Creutzfeldt-Jakob Disease in South West Sydney 2014-2020: An Unusually High Incidence of a Rare Disease.

INTRODUCTION: Creutzfeldt-Jakob disease (CJD), a spongiform encephalopathy, caused by a transmissible misfolded cellular prion protein, is a rapidly progressive, debilitating neurodegenerative disorder with no effective treatment. The estimated global incidence is at 1/million inhabitants. This retrospective study examined the incidence of CJD in South Western Sydney Local Health District (SWSLHD) from 2014 to 2020. BACKGROUND: SWSLHD had an estimated population of 1,038,534 in 2020, with CJD data being limited. METHODS: The New South Wales (NSW) Health Information Exchange (HIE) database, for all admissions with CJD diagnoses in SWSLHD, between 2014 and 2020, was reviewed according to the WHO diagnostic criteria, consistent with the Australian national CJD registry. Only probable CJD cases were included. Incidence was calculated based on the projected SWSLHD population. RESULTS: Thirty-five patients, diagnosed with CJD, were identified. Each was evaluated by 2 independent investigators, including clinical presentation, MRI, EEGs, 14-3-3, and RT-QuIC results, before assigning CJD-probable status. Four failed the CJD criteria and were excluded. Of the 31 CJD-probable cases, most (59%) were male and older (37%, range 61-70 years). The incidence rate peaked at 9/million in 2017 and was above 2/million, throughout the 7 years, with an average of 4.859/million/year. CONCLUSIONS: The incidence of CJD, in SWSLHD, exceeds the national average of 1/million. Cost-effective, adequate diagnostic and screening tools, implementable over a large population, will become increasingly essential.

A Diagnostic Dilemma of White Matter Lesions and Cerebral Oedema without Identifiable Cause-A Neurological Conundrum.

INTRODUCTION: This paper describes a case of bi-frontal vasogenic oedema associated with bilateral frontal lobe and left parietal lobe white matter lesions where extensive investigations, including brain biopsy, failed to establish a diagnosis. CASE REPORT: A 67-year-old female presented with three weeks' history of memory loss, fatigue, insomnia, nausea, and occasional dysphasia. Physical examination was unremarkable, yet cerebral CT and MRI showed bilateral frontal lobe vasogenic oedema. Extensive investigations, including: biochemical; radiological; immunological; microbiological; haematological; histopathological; and cytological, failed to establish a confirmed diagnosis. A multidisciplinary team could not achieve a consensus for this atypical presentation. Brain biopsy was unusual, showing destructive inflammatory and subtly granulomatous disease, but an exhaustive list of auxiliary tests could not confirm a cause, and consensus favoured glial fibrillary acidic protein (GFAP) autoimmune encephalopathy. DISCUSSION: A definitive diagnosis could not be established for this patient despite a gamut of investigations. Although some of the presenting features were consistent with GFAP astrocytopathy, initial staining of the patient's CSF for neuronal antibodies was negative. Her symptoms and radiological changes of brain imaging improved without any corticosteroid therapy. CONCLUSIONS: Through this case report, the aim is to add to the repository of neurological sciences in the hope that future similar presentations could potentially lead to discovery of a new aetiology or contribute towards better understanding of an existing disease process.

Culture of Transplant Perfusate Using BACTEC Technology and Antibiotic Prophylaxis Influences Wound Complications Within a Kidney Transplant and SPK Transplant Cohort.

PURPOSE: Routine screening for microbial contamination in organ recovery perfusion transport solution (ORPTS) is by microbiological culture without broth enrichment. Our aim was to examine the clinical utility of broth enrichment of perfusion solution, through use of BACTEC (Becton Dickinson) blood culture media, in preventing wound complications for transplant recipients in comparison with culture without enrichment. METHODS: We prospectively collected samples of ORPTS of 395 kidney (n = 250) or simultaneous pancreas-kidney (SPK, n = 145) donors over a 7-year period. Results of culture with and without broth enrichment (n = 285) using BACTEC blood culture media were examined to compare the sensitivity of BACTEC with non-BACTEC methods. We then conducted a paired analysis of 110 recipients with both BACTEC and non-BACTEC culture organ perfusion media. We examined the rates of wound infection and whether the use of targeted antimicrobials reduced infections in the BACTEC group and recipients with both types of cultures. RESULTS: Of 395 patients with cultures of ORPTS, first, the results of 79 cultures performed using BACTEC media only were compared with 206 non-BACTEC cultures (n = 285). Second, 110 cultures were performed using both methods. For the first part of the study, BACTEC media detected significantly greater microbial growth than non-BACTEC methods (n = 79, 64.6% vs n = 206, 14.6%; P < .001). In the 110 patients with both BACTEC (52.3%) and non-BACTEC cultures (9.9%), there was significantly higher sensitivity of the BACTEC method (P < .001); 68.2% of these patients had antimicrobial cover in the days immediately following transplant sufficient to cover the cultured organism. In the patients with appropriate antimicrobial cover, the rate of recipient wound infection was significantly reduced (P = .003). CONCLUSIONS: Routine screening of ORPTS with BACTEC broth enrichment should always be employed. When paired with antimicrobial prophylaxis, it has the potential to significantly reduce the risk of recipient wound infection.

Mixed gonococcal infections in a high-risk population, Sydney, Australia 2015: implications for antimicrobial resistance surveillance?

OBJECTIVES: Previous studies have shown that mixed-strain gonococcal infections can occur. However, it remains unclear whether such infections impact upon the reliability of Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance. In this study, we aimed to resolve this question by intensively sampling isolates from gonorrhoea-positive specimens in a high-risk population in Sydney, Australia. METHODS: A total of 615 N. gonorrhoeae isolates, originating from 63 clinical samples (31 rectal swabs and 32 throat swabs), were characterized. All isolates were subject to N. gonorrhoeae identification, antimicrobial susceptibility testing and genotyping by SNP-based MLST. RESULTS: Only 2 of the 63 (3.2%) samples provided evidence of mixed-strain infections. These comprised two rectal swabs that harboured isolates of different SNP-based MLST genotypes; however, the AMR susceptibility profiles of the different genotypes from these samples were indistinguishable. Within-sample differences in the AMR susceptibility profiles were observed for a further seven samples; however, the differences were not considered significant; MIC values were typically within a 2-fold difference or were close to test breakpoints. CONCLUSIONS: Results of this study provide further evidence that mixed-strain gonococcal infections do occur, although at low prevalence. Our data indicate that at a population level such infections are unlikely to impact significantly upon N. gonorrhoeae AMR surveillance.

A comparison of agar dilution with the Calibrated Dichotomous Sensitivity (CDS) and Etest methods for determining the minimum inhibitory concentration of ceftriaxone against Neisseria gonorrhoeae.

OBJECTIVES: The objective of this study was to compare the Calibrated Dichotomous Sensitivity (CDS) based agar dilution (CDS AD) method with the Etest (bioMérieux SA) methods using 2 method protocols for determining the minimum inhibitory concentration (MIC) of ceftriaxone against Neisseria gonorrhoeae. The two method protocols were the manufacturer's protocol for which the Clinical and Laboratory Standards Institute (CLSI) interpretative criteria for Neisseria gonorrhoeae could be applied, and the CDS-adapted protocol. Comparability of MIC data is critical for situation analysis and monitoring trends in global antimicrobial analysis. METHODS: Two hundred and forty eight clinical isolates of N. gonorrhoeae and the World Health Organisation (WHO) N. gonorrhoeae reference strains were tested using the three methods. RESULTS: When compared, CDS AD and CDS Etest gave a regression R(2) value of 94%, the Pearson's correlation coefficient was 97% and a paired comparison within one log2 dilution was 98%. The CDS AD and the Etest (CLSI) comparison gave a regression R(2) value of 90%, a Pearson's correlation coefficient of 95% and a paired comparison within one log2 dilution was 98%. The comparison of the CDS Etest and CLSI Etest gave a regression R(2) value of 91%, a Pearson's correlation coefficient of 95% and a paired comparison within one log2 dilution of 99%. Importantly, there was robust agreement between all three methods for the categorization of susceptibility of Neisseria gonorrhoeae isolates using the WHO nominated breakpoint for decreased susceptibility to ceftriaxone (≥0.125 µg/mL). CONCLUSIONS: The CDS Etest method is comparable to agar dilution and the Etest methods for determining the MIC of ceftriaxone against N. gonorrhoeae.

The implications of endemic IMP-4 carbapenemase for clinical laboratory susceptibility testing.

A local predominance of carbapenemase producing Enterobacteriaceae with low minimum inhibitory concentrations (MIC) to meropenem prompted a review of methods available for carbapenemase detection. We report on results using two selective media, temocillin discs, CarbaNP test, GeneXpert Carba-R assay and an in-house PCR assay.

Molecular epidemiology of Neisseria gonorrhoeae using multi-antigen sequence typing and pulse-field gel electrophoresis in highly endemic Western Australian populations.

BACKGROUND: The remote and indigenous populations of Western Australia (WA) have one of the highest notification rates of gonorrhoea in the world. Despite this, the low rate of antimicrobial resistance in Neisseria gonorrhoeae from these regions permits the use of amoxycillin as empirical therapy. We describe the first molecular epidemiological study of gonococci isolated from this population using two different typing platforms. METHODS: Pulse-field gel electrophoresis (PFGE), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) and antimicrobial susceptibility tests were performed on 128 consecutive N. gonorrhoeae isolates cultured between January 2011 and December 2013. To highlight clusters isolates were evaluated based on their tbpB sequence types. RESULTS: No predominant NG-MAST or PFGE types were found. A total of 67 distinct PFGE pulsotypes were identified amongst the 128 isolates in this study with 20 PFGE pulsotypes representing 78 isolates. A total of 59 NG-MAST sequence types were found, represented by 45 porB alleles and 28 tbpB alleles with 13 tbpB genomogroups from 45 NG-MAST sequence types. TbpB genomogroup 29, represented by 45 isolates, was by far the most common genomogroup overall. CONCLUSIONS: Results from this study suggest that gonococcal epidemiology in WA is quite different between remote regions and major population centres and, in some cases, geographically restricted. It is likely that isolates originating from endemic regions of WA mostly represent independent, small sexual networks with an infrequent interchange between other communities and regions. Given the high rate of antimicrobial resistance elsewhere in Australia, ongoing surveillance is essential to ensure the enduring efficacy of amoxycillin empiric use in the remote regions of WA.

Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance.

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.

Molecular approaches to enhance surveillance of gonococcal antimicrobial resistance.

The best available data indicate that the world is heading towards a pandemic of extensively drug-resistant Neisseria gonorrhoeae. At the same time, clinical microbiology laboratories have moved away from using culture-based methods to diagnose gonorrhoea, thus undermining our ability to detect antimicrobial resistance (AMR) using current technologies. In this Opinion article, we discuss the problem of N. gonorrhoeae AMR, particularly emerging resistance to the cephalosporin ceftriaxone, outline current concerns about the surveillance of N. gonorrhoeae AMR and propose the use of molecular methods on a large scale to systematically enhance surveillance.

Non-culture Neisseria gonorrhoeae molecular penicillinase production surveillance demonstrates the long-term success of empirical dual therapy and informs gonorrhoea management guidelines in a highly endemic setting.

OBJECTIVES: Unlike most of the world, penicillin resistance in Neisseria gonorrhoeae from remote regions of Western Australia (WA) with high gonorrhoea notification rates has not increased despite many years of empirical oral therapy. With the advent of non-culture molecular diagnosis of gonorrhoea and the consequent decline in culture-based susceptibility, it is imperative to ensure the ongoing reliability of combination oral azithromycin, amoxicillin and probenecid for uncomplicated gonorrhoea in this setting. PCR-based non-culture N. gonorrhoeae antimicrobial resistance surveillance for penicillinase production was therefore employed. METHODS: Genital and non-genital specimens that were PCR-positive for N. gonorrhoeae were assessed for penicillinase production by detection of the N. gonorrhoeae TEM-1 plasmid using specific real-time PCR. RESULTS: In remote regions of WA where gonorrhoea is highly endemic, <5% of N. gonorrhoeae isolates were penicillinase-producing. This contrasts with rates of up to 20% observed in the more densely populated metropolitan and rural regions. CONCLUSIONS: In the era of molecular diagnosis of gonorrhoea, non-culture-based antimicrobial resistance surveillance proved useful when developing evidence-based guidelines for the clinical management of locally acquired gonorrhoea in highly endemic regions in WA. The continued efficacy of combination oral amoxicillin, probenecid and azithromycin therapy despite many years of use in a setting highly endemic for gonorrhoea may explain the low rate of penicillin resistance in these remote regions and supports the concept of adding azithromycin to ß-lactam antibiotics to help delay the emergence of multiresistant N. gonorrhoeae.

Direct urine polymerase chain reaction for chlamydia and gonorrhoea: a simple means of bringing high-throughput rapid testing to remote settings?

UNLABELLED: Background Rapid point-of-care tests (POCTs) for chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) have the potential to confer health benefits in certain populations even at moderate sensitivities; however, suitable POCTs for these organisms are currently lacking. METHODS: In this study, we investigated the use of direct urine polymerase chain reaction (PCR), with the view of implementing a simplified PCR strategy for high-throughput chlamydia and gonorrhoea screening in remote settings. Briefly, a simple dilution of the urine was performed before adding it directly to a real-time PCR reaction. The method was evaluated using 134 stored urine specimens that had been submitted for chlamydia and gonorrhoea testing and had been tested using a commercial C. trachomatis and N. gonorrhoeae PCR method. These included samples that were PCR-positive for chlamydia (n=87), gonorrhoea (n=16) or both (n=2). Direct urine testing was conducted using previously described in-house real-time PCR methods for C. trachomatis and N. gonorrhoeae as well as for recognised N.gonorrhoeae antimicrobial resistance mechanisms. RESULTS: The overall sensitivities and specificities of the direct urine PCR were 78% and 100% for chlamydia, and 83% and 100% for gonorrhoea. N.gonorrhoeae penicillin and quinolone resistance mechanisms were characterised in 14 of the 18 N. gonorrhoeae-positive samples. CONCLUSIONS: The results of this study show that the simplified PCR strategy may be a feasible approach for rapid screening and improving chlamydia and gonorrhoea treatment in remote settings.

Real-time PCR genotyping of Neisseria gonorrhoeae isolates using 14 informative single nucleotide polymorphisms on gonococcal housekeeping genes.

OBJECTIVES: Neisseria gonorrhoeae multilocus sequence typing (MLST) is a key tool used to investigate the macroepidemiology of gonococci exhibiting antimicrobial resistance (AMR). However, the utility of MLST is undermined by the high workload and cost associated with DNA sequencing of seven housekeeping genes. In this study, we investigated single nucleotide polymorphism (SNP)-based profiling as a means of circumventing these problems. METHODS: A total of 14 SNPs were selected following in silico analysis of available N. gonorrhoeae MLST sequence data. Real-time PCR methods were developed for characterization of each SNP and applied to 86 N. gonorrhoeae isolates exhibiting a range of ceftriaxone MICs. Twenty-one isolates had previously been characterized by MLST. The ability of the real-time PCR methods to generate SNP profiles and of the 14 SNP profiles to predict MLST types were assessed. RESULTS: In silico analysis of the 217 different MLST types available on the Neisseria web site showed 181 different 14 SNP profiles (Simpson's index of diversity = 0.998). When the real-time PCR methods were applied to the isolates, 29 different 14 SNP profiles were obtained for 83 isolates. Predicted MLST types were consistent with those for the 21 isolates previously characterized by MLST. For 46 isolates with raised ceftriaxone MICs (≥ 0.03 mg/L), there were 14 different 14 SNP profiles observed, with two profiles accounting for more than half of these isolates. CONCLUSIONS: The 14 SNP real-time PCR profiling approach is a simple and cost-effective alternative to N. gonorrhoeae MLST and could be used to complement current typing schemes in N. gonorrhoeae AMR investigations.

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.

Protocol for the molecular detection of antibiotic resistance mechanisms in Neisseria gonorrhoeae.

Gonorrhoea is no longer an easily treatable ailment but rather is now a challenging disease in terms of antimicrobial resistance (AMR) with treatment options rapidly diminishing. The causative agent of gonorrhoea, Neisseria gonorrhoeae, has managed to develop resistance to almost every single drug used against it with the sole exception of extended spectrum cephalosporins. The situation is further exacerbated by the fact that not only are the rates of gonococcal infections on a steady rise globally, but tracking AMR is being undermined by the growing popularity of molecular methods at the expense of traditional bacterial culture in diagnostic laboratories. Recently, concerns have been raised over the emergence of a multi-resistant gonococci and the potential for untreatable gonorrhoea. Maintaining optimal epidemiological surveillance of gonococcal AMR remains an important aspect of gonorrhoea control. The development of molecular tools for tracking AMR in N. gonorrhoeae has the potential to further enhance such surveillance. In this chapter, we discuss nucleic acid amplification-based detection of AMR in gonorrhoea with a particular emphasis on chromosomal-mediated resistance to beta-lactam antibiotics.

The ticking time bomb: escalating antibiotic resistance in Neisseria gonorrhoeae is a public health disaster in waiting.

From a once easily treatable infection, gonorrhoea has evolved into a challenging disease, which in future may become untreatable in certain circumstances. International spread of extensively drug-resistant gonococci would have severe public health implications. It seems clear that under the current treatment pressure from extended-spectrum cephalosporins, and owing to Neisseria gonorrhoeae's remarkable evolutionary adaptability, further rise of ceftriaxone-resistant strains around the world is inevitable. Simply increasing the doses of extended-spectrum cephalosporins will likely prove ineffective in the long run, and has been a lesson learnt for all single-agent therapies used for gonorrhoea to date. We recommend that dual therapy, especially those consisting of extended-spectrum cephalosporins and azithromycin, be adopted more widely and complemented by strengthening of antimicrobial resistance surveillance. Unless there is urgent action at international and local levels to combat the problem of N. gonorrhoeae antimicrobial resistance, we are in for gloomy times ahead in terms of gonorrhoea disease and control.

Enhanced gonococcal antimicrobial surveillance in the era of ceftriaxone resistance: a real-time PCR assay for direct detection of the Neisseria gonorrhoeae H041 strain.

OBJECTIVES: Recent emergence of the extensively drug-resistant Neisseria gonorrhoeae H041 strain in Japan raises concerns that gonorrhoea may soon become untreatable and emphasizes the need for enhanced surveillance. In this study we developed a real-time PCR assay for direct detection of the H041 strain. METHODS: Two real-time PCR assays for detection of the penA gene of the H041 strain, H041-PCR1 and H041-PCR2, were developed and evaluated in parallel. Assay performance was assessed using a panel of pathogenic and commensal Neisseria species (n = 167 strains) including the N. gonorrhoeae H041 strain and clinical specimens (n = 252) submitted for sexual health screening. The detection limits of the assays were compared with a standard N. gonorrhoeae real-time PCR method. RESULTS: Both the H041-PCR1 and H041-PCR2 assays correctly detected the N. gonorrhoeae H041 strain and provided negative results for all other N. gonorrhoeae strains. However, only the H041-PCR2 assay proved to be specific when applied to the non-gonococcal Neisseria species and clinical samples. False-positive results in the H041-PCR1 included cross-reactions with two Neisseria subflava isolates and eight clinical specimens. DNA sequencing of these N. subflava strains revealed the presence of the penicillin-binding protein 2 Ala328Thr alteration previously only observed in the N. gonorrhoeae H041 strain. CONCLUSIONS: The H041-PCR2 assay is suitable for direct detection of the N. gonorrhoeae H041 ceftriaxone-resistant strain in cultured and non-cultured samples.

Enhancing gonococcal antimicrobial resistance surveillance: a real-time PCR assay for detection of penicillinase-producing Neisseria gonorrhoeae by use of noncultured clinical samples.

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.