|Year : 2017 | Volume
| Issue : 1 | Page : 11-14
A study on prevalence and antibiotic sensitivity testing methods for Neisseria gonorrhoeae isolates among female outpatients of sexually transmitted infection clinic
Swapna Muthusamy1, Selvi Elangovan2
1 Department of Microbiology, Sri Venkateshwaraa Medical College Hospital and Research Centre, Puducherry, India
2 Department of Microbiology, Stanley Medical College, Chennai, Tamil Nadu, India
|Date of Web Publication||15-Feb-2017|
Department of Microbiology, Sri Venkateshwaraa Medical College Hospital and Research Centre, Ariyur, Puducherry - 605 102
Source of Support: None, Conflict of Interest: None
CONTEXT: Gonorrhoea is the second most common sexually transmitted infection caused by the bacterium Neisseria gonorrhoeae. Though asymptomatic infections are common in females, adverse outcome is a long list and they serve as hidden sources for the transmission of infection.
AIMS: To study the prevalence of N. gonorrhoeae infection among female outpatients and to compare Clinical Laboratory Standards Institute (CLSI) and calibrated dichotomous sensitivity (CDS) methods for antibiotic sensitivity testing.
SETTINGS AND DESIGN: Cross-sectional study.
SUBJECTS AND METHODS: Total number of subjects included in the study was 130. Two endocervical swabs were collected from each subject during speculum examination. Gram's stain, culture and antibiotic sensitivity test were performed as per CLSI and CDS methods.
STATISTICAL ANALYSIS USED: Results were analysed by Chi-square test using IBM-SPSS Statistics-22 statistical package.
RESULTS: Among the 130 females studied, three were positive for N. gonorrhoeae. Gram's stain and culture were equally sensitive. According to CLSI method, all the isolates were sensitive to spectinomycin, while other drugs showed 33% resistance. CDS method detected 33% resistance to ciprofloxacin and the rest of the antibiotics were 100% sensitive.
CONCLUSIONS: Prevalence of N. gonorrhoeae was 2.3%. Gram's stain has got similar sensitivity as that of culture. CLSI method showed only spectinomycin was exclusively sensitive. CDS method showed all the tested drugs were sensitive except ciprofloxacin.
Keywords: Calibrated dichotomous sensitivity test, Neisseria gonorrhoeae, sexually transmitted infection
|How to cite this article:|
Muthusamy S, Elangovan S. A study on prevalence and antibiotic sensitivity testing methods for Neisseria gonorrhoeae isolates among female outpatients of sexually transmitted infection clinic. Int J Health Allied Sci 2017;6:11-4
|How to cite this URL:|
Muthusamy S, Elangovan S. A study on prevalence and antibiotic sensitivity testing methods for Neisseria gonorrhoeae isolates among female outpatients of sexually transmitted infection clinic. Int J Health Allied Sci [serial online] 2017 [cited 2022 Aug 11];6:11-4. Available from: https://www.ijhas.in/text.asp?2017/6/1/11/200195
Gonorrhoea is a sexually transmitted infection (STI) caused by Neisseria More Details gonorrhoeae. Infection is usually asymptomatic in females unlike males where profuse urethral discharge is the commonest presentation. Gonorrhoea has been associated with increased risk of human immunodeficiency virus acquisition., Molecular methods have been developed for diagnosis of gonorrhoea however, microscopy and culture remains the routinely employed options. Complications can be categorised into maternal, neonatal and disseminated infection, the prevalence of which is about 0.5–3% of the total infection. Gonococcal cervicitis can lead on to complications like infertility, ectopic pregnancy, opthalmia neonatorum, gonococcal arthritis, meningitis, endocarditis,, Disseminated gonococcal infection , and the commonest complication is pelvic inflammatory disease.,N. gonorrhoeae is developing resistance to various classes of antibiotics available for treatment and the emergence of fluoroquinolone resistance has made cephalosporins as the widely followed single option according to Centers for Disease Control and Prevention (CDC) guidelines., This study has been carried out to detect the prevalence of gonococcal cervicitis in our locality, compare Gram's stain and culture for the diagnosis and Clinical Laboratory Standards Institute (CLSI), calibrated dichotomous sensitivity (CDS) methods for antibiotic sensitivity testing of N. gonorrhoeae.
| Subjects and Methods|| |
This is a cross-sectional study conducted in the Department of Microbiology involving sexually active female outpatients attending STI clinic of our hospital. Women with complaints of vaginal discharge, lower abdominal pain, dysuria or menstrual irregularities were included. Women in menstruation or with genital prolapse, with history of antibiotic intake within 2 weeks prior to the visit were excluded from the study. Ethical and research clearance for the study were obtained from the Institutional Ethical Committee and informed consent was obtained from the patients before enrollment into the study. Results were analysed by Chi-square test using International Business Machines Corporation, Newyork- statistical package for social sciences version 22. Procedure was explained to the patient and after wearing sterile gloves ectocervix was cleaned and samples were collected from the endocervical canal using two sterile cotton swabs.
Clean grease free glass slide was taken and smear was prepared by rolling the swab on the slide in one direction. Gram's stain was performed and smear was examined for epithelial cells, pus cells, organisms and their location whether extracellular or intracellular. Neisseria were seen as Gram-negative, intracellular cocci arranged in pairs [Figure 1]. Another swab was inoculated on chocolate agar and modified Thayer Martin medium with vancomycin, vancomycin to inhibit Gram-positive bacteria, colistin to inhibit Gram-negative bacteria, nystatin to inhibit yeasts and molds and trimethoprim to inhibit swarming of Proteus in “Z” pattern, then cross streaked with a sterile loop and incubated at 37°C. A moist cotton ball and a lighted candle were placed inside the jar to provide humidity and CO2 atmosphere. The plates were examined after 24 h and if no growth, were re-examined at 48 h. Presence of small pin point, 0.5–1 mm diameter, smooth, translucent, raised convex colonies were suggestive of Neisseria [Figure 2]. Gram-stain, superoxol test and oxidase tests were done for suspected colony and it showed typical Gram-negative diplococci that was superoxol and oxidase test positive. Antibiotic sensitivity test was performed according to CLSI guidelines.
|Figure 1: Gram's stain of endocervical smear showing plenty of pus cells and Gram-negative intracellular diplococcisuperoxol|
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The isolates were sent to Regional STD Teaching Training and Research Centre, Vardhman Mahavir Medical College and Safdarjang Hospital, New Delhi where confirmation and antibiotic sensitivity testing by CDS method were performed. The strength of discs used in CLSI methods were penicillin 10 IU, tetracycline 30 µg, ceftriaxone 30 µg, ciprofloxacin 5 µg and spectinomycin 100 µg whereas the strength of discs in CDS method were penicillin 0.5 IU, tetracycline 10 µg, ceftriaxone 0.5 µg, ciprofloxacin 1 µg and spectinomycin 100 µg.,
| Results|| |
This study included 130 endocervical swab taken from sexually active females, majority of them belonged to 30–39 years of age. Among 130 female subjects, three of them had N. gonorrhoeae demonstrated by both Gram's stain and culture [Table 1]. Antibiotic sensitivity testing of the three isolates by CLSI and CDS methods showed similar results for spectinomycin, ciprofloxacin and dissimilar results for penicillin, tetracycline and ceftriaxone According to CLSI, 33% of N. gonorrhoeae isolates were resistant to penicillin, tetracycline, ciprofloxacin and ceftriaxone [Table 2]. As per CDS method, resistance was seen only for ciprofloxacin (33%). Since CDS is the World Health Organization (WHO) recommended method for antibiotic sensitivity testing of N. gonorrhoeae, resistance was calculated by taking CDS as the reference method. Isolates of N. gonorrhoeae were sensitive to all the five tested drugs except ciprofloxacin for which showed 33% resistance.
|Table 2: Comparison of antibiogram by Central Laboratory Standards Institute and calibrated dichotomous sensitivity test methods (n=3)|
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| Discussion|| |
Infective cervicitis is a STI of females mainly caused by N. gonorrhoeae and Chlamydia trachomatis. N. gonorhoeae is the second common STI next to Chlamydia.
Our study included 130 subjects belonging to 15–49 years of age and were categorized based on decades. It was found that more than half of females were in 30–39 years age decade. A study done by Hailemariam et al. showed that 20–24 years was the most common age group in their study (33%). According to CDC, the highest rates of gonorrhoeal infections were observed among women aged 20–24 years. Prevalence of gonococcal cervicitis in our study was 2.3%, which is different from the studies by Rao et al. who showed 0% prevalence and Das et al. who found the prevalence to be 14.14%. Differences could be explained by the variations in the study population and geographical area. Our study was carried out in a tertiary care hospital in major city of Tamilnadu among female outpatients, whereas the study by Rao et al. included females in three different villages of Karnataka which showed nil prevalence and Das et al. included women of redlight area in Mumbai and Hyderabad which showed higher prevalence of 14.14%. In our study, both Gram-stain and culture detected equal number of cases [Table 1]. Gram-stain though being simple test showed 100% correlation with culture. This is different from the study done by Hansen et al. where endocervical Gram-stain had only 47% sensitivity to diagnose gonococcal cervicitis. This may be due to the low positivity of our study which was not sufficient to show a statistical significance.
CDS is the WHO recommended method for antibiotic sensitivity testing of N. gonorrhoeae. Our study tried to detect whether CLSI method could yield similar results as that of CDS since low potency discs are not easily available for CDS testing. The comparison of antibiogram by CLSI and CDS methods showed that there were differences in the detection of resistance by the two methods. According to CLSI method out of three isolates tested, two were sensitive to all the drugs but third isolate was resistant to all the drugs except spectinomycin. The same three isolates when tested with CDS method showed that all the three isolates were sensitive to all drugs except one isolate showing resistance to ciprofloxacin.
In a study done by Singh et al., E test minimum inhibitory concentration was taken as gold standard and CLSI and CDS methods were compared with E test. They found that CDS method showed higher correlation with E test than CLSI method. Disparities were more for penicillin, tetracycline and ciprofloxacin. Our study compared CLSI and CDS methods and found that interpretation of penicillin, tetracycline and ceftriaxone results varied between the two methods where one method was showing sensitive while the other was showing resistance. CLSI method holds good for spectinomycin and ciprofloxacin. Percentage of resistance was calculated by taking CDS method as the effective method which detected that ciprofloxacin resistance was 33% and other antibiotics were 100% sensitive [Table 2]. Singh et al. study showed that penicillin resistance was 35.6%, ciprofloxacin resistance was 83.4%, ceftriaxone and spectinomycin resistance were 0% and tetracycline resistance was 10.2%. In a study by Khaki et al., only 1% of the isolates were sensitive to ciprofloxacin and penicillin, 46% were sensitive to tetracycline and all the isolates were sensitive to ceftriaxone and spectinomycin. In our study isolation of N. gonorrhoeae was low to detect a significant difference in the percentage of resistance to various antibiotics.
The convincing point to be noted is spectinomycin resistance is not seen with CDS as well as CLSI method, which is rarely encountered in Indian isolates. In a study done by Bala et al., spectinomycin resistance has been reported in one isolate out of 382 isolates. Spectinomycin is not commonly prescribed in India and this could be the reason for the retention of sensitivity to this drug. This study raises the doubt whether the widely followed CLSI method for antibiotic sensitivity testing of other bacteria holds good for N. gonorrhoeae as well. However it cannot be concluded as the number of isolates tested in our study was very minimal. Patients were treated with single oral dose of cefixime 400 mg and azithromycin 1 g. They were requested to come for follow-up after 1 week during which patients showed clinical improvement. Gram-stain and culture performed during the follow-up visit showed negative results. Limitation of the study is that less prevalent disease has been studied with smaller sample size. Lower rate of isolation is a disadvantage since the comparison did not yield statistical significance. Further studies with larger sample size are needed. Also Contact tracing is an important aspect of any STI prevention. This was not done in our study since most of our study subjects were not willing to reveal their partner's identity.
| Conclusions|| |
Prevalence of N. gonorrhoeae in our study was low. Gram's stain and culture were equally sensitive for detection of N. gonorrhoeae. Comparison of CLSI and CDS method showed that CLSI method reports sensitive as resistant and not vice versa. Therefore CLSI method can be followed in resource poor settings, with the caution that whenever resistance is detected it is advisable to proceed with CDS method of testing for confirmation.
Dr. Manjubala, Regional STD Teaching Training and Research Centre, Vardhman Mahavir Medical College and Safdarjang Hospital, New Delhi for confirming the isolates and reporting CDS method of antibiotic sensitivity testing.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mayor MT, Roett MA, Uduhiri KA. Diagnosis and management of gonococcal infections. Am Fam Physician 2012;86:931-8.
Ding J, Rapista A, Teleshova N, Mosoyan G, Jarvis GA, Klotman ME, et al. Neisseria gonorrhoeae
enhances HIV-1 infection of primary resting CD4 T cells through TLR2 activation. J Immunol 2010;184:2814-24.
Fleming DT, Wasserheit JN. From epidemiological synergy to public health policy and practice: The contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect 1999;75:3-17.
Jain S, Win HN, Chalam V, Yee L. Disseminated gonococcal infection presenting as vasculitis: A case report. J Clin Pathol 2007;60:90-1.
Shetty A, Ribeiro D, Evans A, Linnane S. Gonococcal endocarditis: A rare complication of a common disease. J Clin Pathol 2004;57:780-1.
Akkinepally S, Douglass E, Moreno A. Tricuspid valve gonococcal endocarditis: Fourth case report. Int J Infect Dis 2010;14 Suppl 3:e196-7.
Belkacem A, Caumes E, Ouanich J, Jarlier V, Dellion S, Cazenave B, et al.
Changing patterns of disseminated gonococcal infection in France: Cross-sectional data 2009-2011. Sex Transm Infect 2013;89:613-5.
Chacko MR, Wiemann CM, Smith PB. Chlamydia and gonorrhea screening in asymptomatic young women. J Pediatr Adolesc Gynecol 2004;17:169-78.
Banikarim C, Chacko MR. Pelvic inflammatory disease in adolescents. Semin Pediatr Infect Dis 2005;16:175-80.
Centers for Disease Control and Prevention (CDC). Update to CDC's sexually transmitted diseases treatment guidelines, 2006: Fluoroquinolones no longer recommended for treatment of gonococcal infections. MMWR Morb Mortal Wkly Rep 2007;56:332-6.
Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2010. MMWR Recomm Rep 2010;59:49-55.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-fifth Informational Supplement. CLSI Document M100-S25. Wayne, USA: Clinical and Laboratory Standards Institute; 2015.
Bell SM, Pham JN, Fisher GT. Antibiotic Susceptibility Testing by the CDS Method: A Manual for Medical and Veterinary Laboratories. 5th
ed. Australia: Randwick NSW, South Eastern Area Laboratory Services; 2009. p. 42e58. Available from: http://www.web.med.unsw.edu.au/cdstest
. [Last accessed on 2016 Feb 20].
Brunner A, Nemes-Nikodem E, Mihalik N, Marschalko M, Karpati S, Ostorhazi E. Incidence and antimicrobial susceptibility of Neisseria gonorrhoeae
isolates from patients attending the national Neisseria gonorrhoeae
reference laboratory of Hungary. BMC Infect Dis 2014;14:433.
Hailemariam M, Abebe T, Mihret A, Lambiyo T. Prevalence of Neisseria gonorrhea
and their antimicrobial susceptibility patterns among symptomatic women attending gynecology outpatient department in Hawassa referral hospital, Hawassa, Ethiopia. Ethiop J Health Sci 2013;23:10-8.
Rao PS, Devi S, Shriyan A, Rajaram M, Jagdishchandra K. Diagnosis of bacterial vaginosis in a rural setup: Comparison of clinical algorithm, smear scoring and culture by semiquantitative technique. Indian J Med Microbiol 2004;22:47-50.
Das A, Prabhakar P, Narayanan P, Neilsen G, Wi T, Kumta S, et al.
Prevalence and assessment of clinical management of sexually transmitted infections among female sex workers in two cities of India. Infect Dis Obstet Gynecol 2011;2011:494769.
Hansen KC, Dallabetta GA, Reichart C, Pabst KM, Hook EW, Wasserheit JN. Surrogate markers to diagnose gonococcal and chlamydial cervicitis-comparison of leukocyte esterase dipstick, endocervical gram stain and culture. Sex Trans Dis 1991;18:211-6.
Singh V, Bala M, Kakran M, Ramesh V. Comparative assessment of CDS, CLSI disc diffusion and Etest techniques for antimicrobial susceptibility testing of Neisseria gonorrhoeae
: A 6-year study. BMJ Open 2012;2. pii: e000969.
Khaki P, Sharma A, Bhalla P. Comparison of two disc diffusion methods with minimum inhibitory concentration for antimicrobial susceptibility testing of Neisseria gonorrhoeae
isolates. Ann Med Health Sci Res 2014;4:453-6.
Bala M, Ray K, Gupta SM, Muralidhar S, Jain RK. Changing trends of antimicrobial susceptibility patterns of Neisseria gonorrhoeae
in India and the emergence of ceftriaxone less susceptible N. gonorrhoeae
strains. J Antimicrob Chemother 2007;60:582-6.
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[Table 1], [Table 2]