Diversity of endocervical microbiota associated with genital Chlamydia trachomatis infection and infertility among women visiting obstetrics and gynecology clinics in Malaysia.

The cervical microbiota constitutes an important protective barrier against the invasion of pathogenic microorganisms. A disruption of microbiota within the cervical milieu has been suggested to be a driving factor of sexually transmitted infections. These include Chlamydia trachomatis which frequently causes serious reproductive sequelae such as infertility in women. In this study, we profiled the cervical microbial composition of a population of 70 reproductive-age Malaysian women; among which 40 (57.1%) were diagnosed with genital C. trachomatis infection, and 30 (42.8%) without C. trachomatis infection. Our findings showed a distinct compositional difference between the cervical microbiota of C. trachomatis-infected subjects and subjects without C. trachomatis infection. Specifically, significant elevations of mostly strict and facultative anaerobes such as Streptococcus, Megasphaera, Prevotella, and Veillonella in the cervical microbiota of C. trachomatis-positive women were detected. The results from the current study highlights an interaction of C. trachomatis with the environmental microbiome in the endocervical region.

Liquid Chromatography Mass Spectrometry (LC-MS) analysis in determining the saliva protein of orthodontic patients during retention phase.

BACKGROUND: The biological responses involved during retention phase have been studied for many years but little is known about the effect of saliva proteome during retention phase of post-orthodontic treatment. This study aims to identify the protein profiles during retention phase in relation to biological processes involved by Liquid Chromatography Mass Spectrometry (LC-MS) approach. MATERIAL AND METHODS: A total of 5 ml of unstimulated saliva was collected from each subject (10 non-orthodontic patients and 15 post-orthodontic patients with 6-months retention phase). Samples were then subjected to LC-MS analysis. The expressed proteins were identified and compared between groups. Incisor irregularity for both maxilla and mandible were determined with Little's Irregularity Index at 6-months retention phase. RESULTS: 146 proteins and 135 proteins were expressed in control and 6-months retention phase group respectively. 15 proteins were identified to be co-expressed between groups. Immune system process was only detected in 6-months retention phase group. Detected protein in immune system process was identified as Tyrosine-protein kinase Tec. Statistical significant of incisor irregularity was only found in mandible at 6-months retention phase. CONCLUSIONS: Our study suggests that immune system process protein which is Tyrosine-protein kinase Tec could be used as biomarker for prediction of stability during retention phase of post-orthodontic treatment. Key words:Orthodontics, proteomics, retention, LC-MS, saliva.

Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells.

Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

BACKGROUND: The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. RESULTS: A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. CONCLUSION: Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

Occurrence and characterization of Candida nivariensis from a culture collection of Candida glabrata clinical isolates in Malaysia.

BACKGROUND: Candida nivariensis and C. bracarensis have been recently identified as emerging yeast pathogens which are phenotypically indistinguishable from C. glabrata. However, there is little data on the prevalence and antifungal susceptibilities of these species. OBJECTIVE: This study investigated the occurrence of C. nivariensis and C. bracarensis in a culture collection of 185 C. glabrata isolates at a Malaysian teaching hospital. METHODS: C. nivariensis was discriminated from C. glabrata using a PCR assay as described by Enache-Angoulvant et al. (J Clin Microbiol 49:3375-9, 2011). The identity of the isolates was confirmed by sequence analysis of the D1D2 domain and internal transcribed spacer region of the yeasts. The isolates were cultured on Chromogenic CHROMagar Candida (®) agar (Difco, USA), and their biochemical and enzymic profiles were determined. Antifungal susceptibilities of the isolates against amphotericin B, fluconazole, voriconazole and caspofungin were determined using E tests. Clotrimazole MICs were determined using a microbroth dilution method. RESULTS: There was a low prevalence (1.1 %) of C. nivariensis in our culture collection of C. glabrata. C. nivariensis was isolated from a blood culture and vaginal swab of two patients. C. nivariensis grew as white colonies on Chromogenic agar and demonstrated few positive reactions using biochemical tests. Enzymatic profiles of the C. nivariensis isolates were similar to that of C. glabrata. The isolates were susceptible to amphotericin B, fluconazole, voriconazole and caspofungin. Clotrimazole resistance is suspected in one isolate. CONCLUSION: This study reports for the first time the emergence of C. nivariensis in our clinical setting.

Recent advances targeting innate immunity-mediated therapies against HIV-1 infection.

Early defence mechanisms of innate immunity respond rapidly to infection against HIV-1 in the genital mucosa. Additionally, innate immunity optimises effective adaptive immune responses against persistent HIV infection. Recent research has highlighted the intrinsic roles of apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G, tripartite motif-containing protein 5, tetherin, sterile α-motif and histidine/aspartic acid domain-containing protein 1 in restricting HIV-1 replication. Likewise, certain endogenously secreted antimicrobial peptides, namely α/ß/θ-defensins, lactoferrins, secretory leukocyte protease inhibitor, trappin-2/elafin and macrophage inflammatory protein-3α are reportedly protective. Whilst certain factors directly inhibit HIV, others can be permissive. Interferon-λ3 exerts an anti-HIV function by activating Janus kinase-signal transducer and activator of transcription-mediated innate responses. Morphine has been found to impair intracellular innate immunity, contributing to HIV establishment in macrophages. Interestingly, protegrin-1 could be used therapeutically to inhibit early HIV-1 establishment. Moreover, chloroquine inhibits plasmacytoid dendritic cell activation and improves effective T-cell responses. This minireview summarizes the recently identified targets for innate immunity-mediated therapies and outlines the challenges that lie ahead in improving treatment of HIV infection.

Detection of mecA and ermA genes and simultaneous identification of Staphylococcus aureus using triplex real-time PCR from Malaysian S. aureus strain collections.

A triplex real-time polymerase chain reaction (PCR) assay was used for the simultaneous detection of mecA (methicillin resistance), ermA (erythromycin resistance) and femA (Staphylococcus aureus identification) genes in a single assay. Among 93 clinical S. aureus hospital isolates, there were 48 methicillin-resistant S. aureus (MRSA) and 45 methicillin-sensitive S. aureus (MSSA) isolates. Screening the isolates using the triplex real-time PCR assay, the mecA, ermA and femA genes were detected in all MRSA isolates. The triplex real-time PCR assay was completed within 3h and is a useful genotypic method for detecting the resistance determinants as well as for the identification of S. aureus isolates. These findings will assist the clinical laboratory in identifying these resistance genes and S. aureus rapidly, thus benefiting patient therapy. This study represents a valuable source of information for researchers to study the local antibiotic resistance pattern, which can increase our knowledge of the antibiotic resistance profile, using real-time PCR technology.

Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay.

In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.

Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay.

For rapid identification of methicillin-resistant Staphylococcus aureus, molecular methods are generally targeting mecA and species-specific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment-specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance.