We evaluated toxigenic Clostridium difficile detection by a lateral flow assay for antigen and toxin, an enzyme immunoassay, and two commercial PCR methods. Compared to the cell cytotoxicity neutralization assay and toxigenic culture, both toxin detection methods lacked sensitivity. PCR following combined antigen and toxin detection provided the most useful diagnostic information.

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Introduction The nuc gene encodes a thermonuclease which is present in Staphylococcus aureus but not in coagulase-negative staphylococci (CoNS) and is the target of the rapid phenotypic thermonuclease test. The effect of nuc gene variation in methicillin-resistant S. aureus (MRSA) on the performance of PCR testing has been noted, although there are no reports about the effect of MRSA on the activity of the thermonuclease enzyme. Aim Our goals were to examine the sensitivity and specificity of the thermonuclease test used to distinguish S. aureus from CoNS cultured from blood. In addition, we aimed to assess differences in the sensitivity, specificity and accuracy of the thermonuclease test between methicillin-sensitive S. aureus (MSSA) and MRSA isolates. Methodology We performed a retrospective analysis of 1404 isolates. Each isolate from a positive blood culture was identified as a Gram-positive coccus by microscopy then analysed with the thermonuclease test (Southern Group Laboratory) prior to confirmatory identification using VITEK microbial identification platforms (bioMérieux) and cefoxitin disc diffusion testing. Results Of 1331 samples included in the final analysis, 189 were thermonuclease-positive, of which 176 were identified as S. aureus . Of the 1142 thermonuclease-negative samples, 13 were finally identified as S. aureus , giving a sensitivity of 93.1 % (95 % confidence interval [CI] 88.5–96.3) and specificity of 98.9 % (95 % CI 98.1–99.4). Of the nine proven MRSA samples, eight were thermonuclease-positive, giving a sensitivity of 88.9 % (95 % CI 51.8–99.7). Thermonuclease test accuracy for MSSA and MRSA isolates was 98.1 % (95 % CI 97.2–98.8) versus 98.8 % (95 % CI 98.0–99.3), respectively. Conclusions In the era of increasing use of molecular-based microbiology assays, the thermonuclease test remains a simple, inexpensive and robust test for the presumptive identification of S. aureus cultured from blood, irrespective of methicillin sensitivity.

In some neonatal units, the screening of isolates for antimicrobial-resistant organisms is a matter of routine, with theoretical benefits including the prevention or early detection of outbreaks. This study sought to use whole-genome sequencing (WGS) retrospectively to characterize the genomic epidemiology of Gram-negative organisms obtained from a screening programme in a 32-bed unit providing intensive, high-dependency and special care at City Hospital, Birmingham, UK, identifying occult transmission events and clinically important antimicrobial-resistance (AMR) genes. WGS was performed for 155 isolates collected from rectal and umbilical screening swabs over a 2 month period from 44 individual neonates. Genomic epidemiological analysis showed possible transmission events involving Escherichia coli , Enterobacter cloacae , Klebsiella oxytoca and Klebsiella pneumoniae not detected by routine screening, with eight putative clusters involving different individuals identified. Within phylogenetic clusters, the relatedness of organisms – as determined by the abundance of SNPs – varied widely, indicating that a variety of transmission routes may be implicated. While clinically important AMR genes were not present in the putative transmission clusters, our observation of suspected interspecies horizontal transfer of bla CTX-M-15 within individuals highlights the potential for their spread between organisms as well as individuals in this environment, with implications for surveillance. Our data show that WGS may reveal occult Gram-negative transmission events, demonstrating the potential of sequencing-based surveillance systems for nosocomial pathogens. Challenges remain in understanding how to utilize WGS surveillance to maximum effect in real-world settings.