Kevin Freeman

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.

Enhancing Gonococcal Antimicrobial Resistance Surveillance: a Real-Time PCR Assay for Detection of Penicillinase-Producing Neisseria gonorrhoeae by Use of Noncultured Clinical Samples

ABSTRACT With increasing concerns regarding diminishing treatment options for gonorrhea, maintaining the efficacy of currently used treatments and ensuring optimal Neisseria gonorrhoeae antimicrobial resistance surveillance are of the utmost importance. Penicillin is still used to treat gonorrhea in some parts of the world. In this study, we developed and validated a real-time PCR assay for the detection of penicillinase-producing N. gonorrhoeae (PPNG) in noncultured clinical samples with the aim of enhancing penicillin resistance surveillance. The assay (PPNG-PCR2) was designed to be an indirect marker of penicillinase activity, by targeting a region of sequence predicted to be conserved across all N. gonorrhoeae plasmid types harboring the beta-lactamase gene while not specifically targeting the actual beta-lactamase-encoding sequence. The assay was evaluated by using a total of 118 N. gonorrhoeae clinical isolates and 1,194 clinical specimens, including 239 N. gonorrhoeae-positive clinical samples from which N. gonorrhoeae cells were isolated and for which phenotypic penicillinase results are available. Overall, the PPNG-PCR2 assay provided 100% sensitivity and 98.7% specificity compared to bacterial culture results for the detection of PPNG in clinical specimens. PPNG-PCR2 false-positive results, presumably due to cross-reactions with unrelated bacterial species, were observed for up to 1.3% of clinical samples but could be distinguished on the basis of high cycle threshold values. In tandem with phenotypic surveillance, the PPNG-PCR2 assay has the potential to provide enhanced epidemiological surveillance of N. gonorrhoeae penicillin resistance and is of particular relevance to regions where penicillin is still used to treat gonorrhea.

Further Evaluation of a Rapid Diagnostic Test for Melioidosis in an Area of Endemicity

ABSTRACT Immunochromatographic test (ICT) kits for the rapid detection of immunoglobulin G (IgG) and IgM antibodies to Burkholderia pseudomallei were compared to the indirect hemagglutination (IHA) assay. In 138 culture-confirmed melioidosis cases, sensitivities were 80, 77, and 88% for IHA, ICT IgG, and ICT IgM, respectively. In a prospective study of 160 consecutive sera samples sent for melioidosis serology, respective specificities were 91, 90, and 69, positive predictive values were 41, 32, and 18, and negative predictive values were 99, 98, and 100%. ICT IgM kits are unreliable for diagnosis of melioidosis, but ICT IgG kits may be useful for diagnosing travelers presenting with possible melioidosis who return from regions where melioidosis is endemic.

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target

Aims: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. Methods: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. Results: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. Conclusions: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.

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.

Cryptic-Plasmid-Free Gonococci May Contribute to Failure of cppB Gene-Based Assays To Confirm Results of BD ProbeTEC PCR for Identification of Neisseria gonorrhoeae

We note the recent article by Koenig et al. ([3][1]) which describes discordance between results obtained with the BD ProbeTEC ET System (BDPT) and a cppB gene-based PCR assay for the detection of Neisseria gonorrhoeae . Overall, 22.6% of BDPT-positive assays were not confirmed by the cppB gene-

A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility

OBJECTIVES The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility. METHODS The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available. RESULTS The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples. CONCLUSIONS The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.

Multitarget PCR assay for direct detection of penicillinase-producing neisseria gonorrhoeae for enhanced surveillance of gonococcal antimicrobial resistance

ABSTRACT A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.

Surprisingly Low Seroprevalence of Burkholderia pseudomallei in Exposed Healthy Adults in the Darwin Region of Tropical Australia Where Melioidosis Is Highly Endemic

ABSTRACT In the Darwin region of Australia where melioidosis is highly endemic, only 11/354 (3%) healthy residents were seropositive by indirect hemagglutination assay, despite extensive exposure to Burkholderia pseudomallei. None developed melioidosis, but some described a prior self-limiting illness. This seropositivity rate is much lower than that seen in northeast Thailand, where melioidosis is similarly highly endemic, potentially reflecting important differences between these two locations in the epidemiology of melioidosis.

The influence of target population on nonculture-based detection of markers of Neisseria gonorrhoeae antimicrobial resistance

BACKGROUND With treatment options for gonorrhoea (Neisseria gonorrhoeae) diminishing, strengthening antimicrobial resistance (AMR) surveillance is paramount. METHODS In this study, we investigated polymerase chain reaction (PCR) based methods, in parallel with N. gonorrhoeae multi-antigen sequence typing (NG-MAST), for direct detection of four N. gonorrhoeae chromosomal mechanisms associated with emerging resistance to extended spectrum cephalosporins using noncultured samples: an adenine deletion in the mtrR promoter, a mosaic penicillin-binding protein (PBP) 2, an A501V PBP2 mutation, and alterations at positions 120 and 121 of the porB protein. The PCR assays were validated using a panel of characterised N. gonorrhoeae isolates (n=107) and commensal Neisseria (n=100) species. These PCR assays with NG-MAST were then applied to noncultured clinical specimens from distinct populations in Australia with differing levels of N. gonorrhoeae AMR: the Northern Territory (NT), where resistance has a low population prevalence, and Queensland (Qld), with higher AMR prevalence. RESULTS The real-time PCR assays proved highly sensitive and specific. When applied to the noncultured samples, only 1 out of 50 (2%) samples from NT harboured a resistant mechanism, whereas the Qld samples (n=129) collected over different periods showed progressive acquisition of resistant mechanisms, and these were associated with specific NG-MAST types, including Type 225. CONCLUSIONS The results suggest that our PCR-based methods could be used to rapidly pinpoint incursion of resistant strains into previously unaffected populations. Likewise, our results show that for molecular AMR surveillance, the population being investigated is as important as the genetic mechanisms being targeted.