Prashant Kumar Mishra

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

Seroprevalence of Antibodies against Pkn1, a Novel Potential Immunogen, in Chlamydia trachomatis-Infected Macaca nemestrina and Human Patients

Chlamydia trachomatis (CT) is an important cause of sexually transmitted genital tract infections (STIs) and trachoma. Despite major research into chlamydial pathogenesis and host immune responses, immunoprotection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterized vaccine candidates have shown variable efficacy ranging from no protection to partial protection in vivo. It is therefore a research priority to identify novel chlamydial antigens that may elicit protective immune responses against CT infection. In the present study we assessed the seroprevalence of antibodies against protein kinase1 (Pkn1), DNA ligaseA (LigA), and major outer membrane protein A (OmpA) following natural CT infection in humans and in experimentally induced CT infection in Macaca nemestrina. Antigenic stretches of Pkn1, LigA, and OmpA were identified using bioinformatic tools. Pkn1, LigA, and OmpA genes were cloned in bacterial expression vector and purified by affinity chromatography. Our results demonstrate significantly high seroprevalence of antibodies against purified Pkn1 and OmpA in sera obtained from the macaque animal model and human patients infected with CT. In contrast no significant seroreactivity was observed for LigA. The seroprevalence of antibodies against Pkn1 suggest that nonsurface chlamydial proteins could also be important for developing vaccines for C. trachomatis.

Evaluations of in-house PCR based diagnostic assay using pfoB gene for diagnosis of Trichomonas vaginalis among symptomatic women with vaginal discharge

Background Trichomonas vaginalis is one of the most prevalent non viral sexually transmitted diseases (STDs) in developing countries. Although several PCR based diagnostic tests are available, the performance, accuracy and sensitivity of these assays vary. Therefore, we decided to compare the performance of various diagnostic gene targets (18S rRNA, b tublin, pROS21) of T. vaginalis under similar conditions and also compared an in-house developed PCR assay targeting pfoB gene with these methods.

Evaluating the utility of syndromic case management for three sexually transmitted infections in women visiting hospitals in Delhi, India.

Utility of syndromic case management (SCM) in women visiting obstetrics & gynecology department needs to be evaluated as it is subjective and imperfect. Consequently, antibiotic resistance has accelerated along with increased risk of infection to the partners. To understand the effectiveness and/or inadequacies of SCM, 11000 women were recruited and examined by clinicians for infection by Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Bacterial vaginosis (BV) and others. Amongst these patients, 1797 (16.3%) reported vaginal discharge (VD). Other symptoms included: vaginitis (97%), cervicitis (75%), genital ulcers (60%), abnormal vaginal discharge (55%) and lower abdominal pain (48%). The patients were treated for single or co-infections using pre-packed National Aids Control Program III STI/RTI Kits. However, based on PCR diagnostics, 1453/1797 (81%) subjects were uninfected for NG/TV/CT. Amongst 344 (19%) infected patients, 257 (75%) carried infection with single pathogen (TV/NG/CT) while 87/344 (25%) were co-infected with multiple pathogens. Prevalence of TV, NG & CT was 4%, 7% and 8% respectively. Co-infection with CT + NG was highest, 51% (44/87), whereas, co-infection with CT + TV was 21% and NG + TV was 18% while co-infection with all three pathogens was 1.3%. We conclude that SCM is imprecise and successful intervention requires accurate and confirmatory diagnostic approach.

P3-S1.41 Coinfection of Neisseria gonorrhoeae and Chlamydia trachomatis in symptomatic and asymptomatic women in India: implications in reproductive health

Background Despite the recent advances in diagnosis, Neisseraia gonorrhoeae (NG) and Chlamydia trachomatis (CT) remain leading cause of STDs worldwide and account for STIs morbidity leading to acquisition of HIV and HPV infection. Coinfection of gonococcus and Chlamydia is reported from various countries. In recent study we reported high rate of infection by CT using an in house developed PCR method. A number of studies also suggest increasing rate of antibiotic resistance in NG. Thus we developed a rapid, specific and cost effective diagnostic method to detect prevalence of co infection by NG and CT. Methodology: (1) Unique gene sequence of CT and NG were amplified from gDNA isolated from endocervical swabs. (2) Validation of in house PCR method using Roche AMPLICOR Micro well plate CT/NG kit. (3) Use of molecular beacon to develop easy visual method. (4) Establishment of multiplex PCR (mPCR) for simultaneous detection of CT and NG. Results 360 clinical samples were used to validate in house PCR assay. Discrepancy of the samples was resolved by amplifying genes encoding for outer membrane proteins (rmp for NG and ompA for CT). The resolved PPV and NPV were found to be 94% and 99% for CT, 92.0% and 96% for NG. Molecular beacon probe was used which helped in visualisation of PCR product directly in tube using dark reader which also improved the sensitivity of assay. The overall infection rate by NG was 26% and 8.6% in symptomatic and asymptomatic patients while that of CT was 26.3% and 21% respectively. To make the method easy to use in remote areas with minimum laboratory infrastructure, the in house PCR assay was standardised for detection using dry swabs, with crude DNA preparation and stabilisation of reagents at 4C (up to 6 months) was achieved for easy transportation and storage. Using single PCRs, coinfection by CT and NG was found to be 18% in symptomatic (74/410) and 5 % in asymptomatic patients (18/360). We further developed mPCR for simultaneous detection two pathogens. Sensitivity of in house mPCR was found to be 95% when evaluated against PCR for individual pathogen. Conclusion Detection of both the pathogens in single PCR assay makes it economical both in terms of cost and time. The in house assay is highly sensitive, easy to perform and requires minimum infrastructure as well as technical expertise, making it a better option for routine diagnosis of genital infection in developing countries, which would be of great consequences in disease management.

Prevalence and co-infection study of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis among symptomatic women using PCR assay

Background Sexually transmitted infections (STIs) are one of the major causes of acute illness, infertility, long term disability and death for millions of men, women and infants globally. Trichomonas vaginalis, Neisseria gonorrhea and Chlamydia trachomatis are well established agents of STIs leading to vaginal discharge in women. However, the prevalence and co infection patterns among symptomatic women is lacking in India. The present study aims to determine the prevalence and co infection of Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis, in women with vaginal discharge attending OPD, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi.

Identification of putative secretory and membranous proteins of Chlamydia trachomatis using bioinformatics tools

Background Intuitive understanding of hypothetical genes’s signal peptide, transmembrane helix (TH) domain and LipoP box of any pathogenic microorganisms is a prerequisite for the rational development of drugs and vaccine candidates. A protein harboring these sequences may serve as receptor and conveyer for various biostimulations generated during host-pathogen interaction. Identification and annotation of these proteins may help in designing of novel drug or developing new vaccine candidates to control CT infections.

P2.137 Syndromic Management of Vaginal Discharge: Is It Justified?

Introduction Vaginal discharge constitutes an important symptom in women attending gynaecology outpatient clinics. Some of these cases are due to sexually transmitted organisms such as N. gonorrheae, C. trachomatis and T. vaginalis and can lead to poor reproductive health and co-infection with HIV. The control of these infections can lead to substantial reduction in the transmission of HIV. Given the high cost of diagnosis, National Aids Control Organisation advocates syndromic management of patients with genitourinary complaints and treatment with drugs that target the most frequent etiological agents. However, it can lead to emergence of antibiotic resistant strains due to overtreatment. Therefore, we tried to find out prevalence of these three pathogens in cases of symptomatic vaginal discharge and to determine the number of patients who were over-treated following syndromic approach. Methods 320 non-pregnant women (16 – 60 years) attending the gynaecology outpatient clinic of VMMC & Safdarjung Hospital, New Delhi, with complaint of vaginal discharge and clinically suspected to be infected by N. gonorrhoeae, C. trachomatis & T. Vaginali were recruited. Endocervical swabs were used for detection of these organisms by PCR. Results Of the 320 subjects 24 (7.5%) were positive for C. trachomatis, 19 (5.9%) for N. gonorrhoeae & 13 (4.6%) for T. vaginalis. Furthermore, 8(2.5%) patients had co-infection of C. trachomatis & N. gonorrhoeae, 07(2.1%) had co-infection of C. trachomatis & T. vaginalis, 07(2.1%) had N. gonorrheae & T. vaginalis and 06 (1.8%) cases had C. trachomatis, N. gonorrheae & T. vaginalis. Infection was found in 49/320 patients with infection rate of 15.3%. Since all the patients (320) were treated the overtreatment rate was 84.6%. Conclusion Cost-effective technology for simultaneous detection of these pathogens is urgently required in developing countries so that all clinically suspicious cases of vaginal discharge are given treatment only after confirmed diagnosis.

P2.076 Development of a Molecular Beacon-Mediated Diagnostic Probe Assays For the Detection of Trichomonas Vaginalis in Dry Swabs Specimens of Symptomatic Women

Introduction Trichomonas vaginalis (TV) is a protozoan parasite that infects the genitourinary tract of 250 million individuals annually, leading to trichomoniasis. The Infection is associated with preterm delivery, low foetus birth weight and increased susceptibility to other STDs as well as increased HIV acquisition. Infection with Trichomonas is globally underestimated because of ineffective screening protocols and under equipped pathological laboratories especially in developing countries. As a consequence, trichomoniasis is associated with poor reproductive health, with numerous clinical squeal and complications. In developing countries like India, due to forbidden cost of commercial kits, syndromic management is preffered. Hence, a cost effective and quick diagnostic assay is urgently required. The present study is an attempt to develop an inexpensive, specific and sensitive quantitative assay for Trichomonas vaginalis. Methods Specimens were retrieved consecutively from patients with vaginal complaints. In-house primers and molecular TV beacon (Tv-B) were designed to detect the presence of unique regions in the genome of Trichomonas in clinical isolates. The sensitivity and specificity of the inhouse primers were evaluated against published primers and the method was validated against qPCR based commercial kit using DNA isolated from 802 dry clinical swabs. Results In-house designed PCR based assay for detection of trichomonas was highly sensitive as could detect as low as 10fg of genomic DNA (3–5 pathogens). Using molecular beacon, Tv-B, 83 women (10.3%) tested positive for trichomonas out of 802 women (age 15 yrs –55 yrs). The assay was extremely specific and sensitive (99.25% and 94.64% respectively). The PPV was found to be 94% and NPV was 99%. The assay could be used for quantification of load of infection. Conclusion The results demonstrated that in housed developed test for TV is highly specific, sensitive, pocket and user friendly.

P5.105 Identification and Functional Annotation of Secreted Proteins of Chlamydia Trachomatis Using Bioinformatics Tools

Background The large number of sexually transmitted diseases cases caused globally each year by Chlamydia trachomatis has made this organism a World Health Organization priority for vaccine development. Even after a decade of availability of C. trachomatis genome sequence, no promising vaccine has seen the light of the day. This clearly indicates the challenges in discovering new vaccines against this organism but also suggests a gap in our current understanding of Chalmydial biology. We attempt to bridge this gap by carrying out extensive annotation of hypothetical proteins of C. trachomatis and further identification of candidate genes that might be involved during the immune response against this organism. In this study, we have shortlisted proteins secreted by the general export pathway of C. trachomatis from among the hypothetical proteins of this organism with an aim to identify novel vaccine candidate gene/s. Methodology Characterization of the proteins was carried out using various Bioinformatic tools, Pfam, Tigrfam, Scanprosite, CDD, Signal P, SigPred, TMPred, TMHMM and Lipo P. Results 336 hypothetical proteins were deduced from the C. trachomatis genome and were analysed with several software tools for functional annotation. Next we identified the hypothertical proteins are secreted via the general export pathway (GEP) using bioinformatics approach. We were able to classify the shortlisted proteins into three broad categories as outer membrane proteins, secretory proteins and lipoproteins. These shortlisted candidate proteins could possibly induce protective immunity or elicit immune responses of diagnostic value. Few of them can further turn out to be good vaccine candidate genes as well. Conclusion The identification of novel secreted proteins of C. trachomatis opens the way to studies on their subcellular localization and to the immunological characterization of these proteins to define their potential for immunological diagnosis and/or vaccine design.