MAJOR ARTICLE. It recently has been shown that as many as 42% of endocervical swab samples obtained from female sex

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1 MAJOR ARTICLE Treatment Failure with the Use of Ciprofloxacin for Gonorrhea Correlates with the Prevalence of Fluoroquinolone-Resistant Neisseria gonorrhoeae Strains in Bangladesh Motiur Rahman, 1 Ashraful Alam, 2 Khairun Nessa, 1 Shamsun Nahar, 1 Dilip K. Dutta, 1 Lubna Yasmin, 3 Shirajum Monira, 1 Zafar Sultan, 1 Shahnewaz Alam Khan, 4 and M. John Albert 1,a 1 International Centre for Diarrhoeal Disease Research, 2 Bangabandhu Sheikh Mujib Medical University, 3 Department of Biochemistry, Dhaka University, and 4 Concern Bangladesh, Dhaka, Bangladesh Although ciprofloxacin is one of the recommended drugs of choice for the treatment of gonorrhea, in vitro resistance to this drug has been observed in surveillance studies and case reports from many parts of the world, including Bangladesh. However, to our knowledge, there have been no prospective studies of the correlation between in vitro response to the drug and treatment outcome. Therefore, a prospective study of 217 female sex workers in Dhaka, Bangladesh, was conducted to examine the correlation between the in vitro response of Neisseria gonorrhoeae and the outcome of ciprofloxacin treatment. Overall, 37.8% of the gonococcal isolates recovered from female sex workers were resistant to ciprofloxacin, and there was a good correlation between in vitro resistance and treatment failure. These findings suggest that in vitro resistance to ciprofloxacin is predictive of clinical treatment failure in patients with gonorrhea. Although there has been a sharp decline in the incidence of gonococcal infection among individuals in developed countries during the past decade, gonorrhea remains one of the most common sexually transmitted infec- Received 7 April 2000; revised 2 August 2000; electronically published 9 March Written, informed consent was obtained from all subjects enrolled in the study. The study was approved by the ethical review committee of International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B; Dhaka, Bangladesh). Financial support: This research was funded by the ICDDR, B: Centre for Health and Population Research, which is supported by countries and agencies that share its concern for the health problems in developing countries (grant # ). Current donors providing unrestricted support include the aid agencies of the governments of Australia, Bangladesh, Belgium, Canada, Japan, The Netherlands, Sweden, Sri Lanka, Switzerland, the United Kingdom, and the United States; and the United Nations Children s Fund. a Present affiliation: Department of Microbiology, Faculty of Medicine, Kuwait University, Safat. Reprints or correspondence: Dr. Motiur Rahman, Laboratory Sciences Division, ICDDR, B, GPO Box 128, Dhaka 1000, Bangladesh (motiur@icddrb.org). Clinical Infectious Diseases 2001; 32: by the Infectious Diseases Society of America. All rights reserved /2001/ $03.00 tions among individuals in developing countries and is a global health problem [1]. According to the World Health Organization (WHO), there are 62 million new cases of gonorrhea worldwide annually, and most of the cases are believed to occur in regions of Southeast Asia, including Bangladesh [2]. Strategies for the control of gonorrhea have relied on the use of highly effective and, often, single-dose therapy administered at the time of diagnosis. In response to changes in gonococcal susceptibility, in 1993 the Centers for Disease Control and Prevention (CDC; Atlanta) advocated the use of third-generation cephalosporins or fluoroquinolones as first-line therapy for uncomplicated cases of gonorrhea [3, 4]. Since that time, the fluoroquinolone ciprofloxacin has been extensively used in Bangladesh as first-line therapy for uncomplicated and suspected cases of gonococcal infections. It recently has been shown that as many as 42% of endocervical swab samples obtained from female sex 884 CID 2001:32 (15 March) Rahman et al.

2 workers (FSWs) in Bangladesh had culture results that were positive for Neisseria gonorrhoeae [5]. Subsequent studies of the antimicrobial susceptibility of gonococcal isolates recovered from FSWs showed that 11.7% of the isolates were resistant to ciprofloxacin (MIC, 1.0 mg/ml) and that 26.6% had reduced susceptibility to ciprofloxacin (MIC, mg/ml) [6]. Although a successful outcome of antimicrobial therapy is conditioned by a number of factors, a good correlation between the level of in vitro susceptibility and microbiological cure is essential for the prediction of treatment outcome. In the literature, reports of quinolone-resistant N. gonorrhoeae are from either systematic surveillance [7, 8] or sporadic case reports [9 13]. Moreover, the National Committee for Clinical Laboratory Standards (NCCLS) and CDC breakpoints for quinolone resistance in N. gonorrhoeae are not based on clinical trial data. Also, retrospective studies have suggested that the correlation between treatment failure and in vitro susceptibility is not consistent for azithromycin, ciprofloxacin, and ofloxacin [14 16], because treatment failure was observed in patients who had isolates with MIC values that were within ranges that indicated susceptibility to azithromycin and ofloxacin [14, 15]. We therefore conducted a prospective study to assess the correlation between in vitro response to, and treatment outcome with, ciprofloxacin in FSWs from Bangladesh who had uncomplicated cases of gonorrhea. SUBJECTS, MATERIALS, AND METHODS The study was conducted from September 1998 through February 1999 among FSWs in Dhaka, the capital of Bangladesh. The subjects enrolled in the study were FSWs who were not currently involved in the sex trade; they were recruited from a rehabilitation center where they were participating in a government rehabilitation program. All FSWs who were years old, regardless of whether they had symptoms of sexually transmitted infections, were eligible for enrollment in the study. Subjects were excluded from the study if they were pregnant, were lactating, had concomitant systemic illnesses, or had been receiving antimicrobial therapy during the preceding 2 weeks. The participants were informed of the study, and written consent was obtained. A female physician performed a genital examination, including examination with use of a speculum, and an endocervical swab sample was collected for culture of N. gonorrhoeae. After gonorrhea was diagnosed on the basis of culture results, the FSWs were treated with a single, orally administered dose of 500 mg of ciprofloxacin. FSWs who had symptoms of sexually transmitted infections and who had culture results that were negative for gonorrhea were treated according to WHO syndromic management guidelines [17]. After 8 10 days, a second endocervical swab sample was collected from all FSWs who had positive culture results and who stayed in rehabilitation. Those patients who had positive culture results but from whom a second endocervical swab sample could not be collected were excluded from the study. Subjects were considered to be cured (defined as treatment success ) if the second endocervical swab sample tested negative for N. gonorrhoeae. If the endocervical swab sample remained culture positive, the subject was considered to have treatment failure. Treatment failure was not due to reinfection, because the subjects stayed in the rehabilitation center and, as a result of close supervision, did not have sexual contact for reinfection. The lack of sexual contact between the time of therapy and posttreatment performance of culture was further verified by patients responses to interviews. Patients who were found to be infected with a ciprofloxacin-resistant isolate were treated with a single im injection of 250 mg of ceftriaxone, and all patients who had culture-positive gonorrhea were later treated for chlamydial infection, as recommended by WHO [17]. Isolation of N. gonorrhoeae. An endocervical swab sample was streaked on modified Thayer-Martin agar (BBL Microbiology Systems). N. gonorrhoeae was identified on the basis of colony morphological findings; Gram staining; and oxidase, catalase, and sugar oxidation tests. Isolates were further confirmed by means of PCR analysis done with the use of primers that amplified a 390-bp region of the gonococcal cryptic plasmid [18]. Isolates were stored at 70 C in trypticase soy broth with 20% glycerol, for further study. MICs. MICs of penicillin, tetracycline, ciprofloxacin, ofloxacin, norfloxacin, ceftriaxone, and spectinomycin were determined by means of an agar dilution method, as described elsewhere [19]. The 2-fold serial dilutions of the antibiotics that were used were as follows: penicillin, mg/ml; tetracycline, mg/ml; ciprofloxacin, mg/ml; ofloxacin, mg/ml; norfloxacin, mg/ml; ceftriaxone, mg/ml; and spectinomycin, mg/ml. The diluted antimicrobial agents were incorporated into GC agar base (Becton Dickinson) supplemented with 1% Isovitalex (Becton Dickinson). Plates were inoculated with 10 4 cfus of bacteria and were incubated in 5% CO 2 for 36 h. Five N. gonorrhoeae reference strains (WHO strains A E) with known MICs were included in each test for purposes of quality control. The tests were performed 3 times, and the mean value of the test results was obtained. The end point was defined as the lowest concentration of an antimicrobial agent that completely inhibited growth. The antimicrobial susceptibility was judged according to MIC breakpoint criteria defined by the NCCLS [20]. b-lactamase test. All isolates were tested for b-lactamase production by means of a paper acidimetric method, as described elsewhere [21]. Serogroup and serovar analysis. The serogroups of the isolates were determined by use of a coagglutination test (Phad- Treatment Failure in Patients with Gonorrhea CID 2001:32 (15 March) 885

3 ebact Monoclonal GC kit; Boule Diagnostics AB) done according to the manufacturer s instructions. The serovar of each isolate was also determined by means of a coagglutination method, by use of a panel of 14 monoclonal antibodies to protein I (Boule Diagnostics AB). PCR restriction fragment length polymorphism of the isolates. Chromosomal DNA was extracted from overnight cultures of the isolates, as described elsewhere [22]. The por gene of the isolates was amplified by PCR analysis done with the use of specific primers from the conserved region of the gene. The primers used were POR1 (ATG AAA AAA TCC CTG ATT GCC C) and POR2 (TTA GAA TTT GTG GCG CAG A) [23]. Amplification of the por gene consisted of 35 cycles of 1 min at 94 C, 2 min at 45 C, and 3 min at 70 C. An initial step and a final step of 5 min at 94 C and 10 min at 72 C, respectively, were included. The amplified products were analyzed by means of 1% agarose gel electrophoresis. The amplified product was kb for isolates that expressed protein I/A (PIA), compared with an amplified product of 1.1 kb for isolates that expressed protein I/B (PIB) [24]. Fifteen microliters of the PCR product were digested with Msp A1I restriction enzyme (Promega) and was run on a 6% nondenaturing polyacrylamide gel. The banding patterns were analyzed after ethidium bromide staining was done. RESULTS Prevalence of gonorrhea and treatment failure. A total of 217 FSWs were enrolled in the study. Of these FSWs, 80 (36.9%) had culture results that were positive for gonorrhea and received treatment with ciprofloxacin. A second endocervical swab sample was collected from 66 FSWs; samples could be obtained from only 66 women, because the other women left the rehabilitation center early. The second specimen tested positive for gonorrhea in 25 (37.9%) of these 66 FSWs. Antimicrobial susceptibility by agar dilution. The antimicrobial susceptibilities of isolates from both groups of patients (those with treatment failure and those with treatment success) are shown in figure 1. A total of 95% (39 of 41), 98% (40 of 41), and 98% (40 of 41) of the isolates in the group of patients with treatment success had susceptibility to ciprofloxacin, ofloxacin, and norfloxacin, respectively, and 5% (2 of 41), 2% (1 of 41), and 2% (1 of 41) had reduced susceptibility to ciprofloxacin, ofloxacin, and norfloxacin, respectively (figure 1A). On the other hand, 96% (24 of 25) of the isolates from patients with treatment failure were resistant to ciprofloxacin, ofloxacin, and norfloxacin, and 4% (1 of 25) had reduced susceptibility to all of these drugs (figure 1B; P!.001). A total of 56% (14 of 25) and 72% (18 of 25) of the isolates from the patients who had treatment failure were resistant to penicillin and tetracycline, compared with 44% (18 of 41) and 59% (24 Figure 1. Antimicrobial susceptibility of Neisseria gonorrhoeae strains isolated from female sex workers who responded to therapy with ciprofloxacin, 500 mg (A), and from those who failed to respond to treatment (B), as determined by use of the agar dilution method. Susceptible strains (white bars), intermediately susceptible strains or strains with reduced susceptibility (hatched bars), and resistant strains (black bars) are shown. of 41) of the isolates, respectively, from the patients that had successful treatment of infection ( P 1.05). The distribution of MICs of ciprofloxacin for the isolates from both patient groups is shown in figure 2. For 95% of the isolates from patients who had treatment success, the MICs were mg/ml. On the other hand, for 96% of the isolates from patients who had treatment failure, the MICs were 1 32 mg/ml. Almost all of the isolates in both groups were susceptible to ceftriaxone and spectinomycin. The antimicrobial susceptibilities of the pretreatment and posttreatment isolates from patients who had treatment failure were compared. The MICs for pre- and posttreatment isolates were found to be similar. Penicillinase-producing N. gonorrhoeae. Of the isolates, 15% (10 of 66) were penicillinase-producing N. gonorrhoeae; 22% (9 of 41) of these were from patients with treatment 886 CID 2001:32 (15 March) Rahman et al.

4 Figure 2. Distribution of MICs of ciprofloxacin for Neisseria gonorrhoeae isolates from patients with treatment success (white bars) or failure (black bars). The breakpoint criteria used for assessment of the isolates were those recommended elsewhere [20, 21]. success, and 4% (1 of 25) were from patients with treatment failure. Serogroup and serovar analysis. To determine the clonal relation among the isolates, and to confirm that treatment failure was due to resistance of the primary isolates to ciprofloxacin, rather than to reinfection, serogroup and serovar analyses were performed for all isolates. All paired isolates were found to be of identical serogroup and serovar, as determined by means of a coagglutination method that used a panel of 14 monoclonal antibodies to protein I. Of the 25 paired isolates from patients who had treatment failure, 4 (16%) belonged to serogroup A and 21 (84%) belonged to serogroup B. Of the isolates from patients who had treatment success, 54% (22 of 41) belonged to serogroup A and 46% (19 of 41) belonged to serogroup B. All isolates, from both groups of patients, that expressed PIA belonged to the Arst serovar. Serovar Bypts was the most common serovar, occurring in 15% of isolates (10 of 66) from both patient groups. PCR restriction fragment length polymorphism analysis of the isolates. To further establish that treatment failure was due to resistance of the primary isolates to ciprofloxacin, rather than to reinfection with resistant isolates of same serovar, the por gene was amplified from each pair of isolates by use of PCR and was cleaved with the Msp A1I restriction enzyme. All paired isolates were found to be identical and to harbor either PIA or PIB, and they showed identical digestion patterns (figure 3). DISCUSSION One-third of the FSWs that were examined had culture results that were positive for N. gonorrhoeae, which is in agreement with findings from our previous studies [5, 6]. Control of gonococcal infection is important, considering its associated complications and sequelae and its role in facilitating acquisition and transmission of HIV [25]. Ciprofloxacin and ofloxacin are currently recommended by the WHO and the CDC for the treatment of uncomplicated gonorrhea. Ciprofloxacin has been used extensively in Bangladesh because it is relatively cheap and effective and because only a single, orally administered dose is required. In a recent retrospective study, success with the use of ciprofloxacin treatment for resistant isolates and for reduced-susceptibility isolates was found to be 40% and 92%, respectively; however, treatment failure has also been reported in patients who are infected with isolates that exhibit reduced susceptibility to ciprofloxacin [15]. These findings may suggest that in vitro susceptibility to ciprofloxacin is not well correlated with treatment outcome. In our prospective study, treatment with 500 mg of ciprofloxacin failed in more than one-third of the patients, and in 96% of these cases, the isolate was found to be resistant to ciprofloxacin, ofloxacin, and norfloxacin. On the other hand, 95% of isolates from patients who responded to therapy were susceptible to ciprofloxacin, ofloxacin, and norfloxacin. Therefore, there was a good correlation between treatment outcome and in vitro susceptibility to ciprofloxacin. However, among the isolates with reduced susceptibility (MIC, mg/ml), treatment success was observed in 67% (2 of 3) of the cases with an MIC of 0.25 mg/ml, and treatment failure was observed in 33% (1 of 3) of the cases with an MIC of 0.5 mg/ml. A similar finding was reported elsewhere [26]. One of the major problems associated with the use of prospective studies for determination of the correlation between in vitro susceptibility and microbiological cure among FSWs with gonorrhea is that subjects often are reinfected. FSWs in the present study did not have the opportunity to be reinfected; Treatment Failure in Patients with Gonorrhea CID 2001:32 (15 March) 887

5 Figure 3. A, PCR amplified por genes of paired isolates of Neisseria gonorrhoeae that were collected before and after treatment, after separation on 1% agarose gel and staining with ethidium bromide were done. Primers POR1 and POR2 amplified a kb amplicon for the por gene that expressed protein I/A (PIA) and a 1.1-kb amplicon for the por gene that expressed protein I/B (PIB). Lanes 1, 3, 5, 7, and 9 denote pretreatment isolates 4, 15, 27, 52, and 59, respectively. Lanes 2, 4, 6, 8, and 10 denote posttreatment isolates from the corresponding patients. Lane M, a molecular-weight-marker Fx 174 DNA/HaeIII fragment. B, Restriction fragment length polymorphism patterns of Msp A1I digested, PCR-amplified por genes from pre- and posttreatment N. gonorrhoeae isolates. Lanes 1, 3, 5, and 7 indicate pretreatment isolates 4, 15, 52, and 57, respectively. Lanes 2, 4, 6, and 8 indicate posttreatment isolates from corresponding patients. Lane M, a molecular-weight-marker 100-bp DNA ladder. they were in a rehabilitation center and were no longer involved in the sex trade. All paired isolates in this study showed identical serogroup, serovar, and restriction enzyme digestion patterns, which indicated the presence of persistent infections only. The frequency of infection with fluoroquinolone-resistant strains has increased dramatically since the early 1990s. It is interesting to note that, in our previous study, 11.7% of the gonococcal isolates were resistant, and 26.6% had reduced susceptibility to ciprofloxacin [6]. However, in the current study, 37.8% of the isolates were resistant and 5% had reduced susceptibility to ciprofloxacin, which is a significant increase in resistance in the relatively short period of a year. Resistance to ciprofloxacin is chromosomally mediated, affects all the members of the fluoroquinolone group of antibiotics, and apparently is incremental. Resistance to fluoroquinolone in Neisseria species occurs as a result of a point mutation in the DNA gyrase (gyra) gene and the topoisomerase IV (parc) gene. The significant number of FSWs who failed to respond to treatment with ciprofloxacin (37.8%) in our study suggests that FSWs in Bangladesh are exposed to ciprofloxacin and that the resistance might have developed under selective antimicrobial pressure. However, the possibility of introduction of resistant strains from outside sources cannot be ruled out. Of the 25 isolates from patients with treatment failure in the present study, 18 (72%) had chromosomally mediated resistance to penicillin, tetracycline, or both, which indicates that ciprofloxacin-resistant strains are more prevalent in isolates that have chromosomally mediated resistance to penicillin and tetracycline. Similar patterns have been reported in the United States, Thailand, and Bangladesh [5, 27, 28]. On the other hand, 22% (9 of 41) of the isolates from patients with treatment success were penicillinase-producing N. gonorrhoeae, compared with 4% (1 of 25) of those from patients who had treatment failure. The frequent appearance of elevated MICs of ciprofloxacin in strains with chromosomally mediated resistance to penicillin and tetracycline remains unexplained, because the mechanism of resistance to ciprofloxacin is different from the mechanisms of resistance to penicillin and tetracycline. It is important that the clinical relevance of in vitro resistance to antimicrobial agents that are commonly used for the treatment of gonorrhea be defined. This prospective study showed that antimicrobial susceptibility tests are useful for choosing an antimicrobial agent and for predicting treatment outcome in patients with gonorrhea. Therefore, susceptibility tests for ciprofloxacin in vitro had a good predictive value for the outcome of antimicrobial therapy in our study. Moreover, our study showed an increasing resistance of N. gonorrhoeae isolates to ciprofloxacin in the city of Dhaka. Therefore, the situation should be monitored, and appropriate alternative drugs should be considered for the treatment of gonorrhea. The isolates that were tested demonstrated high rates of susceptibility to the broad-spectrum cephalosporin ceftriaxone and to spectinomycin, with susceptibility rates of 95% and 100%, respectively. Therefore, ceftriaxone and spectinomycin should be the alternative drugs of choice for the treatment of gonorrhea. Acknowledgments We thank Concern Bangladesh, for their cooperation in specimen collection; Aklima Begum and Nargis Akther, for their assistance; J. W. Tapsall (Prince of Wales Hospital, Sidney, Australia), for providing us with WHO reference strains of N. gonorrhea; and Josef Bogaerts and V. I. Mathan, for their careful review of this article. 888 CID 2001:32 (15 March) Rahman et al.

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