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Molecular Epidemiology of Recently Emergent Ciprofloxacin-Resistant Neisseria gonorrhoeae in South Africa

Moodley, Prashini MBChB, PhD*; Martin, Iona M. C. PhD; Pillay, Keshree MBChB*; Ison, Catherine A. PhD; Sturm, A Willem MD, PhD*

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Sexually Transmitted Diseases: June 2006 - Volume 33 - Issue 6 - p 357-360
doi: 10.1097/01.olq.0000194581.02022.f0
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THERE IS WIDESPREAD EVIDENCE linking the classic sexually transmitted infections (STIs) with enhanced transmission of human immunodeficiency virus (HIV).1,2 As such, the effective control of the former should be a priority in any program that addresses the acquired immunodeficiency syndrome pandemic.2–4 The management and control of STIs is, however, multifaceted and needs to be tailored to the needs of different areas.2

The syndromic approach for the management of STIs is advocated in developing countries with a high prevalence of these infections.5,6 Guidelines for this approach need regular review based on area-specific periodic monitoring of the prevalence of STI pathogens and their susceptibility profiles.7 Molecular typing of organisms is an important tool for surveillance because it informs on possible patterns of transmission. Timely action based on data generated through such surveillance and genotyping could result in the control of outbreaks with newly introduced resistant organisms, or it may result in differential treatment guidelines within a country.

The syndromic approach for the management of symptomatic STIs has been practiced in South Africa for close to 10 years. Ciprofloxacin has been used for the treatment of potential infection with Neisseria gonorrhoeae in men presenting with male urethritis syndrome and in nonpregnant women presenting with vaginal discharge syndrome.

An increase in the minimum inhibitory concentrations (MICs) of ciprofloxacin in N gonorrhoeae has been previously reported from South Africa.8 MICs in the resistant range (1 mg/l) were also reported, although this did not result in clinical failure.9

In September 2003, clinical failures in patients treated with ciprofloxacin for infection with N gonorrhoeae were observed in Durban.10 This prompted us to perform a study at a local STD clinic where we determined the susceptibility profile of N gonorrhoeae among patients presenting with male urethritis syndrome.

Materials and Methods

Setting

The study was conducted in the STD clinic at the Prince Cyril Zulu Communicable Disease Centre in Durban, South Africa.

Patients

Due to administrative difficulties, the duration of the study was limited to the month of November in 2003. We therefore recruited patients from whom the yield of N gonorrhoeae would be the highest (i.e., consecutive male patients presenting with a visible urethral discharge).

Specimen Collection and Isolation of N gonorrhoeae

A Dacron swab was inserted 2 to 3 cm into the urethra and withdrawn while rotating. New York City plates (Oxoid Ltd. Basingstoke, Hampshire, England) were inoculated on site. Plates were transported in candle jars to the laboratory within 3 hours and incubated at 37°C in 5% CO2 for 48 hours. Suspected colonies of N gonorrhoeae were identified by means of Gram staining, oxidase test, β-galactosidase, hydroxy-prolylaminopeptidase, γ-glutamylaminopeptidase, and acid production from glucose, lactose, and maltose.

Antimicrobial Susceptibility Testing

MICs of penicillin, tetracycline, spectinomycin, ciprofloxacin, and ceftriaxone were determined by means of the agar dilution method. Twofold serial dilutions of antibiotics were added to molten GC agar base (Oxoid Ltd.) supplemented with 1% isovitalex at a temperature of 45°C. After solidification, these plates were seeded with 104 cfu/spot of bacteria by means of a multipoint inoculator and incubated at 37°C in CO2 for 24 hours. N gonorrhoeae (ATCC 49,226) was used as the control. The antimicrobial susceptibility was judged using breakpoint criteria defined by the NCCLS.11 Susceptibility to penicillin and tetracycline were defined as follows: Susceptibility to tetracycline and penicillin were defined as follows: tetracycline susceptible isolates (TET-S): MIC ≤0.25 mg/l; tetracycline intermediate isolates (TING): MIC 0.5–1 mg/l; chromosomally mediated tetracycline resistant N gonorrhoeae (CMTRNG): MIC 2 to 8 mg/l; high-level tetracycline resistant N gonorrhoeae (TRNG): MIC ≥16 mg/l; penicillin susceptible isolates (PEN-S): MIC ≤0.06 mg/l; penicillin intermediate isolates (PING): MIC 0.125–1 mg/l; chromosomally mediated penicillin resistant N gonorrhoeae (CMPRNG): MIC ≥2 mg/l in the absence of β lactamase production. Strains testing positive for β-lactamase production by the chromogenic cephalosporin method are referred to as penicillinase producing N gonorrhoeae (PPNG).

Genotyping

Based on MICs, a selection of isolates from the current study (CR study numbers) was typed using N gonorrhoeae multiantigen sequence typing (NG-MAST).12 This included all resistant isolates, a random selection of susceptible isolates, and those with intermediate susceptibility. In addition, isolates from a study done in Kwamsane (C or U study numbers) in 200210 were also typed. Briefly, internal regions of the por and tbpB genes were amplified by PCR, and both strands of DNA were sequenced using an ABI 3,700. Sequences were aligned, edited, and trimmed to a fixed length from conserved positions, as previously described.12 Alleles were assigned to each por and tbpB sequence and the corresponding sequence type (ST) assigned from the combination of the alleles at the 2 loci using the NG-MAST website.13 (eg: por-158 and tbpB-4 gives ST 217). Clusters of isolates, defined as more than 1 isolate having the same ST, were identified. Alignments of trimmed sequences were performed using ClustalX.14

Ethics

Approval was obtained from the ethics committee at the University of KwaZuluNatal.

Results

One hundred thirty-nine N gonorrhoeae isolates were obtained from different patients.

Table 1 shows the susceptibility patterns to the antimicrobials tested. Ninety-nine (71%) isolates were TRNG. The 31 (22%) ciprofloxacin-resistant isolates all demonstrated a ciprofloxacin MIC ≥2 mg/l and were also all TRNG. Apart from 2 (1%) isolates that had MICs of 0.125 mg/l, none displayed intermediate susceptibility to ciprofloxacin. Although all isolates tested were susceptible to ceftriaxone, 131 (94%) displayed increased MICs to this drug. Of these, 79 (60%) were PING, 7 (5%)were CMPRNG, and 41 (31%) were PPNG. All isolates were susceptible to spectinomycin.

T1-5
TABLE 1:
Activity of Antimicrobial Agents on N gonorrhoeae Isolates (N = 139) From Durban, KwaZuluNatal in November 2003

Forty-nine isolates were selected for genotyping. The por gene in 1 and tbpB gene in 2 could not be amplified. The results are therefore based on 46 isolates, of which 39 (6 susceptible, 2 intermediate, and 31 resistant) were from the current study and 7 (3 susceptible and 4 resistant) from the 2002 study. The ciprofloxacin MICs and the ST are shown in Table 2. Typing of the 46 isolates revealed 4 ST clusters: ST 217 (20 isolates), ST 520 (2 isolates), ST 524 (4 isolates), and ST 531 (2 isolates). The remaining 18 STs comprised single isolates. Three of the STs within this dataset have been previously described and are documented at the NG-MAST website.13

T2-5
TABLE 2:
NG-MAST-Based Genotypes of N gonorrhoeae Isolates From KwaZuluNatal

ST 217 was designated por and tbpB alleles 158 and 4, respectively. STs 525, 526, and 527 were closely related to ST 217, having identical tbpB alleles with only a single base-pair difference between their respective por alleles and por allele158. The isolate designated ST 534 differed by 1 base pair in the por allele from the isolates belonging to cluster ST 524.

Discussion

The overall merits of surveillance-directed syndromic management (SM) of STIs in developing areas is unquestionable.15 In South Africa, surveillance to monitor susceptibility patterns of N gonorrhoeae is currently not a regular feature of the national STI control program and occurs mainly in the context of research settings. However, data generated from these limited studies should be a signal for action when significant changes are observed.

The Prince Cyril Zulu Communicable Disease Centre hosts one of the largest STD clinics in KwaZuluNatal. This study revealed that 22% of men with gonococcal urethritis seen at this clinic during a period of 1 month harbored a quinolone-resistant isolate. The current recommendation for the management of male urethritis and female discharge (nonpregnant) is ciprofloxacin and doxycycline (plus metronidazole in women). A patient that fails to respond at the end of 1 week is advised to return to the clinic, when second-line treatment with ceftriaxone is administered. Given the volume of patients seen at this clinic, as well as the high prevalence of resistant organisms among the clinic attendees, this practice is far from optimal. Postponing effective treatment by 1 week in an area with a high prevalence of HIV and gonorrhea has obvious implications in terms of transmission of both organisms. Nonresponding gonococcal disease increases the duration of mucosal inflammation, which in turn increases the likelihood of transmission of HIV.2 In addition, the initial inappropriate treatment of the gonococcal infection increases the time period during which these patients are infective with gonococci, resulting in preferential transmission of the resistant organisms.

Ceftriaxone and spectinomycin are recommended for the treatment of infection with N gonorrhoeae in pregnancy or in those who fail to respond to treatment with ciprofloxacin. Although resistance of N gonorrhoeae to ceftriaxone has not been detected, an increase in MIC values (≥0.007 mg/l) was observed in about 5% of isolates in 1995, increasing to 50% in 1999/2000,8 with a further rise to 94% in this study. This correlated with increased penicillin MICs in the absence of β-lactamase production. This trend has also been reported from other areas of the world.16–18 However, we do not show the previously described relationship between CRMNGs to tetracycline18 and increased ceftriaxone MICs. This observation is likely to be masked by the high prevalence of TRNG isolates in our area.

An increase in MICs was initially observed for spectinomycin from 1995 to 1999.8 This appears to have now stabilized, with almost all isolates displaying MICs of 16 mg/l and 32 mg/l. A 1-step chromosomal mutation that confers complete resistance has previously been reported.19 However, a resistance mechanism resulting in a gradual increase of spectinomycin resistance has not been described so far. The advised breakpoint for susceptibility is 32 mg/l. With the MIC values of our isolates now bordering on resistance, an efficacy study of spectinomycin in our patient population is warranted.

Because genotyping was performed on a limited number of isolates, concrete epidemiologic conclusions cannot be drawn. ST 217 constitutes our largest cluster of isolates. Of interest is that the resistant isolates obtained from patients in KwaMsane in 2002 were part of the ST 217 cluster. This is not surprising because KwaMsane is a periurban settlement situated along a major road that links directly with Durban. Travel between the 2 areas is relatively easy by means of minibus taxis. Quinolone-resistant N gonorrhoeae (QRNG) isolates with ST 217 have previously been reported from Europe.13 The ease of travel around the world allows for the introduction of foreign strains into a community. Durban and Richards Bay (which draws its workforce from surrounding areas, including Kwamsane) are major ports in Africa, and passing sailors form part of the clientele of sex workers from these areas. The Gauteng goldmines also draw its workforce in the form of migrant workers from the Kwamsane area, and bidirectional spread of isolates will occur.

One could speculate that the minor variation between the por allele 158 of the largest cluster ST 217 and the por alleles of STs 525, 526, and 527 suggests rapid evolution from allele 158. A 1-base-pair variation in the por allele was also seen between the cluster of isolates designated ST 524 and the isolate designated ST 534. The clustering of isolates suggests that endemic transmission is now occurring. However this observation requires verification by other typing methods.

The large number of different STs comprising single ciprofloxacin-resistant isolates suggests that these resistant isolates might not be from a single clonal source, but rather represent local emergence of resistance, multiple cases of importation, or both. In addition to the variation in STs seen among the resistant isolates, the mix of susceptible and resistant isolates seen within clusters of ST 520 and ST 53 suggests local de novo resistance development. Although isolates with ST 217 have previously been seen in Europe, it cannot be determined whether the resistance seen in South Africa is the result of the introduction of already resistant strains into the area with subsequent spread, independent resistance development in the area, or both. More extensive typing of national and international N gonorrhoeae isolates needs to be performed to inform on the transmission patterns of resistant organisms in South Africa.

It is over a year ago that clinical failure following quinolone treatment for gonorrhea was first seen. It is highly likely that the unchecked presence of ciprofloxacin-resistant isolates of N gonorrhoeae, in a highly mobile population with complex sexual networks and rapid reinfection,20 for such a long period has resulted in widespread dissemination. The recommendation from WHO is to use drugs that are 95% effective as empirical treatment for the management of STIs.6 In KwaZuluNatal, our isolates now show 22% resistance. This calls for a rapid nationwide surveillance and a review of the SM guidelines with a view to replace ciprofloxacin as first-line treatment in areas where ≥5% of uncomplicated urogenital infections caused by N gonorrhoeae are not cured.

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