Achchhe Lal Patel

Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis

BackgroundScreening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world.MethodsPrevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay.ResultsWe detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission.ConclusionsThe in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.

Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

Diagnosis of Neisseria gonorrhoeae Using Molecular Beacon

Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries.

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.

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.

Multi-centric validation of an in-house-developed beacon-based PCR diagnostic assay kit for Chlamydia and Neisseria and portable fluorescence detector

Objective. The development of an accurate, sensitive, specific, rapid, reproducible, stable‐at‐room‐temperature and cost‐effective diagnostic kit, and a low‐cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries. Methods. We developed the ‘Chlamy and Ness CT/NG kit’ based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi‐centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)‐based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 °C and the inter‐day reproducibility of results were also analysed. Results. The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the ‘inter‐rater’ &kgr;=0.976 for Chlamydia and &kgr;=0.943 for Neisseria. Conclusion. Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource‐limited settings of developing countries.

O4-S2.02 Seroprevalence of novel immunogens of chlamydia trachomatis and their cytokine response in PBMC Cells under in vitro conditions

Background Chlamydia trachomatis (CT) is an obligate intracellular parasite which causes STD and trachoma. Despite major research into Chlamydial pathogenesis and host immune responses, immuno-protection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterised vaccine candidates in general have shown variable efficacy ranging from no protection to partial protection. It is therefore a research priority to identify novel Chlamydial antigens that may elicit protective immune responses. Objectives The goal of the present study was to assess the seroprevalence to pkn1and DNA j following natural CT infection in human. The prospects of pkn1 as a Type third secretion substrate and DNA j a non surface Chlamydial protein as potential antigen, prompted us to explore the immunogenic potential of both protein. Methods pkn, DNA j and ompA were cloned in bacterial expression vector pTrcHis. Ni+-NTA affinity chromatography was used to purify the recombinant proteins. Antigenic stretches of Pkn1, DNA j and OmpA were identified using Bcepred web server, designed for identification of subunit vaccine candidate by Bioinformatics Centre of IMTECH Chandigarh, India. To validate the bioinformatics based analysis, sera of human patient were used to determine seroreactivity of pkn1 and DNA j proteins. OmpA was used as a positive control during the study. Results Present study showed a high seroprevalence of antibodies against Pkn1 and OmpA (p<0.001) in sera of humans infected with CT. while, no antibodies were observed for DNA j. Our studies have shown an association between release of TNF-α and IFN-y levels upon stimulation of PBMC with Pkn1 and OmpA. Cytokine expression profiling (IL-1ß, TNF-α, IL-2 , IL10 and IFN-y) of Human PBMCs in response to Pkn1 stimulation demonstrate for the first time that Pkn1 is a novel immunodominant Chlamydial antigen that is capable of influencing both Th1 and Th2 immune responses by stimulating the release of both Humoural and Cell-mediated regulatory cytokines. Conclusions Our study demonstrated strong serological responses to Pkn1 and major outer membrane in natural human infection suggesting the role of pkn1 in immune response. Studies are in progress to check how the immunomodulation by Pkn1 alters host-pathogen interactions.