Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific and South East Asian regions, 2007-2008.

Long-term surveillance of antimicrobial resistance in Neisseria gonorrhoeae has been conducted in the World Health Organization (WHO) Western Pacific Region (WPR) to optimise antibiotic treatment of gonococcal disease since 1992. In 2007 and 2008, this Gonococcal Antimicrobial Surveillance Programme (GASP) was enhanced by the inclusion of data from the South East Asian Region (SEAR) and recruitment of additional centres within the WPR. Approximately 17,450 N. gonorrhoeae were examined for their susceptibility to one or more antibiotics used for the treatment of gonorrhoea by external quality controlled methods in 24 reporting centres in 20 countries and/or jurisdictions. A high proportion of penicillin and/or quinolone resistance was again detected amongst isolates tested in North Asia and the WHO SEAR, but much lower rates of penicillin resistance and little quinolone resistance was present in most of the Pacific Island countries. The proportion of gonococci reported as 'resistant', 'less susceptible' or 'non-susceptible' gonococci to the third-generation cephalosporin antibiotic ceftriaxone lay in a wide range, but no major changes were evident in cephalosporin minimal inhibitory concentration (MIC) patterns in 2007-2008. Altered cephalosporin susceptibility was associated with treatment failures following therapy with oral third-generation cephalosporins. There is a need for revision and clarification of some of the in vitro criteria that are currently used to categorise the clinical importance of gonococci with different ceftriaxone and oral cephalosporin MIC levels. The number of instances of spectinomycin resistance remained low. A high proportion of strains tested continued to exhibit a form of plasmid mediated high level resistance to tetracyclines. The continuing emergence and spread of antibiotic resistant gonococci in and from the WHO WPR and SEAR supports the need for gonococcal antimicrobial resistance surveillance programs such as GASP to be maintained and potentially expanded.

Experience with an external quality assurance scheme for antimicrobial susceptibility testing of Neisseria gonorrhoeae in India, 2001-2007.

Antimicrobial resistance (AMR) in Neisseria gonorrhoeae compromises patient treatment and disease control. Epidemiologically based surveillance of AMR in gonococci is needed to optimize standard treatment regimens. Validation of AMR surveillance data depends on external quality assurance schemes (EQAS). AMR surveillance data quality in India during 2001-2007 was assessed by participants testing panels of reference strains and repeated re-challenge with identical controls, accompanied by educative feedback. Overall, correct results were obtained for 944 (82%) of 1030 tests performed for five 'core' antibiotics. Aggregated error rates decreased from 33% (123 tests) in 2001 to 4.4% (180 tests) in 2007 with improvements in individual laboratory performance. Cephalosporin test results produced high error rates without improvement. Reference centre and network laboratory collaboration produced marked improvements in test performance through annual EQAS integrating proficiency testing and participant education. More frequent EQAS cycles would assist this process. These experiences may be applicable in similar settings elsewhere.

Implications of current recommendations for third-generation cephalosporin use in the WHO Western Pacific Region following the emergence of multiresistant gonococci.

To ascertain recommendations for the treatment of gonorrhoea in the WHO Western Pacific Region (WPR) following the emergence of "cephalosporin-resistant" Neisseria gonorrhoeae and to relate these to clinical and laboratory measures directed towards disease and antibiotic resistance control. WHO WPR Gonococcal Antimicrobial Resistance Programme members provided data on the type, dose and source of third-generation cephalosporins recommended for the treatment of gonorrhoea. Ceftriaxone was recommended more widely (11/15 respondents) than cefixime (five centres). No cephalosporins were recommended in three jurisdictions. One other oral (ceftibuten) and injectable (cefodizime) agent was recommended. Uniform (400 mg) doses of cefixime were recommended but ceftriaxone regimens ranged between 125 mg and 1 g, with nine of 11 respondents using a 250 mg dose. Both generic and proprietary preparations were widely used. Third-generation cephalosporins are widely recommended for the treatment of gonorrhoea in the WPR, with injectable ceftriaxone more extensively so than oral cefixime and in an expanded dose range. Few other cephalosporins were recommended. Current knowledge suggests that the trend towards ceftriaxone treatment in higher doses may decrease the impact of the circulation of "cephalosporin-resistant" gonococci in the WPR. These recommendations represent public sector practice only and of themselves are unlikely to contain the further spread of "cephalosporin-resistant" gonococci because of the general clinical use of cephalosporins. Optimisation of strategies for laboratory detection of third-generation cephalosporin resistance can be simplified in the WPR because of the restricted spectrum of cephalosporins recommended. Additional efforts are urgently required for both disease and antibiotic resistance control in gonorrhoea.

Analysis of trends in antimicrobial resistance in Neisseria gonorrhoeae isolated in Australia, 1997 2006.

OBJECTIVES: To analyse trends in antimicrobial resistance (AMR) in Neisseria gonorrhoeae (GC) isolated in Australia between 1997 and 2006 and to identify factors influencing emergence and spread of AMR in GC. METHODS: AMR data were generated in reference laboratories in each state and territory of Australia using the methods of the Australian Gonococcal Surveillance Programme from a comprehensive sample of GC. Trends in the proportion of strains resistant to penicillin, ciprofloxacin, spectinomycin and ceftriaxone or with high-level tetracycline resistance (TRNG) were determined from aggregated national data and were also disaggregated by region. Further analyses of additional AMR, demographic, transmission and antibiotic use data were also performed. RESULTS: More than 36,000 GC were examined. Significant increases in resistance to penicillin and ciprofloxacin and in TRNG occurred in national data and in urban populations. Approximately half of the GC tested in larger urban centres were penicillin and/or ciprofloxacin resistant by 2006. These high rates of resistance arose despite low (penicillin) or absent (ciprofloxacin) exposure. In contrast, in rural and remote areas with very high disease rates and high rates of penicillin use, <5% of GC tested were penicillin (or quinolone) resistant. No spectinomycin-resistant GC were detected. Low numbers of GC with raised MICs of ceftriaxone were present in urban centres each year from 2001 onwards. CONCLUSIONS: Significant increases in AMR in GC occurred in parts of Australia in the 10 years to 2006. The data suggest that the AMR seen in GC in urban populations were the result of their repeated importation into Australia and ultimate introduction into established sexual networks rather than originating de novo or as a result of selection by antibiotic use or misuse.

The phase-variable allele of the pilus glycosylation gene pglA is not strongly associated with strains of Neisseria gonorrhoeae isolated from patients with disseminated gonococcal infection.

The Neisseria gonorrhoeae pglA gene has two alleles, one of which is phase variable. A previous study reported that all disseminated gonococcal infection (DGI) isolates contained the phase-variable allele and proposed a causal link. In the present study of 81 strains no absolute correlation between DGI and the phase-variable pglA allele was observed.

Global transmission of prolyliminopeptidase-negative Neisseria gonorrhoeae strains: implications for changes in diagnostic strategies.

BACKGROUND: Species confirmation of Neisseria gonorrhoeae is commonly performed with biochemical kits, rely on the activity of the enzyme prolyliminopeptidase (PIP). This enzyme has previously been considered to be almost universally present in N gonorrhoeae. However, increasing numbers of N gonorrhoeae isolates lacking PIP activity have been identified. OBJECTIVES: To investigate the possibility of a widespread transmission of one or several N gonorrhoeae PIP-negative strains among several countries worldwide. METHODS: PIP-negative N gonorrhoeae isolates cultured from 2001 to 2004 in Australia, New Zealand and Scotland were comprehensively characterised and compared with previous data from England and Denmark. All isolates were characterised by antibiotic susceptibility testing, serovar determination, pulsed-field gel electrophoresis (PFGE), opa-typing, sequencing of the entire porB gene and N gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Most (83%) of the viable Australian isolates, and all the New Zealand and Scottish isolates were assigned serovar IB-4, with similar antibiograms, nearly identical porB1b gene sequences, identical (ST210) or highly related (ST292, ST1259) NG-MAST STs, and indistinguishable or related PFGE fingerprints as well as opa-types. The isolates showed characteristics indistinguishable or highly related to the previously described English and Danish outbreak strain. CONCLUSIONS: A comprehensive characterisation indicates a widespread dissemination, mainly among men who have sex with men (MSM), of indistinguishable and highly related genotypes that have evolved from a single N gonorrhoeae PIP-negative serovar IB-4 strain among several countries worldwide. An increased awareness of PIP-negative N gonorrhoeae strains is crucial and changes in the diagnostic strategies may need to be considered.

Prevalence of antimicrobial resistance in Neisseria gonorrhoeae in an HIV/AIDS clinic.

Antimicrobial resistance in Neisseria gonorrhoeae is increasing in the Pacific region. The standard antibiotic used in urban Australia is intramuscular ceftriaxone. Isolates with reduced sensitivity are being isolated; however, resistance to ceftriaxone has yet to occur. Continued surveillance of antimicrobial resistance in the pathogen, with communication between clinic and laboratory, is needed to ensure optimal treatment of infection.

Increasing resistance to ciprofloxacin and other antibiotics in Neisseria gonorrhoeae from East Java and Papua, Indonesia, in 2004 - implications for treatment.

We examined gonococci isolated in 2004, in East Java and Papua, Indonesia, to review the suitability of ciprofloxacin-based and other treatment regimens. Gonococci from the two provinces were tested in Sydney for susceptibility to penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, gentamicin, azithromycin and rifampicin. Of 163 gonococcal isolates from East Java (91) and Papua (72), 120 (74%) of gonococci, 62 (68%) and 58 (80%) from East Java and Papua, respectively, were penicillinase-producing gonococci and 162 displayed high-level tetracycline resistance. Eighty-seven isolates (53%) were ciprofloxacin resistant, 44 (48%) from East Java and 43 (60%) from Papua. All isolates were sensitive to cefixime/ceftriaxone, spectinomycin and azithromycin. Minimum inhibitory concentrations of gentamicin were in the range 0.05-8 mg/L. Sixty-nine gonococci (42%) showed combined resistance, to penicillin, tetracycline and quinolones. Quinolone resistance has now reached unacceptable levels, and their use for the treatment of gonorrhoea in Indonesia should be reconsidered.

A cluster of culture positive gonococcal infections but with false negative cppB gene based PCR.

OBJECTIVES: To describe the prevalence and characteristics of isolates of Neisseria gonorrhoeae grown from urine samples that produced negative results with nucleic acid amplification assays (NAA) targeting the cppB gene. METHODS: An initial cluster of culture positive, but cppB gene based NAA negative, gonococcal infections was recognised. Urine samples and suspensions of gonococci isolated over 9 months in the Northern Territory of Australia were examined using cppB gene based and other non-cppB gene based NAA. The gonococcal isolates were phenotyped by determining the auxotype/serovar (A/S) class and genotyped by pulsed field gel electrophoresis (PFGE). RESULTS: 14 (9.8%) of 143 gonococci isolated were of A/S class Pro(-/)Brpyut, indistinguishable on PFGE and negative in cppB gene based, but not other, NAA. CONCLUSIONS: This cluster represents a temporal and geographic expansion of a gonococcal subtype lacking the cppB gene with consequent loss of sensitivity of NAA dependent on amplification of this target. Gonococci lacking the cppB gene have in the past been more commonly associated with the PAU-/PCU- auxotype, a gonococcal subtype hitherto infrequently encountered in Australia. NAA based on the cppB gene as a target may produce false positive as well as false negative NAA. This suggests that unless there is continuing comparison with culture to show their utility, cppB gene based NAA should be regarded as suboptimal for use either as a diagnostic or supplemental assay for diagnosis of gonorrhoea, and NAA with alternative amplification targets should be substituted.

Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay.

OBJECTIVE: Development of a rapid, sensitive, and accurate assay for confirmation of Neisseria gonorrhoeae in clinical samples. METHOD: Two real time polymerase chain reaction (PCR) assays, developed on the LightCycler for amplification of the N gonorrhoeae cppB gene, were utilised for confirmation of this bacterial pathogen in samples positive by the Roche Cobas Amplicor assay. Performance characteristics of the two assays were compared with other commercial nucleic acid amplification assays, including the Abbott LCx and Roche 16S rRNA tests. RESULTS: All related Neisseria as well as other bacterial species tested negative by both cppB gene based assays, whereas 120 N gonorrhoeae clinical isolates from various geographical regions gave in positive results. Both assays had a sensitivity of one copy per reaction. 122 clinical samples positive and another 50 samples negative for N gonorrhoeae by Roche Cobas Amplicor were selected from a specimen pool of more than 3000 women tested previously. Overall, 73 of 122 (59.8%) samples were confirmed as positive. The two real time assays had sensitivities of 99% and 100% and specificities of 98% and 100%, respectively. The 16S and LCx assays produced similar results to the real time assays, indicating a similar sensitivity to and specificity of both real time assays. CONCLUSION: The data from this study highlight the need to confirm N gonorrhoeae positive Cobas Amplicor PCR results as an important part of the testing algorithm of all diagnostic laboratories utilising this assay.

Surveillance of antibiotic resistance in invasive isolates of Neisseria meningitidis in Australia 1994-1999.

A total of 1434 strains of Neisseria meningitidis isolated from cases of invasive meningococcal disease (IMD) in Australia between 1994 and 1999 were examined by standard methods for susceptibility to antibiotics used for treatment and prophylaxis. The proportion of isolates fully susceptible to penicillin decreased from 45% in 1994 to 26% in 1999 (P<0.001). All the other isolates were less sensitive to penicillin except for two meningococci with a penicillin MIC of 1 mg/l. The geometric mean penicillin MIC increased from 0.045 to 0.065 mg/l from 1994 to 1999. There was no significant difference in the geometric mean penicillin MICs of serogroup B and serogroup C meningococci. Penicillin susceptibility was significantly associated with a poorer outcome. Isolates from survivors of IMD had a higher geometric mean penicillin MIC (0.06 mg/l) than those from fatal cases (0.048 mg/l) (P< 0.001). This suggests that factors other than the decrease in susceptibility to penicillin observed were more relevant to outcome in IMD. All isolates were fully susceptible to ceftriaxone. Rifampicin resistance was infrequent (eight isolates in 6 years) and sporadic. A single isolate had decreased quinolone susceptibility. Despite the significant shift in susceptibility to penicillin recorded, this group of antibiotics remains a suitable treatment for IMD in Australia.

Confirmation of invasive meningococcal disease by single point estimation of IgM antibody to outer membrane protein of Neisseria meningitidis.

OBJECTIVES: The sensitivity of laboratory confirmation of invasive meningococcal disease (IMD) by culture or PCR is affected by prior antibiotic treatment and decreasing use of early lumbar puncture. Serological diagnosis of IMD is not widely used because of reliance on paired serum samples. The application of single point estimations of IgM antibodies in the diagnosis of IMD was explored. DESIGN: Outer membrane proteins from a mix of commonly encountered meningococcal serotypes were partially purified and used as an antigen in an enzyme immunoassay for the detection of IgM antibody. The cut-off for the assay was derived using sera from blood bank donors and the accuracy then evaluated with sera from patients with culture-confirmed IMD, other bacterial infections and culture-proven nasopharyngeal colonisation with Neisseria meningitidis. RESULTS: The coefficient of variability of the assay was < 10% in negative, mid- and high-range positive sera and the specificity of the assay was at least 93%. In sera collected from 49 adult patients at various times after positive blood or CSF culture-confirmed IMD, the assay had a sensitivity of 100% in specimens collected between 5 and 18 days. At the time of isolation of meningococci from either blood or CSF, eight of 29 sera were IgM-positive, but beyond 70 days no positive results were detected. No differences were seen in the IgM responses in patients from whom different serogroups of N. meningitidis were recovered. CONCLUSIONS: Serological examination by single point IgM enzyme immunoassay (EIA) offers the possibility of an expanded laboratory confirmation of IMD in adults for samples taken between 5 and 18 days after onset.

Characteristics of homosexually-active men with gonorrhoea during an epidemic in Sydney, Australia.

Our objective was to investigate an epidemic of gonorrhoea among homosexually-active men in Sydney. Demographic and behavioural data on all homosexually-active men diagnosed with gonorrhoea (any site) at the Sydney Sexual Health Centre (SSHC) from 1992 through 1998 were reviewed. The men diagnosed with anal gonorrhoea were then compared with all homosexually-active men who tested negative for anal gonorrhoea or who were not tested for anal gonorrhoea at the SSHC between 1996 and 1998. Data on HIV status and country of birth of men diagnosed with anal gonorrhoea during 1998 at the Taylor Square Private Clinic were also reviewed. Over the period 1992 to 1998, homosexually-active men diagnosed with gonorrhoea at SSHC tended to become older at the time of diagnosis (median age 26.5 years in 1992 up to 31.0 years in 1998), indicating a cohort effect in the clinic population due to service reductions. When compared with men who tested negative for anal gonorrhoea at SSHC between 1996 and 1998, those with anal gonorrhoea were more likely to have anogenital symptoms (adjusted odds ratio [OR] 2.3), to have had a past history of gonorrhoea (OR 3.1), to present as a contact of gonorrhoea (OR 8.6), to have used condoms less consistently (OR 2.3), to be HIV positive or of unknown HIV status (OR 3.2), and to have been born in an English-speaking country other than Australia (OR 2.9). The last feature was not observed at the private clinic. In conclusion, the gonorrhoea epidemic was linked to public health service reductions, though it seems unlikely to be the only factor. Homosexually-active men with anal gonorrhoea had well established behavioural risk factors while men with concurrent HIV infection were over-represented. Given the role of gonorrhoea in promoting the spread of HIV infection, a National Sexual Health Strategy--closely linked to the National HIV/AIDS Strategy--is due.

Correlation of in vitro susceptibilities to newer quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC.

The in vitro activities of ciprofloxacin, trovafloxacin, moxifloxacin, and grepafloxacin against 174 strains of Neisseria gonorrhoeae isolated in Sydney, Australia, were determined. The strains included 84 quinolone-less-sensitive and -resistant N. gonorrhoeae (QRNG) strains for which ciprofloxacin MICs were in the range of 0.12 to 16 microg/ml. The QRNG included strains isolated from patients whose infections were acquired in a number of countries, mostly in Southeast Asia. The gyrA and parC quinolone resistance-determining regions (QRDR) of 18 selected QRNG strains were sequenced, and the amino acid mutations observed were related to the MICs obtained. The activities of moxifloxacin and grepafloxacin against QRNG were comparable to that of ciprofloxacin. Trovafloxacin was more active than the other quinolones against some but not all of the QRNG strains. Increments in ciprofloxacin resistance occurred in a step-wise manner with point mutations initiated in gyrA resulting in amino acid alterations Ser91-to-Phe, Ser91-to-Tyr, Asp95-to-Gly, and Asp95-to-Asn. Single gyrA changes correlated with ciprofloxacin MICs in the range 0.12 to 1 microg/ml. The Ser91 changes in GyrA were associated with higher MICs and further QRDR changes. QRNG strains for which ciprofloxacin MICs were greater than 1 microg/ml had both gyrA and parC QRDR point mutations. ParC alterations were seen in these isolates only in the presence of GyrA changes and comprised amino acid changes Asp86-to-Asn, Ser87-to-Asn, Ser87-to-Arg, Ser88-to-Pro, Glu91-to-Lys, and Glu91-to-Gln. QRNG strains for which MICs were in the higher ranges had double GyrA mutations, but again only with accompanying ParC alterations. Not only did the nature and combination of GyrA and ParC changes influence the incremental increases in ciprofloxacin MICs, but they seemingly also altered the differential activity of trovafloxacin. Our findings suggest that the newer quinolones of the type examined are unlikely to be useful replacements for ciprofloxacin in the treatment of gonorrhea, particularly where ciprofloxacin MICs are high or where resistance is widespread.

Annual report of the Australian Gonococcal Surveillance Programme, 1999.

The primary aim of the Australian Gonococcal Surveillance Programme (AGSP) is to monitor the antibiotic susceptibility of Neisseria gonorrhoeae. In 1999 the AGSP examined 3,740 isolates of gonococci from all States and Territories. The rates and sites of infection and antibiotic susceptibility patterns varied considerably between regions, reflecting the considerable differences between non-urban and urban gonorrhoea in Australia. Resistance to the penicillin and quinolone groups of antibiotics was highest in urban centres. Although penicillins remained suitable for use in many parts of non-urban Australia, enhanced surveillance is required as levels of resistance increase. Endemic transmission of quinolone-resistant gonococci (QRNG) in homosexually active men increased substantially in New South Wales and Victoria where more then 90% of all QRNG were found. QRNG in other centres continued to be isolated mostly from overseas travellers and at a low frequency. All isolates remained sensitive to spectinomycin and ceftriaxone. A further increase in the number of gonococcal isolates from homosexually active men was recorded in New South Wales and Victoria. Strains examined in South Australia, New South Wales and Victoria were predominantly from male patients and rectal and pharyngeal isolates were common. In other centres the male to female ratio of cases was lower, and most isolates were from the genital tract in rates similar to those occurring in previous years. The impact of non-culture based detection methods will adversely affect the ability of the AGSP to monitor trends in gonococcal disease in future years.