Rafiq Khanani ( Department of Pathology, Sindh Medical College, Karachi. )
A. Razak Memon ( Department of Pathology, Sindh Medical College, Karachi. )
Rasool Bux Shaikh ( Department of Pathology, Sindh Medical College, Karachi. )
Ghulam Ali ( Department of Pathology, Sindh Medical College, Karachi. )
Mubashir Shaikh ( Department of Pathology, Sindh Medical College, Karachi. )
Tariq Sandila ( Department of Pathology, Skin arid Social Hygiene Centre, Karachi. )
Zahid Hussain ( Department of Pathology, Sindh Medical College, Karachi. )
Nighat Parveen ( Department of Pathology, Skin arid Social Hygiene Centre, Karachi. )
Urethral or cervical swab of 255 patients attending Skin and Social Hygiene Centre and found positive for gram negative Intracellular diplococol on direct microscopy were inoculated on Modified New York City (MNYC) medium and chocolate (heated blood) agar for Isolation of neisseria gonorrhea. Growth of N. gonorrhea was obtained in 134(52.5%) cases. These strains were tested for penicillin susceptibility by disc diffusion method and for the production of B-lactamase by rapid penicillinase paper strip test and rapid chromogenic cephalasporin method. Penicillin resistance was found in 31(23%) strains, of which twelve (9%) were B- lactamase producers (PPNG), the remaining 19 (14%) strains were penicillin resistant B-lactamase negative (Pen RB Neg). We conclude that PPNG as well as other penicillin resistant strains (Pen RB Neg) of neisseria gonorrhea are prevalent in our country and appropriate changes In the conventional therapeutic regime are desirable (JPMA 44:70, 1994).
Gonorrhea has become one of the most common bacterial infections worldwide. Among the factors responsible for this gonorrhea pandemic are alleged increase in promiscuity, use of oral or parentral contraceptive methods rather than protective contraceptive methods, a large and gradually increasing reservoir of asymptomatic females and males who unknowingly transmit the disease to their sexual partners and decreasing susceptibility of gonococci to commonly used antibiotics1. During the last 30 years, resistance to penicillin G has gradually risen probably by selection of chromosomal mutants2. The problem of resistance of gonococci to penicillins has been further aggravated by emergence of B-lactamase producing gonococci since 19763. This resistance is plasmid mediated and gonococci might have acquired it from haemophilus or other gram negative organisms4 . Focal outbreaks of B-lactamase producing neisseria gonorrhea have been reported from Phillipines, Thailand, California, New York and elsewhere and endemic foci are being established5. This study was conducted to assess the frequency of treatment failures with conventional therapy for gonorrhea.
Material and Methods
Origin of strains:
Skin and Social Hygiene Centre is the only hospital in Karachi specifically designated as a centre for sexually transmitted diseases (STD). This study includes 255 patients who presented to this hospital from March, 1989 to January, 1992 with a history of purulent urethral discharge and showing intracellular gram -ye diplococci on direct microscopy of urethral or cervical smears.
Isolation and identification:
Specimens were collected by two sterile calcium alginate swabs - one swab was used for marking well on chocolate agar which was subsequently spread thinly by a sterile wire loop6. Second swab was seeded by streaking over the surface of the Modified New York City (MNYC) medium while rotating it to ensure that all surfaces of swabs tip come in contact with the medium. Inoculated plates were incubated immediately in a candle jar with white wax candles, transferred to incubator at 35°C and examined after 24 and 48 hours. Characteristic 0.5-1.0 mm, grey to white, opaque, raised and glistening colonies were subjected to oxidase test. Rapid reacting oxidase positive colonies were picked up for gram staining and biochemical confirmation with rapid carbohydrate degradation tests (oxoid).
Susceptibility to penicillin:
It was determined by picking 5-10 colonies from primary plate, emulsifying it in Muller Hinton broth adjusting inoculum size to 108 organisms/mi with 0.5 McFarland Standard and inoculating it on 1% IsoVitalex supplemented GC agar base plate and applying a 6 ug penicillin disc. It was incubated in a candle jar at 35°C. A zone size of c 20mm in diameter at 24-48 hours was suggestive of a penicillin resistant strain7. Control strains obtained from Centres for Disease Control (CDC), Atlanta, Georgia were also included with each batch. All the strains showing resistance to penicillin, i.e., zone diameter of <20 mm were tested for B-lactamase production.
It was determined by (0 Rapid Penicillinase paper Strip test8 and (ii) Rapid Chromogenic Cephalosporin method using nitrocefin impregnated paper discs9. Control strains from CDC viz: penicillin resistant f3-lactamase negative (Pen RB Neg) and Penicillin resistant B-lactamase positive (Pen RB Pos) were also included with each batch. Penicillin resistant strains which were found B-lactamase negative were once again subjected to penicillin susceptibility test for reconfirmation of its penicillin resistance.
Out of 255 cases, growth of neisseria gonorrhea was obtained in 134 (52.5%) cases. Of these 103 (77%) were sensitive and 31 (23%) resistant to penicillin. Production of B-lactamase was detected in 12 (9%) strains (PPNG; Pen RB Positive). Remaining 19 (14%) strains were penicillin resistant B-lactamase negative (Pen RB Negative).
Sexually transmitted diseases (STD) are hyperendemic in many developing countries and their incidence is increasing10. Treatment failures with conventional regimes of penicillin or ampicillin led us to conduct this study to find out susceptibility of neisseria gonorrhea to penicillin, the most widely used drug for treatment of gonorrhea. Present study showed resistance to penicillin in 23% strains, which is similar to 20 to 31% reported in other series11,13. However, it is much lower than that of 41% reported by Gedebou and Tassew14 and 62% reported by Ng et al15. In the present study 12(9%) PPNG strains were isolated. This is comparable to 16% PPNG strains isolated by Chowdhary et al16 14.6% reported by Urabe et al17. . However, the figure is much lower compared to 40-50% PPNG strains reported from USA and Singapore5. The case histories of patients included in the present study indicated that eight (Pen RB Positive) strains isolated were acquired within the country and four strains were imported by sailors. However, due to stigma attached to STDs, concealment of facts by patients, lack of education, limited availability of STD clinics and almost non-existence of contact tracing and epidemiological studies, it is difficult to ascertain whether there are established endemic foci of PPNG and other penicillin resistant strains or these represent importation and only occasional secondary cases. Systematic multicentre studies for isolation, identification and susceptibility of neisseria gonorrhea are needed to formulate recommendations for treatment of gonorrhea, as conventional measures in vogue are bound to result in treatment failures leading to spread of disease and increased incidence of complications of gonorrhea.
We sincerely thank Dr. Tahira Naqvi, Director, Skin and Social Hygiene Centre for help and guidance in patient selection. Our thanks are also due to Miss Amna and Mr. Rustam for technical assistance and to Mr. Muhammad Zamiruddin Kauser for typing the manuscript.
1. WHO Technical Report Series. Beta-Lactamase-Producing neisseria gonorrhea, 1978;616:123-42.
2. Biswaa, GD., Blackman, E.Y. and Sparling. PP. High frequencyconjugal transfer of a gonococcal penicillinase plasmid. J.Bacteriol., 1980; 143:1318-24.
3. Phillips, l.B-lactamase producing penicilin resistant gonococcus. Lancet, 1976;ii:556-57.
4. Sykes, RB. snd Matthew, MJ. Antimicrob. Chemother., 1976;2:115-57.
5. Centers for Disease control. Penicillinase producing neisseria gonorrhea-United States, Worldwide. Morb. Mort.Wkly. Rep., 1979;28:85-87.
6. Reyn, A. Antibiotic sensitivityof gonococcal strains isolated in the South-East Asia and Western Pacific Regions in 1961-68. Bull. WHO., 19694;0:257-62.
7. Thornsberry, C., Gavan, T.L. and Garlach, EN. New developments in antimicrobial agent susceptibility testing. Cumitech 6, American Society for Microbiology, Washington, D.C., pp. 1-2.
8. Jorgensen, J.H., Lee, J.C. and Alexander, GA. Rapid penicillinase paper strip teat for detection of beta-lactamase producing haemophilus influenzse and neisseria gonorrhea. Antimicrob. Agents, Chemother., 1977; 11:1087-88.
9. BBL Microbiology Systems. Cefinase Package insert BBL Microbiology systems. Cock cysville Md., 1986.
10. DeSchryver, A. and Meheua, A. Epidemiology of sexually transmitted diseasea: the global picture. Bull. WHO., 1990;68:639-54.
11. Renkonen, O.V., Sivonen, A., Lassus, A. and Salo, O.P. Current status of the in vitro senaitivityofgonococcus 10 penicillin in Pinland.Acta. Derm. Venereol., 1970;50:151-53.
12. Moatter, T. A study of neisseria gonorrhea in Karachi (thesis). University of Karachi, 1983, pp. 33-35.
13. Seth, A.D., Kolator, B. and Wilkinson, A.E. Sensitivity of neisseris gonorrhea to antibiotics in London 1976-78. Br.J. Vener.Dis., 1979;55:325-28.
14. Gedebou, M. and Tassew, A. Neisseria gonorrhea isolates from Ethiopia: in vitro susceptibility pattern to live antibiotics. Bull. WHO., 1980;58:67-71.
15. Ng. W.S., Anton, P. and Arnold, K. Nisseria gonorrhea strains isolated in Hong Kong: invitro susceptibility to 13 antibiotics. AntimicrobAgents. Chemother., 1981;79:12-17.
16. Chowdhury, M.N.H., Pareek, S.S. and Mshgoub, El-S. Penicillinsse producingneisseria gonorrhea in Riyadh, Saudi Arabia. Br.J.Vener.Dis., 1981;57:256-58.
17. Urabe, S., Yoshida, S. and Mizuguchi, Y. Epidemiology and treatment of gonorrhea caused by penicillinase producing strains of neisseria gonorrhea in Fukuolta, Japan. Br.J.Vener.Dis., 1983; 59:37-40.