B strains in GenBank. The two non-synonymous mutations were located at (i) position 271 in the second constant domain, an adenine (A) to guanine (G) substitution (ACT→GCT), changing the amino acid at position 91 from threonine to alanine (Thr→Ala) in all 26 strains; and (ii) position 887 in the fourth variable domain, a cytosine (C) to thymine (T) substitution (GCA→GTA), changing the amino acid at residue 296 from alanine to valine (Ala→Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1 (China Qinghai Tibetan-1) and CQZ-2 (China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.

Laboratory investigation of a suspected outbreak caused by Providencia stuartii with intermediate resistance to imipenem at a long-term care facility.

Providencia stuartii survives well in natural environment and often causes opportunistic infection in residents of long-term care facilities (LTCFs). Clinical isolates of P. stuartii are usually resistant to multiple antibiotics. The bacterium is also naturally resistant to colistin and tigecycline. Treatment of infections caused by carbapenem-resistant P. stuartii is challenging. During a 15-month period in 2013-2014, four isolates (P1, P2, and P3B/P3U) of P. stuartii showing intermediate resistance to imipenem were identified at a regional hospital in southern Taiwan. They were identified from three patients (P1-P3) transferred from the same LTCF for the treatment of the infection. Pulsed-field gel electrophoresis was used to genotype the isolates. Resistance genes/plasmids and outer membrane proteins were investigated by polymerase chain reaction and sequence analysis. Isolates P1 and P3B/P3U demonstrated similar pulsotypes. All isolates were found to have resistance genes (blaCMY-2, qnrD1, aac(6')-Ib-cr) carried on nonconjugative IncA/C plasmids of different sizes. A single point mutation was identified in the chromosomal gyrA (Ser83Ile) and parC (Ser84Ile) genes of all isolates. Various point mutations and insertion/deletion changes were found in their major outer membrane protein gene ompPst1. Isolates of similar pulsotypes could appear after 15months and caused urosepsis in another resident of the same LTCF. The bacterium may have persisted in the environment and caused opportunistic infection. As LTCF residents are usually vulnerable to infections, surveillance of multidrug-resistant organisms and infection control intervention that have been established in acute-care hospitals to control infections by resistant organisms are apparently as essential in LTCFs.

Chlamydia and ocular adnexal lymphomas: An Indian experience

Chlamydia and ocular adnexal lymphomas: an Indian experienceOcular adnexal lymphomas (OALs) are a heterogeneous group of malignancies, majority being extranodal mucosa-associated lymphoid tissue (MALT) type. Different geographical regions have reported association of Chlamydia with OALs (MALT type). In India, role of Chlamydia in OALs remains unexplored. The aim of this study was to detect Chlamydia and to correlate with clinicopathological features of OALs in India. The clinicopathological features of 41 OAL cases were studied prospectively. Chlamydia DNA was detected by genus specific PCR amplifying major outer membrane protein (MOMP) gene followed by DNA sequencing. Chlamydia immunoexpression was evaluated by immunofluorescence and immunohistochemistry. The results were correlated with clinicopathological features including follow-up and survival. Chlamydia genome was detected in 3/41 (7.3%) OAL cases by PCR. Direct sequencing revealed C. trachomatis in 3 positive cases. Immunofluorescence and immunohistochemistry showed Chlamydia antigen in 5/41 and 1/41 cases respectively. Immunofluorescence demonstrated higher sensitivity than immunohistochemistry. A significant association was observed between Chlamydia positivity and orbital location (P=0.05). Follow-up revealed relapse in 2 Chlamydia positive cases (P=0.056). Our results demonstrate for the first time presence of C. trachomatis genome in 7.3% OAL cases in India. As no other reports are documented, more detailed studies from different regions within India are needed to explore status of Chlamydia in OALs.

Comparison of clinical performance of antigen basedenzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed.

Molecular characteristics of the ompA gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students.

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene (ompA) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in GenBank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The ompA gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information (NCBI) BLAST search and homology analysis. The entire ompA gene sequence was compared with the corresponding gene sequences of serotype B strains available in GenBank. Of the 45 students aged 6-13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening (average age, (9.09±1.63) years; sex ratio, 1.0), accounting for 57.78% (26/45). The cycle threshold values for real-time PCR were 16.79-37.77. Half (13/26) of C. trachomatis-positive students had a bacterial copy number of >10(5). The compliance rate of the ompA gene sequences with the C. trachomatis serotype B strains in GenBank was up to 99%. Two novel genetic mutations were found when the ompA gene was compared with those of the 11 serotype B strains in GenBank. The two non-synonymous mutations were located at (i) position 271 in the second constant domain, an adenine (A) to guanine (G) substitution (ACT→GCT), changing the amino acid at position 91 from threonine to alanine (Thr→Ala) in all 26 strains; and (ii) position 887 in the fourth variable domain, a cytosine (C) to thymine (T) substitution (GCA→GTA), changing the amino acid at residue 296 from alanine to valine (Ala→Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1 (China Qinghai Tibetan-1) and CQZ-2 (China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.

Prevalence of Chlamydia trachomatis and Mycoplasma genitalium in Patients with Benign and Malignant Ovarian Cancer by Nested PCR Method

Background: Chlamydia trachomatis (C. trachomatis) and Mycoplasma genitalium (M. genitalium) are considered factors in cervical and ovarian cancer and are associated with flaky cell carcinoma of the cervix. The role of steady infection, leading to chronic inflammation, in the of ovarian cancer has received very little consideration, although a background of pelvic inflammatory disease (PID) is in a case-control study associate to higher risk for ovarian cancer. C. trachomatis, the most common and important cause of PID in the developed world is the genital and cervical infectious agent. The aim of this study was prevalence of C. trachomatis and M. genitalium in patients with ovarian cancer who referred to Imam Hossein Hospital of Shahid Beheshti University of Medical Sciences, Tehran, Iran. M aterials and Methods: In this descriptive study that was conducted from January 2014 to April 2015, 124 samples were studied which obtained from patients with ovarian cancer who referred to medical centers of Shahid Beheshti University of Medical Sciences. After obtaining samples from ovarian cancer tissue by the pathologist, for extraction DNA, samples were transferred to the laboratory of university. To confirm the presence of C. trachomatis in samples of ovarian cancer, specific primers for the Major Outer Membrane Protein (MOMP) genes of C. trachomais , were designed and used Nested PCR method for detection of M. genitalium . Sequencing was performed on the PCR and Nested PCR product to confirm the presence of C. trachomatis and M. genitalium . R es ults: Out of 124 samples of ovarian cancer, 62 (50%) samples were malignant cancer and 62 (50%) were benign cancer as control group. From 65 malignant samples 14 (22.5%) were Chlamydia trachomatis positive. None of the tissue samples of benign cancer of ovary were positive for C. trachomatis . Notably, none of the 124 ovarian samples were positive in the M. genitalium standard PCR assay. C onclusion: The results suggest that the spread of C. trachomatis in the female with ovarian cancer may be common. This finding reflects a possible role of C. trachomatis in the carcinogenesis of ovarian tumors. C. trachomatis infection may play a relative role in the pathogenesis of ovarian carcinomas or it could facilitate its progression.

Detection of Chlamydia infection in Peromyscus species rodents from sylvatic and laboratory sources.

To determine if Chlamydia muridarum, or other chlamydiae, are enzootic in rodents, we probed a serum bank of wild Peromyscus spp. mice for immunoglobulin G-antibody reactivity to ultraviolet light-inactivated C. muridarum elementary bodies (EBs) using an enzyme-linked immunoassay. Applying a cut-off for a positive reaction of OD(405) nm = 0.1 at a 1:20 dilution, we found titratable antibody reactivity in 190 of 247 specimens surveyed (77%, mean OD(405) = 0.33 ± 0.26, range = 0.11-1.81, median = 0.25). In addition, serum samples were obtained from a colony of specific pathogen-free Peromyscus spp. maintained at the University of South Carolina and six of 12 samples were reactive (50%, mean OD(405) = 0.19 +/- 0.08, range = 0.1-0.32, median = 0.18). Lastly, 40 additional wild Peromyscus spp. were captured in a disparate region of Midwestern USA and 22 serum specimens were reactive (55%, mean OD(405) = 0.22 +/- 0.11, range = 0.1-0.48, median = 0.2). Specificity of selected reactive sera for chlamydial antigen was confirmed on Western blot using resolved purified EBs as the detecting antigen. From tissues removed from several mice at necropsy, the gene for chlamydial 16S ribosomal ribonucleic acid (rRNA) was amplified by polymerase chain reaction (PCR). Positive samples of 16S rRNA were subjected to additional PCR for the major outer membrane protein gene (ompA). The amplicons of three select ompA positive samples were sequenced with ≥99% homology with C. muridarum. Our findings indicate that chlamydial infection is enzootic for Peromyscus spp., and that C. muridarum, or a closely related species or strain, is likely the agent in the tested rodent species.

Molecular characterization and Bioinformatics analysis of 31kDa major outer membrane protein gene of Brucella melitensis, Malaysian isolate

Molecular characterization and Bioinformatics analysis of 31kDa major outer membrane protein gene of Brucella melitensis, Malaysian isolate

DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases.

This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.