Australian Gonococcal Surveillance Programme Research Articles

A national quality assurance survey of Neisseria gonorrhoeae testing

The aims of this study were to (1) conduct a national survey of Neisseria gonorrhoeae identification by National Neisseria Network (NNN) reference laboratories contributing data to the Australian Gonococcal Surveillance Programme and (2) determine the prevalence in Australia of strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays for confirmation of gonococcal nucleic acid amplification tests. Gonococcal clinical isolates referred to NNN laboratories for the first half of 2012 were screened using in-house real-time PCR assays targeting multicopy opa, porA pseudogene and cppB genes. There were 2455 clinical gonococcal isolates received in the study period; 98.6 % (2420/2455) of isolates harboured all three gene targets, 0.12 % (3/2455) were porA-negative, 0.04 % (1/2455) opa-negative and 1.14 % (28/2455) cppB-negative by PCR. Notably, no isolates were simultaneously negative for two targets. However, three isolates failed to be amplified by all three PCR methods, one isolate of which was shown to be a commensal Neisseria strain by 16S rRNA sequencing. Using PCR as the reference standard the results showed that (1) identification of N. gonorrhoeae isolates by NNN laboratories was highly specific (99.96 %) and (2) strains of N. gonorrhoeae lacking gene sequences commonly targeted by in-house PCR assays are present but not widespread throughout Australia at this point in time.

Neisseria meningitidis with reduced susceptibility to quinolones in Singapore

Sir, Ciprofloxacin is commonly prescribed as a single-dose chemoprophylactic agent for close contacts of patients with meningococcal infection. Neisseria meningitidis with reduced susceptibility to fluoroquinolones has recently emerged, with sporadic cases reported in France, Australia, Argentina, Spain, Hong Kong and Israel. Recently an outbreak of serogroup A emerged in India, and a cluster of three cases of serogroup B in the USA. To our knowledge, failure of ciprofloxacin prophylaxis due to quinolone resistance has not been reported To date, reduced susceptibility to fluoroquinolones in meningococci has remained rare, with 10 unrelated strains found in an analysis of 5300 isolates in Spain, and only 3 from .5000 isolates in France. Most mutations have arisen in GyrA. However, mutations have also been detected in GyrB and ParE, although their significance remains uncertain. In addition, in vitro changes have been induced in ParC and resistance to quinolones has also been reported through an efflux mechanism. – 4 Following the isolation of N. meningitidis with reduced susceptibility to ciprofloxacin from a 36-year-old man with meningitis in 2008, all stored viable clinical isolates from Singapore’s three largest hospitals (Tan Tock Seng, Singapore General and National University hospitals) were screened for reduced susceptibility to nalidixic acid and rifampicin by disc diffusion, using the CLSI guidelines. Ciprofloxacin MICs were determined by agar plate dilution for isolates with reduced susceptibility to nalidixic acid, using the Australian Gonococcal Surveillance Programme (AGSP) guidelines. MICs obtained with this method give identical results for identical GyrA changes in Neisseria gonorrhoeae and N. meningitidis. All isolates for which the MIC is .0.03 mg/L either have changes in the quinolone resistance determining region (QRDR) or an increased efflux of quinolones, while those for which the MIC is ,0.03 mg/L do not. In the absence of clinical correlation data, an MIC of .0.03 mg/L is taken to indicate reduced susceptibility by both the AGSP and CLSI methods. Multilocus sequence typing (MLST) was carried out using protocols available on the Neisseria MLST website. To characterize the mechanisms of resistance to ciprofloxacin, we sequenced the QRDRs of GyrA, GyrB, ParC and ParE of isolates with a ciprofloxacin MIC of .0.03 mg/L. Penicillin MICs were determined with Etests (AB BIODISK, Solna, Sweden) for all isolates, using Mueller–Hinton agar with 5% sheep blood and interpreted according to CLSI criteria for agar dilution. Of 75 isolates collected between 2000 and 2008, two (3%) were resistant to nalidixic acid, a Group C isolate from 2008 and a Group B isolate from 2002. Both isolates had a raised ciprofloxacin MIC of 0.25 mg/L. All 75 isolates were susceptible to rifampicin. There were 13 isolates (17%) intermediate and 2 (3%) resistant to penicillin (MICs of 0.38 and 0.47 mg/L). MLST confirmed that the two isolates with reduced quinolone susceptibility were unrelated. The 2008 isolate was identified as sequence type (ST)-4821. The 2002 isolate was a new ST with alleles 4, 10, 76, 71, 13, 2, 75 although the aroE allele had one nucleotide different from allele 76 (C to T at position 39). The 2008 ST-4821 isolate had the most common GyrA substitution of threonine to isoleucine at position 91. The 2002 isolate had three mutations detected in GyrA: Thr91!Val; Asn103!Asp; and Ala105!Ser. To our knowledge, the Thr91!Val substitution has not been previously described in N. meningitidis. Neither of the isolates had changes in GyrB, ParC or ParE. Similarities in the mechanism of quinolone resistance in meningococci and gonococci, have prompted concerns that resistance will climb to mirror that of gonococci. Resistance to quinolones among N. gonorrhoeae is now so common that use of ciprofloxacin for the treatment of gonorrhoea is no longer recommended by the US Centers for Disease Control and Prevention. In Singapore, ciprofloxacin resistance among gonococci reached an alarming 61.8% in 2006 (74% in 2008; A. L. Tan, unpublished data). Ciprofloxacin is a prescription-only medicine in Singapore, but is in widespread use. An outbreak of meningococcal meningitis with reduced susceptibility to quinolones has already been seen in India in 2005, a country with liberal use of ciprofloxacin. Although our data are from small numbers, as meningococcal infections are uncommon in Singapore (usually ,10 cases per year), the 3% rate of quinolone resistance in this study is remarkably high when compared with other reports. Surveillance for reduced susceptibility and judicious quinolone use is forever increasing in importance but our local resistance data for the related N. gonorrhoeae may suggest the future is already written.

Antimicrobial susceptibility profile of <i> <i>Neisseria</i> gonorrhoeae</i> at STI clinic

A total of 100 consecutive patients who attended a sexually transmitted infections clinic were studied. Thirteen had gonococcal urethritis, of which 10 showed growth of Neisseria gonorrhoeae on culture. All the isolates were tested for antimicrobial susceptibility by Australian Gonococcal Surveillance Programme (AGSP) method and beta lactamase production by chromogenic cephalosporin test. Four patients were co-infected with each of the following: HIV, HBV and Chlamydia trachomatis . Gonococcal urethritis (13%) was found more in male patients. Ten percent gonococcal isolates were penicillinase-producing N. gonorrhoeae , and another 10% were tetracycline-resistant N. gonorrhoeae .

ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF NEISSERIA GONORRHOEAE AT STI CLINIC

A total of 100 consecutive patients who attended a sexually transmitted infections clinic were studied. Thirteen had gonococcal urethritis, of which 10 showed growth of Neisseria gonorrhoeae on culture. All the isolates were tested for antimicrobial susceptibility by Australian Gonococcal Surveillance Programme (AGSP) method and beta lactamase production by chromogenic cephalosporin test. Four patients were co-infected with each of the following: HIV, HBV and Chlamydia trachomatis. Gonococcal urethritis (13%) was found more in male patients. Ten percent gonococcal isolates were penicillinase-producing N. gonorrhoeae, and another 10% were tetracycline-resistant N. gonorrhoeae.

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

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. 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. 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. 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 incidence of gonorrhoea and the antibiotic sensitivity of gonococci in Australia, 1981-1991. The Australian Gonococcal Surveillance Programme.

To review and analyse the changing incidence of gonorrhoea and the increasing antibiotic resistance in gonococci in Australia from 1981 to 1991. Use of data from the sample of gonorrhoea in Australia examined by the Australian Gonococcal Surveillance Programme (AGSP), a continuing long-term multi-centric study of gonococcal disease and gonococcal susceptibility to antibiotics, over the period 1 July, 1981 to 30 June, 1991. Over 32,000 cases and strains from defined sources were examined in the 10 year study period. The number of cases of gonorrhoea in the sample decline from a peak of 6599 in 1982-1983 to 1121 in the final year under review, a reduction of 83%. Periods when greater than average reductions in incidence occurred in different groups were noted. Ano-rectal gonorrhoea in men decreased sharply in 1985 during an overall decline of 92.5% recorded between 1 July, 1981 to 30 June, 1987. However, the incidence of ano-rectal cases in males rose in subsequent years while gonorrhoea, overall, continued to decrease and at a greater rate after 1985. Antibiotic resistance in gonococci in Australia was manifested both as a progressive increase in the levels of intrinsic resistance to the penicillins and through the appearance and spread of penicillinase-producing N gonorrhoeae (PPNG). At the end of the review period in June, 1991, 8.8% of gonococcal isolates in Australia showed high levels of intrinsic resistance to the penicillins and 13% of strains were PPNG. These separate mechanisms of resistance appeared at different times in different parts of Australia, and their importance also varied throughout the country. Most infections with PPNG were acquired by men overseas whereas most women with PPNG were infected locally. Endemic spread of PPNG was a significant problem in Sydney and Melbourne, but decreased in importance in the later years of the study. In the past decade a large reduction in the incidence of gonorrhoea and, by implication, other STDs has occurred in the past decade in Australia. In some groups of patients the decline in incidence is continuing while in others a slight increase has been noted. Resistance to antibiotics of gonococci in Australia was mainly restricted to the penicillins, but through both chromosomal and plasmid-mediated mechanisms. This resistance was seen particularly in Sydney and Melbourne where endemic cycles of transmission of PPNG were established, and in infected travellers from S-E Asia in other centres. Valid and comparable, and regionally relevant data are a continuing requirement for assessing and modifying antibiotic treatment regimens for gonococcal disease.

Strain characteristics and antibiotic susceptibility of isolates of Neisseria gonorrhoeae causing disseminated gonococcal infection in Australia. Members of the Australian Gonococcal Surveillance Programme.

The auxotype (A) and serovar (S) distribution and antibiotic and serum sensitivity of 22 strains of Neisseria gonorrhoeae isolated from blood and joints were determined. With one exception, these strains from disseminated gonococcal infections (DGI) belonged to one of 4 serovars of the IA serogroup and were resistant to killing by normal human serum. The auxotype distribution of these Australian strains differed significantly from that reported elsewhere in that 17 of the 22 isolates were proline requires, but none were of the AHU auxotype. This lack of the AHU auxotype in the DGI strains in Australia was explained by the virtual absence of AHU requirers in a sample of 1560 mucosal strains isolated in Sydney and Darwin from 1987 to 1990. The A/S distribution of these mucosal isolates also helped to account for the low (0.12) percentage of DGI strains in isolates examined by the Australian Gonococcal Surveillance Programme (AGSP) from 1981 to 1991, and the differences in the rates of DGI in Sydney (0.08%) and Darwin (0.87%). There was a relative lack of the IA serogroup strains which are mostly responsible for DGI in the mucosal isolates from Sydney (15% of all strains) but a higher proportion of these serovars (40%) in the Darwin sample. There were 46 cases of DGI in data from the AGSP, 29 of these being women. Seven of the cases diagnosed in Australia were infected with penicillinase-producing gonococci suggesting that antibiotics other than the penicillins should now be used for this condition in this region.

Use of a quality assurance scheme in a long-term multicentric study of antibiotic susceptibility of Neisseria gonorrhoeae.

Experience with a quality control programme for a multicentric collaborative study of antibiotic susceptibility of Neisseria gonorrhoeae is described. The Australian Gonococcal Surveillance Programme (AGSP) has monitored the antibiotic sensitivity of gonococci by surveying results of quantitative sensitivity tests performed in a number of centres throughout the country since 1981. A standardised system of testing by means of agar plate dilution was adopted to ensure uniformity of methods and results and to permit temporal and regional differences in antibiotic sensitivity to be identified and compared. A control programme was developed to provide quality assurance regarding each laboratory's performance. Over a period of nine years more than 3600 MIC determinations on control strains, examined as unknowns, were performed. An overall error rate of 3.1% was observed. Higher error rates were noted in the beginning of the programme but these declined as the study progressed. In addition repeat and duplicate testing of strains was performed. The results of these tests also indicated an improvement in performance over the study period. Close attention to all aspects of the testing procedure reduced the number of errors recorded. At the beginning of the programme, various factors were investigated as possible causes of error. The formulation and preparation of media were found to be potential sources of result variation. Once these variables were controlled, lack of experience with end-point determination and errors in preparation of dilutions of antibiotic solutions emerged as the commoner causes of discrepancy. In the experience of the AGSP, a long-term multicentric evaluation of gonococcal sensitivity can achieve accurate and reproducible results through standardisation of methods and application of a continuing quality assurance scheme.

Changing epidemiology of gonococcal disease in Australia 1981-1988

In Australia dramatic changes have recently occurred in the incidence and patterns of gonococcal infections. These changes were consequent upon increasing awareness of HIV infection and its transmission and were monitored by the Australian Gonococcal Surveillance Programme (AGSP). This is a continuing laboratory based scheme which has surveyed and reported on more than 30,000 gonococcal infections in an unchanged sample examined since 1981. A significant decrease in the incidence of gonorrhoea has been observed with a decline from the peak annual rate of 6600 infections in 1982-83 to 1675 cases in 1987-88 representing a 75% reduction in overall numbers. Rapid rates of decline were noted in periods in 1983, 1985 and 1987. Further, the rates of decrease were different in men and women so that the male:female ratio of the disease changed from 3.4 in 1981-82 to 2.3 in 1987-88. A disproportionate decrease in rectal isolates occurred in male patients with the most rapid decline occurring in 1985. The rate of rectal gonorrhoea was then only 10% of that recorded in 1981-82. No further decrease in this rate has been observed subsequently, an observation with obvious relevance to HIV transmission. In women, increases in the rates of pharyngeal gonorrhoea were also observed. In Australia reliable statistics on sexually transmissible diseases were for many years unavailable or unreliable. The AGSP statistics on gonorrhoea help to provide this information and also reflect changes occurring in the wider spectrum of venereal diseases.

Changing patterns in gonococcal infection in Australia, 1981-1987. Australian Gonococcal Surveillance Programme.

Data from the Australian Gonococcal Surveillance Programme (AGSP) were used to analyse changes in the patterns of gonococcal disease in Australia over the period July 1, 1981 to June 30, 1987. The AGSP is a continuing multicentric study of aspects of gonococcal disease and bases its analyses on laboratory-confirmed cases of gonorrhoea. The number of gonococcal isolates was highest in 1982-1983, when 6599 strains were cultured, but it fell in each subsequent year of the study until fewer than half this number was isolated in 1986-1987. The decrease in the number of isolated strains was proportionately-greater in men than in women, but in spite of this, the male-to-female ratio of disease in Australia remains unacceptably-high at two to one. In male patients, the decrease in the incidence of rectal isolates from 11.7% of all strains in 1981-1982 to 1.8% of all strains in 1986-1987 particularly was noteworthy. In female patients, pharyngeal isolates constituted an increasing proportion of strains and rose from 4.6% of all strains in 1981-1982 to 9.0% of all strains in 1986-1987; this has implications for the diagnosis and management of gonococcal disease in this group.

Penicillin sensitivity of gonococci in Australia: development of Australian gonococcal surveillance programme. Members of the Australian Gonococcal Surveillance Programme.

We describe the development of the Australian gonococcal surveillance programme from February 1979 to July 1982. Participants in the programme were the major public health laboratories in each state, which perform quantitative sensitivity tests on about 5400 strains of Neisseria gonorrhoeae yearly. Participating laboratories conducted sensitivity tests by a standardised technique which is described, and the results from each centre were collated quarterly. Temporal and regional differences in gonococcal sensitivity patterns were noted and, in the twelve months ending June 1982, there was a trend towards a decrease in gonococci sensitive to penicillin. Increasing numbers of penicillinase producing gonococci were found, and the isolation rates of these strains varied in different regions at different times.