Seema Sood

Monitoring antimicrobial resistance in Neisseria gonorrhoeae in selected countries of the WHO South-East Asia Region between 2009 and 2012: a retrospective analysis

Objective The aim of the present study was to retrospectively analyse the data reported on antimicrobial resistance (AMR) in Neisseria gonorrhoeae in six South-East Asia Region countries from 2009 to 2012 following the revitalisation of the WHO global Gonococcal Antimicrobial Surveillance Program (GASP). Methods AMR data were generated for 7 antibiotics of 4675 isolates in 18 focal point laboratories using the calibrated dichotomous sensitivity (CDS) or Clinical and Laboratory Standards Institute (CLSI) methods and minimal inhibitory concentration testing by Etest in some of the centres. The results were interpreted using the breakpoints recommended. Results High-level resistance to traditional antibiotics, penicillin (25% to 100%) and tetracycline (10% to 100%) and the previously recommended ciprofloxacin (38% to 100%) was observed in all the countries. Overall, >90% of less susceptible and resistant isolates to penicillin and ciprofloxacin were identified from 15 laboratories. Decreased susceptibility to ceftriaxone and cefpodoxime was reported by nine and eight centres, respectively. Resistance to spectinomycin (0.6% to 10.5%) and azithromycin (<5%) was reported only by three centres. The increasing trends of resistance towards penicillin, tetracycline and ciprofloxacin were demonstrated in Bhutan, India, Sri Lanka and Thailand, and no large intercountry variations were evident. Insignificant trends in decreased susceptibility towards ceftriaxone were reported. Conclusions Expansion of the WHO GASP facilitated enhanced AMR surveillance to meet the ongoing challenges of control of gonococcal AMR. The results highlight that the emergence of decreased susceptibility to ceftriaxone and resistance to spectinomycin and azithromycin will unavoidably lead to loss of therapeutic options, and a search for new effective agents needs to be initiated to respond to the emergence of resistant isolates.

The role of a commercial enzyme immuno assay antigen detection system for diagnosis of C. trachomatis in genital swab samples.

In the present pilot study, endocervical and urethral swabs collected from 100 patients attending sexually transmitted disease (STD) clinics and regional centre for STD in two referral hospitals in New Delhi were analyzed by enzyme immune assay (EIA), polymerase chain reaction (PCR) and direct fluorescent antibody (DFA) for detection of C. trachomatis. It was found that EIA could detect a very low number of cases (3/100) as against DFA (11/100) and PCR (9/100). Thus, in spite of the widespread availability, lower cost and ease of performance of the enzyme-linked-immunosorbent serologic assay, the present study highlights the need to employ sophisticated diagnostic tools like DFA and PCR for detection of Chlamydia trachomatis in STD patients.

A pilot study for diagnosis of genital Chlamydia trachomatis infections by polymerase chain reaction among symptomatic Indian women.

BACKGROUND Chlamydia trachomatis is the most common bacterial etiology of sexually transmitted infection. AIM A pilot study was designed using PCR for amplification and detection of a specific 517 bp sequence of the common endogenous plasmid of C. trachomatis from clinical swab specimens obtained from symptomatic female patients attending STD clinics of AIIMS and Regional STD Teaching, Training & Research Center, Safdarjang Hospital, New Delhi. METHODS 97 patients were recruited in the study, and endocervical swabs were collected following standard procedures. The samples were analyzed by PCR and direct fluorescence antibody (DFA) for detection of C. trachomatis, and the sensitivity, specificity, PPV and NPV of PCR were calculated taking DFA as gold standard. RESULTS Out of 97 samples tested, 9 were positive for C. trachomatis by PCR. 1 PCR positive patient was negative by DFA although a total of 11 patients were positive by DFA. The sensitivity, specificity, PPV and NPV of PCR with reference to DFA was 72.73%, 98.84%, 88.89% and 96.59%, respectively. This PCR had high specificity and NPV for detection of C.trachomatis. CONCLUSIONS In light of the introduction of enhanced syndromic approach, which involves the use of laboratory techniques (wherever possible) to confirm clinical diagnosis, a diagnostic PCR with high specificity and NPV is particularly valuable for determination of etiological diagnosis and hence contribute to judicious use of antimicrobials in the community.

Evaluation of an opa gene-based nucleic acid amplification test for detection of Neisseria gonorrhoeae in urogenital samples in North India

Due to the poor positive predictive value of nucleic acid amplification tests (NAATs) for gonorrhoea when applied to a low-prevalence setting, current guidelines recommend the use of supplementary polymerase chain reaction (PCR) targeting a different gene for confirmation of true positives in urogenital specimens. This study sought to standardize and evaluate performance of an in-house opa gene-based PCR assay for gonorrhoea compared to assays targeting the porA pseudogene and 16S rRNA gene. Four hundred samples (300 endocervical, 100 urethral swabs) from patients attending STD clinics in New Delhi, India were used. The sensitivity, specificity, positive predictive value and negative predictive value of the opa-based PCR were 100%, 97·9%, 89·5% and 100%, respectively. In females, the use of NAATs provided enhanced diagnosis of gonorrhoea.

Comparative analysis of syndromic case management and polymerase chain reaction based diagnostic assays for treatment of Neisseria gonorrhoeae, Chlamydia trachomatis and genital mycoplasmas in patients of genitourinary discharge

To respond to the situation of high prevalence and need for effective treatment for sexually transmitted infections (STIs) in low-resource settings, syndromic diagnostic approach was recommended by the World Health Organization and was adopted by National AIDS Control Organization at the primary health centre level. A retrospective study was undertaken in symptomatic patients attending an STI clinic to validate the syndromic approach for genitourinary discharge syndrome. For aetiological diagnosis, culture and/or polymerase chain reaction was used. An infective aetiology could be established in only 20% (106 of 530) patients. The present data call for an early appraisal and review of the diagnostic policy by national authorities on syndromic case management.

Gonococcal opa gene as a diagnostic target for nucleic acid amplification tests in Indian Population

The switch from conventional to molecular methods for the laboratory diagnosis of gonorrhoea is inspired by difficulties associated with cultivation coupled with constraints with regard to its sensitivity. In an endeavour to keep pace, we have developed an in-house polymerase chain reaction (PCR) targeting the opa gene of Neisseria gonorrhoeae using self-designed primers (GenBank accession no. for primers: PUID 9716120 SNUM 2706 Ng_opa). The sensitivity, specificity, positive predictive value and negative predictive value for this assay were found to be 100% (95% confidence interval [CI] 92.5–100), 97.9% (95% CI 95.6–99.1), 89.5% (95% CI 79.1–95.3) and 100% (95% CI 98.6–100), respectively.[1] We have been using this as the supplemental assay in conjunction with 16S ribosomal PCR for the detection of N. gonorrhoeae in urogenital samples. This is in compliance with 2002 CDC guidelines that prescribe routine repeat testing of all positive specimens. Recent CDC guidelines (2014) recommend retesting a positive genital tract specimen with an alternate target assay, primarily when nucleic acid amplification test used detects non-gonococcal Neisseria species.[2] In light of this advisory, we wanted to further assess the specificity of our assay. Although a few strains of non-Neisseria N. gonorrhoeae sp. were included during standardisation studies,[1] we went a step further to check the specificity of our PCR against a large panel of well-characterised commensal Neisseria sp. These included Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria subflava, Neisseria polysaccharea and Neisseria sicca. In addition, a phenotypically similar organism, Moraxella catarrhalis, which inhabits the upper respiratory tract, was included in the present investigation. Figure 1 shows that our primers detected only gonococcal Figure 1: Polymerase chain reaction results of opa gene. Lane 1: Blank, Lane 2: Control WHO F, Lane 3: 100 bp ladder, Lane 4: Neisseria gonorrhoeae ATCC 49226, Lane 5: Neisseria cinerea THO 148D10, Lane 6: Neisseria sicca ATCC 29193, Lane 7: Neisseria flavescens WARB 148D7, Lane 8: Neisseria lactamica KIRK 148E4, Lane 9: Neisseria sicca 1316 148E5, Lane 10: Neisseria mucosa BLEA 148E6, Lane 11: Neisseria subflava ELFO 148E10, Lane 12: Neisseria polysaccharea 33125 100F10, Lane 13: Moraxella catarrhalis NCTC11020 200J3, Lane 14: Neisseria sicca (clinical isolate), Lane 15: Neisseria sicca (clinical isolate), Lane 16: Neisseria sicca (clinical isolate) *S Mohanty, R Gaind