The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

Simultaneous co-detection of wild-type and vaccine strain measles virus using the BD MAX system.

Despite the reported elimination of measles virus in Australia, importation of cases from endemic countries continues to lead to secondary local transmission and outbreaks. Rapid laboratory confirmation of measles is paramount for individual patient management and outbreak responses. Further, it is important to rapidly distinguish infection from wild-type virus or vaccine strains to guide public health responses. We developed a high throughput, TaqMan-based multiplex reverse-transcription-polymerase chain reaction (PCR) assay using the BD MAX platform (Becton Dickinson) that simultaneously detects measles virus and differentiates between wild-type and vaccine strains without the need for sequencing.

Relentless spread and adaptation of non-typeable vanA vancomycin-resistant Enterococcus faecium: a genome-wide investigation.

Background: VRE are prevalent among patients in ICUs. Non-typeable vanA VRE, due to loss of one of the genes used for MLST (pstS), have increased in Australia, suggestive of a new, hospital-acquired lineage. Objectives: To understand the significance of this lineage and its transmission using WGS of strains isolated from patients in ICUs across New South Wales, Australia. Methods: A total of 240 Enterococcus faecium isolates collected between February and May 2016, and identified by conventional PCR as vanA positive, were sequenced. Isolates originated from 12 ICUs in New South Wales, grouped according to six local health districts, and represented both rectal screening swab (n = 229) and clinical (n = 11) isolates. Results: ST analysis revealed the absence of the pstS gene in 84.2% (202 of 240) of vanA isolates. Two different non-typeable STs were present based on different allelic backbone patterns. Loss of the pstS gene appeared to be the result of multiple recombination events across this region. Evidence for pstS-negative lineage spread across all six local health districts was observed suggestive of inter-hospital transmission. In addition, multiple outbreaks were detected, some of which were protracted and lasted for the duration of the study. Conclusions: These findings confirmed the evolution, emergence and dissemination of non-typeable vanA E. faecium. This study has highlighted the utility of WGS when attempting to describe accurately the hospital-based pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism.

Locally Acquired mcr-1 in Escherichia coli, Australia, 2011 and 2013.

We identified discrete importation events of the mcr-1 gene on incompatibility group IncI2 plasmids in Escherichia coli isolated from patients in New South Wales, Australia, in 2011 and 2013. mcr-1 is present in a small minority of colistin-resistant Enterobacteriaceae and appears not to be established locally.

Rapid Detection of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible S. aureus Directly from Positive Blood Cultures by Use of the BD Max StaphSR Assay.

The BD Max StaphSR assay is an automated qualitative in vitro diagnostic test for the direct detection and differentiation of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). A total of 460 specimens were tested, and the results were compared with standard culture-based identification. MRSA was detected in 48 samples (sensitivity of 100%; positive predictive value [PPV] of 100%). MSSA was detected in 112 samples (sensitivity of 99.1%; PPV of 100%), and 299 samples containing coagulase-negative staphylococcus and nonstaphylococcal species were negative by the BD Max StaphSR assay (specificity of 100%; negative predictive value [NPV] of 99.7 to 100%).

Distribution of acquired AmpC ß-lactamase genes in Sydney, Australia.

Investigation of plasmid-borne AmpC ß-lactamase genes in Escherichia coli and Klebsiella spp. revealed blaCMY-2-like genes predominantly in E. coli and blaDHA genes equally distributed between both species. This distribution remained stable over time, but blaACT/MIR-like genes, initially common in Klebsiella spp., were not identified in more recent isolates.

Emergence of blaKPC carbapenemase genes in Australia.

blaKPC genes encoding resistance to carbapenems are increasingly widely reported and are now endemic in parts of several countries, but only one Klebsiella pneumoniae isolate carrying blaKPC-2 had previously been reported in Australia, in 2010. Here we characterised this isolate, six additional K. pneumoniae and one Escherichia coli carrying blaKPC and another K. pneumoniae lacking blaKPC, all isolated in Australia in 2012. Seven K. pneumoniae belonged to clonal complex (CC) 292, associated with blaKPC in several countries. Five with blaKPC-2 plus the isolate lacking a blaKPC gene were sequence type 258 (ST258) and the seventh was the closely related ST512 with blaKPC-3. The eighth K. pneumoniae isolate, novel ST1048, and the E. coli (ST131) also carried blaKPC-2. blaKPC genes were associated with the most common Tn4401a variant, which gives the highest levels of expression, in all isolates. The ST258 isolates appeared to share a similar set of plasmids, with IncFIIK, IncX3 and ColE-type plasmids identified in most isolates. All K. pneumoniae isolates had a characteristic insertion in the ompK35 gene resulting in a frameshift and early termination, but only the ST512 isolate had a GlyAsp insertion in loop 3 of OmpK36 that may contribute to increased resistance. The clinical epidemiology of blaKPC emergence in Australia thus appears to reflect the global dominance of K. pneumoniae CC292 (and perhaps E. coli ST131). Some, but not all, patients carrying these isolates had previously been hospitalised outside Australia, suggesting multiple discrete importation events of closely related strains, as well as undetected nosocomial spread.

Complete sequence of pJIE143, a pir-type plasmid carrying ISEcp1-blaCTX-M-15 from an Escherichia coli ST131 isolate.

pJIE143 (34 kb), from an Escherichia coli ST131 isolate, carries bla(CTX-M-15) but could not be typed using the standard PCR-based replicon-typing primer set. Complete sequencing revealed a backbone with similarity to IncX plasmids, including a pir-like gene encoding a π-like replication protein and iterons related to those of other IncX plasmids. The 2.971-kb ISEcp1-bla(CTX-M-15)-orf477Δ transposition unit often found within Tn2 is inserted just beyond the end of pir, flanked by 5-bp direct repeats.

Efficient direct extended-spectrum ß-lactamase detection by multiplex real-time PCR: accurate assignment of phenotype by use of a limited set of genetic markers.

The number and diversity of genes potentially complicate genetic approaches to the rapid detection of transmissible extended-spectrum ß-lactamase genes. We developed a robust multiplexed real-time PCR assay based on targets identified in a prior survey and used this to detect relevant genes in 617 consecutive clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae.