A Pilot Study on Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates From Nepal CHINTAMANI CHAUDHARY, MBBS,* FAISAL ARIF HASAN CHAUDHARY, MSC,† AMIT RAJ PANDY, BSC,* NARAYAN KARKI, BSC,* PALPASA KANSAKER, MSC,‡ ANIL K. DAS, MBBS,* JAMES L. ROSS, PHD,§ SARALA MALLA, MBBS,‡ ANOWAR HOSSAIN, MBBS,† GRAHAM NEILSEN, PHD, AND MOTIUR RAHMAN, PHD† DESPITE A SHARP DECLINE IN the incidence of gonococcal infection in developed countries during the last decade, gonorrhea remains one of the most common sexually transmitted infections (STIs) in developing countries and a global health problem. 1 In the absence of effective vaccine, control of gonococcal infection mainly depends on identification and treatment of the infected individuals and reductions in sexual risk behavior. Early and successful antibiotic treatment of gonococcal infection is impor- tant for cure of the patient, prevention of complications, and to reduce transmission. 2 Strategies for control of gonorrhea have relied on the use of highly effective and often single-dose therapy administered at the time of diagnosis. 3 Information on antimicro- bial susceptibility of Neisseria gonorrhoeae is therefore important to guide selection of an appropriate antimicrobial agent. 4 Antimi- crobial resistance in gonococci often spreads rapidly between countries, and infected travelers often appear for treatment in countries distant from the place of contact. 5 Hence, local and regional antimicrobial resistance data are important for manage- ment of gonorrhea. Specific data on the incidence of gonorrhea and antimicrobial resistance of N. gonorrhoeae in Nepal is lacking. A pilot study was conducted to assess the effectiveness of current recommendations for treatment for gonorrhea, which is a single oral dose of 500 mg ciprofloxacin. Materials and Methods Bacterial Strain A total of 16 gonococcal isolates isolated from symptomatic and asymptomatic males (n = 65) and females (n = 280) attending an STI service delivery clinic in Eastern Nepal between May and September 2003 were cultured on modified Thayer-Martin me- dium (MTM). Isolates were presumptively identified as N. gonor- rhoeae by Gram stain, oxidase, and superoxol test. Isolates were stored at -86°C at the National Public Health Laboratory, Kath- mandu, Nepal, and were transported to ICDDR, B: Centre for Health and Population Research, Dhaka, Bangladesh, for confir- mation of the identity by polymerase chain reaction (PCR) and for further analysis. 6 Minimum Inhibitory Concentrations Minimum inhibitory concentrations (MICs) of penicillin (Sigma, St. Louis, MO), tetracycline (Sigma), ciprofloxacin (Bayer, Hamp- shire, U.K.), ceftriaxone (Sigma), spectinomycin (Upjohn, Puurs, Belgium), and azithromycin (Pfizer Inc., CT) for the isolates were determined by the agar dilution method. 7,8 The breakpoint criteria defined by the National Committee for Clinical Laboratory Stan- dards (NCCLS) was used for penicillin, tetracycline, ciprofloxacin, ceftriaxone, spectinomycin and the breakpoint criteria used for azithromycin was MIC 0.25 g/mL for susceptible, 0.5 g/mL for reduced susceptible, and 1 g/mL for resistance. 7–9 Briefly, 1  10 4 cfu of the bacterial suspension was spotted on GC agar plates (Becton Dickinson, MD) containing Kellogg’s supplement and a 2-fold serial dilution of antimicrobial agents using a multi- point inoculator (Mast Diagnostic Ltd., SCAN 114). 6 N. gonor- rhoeae reference strains WHO A, B, C, D, E, G, H with known MICs were included for quality control in each test. Each test was repeated 3 times. Phenotypic Characterization The criteria used for phenotypic characterization of N. gonor- rhoeae were based on plasmid and chromosomally mediated re- sistance to penicillin and tetracycline as described earlier. 10 -lactamase Test All penicillin-resistant isolates were tested for -lactamase pro- duction by a paper acidimetric method as described earlier. 11 This study was conducted at the ICDDR, B: Centre for Health and Population Research with the support of cooperative agreement HRN-A-00 to 96-90005 to 00 from USAID. ICDDR, B acknowledges with gratitude the commitment of USAID to the center’s research efforts. The authors thank AMDA Nepal and Kamala Moktan and Stephanie Suhowatsky from FHI, Nepal, for their cooperation in setting the STI Laboratory in Eastern Nepal. Correspondence: Motiur Rahman, PhD, Laboratory Sciences Division, ICDDR, B, GPO Box-128, Dhaka-1000, Bangladesh, E-mail: motiur@ icddrb.org. Received for publication September 4, 2004, and accepted February 14, 2005. From *AMDA, Kathmandu, Nepal; the †International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh; the ‡National Public Health Laboratory, Kathmandu, Nepal; §Family Health International, Nepal; and Family Health International, Asia and Pacific Field Programs Division, Bangkok, Thailand Sexually Transmitted Diseases, October 2005, Vol. 32, No. 10, p.641-643 DOI: 10.1097/01.olq.0000179906.57604.e3 Copyright © 2005, American Sexually Transmitted Diseases Association All rights reserved. 641