Study on asymptomatic Neisseria gonorrhoeae cases among infertile subjects

Study on asymptomatic Neisseria gonorrhoeae cases among infertile subjects in Tikrit city and its Sinan B. Issa* Samah H. Khalil ** Chateen E. Ali *** Maad M. Shalal **** * Dept. of Medical Microbiology, College of Medicine- Tikrit University ** Dept. of Microbiology, Al-door Technical institute, *** Dept. of Medical Microbiology, College of Dentistry- Tikrit University **** Dept. of Obstetrics and Gynecology, College of Medicine- Al-Nahrain University Abstract Neisseria gonorrhoeae is a gram-negative diplococcus that cause a disease called Gonorrhea. It is a public health problem and is the most common reportable infectious disease. A cross- sectional community based study was held in Tikrit city and its surrounding from Oct. 2007 to the end of Feb. 2008. Seminal fluid specimens and cervical swabs of 38 infertile subjects (20 males and 18 females) who are clinically asymptomatic for gonorrhea were examined via gram stained smear and culture techniques. Also, seminal fluid analyses for all males and ultrasonographies for most females were done. Data were taken from each subject via a specialized questionnaire prepared for this purpose. Three drugs regimens were used for the treatment of positive cases then another seminal fluid specimens or cervical swabs were taken after a week to assess the efficacy of these regimens. The N. gonorrhoeae diplococci were noticed in 23 (60.5%) of direct gram stained smears taken from the infertile subjects, while cultures revealed positive results for 26 (68.4%) cases. The majority of the infertile subjects where at the age group 21-30 years (23 subjects or 60.5 %). The number of positive cases among the primary infertility group was (22) cases or (81.5%), while it is only 4 positive cases (36.4%) among secondary infertility group. History of urethral discharge was reported in 19 (73%) of the total 26 positive N. gonorrhoeae subjects. The first regimen of treatment was efficient in eradicating N. gonorrhoeae from 20 of the 26 positive cases (76.9%); six cases (23.1%) were resistant cases that were treated successfully by the second or third regimen. This high rate of N. gonorrhoeae among infertile subjects in Tikrit city and surrounding emphasize physicians to exclude this important factor on the start of management of infertility, also, the use of traditional regimens of treatment should be re-evaluated during therapy in order to overcome resistance. Introduction Neisseria are gram negative cocci that typically appear in pairs with the opposing sides flattened, imparting a kidney bean appearance. They are non-motile, nonspore forming, and non-acid fast. Their cell wall is typical of Gram negative bacteria (1). Gonorrhea (also called "the clap"), caused by Neisseria gonorrhoeae, is a public health problem and is the most common reportable infectious disease. Gonorrhea is most frequently spread during sexual contact; however, it can also be transmitted from the mother's genital tract to the newborn during birth to cause ophthalmia neonatorum and systemic neonatal infection. The incubation period is usually 2-8 days. In women, the cervix is the most common site of infection, resulting in endocervicitis and urethritis, which can be complicated by pelvic inflammatory disease (PID). In men, infection causes anterior urethritis (2, 3). Gonorrhea is still one of the great public health problems; no truly effective means of control is yet insight. The major reservoir for continued spread is the asymptomatic patients. Screening programs and case contact studies have shown that almost 50% of infected women are 196

asymptomatic or at least do not have symptoms usually associated with venereal infection. Most men (95%) have acute symptoms with infection. Many who are not treated become asymptomatic but remain infectious (1). Infertility affects 10 15% of all couples and gonorrhea is one of the infections most commonly related to infertility (4). Gonorrhea evokes a pyogenic inflammatory reaction characterized by purulent exudates. As the organism replicates, the oxidation-reduction potential of its environment diminish, allowing polymicrobial infections to occur. The acute phase of infection lasts for weeks and if left untreated it turns gradually to a chronic phase. Such cases may represent 60-80% of the total number of infected females. Gonorrhea damages the female reproductive tract by spread of the organism along the mucosa through the cervix to the adnexa, causing a reduction in ciliary activity and the induction of sloughing. Fallopian tubes may get filled with purulent exudate with secondary involvement of the ovaries and adjacent peritoneum. Frequently, secondary bacterial invasion has by that time replaced the original gonococcal infection. In 15% of cases, abscess rupture may occur, with diffuse peritonitis (5). The aim of this study is to detect the asymptomatic N. gonorrhoeae cases among infertile subjects in Tikrit city and its, and to evaluate some available drugs regimens used for its treatment. Materials and methods A cross- sectional community based study was held in Tikrit city and its surrounding from Oct. 2007 to the end of Feb. 2008 to detect the rate of asymptomatic carriers of N. gonorrhoeae among infertile subjects and to evaluate some available drugs regimens used for treatment. Seminal fluid specimens and cervical swabs of 38 infertile subjects (20 males and 18 females; sixteen of them were couples) who are clinically asymptomatic for gonorrhea were examined via gram stained smear under the microscope, and then cultured directly onto chocolate agar plates with elevated CO 2 tension (Candle jar) at 37 ºC for 24-48 hours. Another gram stained smear were obtained from the positive cultured colonies and checked to assure diagnosis and to assess the presence of other microorganisms. Also, seminal fluid analyses for males were obtained before and after treatment for positive cases to assess the sperm motility, viability of the sperms, and the presence of pus cells. Ultrasonographies for most females were done to assess Fallopian tubes condition. Data about period of infertility, history of urethral discharge, previous treatment, and type of infertility (whether primary or secondary) were taken from each subject via a specialized questionnaire prepared for this purpose. Three drugs regimens were used for the treatment of positive cases based on modified CDC s sexually transmitted diseases treatment guidelines, 2006 (6). These are: First regimen: (Ceftriaxone vial 1gm as a single i.m. injection + azithromycin tab. 1gm orally- both in a single dose therapy, followed by Ciprofloxacin 500mg bid for 7 days). Second regimen: (Spectinomycin vial 2gm as a single i.m. injection + azithromycin tab. 1gm orally- both in a single dose therapy). Third regimen: (Cifixime cap. 400mg twice daily for 10 days + azithromycin tab. 1gm orally as a single dose). The first regimen was used as the drug regimen of choice due to its marketing availability. The second and/or third regimens were used when failure of the first regimen occurred. Azithromycin was used to eradicate concomitant Chlamydia infection. To assess the efficacy of these regimens in treating gonorrhea, another seminal fluid specimen (or cervical swab) was taken a week after the end of treatment and cultured again, then stained with gram stain to check for the presence of N. gonorrhoeae. Diagnosis of N. gonorrhoeae was based on its identification by examination of gram stained smear along with the morphological colonial characteristics. Catalase test and DNase test were used also for further confirmation of the diagnosis. Statistical analysis was done using chi-square test via a specialized computer program (SPSS). 197

Results Among 38 infertile subjects included in this study, sixteen where couples while four males and two females couldn t bring their partners with them for examination (i.e. a total of 20 males and 18 females included in this study). The N. gonorrhoeae diplococci were noticed in 23 (60.5%) of direct gram stained smears taken from the infertile subjects, while cultures revealed positive results for 26 (68.4%) cases. Two cases of smear positive result were negative on culture, while five cases were culture positive- smear negative. Among the total culture- positive N. gonorrhoeae cases, 14 (53.8%) were males with 12 (46.2%) females. The age distribution of the subjects on the study where shown in Table [1]. The primary infertility was noticed in 27 subjects (71.1 %), while only 11 (28.9%) were of the secondary infertility type. The majority of the primary infertile cases where at the age group 21-30 years (21 subjects or 77.8 %), while the majority of the secondary infertile cases where at the 31-40 age group of years (8 cases or 72.7%). Data revealed that the number of positive cases among the primary infertility group was (22) cases or (81.5 %), while it is only 4 positive cases (36.4%) among secondary infertility group, as shown in Table [2]. Significant statistical association was noticed between type of infertility and rate of Neisseria gonorrhoeae detection. The periods of infertility where shown on Figure [1]. The majority of cases where those having less than 2 years period of infertility in both primary and secondary infertility cases. Figure [2] shows the distribution of urethral discharge history (with or without previous treatment history) among the positive cases in current study. As seen in the figure, 19 of the positive cases (73%) were having history of urethral discharge. Out of them, only 6 cases (about one-third) were treated for the discharge previously. Data showed that the first regimen of treatment was efficient in eradicating N. gonorrhoeae from 20 of the 26 positive cases (76.9%); six cases (23.1%) were resistant cases that were treated successfully by the second and/or third regimen as shown by Figure [3]. Seminal fluid analysis before treatment revealed that all the positive cases (fourteen positive males) showed diminished sperm motility (less than 50% of the total personal sperms are motile) together with the presence of more than 10-15 pus cell/ HPF. After treatment with our regimens, the second seminal fluid analysis that was taken from each infected male after one week of treatment showed that the sperm activity was improved in 11 individuals (motility increased to 60-65% for each individual). The pus cells also show lower counts on the second analysis for all treated individuals (about 2-6 cells/ HPF). Three cases after the first regimen showed no improvement of seminal fluid analysis regarding sperm viability and activity parameters in addition to positive culture findings for N. gonorrhoeae, which were then treated successfully by the second or the third regimen. Regarding Ultrasonography for females, fallopian tubes occlusion was noticed only in one (10%) female of the 10 positive female cases. Discussion It is generally accepted that rates of gonococcal disease are high in less developed countries (7). In current study, the rate among asymptomatic infertile subjects under study may reflect the size of the problem in the community. This rate was about 68.4% in culture test, while it is 60.5% in gram stained smear test (i.e. non-culture test). These nearly similar results are due to the slight differences between the two tests in sensitivity (i.e. 95% for microscopy and 85-100% for culture techniques) and specificity (i.e. 98-100% for microscopy and 100% for culture techniques) (8). Two methods are used for detecting N. gonorrhoeae, these are culture and nonculture tests. Culture techniques are considered the tests of choice; but nonculture techniques have replaced culture techniques in some instances- as in screening tests (9). So, here we considered the culture technique as the standard method for detecting N. gonorrhoeae presence and the statistical analyses were based on this fact. 198

The age distribution shows that 23 (60.5%) of our subjects were at the age group between 21 and 30 years old, which is the age group of risk, this agreed with Cook et al (10) who suggest the age around 25 years old as the age of risk for acquiring N. gonorrhoeae. The high rate of N. gonorrhoeae found among the primary infertile subjectsin comparison with the secondary infertile subjects may be attributed, perhaps, to the social habits which make the married couples seek children immediately after their marriage, and, so, they may ask for investigations more than those with secondary infertility whom the social pressure on them is less; this may, in turn, lead to the discovery of asymptomatic N. gonorrhoeae cases. This may also explain the two years period of infertility among the majority of infertile couples. Neisseria gonorrhoeae is versatile in resisting attack, for example in its ability to develop resistance to antimicrobials and in the antigenic variability by which it evades host defenses, thus persisting and often causing asymptomatic (and undetected) infection(8). Thus, it is expected that a high rate of urethral discharge history (i.e. 73%) was reported in current study among the infertile subjects. The typical symptoms of gonorrhea in men include purulent penile discharge and dysuria. In women, the common symptoms include odorless vaginal discharge; vaginal bleeding, particularly after intercourse; and dyspareunia (11). The non- specific symptoms among females which may be similar to these of urinary tract infection or fungal infection of the genital tract may be the cause of neglecting treatment (if any) and poor handling with it, which may lead to asymptomatic presentation after months of infection (5). Antibiotic resistance increasingly compromises effective treatment of gonorrhoea. Inexpensive treatment regimens have been rendered ineffective while efficacious ones are often unaffordable (10). Over the past 60 years, Neisseria gonorrhoeae has acquired clinically significant resistance to sulfonamides, tetracyclines, penicillins, and ciprofloxacin (12). Because of this high resistance behavior, modification for the CDC s treatment guidelines combining two regimens (which are the ceftriaxone single injection regimen and the ciprofloxacin regimen) was used in this study. Still, 6 subjects (23.1%) were resistant cases that were followed by managing them with 2nd and/or 3rd regimens. Al-Hatttawi & Ison (13) from UAE, and Radaddi et al from Saudi Arabia (14) also found that there are few ciprofloxacinresistant isolates in Arabic region and that all the isolates were Spectinomycin- sensitive, which agreed with current study. Also, studies from different parts of the world show similar results regarding antibiotic response among N. gonorrhoeae cases (15, 16, 17, and 18). Diemer et al (19) noticed a decrease in sperm motility due to N. gonorrhoeae infection. This agreed with current results, as when N. gonorrhoeae was treated successfully, the motility of sperms was improved. Also, the pus cells were diminished after treatment, which indicates regression of the inflammatory process, as the presence of N. gonorrhoeae will lead to the influx of Neutrophils and the subsequent production of pus cells (20). This high rate of N. gonorrhoeae among infertile subjects in Tikrit city and surrounding emphasizes physicians to exclude this important factor on the start of management of infertility; also, the use of traditional regimens of treatment should be re-evaluated in order to overcome resistance. References 1. Ryan K J, Ray C G. Sherris medical microbiology. 4th edition. McGraw Hill. New York. 2004. 2. Sparling PF, Handsfield HH. Neisseria gonorrhoeae. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 5 th ed. Philadelphia, Pa: Churchill Livingstone; 2000:2242-58. 3. Sparling PF. Gonococcal Infections. In: Cecil RL, Goldman L, Bennett JC, eds. Cecil Textbook of Medicine. ed. Philadelphia, Saunders; 2000:1743-5. Pa: WB 199

4. Vlasak A R. Infections and infertility. Primary Care Update for OBS/GYNS. 2000; 7(5): 200-206. 5. Shaheen R. Infections and infertility. Indian journal for the practising doctor. 2005; 2 (5): 11-12. 6. Center of Disease Control and Prevention (CDC): Morbidity and Mortality Weekly Report (MMWR). 2006; 55 (RR-11). (Serial online) available from URL: http://www.cdc.gov /STD / treatment/ 2006/ rr5511.pdf 7. Piot P, Islam MQ. Sexually transmitted diseases in the 1990s. Global epidemiology and challenges for control. Sex Transm Dis 1994;21[suppl 2] S7 S13. 8. Tapsall J. Antimicrobial resistance in Neisseria gonorrhoeae. WHO collaborating center for STD and HIV. Sydney, Australia. 2001. 9. U.S. Preventive Services Task Force. Screening for gonorrhea: recommendation statement. AHRQ Publication No. 05-0579-A. Rockville, Md.: Agency for Healthcare Research and Quality, May 2005. Accessed online June 7, 2005, at: http://www.ahrq.gov/clinic/uspstf05/go norrhea/gonrs.htm. 10. Cook RL, Hutchinson SL, Ostergaard L, Braithwaite RS, Ness RB. Systematic review: noninvasive testing for Chlamydia trachomatis and Neisseria gonorrhoeae. Ann Intern Med 2005; 142:914-25. 11. Workowski KA, Levine WC. Sexually transmitted diseases treatment guidelines 2002. Centers for Disease Control and Prevention. MMWR Recomm Rep 2002; 51(RR-6)1-78. 12. Wang S.A., Harvey A.B., Conner S.M., et al. Antimicrobial Resistance for Neisseria gonorrhoeae in the United States, 1988 to 2003: The Spread of Fluoroquinolone Resistance. Ann Int Med. 2007; 147 (2): 81-88. 13. Al-Hattawi K, Ison CA. Characteristics of gonococci isolated from men in Dubai. Epidemiol Infect 1996; 116:15 20. 14. Raddadi AA, Zimmo SK, Abdullah SA. In vitro activity of several antimicrobial agents against Neisseria gonorrhoeae in the western region of the Kingdom of Saudi Arabia. Sex Transm Inf 1998; 74:294. 15. Xia M et al. Neisseria gonorrhoeae with decreased susceptibility to ciprofloxacin: pulsed-field gel electrophoresis typing of strains from North America. Antimicrob Agents Chemother 1996;40:2439 2440. 16. Knapp JS et al. Fluoroquinolone resistance in Neisseria gonorrhoeae. Emerg Infect Dis 1997;3:33 37. Crabbe F et al. Cefaclor, an alternative to third generation cephalosporins for the treatment of gonococcal urethritis in the developing world. Genitourin Med 1997;73:506 509. 17. Aubry-Damon H, Courvalin P. Bacterial resistance to antimicrobial agents: selected problems in France, 1996 to 1998. Emerg Infect Dis 1999,5:315 320. 18. Ison CA, Dillon JA, Tapsall JW. The epidemiology of global resistance among Neisseria gonorrhoeae and Haemophilus ducreyi. Lancet 1998; 351[suppl III] 8 11. 19. Diemer T, Huwe P, Ludwig M, Hauck EW, Weidner W. Urogenital infection and sperm motility. Andrologia. 2003; 35 (5): 283-287. 20. Edwards, J. L., and M. A. Apicella. The molecular mechanisms used by Neisseria gonorrhoeae to initiate infection differ between men and women. Clin. Microbiol. Rev. 2004; 17: 965-981. 200

Table (1): The number of the subjects under study regarding age distribution. Type of infertility <20 yrs 21-30 yrs 31-40 yrs 41-50 yrs Total Primary 4 21 2-27 (71.1%) Secondary - 2 8 1 11 (28.9%) Total 4 23 10 1 38 (100%) Table (2): The number of the subjects under study regarding their type of infertility. Type of +ve cases of N. -ve cases of N. infertility gonorrhoeae gonorrhoeae Total Primary 22 5 27 Secondary 4 7 11 Total 26 12 38 P value < 0.05 No. of cases 12 10 8 6 Primary infertility Secondary infertility 4 2 0 <2 yrs 2 to 5 yrs >5 yrs Fig (1): The period of infertility regarding its type. 201

50% 27% No hx. of urethral discharge hx. of urethral discharge with treatment hx. of ur. Discharge without treatment 23% Fig (2): The distribution of urethral discharge among the positive cases 24% 1st regimen of Rx 2nd & 3rd regimens of Rx 76% Fig (3): The no. of cases regarding the response to treatment regimens. 202