Sinan B. Issa, Dept. of Microbiology, College of Medicine, Tikrit University

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1 A study on Mycoplasma and Ureaplasma species and their association with gonorrhea in infertile Sinan B. Issa, Dept. of Microbiology, College of Medicine, Tikrit University Abstract Humans can be infected with Mycoplasma or Ureaplasma, and they may have no symptoms at all. About 38% of infertility can be traced to a previous sexually transmitted infection (STI). A cross- sectional community based study was held in Tikrit city and its surrounding from Jan to the end of Oct to detect the rate of Mycoplasma, and Ureaplasma infections among infertile females in Tikrit city and its surroundings and their association with gonorrhea cases. Cervical swabs of 52 infertile females were examined via gram stained smear under the microscope to detect Neisseria gonorrhoeae cases, and then cultured directly onto PPLO broth media for the detection of Mycoplasma and Ureaplasma species. Data showed that PPLO media revealed positive results in 18 of the cases (34.6%), whereas Neisseria gonorrhoeae was detected in 31 females (59.6%) on gram stained smear and culture techniques. Mycoplasma and Ureaplasma were detected in 16 of these N. gonorrhoeae cases (i.e. more than half of them). Most of N. gonorrhoeae cases were seen among primary infertility, whereas most of the Mycoplasma & Ureaplasma infections were seen in secondary infertility cases. High failure rates of antibiotics was reported among these females with vaginal discharge (69.8%). Emphasis on appropriate antibiotic regimens of new drugs and/or combined drugs therapy are recommended here to overcome the resistance, both with the proper diagnosis to detect these cases of sexual transmitted infections. Introduction Infertility affects 10 15% of all couples (1), It is a condition in which conception does not take place even after one year of regular, unprotected sexual intercourse. It can be due to a variety of factors, and infection is one of the common causes of infertility. Throughout the world, 38% of infertility can be traced to a previous sexually transmitted infection (STI) (2). Mycoplasma and Ureaplasma are unique microbes in that they lack a cell wall, some species can cause genitourinary tract infections (3). Of the eight Mycoplasma species that have been isolated from the human genital tract, Ureaplasma urealyticum and Mycoplasma hominis are found most frequently. After puberty, colonization with Ureaplasmas and M. hominis occurs primarily as a result of sexual contact. The urogenital Mycoplasmas have been associated with Nongonococcal urethritis, pylonephritis, vaginitis, cervicitis and PID. Men and women can be infected with Mycoplasma or Ureaplasma, and they may have no symptoms at all (2). Of women being evaluated for infertility, 40% are infected with Chlamydia, Mycoplasma or Ureaplasma, as are 36% of those with a previous history of uterine infection and 50% of those with tubal blockage. More than 60% had evidence of a past infection (4). Mycoplasma can cause a sub clinical infection that may cause the infected endometrial tissue to inhibit sperm migration by inducing changes in the ciliated cells that line fallopian tubes. Some strains of Ureaplasma urealyticum isolated from infertile women produce a neuraminidase like substance that may interfere with sperm penetration (2). Gonorrhea, caused by Neisseria gonorrhoeae, is a public health problem and is the most common reportable infectious disease. It is one of the infections most commonly related to infertility (1). Gonorrhea is most frequently spread during sexual contact (5). 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 asymptomatic or at least do not have symptoms usually associated with venereal infection.

2 Most men (95%) have acute symptoms with infection. Many who are not treated become asymptomatic but remain infectious (3). The aim of this study is to detect the rate of Mycoplasma, and Ureaplasma infections among infertile females in Tikrit city and its surroundings and their association with gonorrhea cases along with the most appropriate antibiotic regimens used, and to evaluate the possibility of infection with other pathogens in the studied group. Materials and Methods A cross- sectional community based study was held in Tikrit city and its surrounding from Jan to the end of Oct Cervical swabs of 52 infertile females were examined via wet mounts and gram stained smear under the light microscope, and then swabs were cultured directly into PPLO broth media (at ºC for 48 hours) for the detection of Mycoplasma and Ureaplasma, and onto chocolate agar plates with 5-10% CO2 tension (Candle jar) at 37 ºC for hours for isolation of N. gonorrhoeae. Also, a third swab was taken from each subject to be inoculated onto blood agar plates under aerobic condition (overnight incubation at 37ºC) to isolate other possible predominant bacteria noticed. The turbidity seen in the broth media with the change in color from light violet to dark green is indicative of Mycoplasma and/or Ureaplasma growth (figure 1), also the intracellular gram negative diplococci noticed on gram stain with the growth of smooth, small, convex and regular grey colored colonies on the chocolate agar is revealing N. gonorrhoeae infection. Urease positive test result was used for identification of Ureaplasma urealyticum in positive PPLO broth result (3). The presence of pus cells and polymorphs on wet mounts was reported. Data about the 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. Statistical analysis was done using chi-square test via a specialized computer program (SPSS). Results Among 52 infertile women, 15 (28.8%) only were having recent vaginal discharge, other 33 (63.5%) had have previous history of discharge, whereas only 4 women (7.7%) denied having any vaginal discharge during their lives (fig 2). The PPLO media revealed positive results in 18 of the cases (34.6%) (fig 3). Whereas Mycoplasma species were detected in 13 of them (25% of the total cases), only 5 females (9.6% of the total cases) were harboring Ureaplasma urealyticum in their genital tract. Neisseria gonorrhoeae was detected in 31 females (59.6%) on gram stained smear and culture techniques. Mycoplasma and Ureaplasma were detected in 16 of these N. gonorrhoeae cases (i.e. more than half of them), on the other hand, only two infertile females were having Mycoplasma and/or Ureaplasma infection without harboring N. gonorrhoeae in their genitourinary tract (fig 3). The distribution of infected cases regarding the presence of previous or recent vaginal discharge is shown on Table (1). The primary infertility was noticed in 24 females (46.2 %), while 28 females (53.8%) were of the secondary infertility type. Most of N. gonorrhoeae cases were seen among primary infertility, whereas most of the Mycoplasma & Ureaplasma infections were seen in secondary infertility cases (Table 2). Previous treatment with drugs and/or herbal medications for vaginal discharge was reported among these females and showed in fig (5). The results showed that 40 females (78.8%) were taken antibiotics for vaginal discharge but with high failure rate (69.8%), although it is less than herbal regimens which have a failure rate of (83.3%). Table (3) shows the most common antibiotics that was used for treatment of vaginal discharge with their failure rates. Mixed regimens of third generation Cephalosporins (or Spectinomycin) with azithromycin (or Doxycycline) showed the lowest rate of failure (33.3%). Among the 19 infertile females with no gonorrhea and/or Mycoplasma-

3 Ureaplasma infections, 5 females only were having recent vaginal discharge. All of them were having pus cells with polymorphs seen on wet mounts examination which may indicate a recent inflammation due to other causes. Blood agar plates revealed only one case among those five females with recent discharge that is harboring a predominant Klebsiella infection. A mixture of normal flora was isolated from the other females. Discussion Mycoplasmal organisms are usually associated with mucosal surfaces, residing extracellularly in the respiratory and urogenital tracts (6). In current study, the Mycoplasma and/or Ureaplasma infection was detected in about 34% of cases, this result agrees with that of Chutivongse et al (less than 40%) (4). More than 92% of infertile females in the studied group were having either a recent pathological vaginal discharge or have had a previous discharge few months ago. Also, the distribution of infected cases showed in table (1) revealed that the total symptomatic cases with recent vaginal discharge were about 20-30% of the infected cases in all the three bacteria under study. This can reveal the size of the problem in the community and the need for screening programs to assess it and to assess the asymptomatic cases. Gonorrhea caused by Neisseria gonorrhoeae 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 developed gradually into a chronic phase. Such cases may represent 60 to 80 percent of the total number of infected females. 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 (2,7). In current study, the asymptomatic cases of gonorrhea showed similar high percentage (71%). 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 diagnosis of asymptomatic N. gonorrhoeae cases. While the higher rate of Mycoplasma & Ureaplasma infection among the secondary infertile cases may be attributed, perhaps, to the rapid and high infectivity of these bacteria that may be transmitted by simple genital examination during periodic clinical examination or during labor, besides the sexual contact route of transmission (8). Antibiotic resistance increasingly compromises effective treatment of gonorrhoea. Inexpensive treatment regimens have been rendered ineffective while efficacious ones are often unaffordable (9). Over the past 60 years, Neisseria gonorrhoeae has acquired clinically significant resistance to sulfonamides, tetracyclines, penicillins, and ciprofloxacin (10). Resistance to erythromycins was noticed in 75%. M. hominis is known to be resistant to erythromycins (3, 11) and is probably has been incriminated here. Among the five cases that were detected having recent vaginal discharge, 4 of them were not diagnosed; although symptoms and signs of infection were noticed (pus cells and polymorphs in wet mount examination). This may be due to Chlamydia trachomatis infection as one of the common causes of urogenital infections and infertility (12, 13). Tuberculosis need to be evaluated here (12). Conclusions The study shows high rate of Mycoplasma/ Ureaplasma and N. gonorrhoeae infections among infertile females, with high rate of antimicrobial resistance. Emphasis on appropriate antibiotic regimens of new drugs and/or combined drugs therapy are recommended here to overcome the resistance, both with the proper diagnosis to detect these cases of sexual transmitted infections.

4 References 1. Vlasak A R. Infections and infertility. Primary Care Update for OBS/GYNS. 2000; 7(5): Shaheen R. Infections and infertility. Indian journal for the practising doctor. 2005; 2 (5): Drew WL. Mycoplasma and Ureaplasma. In: Ryan K J, Ray C G. Sherris Medical Microbiology. 4th edition. McGraw Hill. New York. 2004; pps: S Chutivongse, M Kozuhnovak, J Annus, ME Ward, JN Robertson, W Cates, PJ Rowe, TMM Farley. Tubal infertility: Serologic relationship to past chlamydial and gonococcal infection. Sexually Transmitted Diseases, 1995: 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. 5th ed. Philadelphia, Pa: Churchill Livingstone; 2000: Ken B Waites. Mycoplasma infection. Emedicine electronic magazine. Updated mar Available from URL: le/ overview 7. Tapsall J. Antimicrobial resistance in Neisseria gonorrhoeae. WHO collaborating center for STD and HIV. Sydney, Australia Kayser FH, Bienz KA, Eckert J, Zinkernagel RM. Kayser medical microbiology. Thieme Stuttgart. New Yourk, 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: 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): Taylor- Robison D. Antibiotic Susceptibilities of Mycoplasma and treatment of Mycoplasma infections. Journal of Antimicrobial chemotherapy 1997; 40: CDC. Sexually Transmitted Disease Surveillance, Atlanta, GA: U.S. department of Health and Human Services, CDC, National Center for HIV, STD, and TB Prevention; U.S Preventive Services Task Force. Screening for Chlamydial infection: recommendations and rationale. American journal of Preventive Medicine 2001; 20 (suppl 3): Table (1): Shows the distribution of vaginal discharge according to the type of infection. Type of infection With Recent Vaginal Discharge Without or with history of vaginal discharge % of symptomatic cases (i.e. with recent discharge) Total isolates N. gonorrhoeae % 31 Mycoplasma spp % 13 U. urealyticum % 5 P value < 0.05

5 Table (2): Shows the infertility cases distributed according to the type of infection. Type of infection Primary Secondary infertility cases infertility cases Total isolates N. gonorrhoeae Mycoplasma spp U. urealyticum P value < 0.05 Table (3): Shows the distribution of cases according to the type of drugs used for the vaginal discharge. Percentage Drug used of failure No. of drug administration No. of failure cases Quinilones % Cephalosporins (3rd generation) % Spectinomycin % Azithromycin or Erythromycin % Tetracycline or Doxycycline % Mixed regimens % Other drugs % Total % Fig (1): PPLO media used for isolation of Mycoplasma and Ureaplasma. Left tube (positive) shows the change in color compared with the no change of color seen in the right tube (negative)

6 Fig (2): The distribution of cases according to the vaginal discharge. Fig (3): The distribution of cases according to the positive and negative results on PPLO media. Fig (4): The number of infected cases in current study.

7 Fig (5): Illustrates the distribution of cases according to the type of medication taken, with number of failure cases.