A novel molecular assay conducted on the BD Max system to facilitate reflex testing for vanA and vanB in clinical isolates of enterococci.

Infections caused by vancomycin-resistant enterococci (VRE) are common. Real-time PCR assays targeting vanA and vanB facilitate screening of patients in healthcare settings to limit the risk of dissemination, especially amongst those at high-risk of infection or with limited treatment options. Such assays are commonly performed as reflex testing procedures where they augment phenotypic techniques and shorten turnaround time to benefit timely clinical management. 'Random access' and 'sample-to-result' real-time PCR platforms are suited for this application as they are of low complexity and less technically demanding. Modelled on these attributes, we configured a real-time PCR assay (VRE BD) for detection of vanA/B in clinical isolates of enterococci, adapted for the BD Max System (Becton Dickinson). We applied an unconventional approach by testing suspensions of microorganisms in water to circumvent the traditional pre-analytical genomic extraction process. Our objective of this study was to assess the performance of this assay for detection of VRE in cultures by validating against a traditional real-time PCR assay based on the LightCycler 2.0 platform (Roche, VRE RO). A high level of analytical sensitivity and specificity (≥99.0%) for both genes was obtained when testing suspensions derived from blood agar. Results for suspensions obtained from chromID VRE (Edwards Group) showed a similar level of performance for vanA detection (100%), but not for the vanB target (≥90.9%) where a lesser number of isolates were available for testing. However, our results for VRE detection in isolates from these media were repeatable and reproducible, and equated to positive and negative predictive values of ≥95.2% and ≥97.8%, respectively. Furthermore, the VRE BD assay was also able to accurately detect VRE in clinical and spiked BacT/ALERT (bioMérieux) blood cultures. Thus, the technical simplicity, short turnaround time and robustness of this high performing assay for VRE is suitable for reflex testing. In addition, the format developed for the BD Max platform has potential application for reflex testing other molecular targets of clinical importance.

A vanA vancomycin-resistant Enterococcus faecium ST80 outbreak resulting from a single importation event.

BACKGROUND: A marked genotype shift among vancomycin-resistant Enterococcus faecium (VREfm) from vanB to vanA in Australia between 2011 and 2015 is a well-known phenomenon. It is hypothesized that this was caused by multiple independent clones emerging simultaneously in different settings and/or regions. OBJECTIVES: To gain insights into the circumstances surrounding the shift from vanB to vanA VREfm in one Australian hospital. METHODS: The genomes of 69 vanA VREfm isolates from St George Hospital collected between 2009 and 2018 were studied. An expansion of ST80 vanA VREfm was noted following a single introduction. ST80 isolates were thus further characterized using hybrid sequencing and contextualized through comparisons with other published Australian ST80 isolates. Phylogenies were constructed with plasmid sequences compared with the index isolate. RESULTS: The 2011 expansion of ST80 vanA VREfm isolates in our institution originated from the 2009 index isolate, from a patient transferred from overseas. Phylogenetic analysis with other Australian ST80 vanA VREfm isolates showed that the 2011 expansion event was unique, with limited spread to adjacent local health districts. Plasmid analysis showed multiple variants, which can also be traced back to the 2009 isolate, consistent with ongoing plasmid adaptation over time. CONCLUSIONS: These findings confirm an expansion event following a VREfm introduction event leading to a sustained clonal and plasmid outbreak over several years. Moreover, it demonstrates the complexity of countrywide replacement events. This study also highlights the use of hybrid sequencing in establishing an epidemiological relationship to the index isolate that was initially inapparent.

A simple and expedient PCR format for rapid molecular screening of methicillin-resistant Staphylococcus aureus in Amies gel swabs.

Screening patients for carriage of methicillin-resistant Staphylococcus aureus (MRSA) is commonly undertaken in hospital laboratories using phenotypic methods. This work is labour-intensive, costly and may take several days to complete. We report on the validation of a novel rapid screening approach for direct testing of Amies gel swabs for MRSA. The method is based on two quantitative real time-PCR (qRT-PCR) assays for the detection of the nuc and mecA genes of MRSA. Based on SYBR Green technology, the assays use significantly less reagents than conventional qRT-PCR methods and are applied to testing templates derived directly from aqueous suspensions of swabs. Notwithstanding the occurrence of false-positives due to non-specific fluorescence generated by the SYBR Green dye, the novel assays showed a high negative predictive value enabling earlier reporting of negative findings and selection of swabs for confirmatory phenotypic testing for MRSA. In a blinded trial of 461 swabs, of which 34 (7.4%) were previously shown to be culture-positive for MRSA, the novel assays selected 121 (26.2%) swabs (inclusive of the known MRSA-positive swabs) for phenotypic testing. This enabled early reporting of negative findings for 340 (73.8%) of the 461 swabs tested. Application of this method has implications for screening strategies for large laboratories whilst achieving cost benefits.

Plesiomonas shigelloides Revisited.

After many years in the family Vibrionaceae, the genus Plesiomonas, represented by a single species, P. shigelloides, currently resides in the family Enterobacteriaceae, although its most appropriate phylogenetic position may yet to be determined. Common environmental reservoirs for plesiomonads include freshwater ecosystems and estuaries and inhabitants of these aquatic environs. Long suspected as being an etiologic agent of bacterial gastroenteritis, convincing evidence supporting this conclusion has accumulated over the past 2 decades in the form of a series of foodborne outbreaks solely or partially attributable to P. shigelloides. The prevalence of P. shigelloides enteritis varies considerably, with higher rates reported from Southeast Asia and Africa and lower numbers from North America and Europe. Reasons for these differences may include hygiene conditions, dietary habits, regional occupations, or other unknown factors. Other human illnesses caused by P. shigelloides include septicemia and central nervous system disease, eye infections, and a variety of miscellaneous ailments. For years, recognizable virulence factors potentially associated with P. shigelloides pathogenicity were lacking; however, several good candidates now have been reported, including a cytotoxic hemolysin, iron acquisition systems, and lipopolysaccharide. While P. shigelloides is easy to identify biochemically, it is often overlooked in stool samples due to its smaller colony size or relatively low prevalence in gastrointestinal samples. However, one FDA-approved PCR-based culture-independent diagnostic test system to detect multiple enteropathogens (FilmArray) includes P. shigelloides on its panel. Plesiomonads produce ß-lactamases but are typically susceptible to many first-line antimicrobial agents, including quinolones and carbapenems.

Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample inhibition and suitability for detection of low levels of target DNA in random and multiple samples.

Prevalence of viruses in stool of premature neonates at a neonatal intensive care unit.

AIM: Premature neonates represent a population highly vulnerable to infection. This study aims to profile viral colonisation of gut and the associated clinical manifestations among premature neonates admitted to a neonatal intensive care unit (NICU) in Australia. METHODS: In a cohort of 75 premature neonates born at less than 32 weeks gestation, who were followed for 4 weeks following admission to a NICU in Sydney, Australia, multiplex polymerase chain reaction assays were used to determine viral presence in stool, and clinical data were examined. RESULTS: Overall, viral RNA or DNA was detected in 24/419 (5.7%) of specimens, including norovirus genogroup 2 (1.9%), enterovirus (1.2%), herpes simplex virus-2 (1.2%), cytomegalovirus (0.7%), Epstein-Barr virus (0.5%) and rotavirus (0.2%). Viral infection was detected in 13/75 (17%) of premature neonates at some time point, including five (7%) neonates shedding more than one type of virus in stool. A higher rate of infection was observed among premature neonates with intrauterine growth restriction (56%) compared with those infants born appropriate for gestational age (12%. P = 0.006). CONCLUSION: The overall viral detection rate in stool of 5.7% (affecting 17% of neonates) indicates viral infections are an important health risk for premature infants in NICU.

Norovirus GII.4 variant 2006b caused epidemics of acute gastroenteritis in Australia during 2007 and 2008.

BACKGROUND: Over the last decade, four epidemics of norovirus-associated gastroenteritis have been reported in Australia. These epidemics were characterized by numerous outbreaks in institutional settings such as hospitals and nursing homes, as well as increases in requests for NoV testing in diagnostic centers. During 2007 and 2008, widespread outbreaks of acute gastroenteritis were once again seen across Australia, peaking during the winter months. OBJECTIVES: The primary objective of this study was to characterize two winter epidemics of NoV-associated gastroenteritis in 2007 and 2008 in Australia. Following this, we aimed to determine if these epidemics were caused by a new GII.4 variant or a previously circulating NoV strain. STUDY DESIGN: NoV-positive fecal samples (n=219) were collected over a 2-year period, December 2006 to December 2008, from cases of acute gastroenteritis in Australia. NoV RNA was amplified from these samples using a nested RT-PCR approach targeting the 5' end of the capsid gene, termed region C. Further, characterization was performed by sequence analysis of the RdRp and capsid genes and recombination was identified using SimPlot. RESULTS: From 2004 to 2008, peaks in the numbers of NoV-positive EIA tests from the Prince of Wales Hospital Laboratory correlated with the overall number of gastroenteritis outbreaks reported to NSW Health, thereby supporting recent studies showing that NoV is the major cause of outbreak gastroenteritis. The predominant NoV GII variant identified during the 2007-2008 period was the GII.4 pandemic variant, 2006b (71.51%, 128/179), which replaced the 2006a variant identified in the previous Australian epidemic of 2006. Four novel GII variants were also identified including the three GII.4 variants: NoV 2008, NoV Osaka 2007 and NoV Cairo 2007, and one novel recombinant NoV designated GII.e/GII.12. CONCLUSION: The increase in acute gastroenteritis outbreaks in 2007 and 2008 were associated with the spread of the NoV GII.4 variant 2006b.

Trichomonas vaginalis: underdiagnosis in urban Australia could facilitate re-emergence.

OBJECTIVES: Trichomonas vaginalis (TV) has a low profile in urban sexually transmitted infection (STI) clinics in many developed countries. The objective of this study was to determine the true prevalence of TV in an Australian urban sexual health setting using sensitive molecular diagnostic techniques. METHODS: A cross-sectional study investigating the aetiology of cervicitis in women attending two urban sexual health clinics in Sydney, Australia, enrolled 356 consecutive eligible women from 2006 to 2008. The diagnostic yield from the standard clinical practice of discretionary high vaginal wet preparation microscopy in women with suspicious vaginal discharge was compared with universal use of nested PCR for TV of cervical samples. RESULTS: TV was detected by PCR in 17/356 women (4.8%, 95% CI 2.8 to 7.5%), whereas only four cases (1.1%, 95% CI 0.3 to 2.8%) were detected by discretionary wet preparation microscopy. Eleven of the 17 women (p=0.003) were of culturally and linguistically diverse background. Additionally, cervicitis was found to be significantly associated with TV, RR 1.66 (1.14 to 2.42), p=0.034. CONCLUSIONS: Traditional TV-detection methods underestimate TV prevalence in urban Australia. The TV prevalence of 4.8% by PCR testing in this study exceeds previously reported urban Australian TV rates of <1%. An increase in trichomoniasis-associated adverse reproductive outcomes and enhanced HIV transmission poses a salient public health threat. Accordingly, TV warrants a higher profile in urban STI clinic settings in developed countries, and we suggest that priority be given to development of standardised molecular TV detection techniques and that these become part of routine STI testing.

Multiplex PCR testing detection of higher-than-expected rates of cervical mycoplasma, ureaplasma, and trichomonas and viral agent infections in sexually active australian women.

Knowing the prevalence of potential etiologic agents of nongonococcal and nonchlamydial cervicitis is important for improving the efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs), designated VDL05, VDL06, VDL07, and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis, including cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A to E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual-health clinic in Sydney, Australia, during 2006 and 2007. The agents detected alone or in combination in all cervical swabs (percentage of total swabs) included CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and group B streptococci (0.4). Adenovirus species A to E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections.

Norovirus excretion in an aged-care setting.

Norovirus genogroup II excretion during an outbreak of gastroenteritis was investigated in an aged-care facility. Viral shedding peaked in the acute stage of illness and continued for an average of 28.7 days. The viral decay rate was 0.76 per day, which corresponds to a viral half-life of 2.5 days.

Epidemics of gastroenteritis during 2006 were associated with the spread of norovirus GII.4 variants 2006a and 2006b.

BACKGROUND: Acute gastroenteritis is commonly associated with norovirus genogroup II (GII) infection. Norovirus GII has 17 classified genotypes (GII.1-GII.17), but only 1 norovirus genotype (GII.4) is associated with global epidemics of gastroenteritis. In 2006, an increase in global norovirus activity was observed. METHODS: During the period from December 2005 through August 2006, a total of 231 fecal samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. Norovirus RNA was amplified and sequenced to determine norovirus genotype and relatedness to known epidemic norovirus GII.4 variants. RESULTS: Two GII.4 variants, designated 2006a and 2006b, were identified in 61.8% and 11.3%, respectively, of the 186 cases investigated. Norovirus 2006a and 2006b have also been implicated as the predominant causes of norovirus-associated gastroenteritis across Europe in 2006. CONCLUSIONS: The global increase in norovirus-associated gastroenteritis in 2006 was linked to the emergence of 2 novel GII.4 variants, 2006a and 2006b.

Lactose malabsorption in the elderly: role of small intestinal bacterial overgrowth.

OBJECTIVE: The prevalence of lactose malabsorption (LM) is increased in the elderly, although the mechanisms responsible are still a matter of speculation. The objective of this study was to investigate the possible roles of reduced functional small intestinal absorptive area, lactase deficiency and small intestinal bacterial overgrowth (SIBO). MATERIAL AND METHODS: Twenty Caucasian (Anglo-Celtic), asymptomatic, well-nourished, elderly volunteers (median age 79 years, range 70-94 years) with no clinically apparent predisposition to SIBO underwent a 50 g lactose breath hydrogen test (LBHT) and mannitol absorption test, the latter as an index of functional small intestinal absorptive area. Those with LM additionally underwent bacteriological assessment of small intestinal secretions and mucosal biopsy, to assess the contribution of SIBO and lactase deficiency, respectively, to the pathogenesis of LM in individual cases. The prevalence of SIBO was also determined in elderly subjects without LM. Twenty asymptomatic younger subjects (median age 29 years, age range 18-35 years) served as controls. All subjects were "hydrogen producers" in response to lactulose. RESULTS: LM was evident in 10/20 (50%) elderly subjects and 1/20 (5%) younger subjects (p=0.003). Mannitol absorption did not differ significantly in elderly and younger subjects or in elderly subjects with and without LM. SIBO was documented in 9/10 (90%) elderly subjects with LM; eradication was associated with resolution of LM. Lactase deficiency was evident in only one elderly subject with LM. SIBO was evident in 2/10 (20%) elderly subjects without LM (p=0.005 compared to those with LM). Lactulose breath hydrogen test identified only 2/11 (18%) elderly subjects with SIBO. CONCLUSIONS: Increased prevalence of LM in the elderly is mostly due to clinically non-apparent SIBO, rather than mucosal factors. The lactulose breath hydrogen test cannot be relied upon to identify elderly subjects with SIBO, even in those without an anatomical predisposition.

Dynamics of appearance and expansion of a prolyliminopeptidase- negative subtype among Neisseria gonorrhoeae isolates collected in Sydney, Australia, from 2002 to 2005.

Recent studies have demonstrated a wide geographic circulation of isolates of Neisseria gonorrhoeae that fail to produce prolyliminopeptidase (PIP). Tests based on the production of this enzyme are important elements of a number of identification systems for gonococci. We documented the appearance, expansion, and contraction of subtypes of 165 PIP-negative gonococci detected in an extended and systematic sample of the 3,926 N. gonorrhoeae isolates collected in Sydney, Australia, from July 2002 to September 2005. Their appearance, peak, and decline followed an "epidemic" curve. At the peak of their prevalence in 2003, PIP-negative gonococci comprised 22% of all isolates. Closely related phenotypes accounted for 162/165 of the PIP-negative gonococci. Algorithms for confirmation of N. gonorrhoeae should take account of the temporal and geographic variability of gonococci by utilizing two or more distinct confirmatory methods.

Genetic diversity of Sapovirus in children, Australia.

Sapovirus was detected in 7 of 95 stool specimens from children with gastroenteritis of unknown etiology in Sydney, Australia, from August 2001 to August 2002 and from February 2004 to August 2004, by using reverse transcription-polymerase chain reaction. Sequence analysis of the N-terminal capsid region showed all human sapovirus genogroups.