Divya Sachdev

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.

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.

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.

O21.5 Understanding the Molecular Mechanism of mtrR in the Regulation of Antimicrobial Resistance in Neisseria Gonorrhoeae Using in Vitro and In Silico Studies

Background Neisseria gonorrhoeae, a major STD causing pathogens, tends to pose high burden of morbidity that is borne disproportionately by women and infants with approximately 2/3rd of cases from developing countries. In the absence of appropriate vaccine and rapid, easy, economical test, antibiotic therapy is recommended for treatment on the basis of clinical symptoms. This has led to the emergence of antibiotic resistant strains. Since increasing antimicrobial resistance makes Neisseria as super bug, we have tried to elucidate the mechanism of development of antibiotic resistance. Methods Mutational analysis of mtrR gene and its DNA binding site was carried out for 28 clinical isolates resistant to multiple drugs. Wild type and mutant mtrR were cloned, expressed and purified. Fluorescence assay and electrophoretic mobility shift assay (EMSA) were carried out to study the effect of mutations in MtrR on its biological activity. Using discovery studio, structure of MtrR was modelled in-silico to understand how mutations affect its interaction with DNA. Results Mutations in DNA binding domain (G45D) and dimerization domain of MtrR (H105Y) as well as in promoter region of MtrR (A/T deletion) were observed in clinical isolates (n = 28). EMSA and fluorimetric results suggest decreased binding of mutant MtrR with its promoter. In silico modelled structure of wild type and mutant MtrR proteins suggest altered conformation of the mutant protein. Altered conformation leads to difference in the posture of homodimer formed and increased centre to centre distance of helix 1 and helix 1’ in two monomers of mtrR. In silico analysis of protein-DNA complex suggest that this increased distance cause altered binding of the mutant with DNA. Conclusions Mutations in mtrR result is altered conformation of the protein leading to its decrease binding to DNA. This leads to enhanced expression of MtrCDE efflux pump resulting in increased efflux of drug.

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.

Mutation pattern in the genome of Neisseria gonorrhoeae and its association with multidrug-resistant isolates from Delhi, India

Mutations in PenA, PorB, MtrE and MtrR genes responsible for antimicrobial resistance were checked in 27 drug-resistant clinical isolates of Neisseria gonorrhoeae (NG). Phenotype PIB (88.88%) and mutation at G120 and A121 positions of porB were recurrent. N122K, a novel mutation, was observed in PorB in three resistant isolates. Substitution H105Y in MtrR was widespread (37% of clinical isolates). The presence of a novel mutation (L33V), along with G45D mutation in MtrR, was associated with less-resistant isolates, in contrast to isolates with G45D mutation alone. African-type penicillinase-producing NG plasmid was observed most frequently (17/27) in penicillin-resistant isolates.

P1.016 Molecular Analysis of Antimicrobial Resistant Neisseria Gonorrhoea Isolates from Delhi, India: A Functional Genomics Approach

Background In India, knowledge regarding N. gonorrhoeae antimicrobial resistance profiling is limited, and data concerning genetic characteristics of N. gonorrhoeae is also lacking. Herein, we investigated the genetic resistance determinants for various antimicrobials used against N. gonorrhoeae isolated in Delhi, India. Various studies have shown that this resistance towards antimicrobials could be either plasmid or chromosomal mediated involving mutations in various genes. Methods Molecular basis of plasmid and chromosomal mediated antimicrobial resistance was analysed by amplifying and sequencing the most target genes, pen A and por B, of N. gonorrhoeae. Attempts have been made to in-silico model the structure of mutant PenA to understand how mutations in these genes affect the drug binding. A PCR assay was also carried out to analyse the penicillinase producing N. gonorrhoeae (PPNG). Results Out of the 40 clinical isolates of N. gonorrhoeae studied which were resistant to various antimicrobials, twenty eight isolates showed high resistance to penicillin (3–32µg/ml). These resistant isolates were PPNG positive (70%; 28/40) and predominantly harboured the African type of PPNG plasmid. Only two isolates carried the Asian type of plasmid. Mutations were also observed in penA and porB genes which correlate their effects on drug resistance. Through in silico modelling studies, we were able to even show that a single point mutation at G452S in penA gene changed the susceptibility of N. gonorrhoeae towards penicillin and tetracycline. Conclusions This study clearly shows a cumulative effect of increasing mutations with subsequent increase in resistance towards various antimicrobials. Presence of both African and Asian type of penicillinase producing plasmid gives an indication of extensive travel of patients affected with Gonorrhea. Our in silico modelling studies of mutant proteins provide new insights to access increasing antimicrobial resistance among Neisseria gonorrhoeae.