Molecular versus culture-based testing for gastrointestinal infection.

PURPOSE OF REVIEW: Molecular-based diagnostic methods for the detection of gastrointestinal pathogens are becoming increasingly commonplace in microbiology laboratories. This review aims to summarize recent developments in this field and discuss the clinical application and limitations of implementing these techniques. RECENT FINDINGS: Recent evaluations of multiplex PCR assays show increased sensitivity whenever compared with standard microbiological culture-based methods. In addition to shorter turnaround times, assays can detect an increased repertoire of pathogens from a single specimen and provide useful information for infection prevention and control practices. There are many limitations, however, associated with their use, including clinical interpretation of results and lack of concordance between different test panels. Newer technologies, such as metagenomic analysis, can provide comprehensive information useful to both patient management and public health surveillance. SUMMARY: Molecular techniques are capable of replacing culture in the diagnosis of gastrointestinal infections. Whether all positive results, however, represent true infection is still debateable, as is the clinical significance of identifying more than one pathogen. As it currently stands, microbiological culture remains vital for public health surveillance, monitoring antibiotic resistance and managing outbreaks.

Detection of macrolide resistant Mycoplasma pneumoniae in England, September 2014 to September 2015.

Mycoplasma pneumoniae infection can cause pneumonia, particularly in children. Global increase in macrolide-resistant M. pneumoniae is of concern due to limited therapeutic options. We describe the detection of macrolide resistance-conferring mutations in 9.3% of 43 clinical specimens where M. pneumoniae was detected in England and Wales from September 2014‒September 2015. This study aims to impact by highlighting the presence of macrolide resistance in M. pneumoniae positive patients, promoting increased clinical vigilance.

Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study.

BACKGROUND: Every year approximately 5000-9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are 'blocked' by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients. OBJECTIVE: To evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens: Clostridium difficile, Campylobacter spp., Salmonella spp. and norovirus. DESIGN: A retrospective study of fixed numbers of samples positive for C. difficile (n = 200), Campylobacter spp. (n = 200), Salmonella spp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out. SETTING: MassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts. MAIN OUTCOME MEASURES: Sensitivity and specificity to detect C. difficile, Campylobacter spp., Salmonella spp., and norovirus. RESULTS: Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200 Campylobacter spp., 199 C. difficile, 60 S. enterica, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the MassCode assay, sensitivities for each organism compared with standard microbiological testing ranged from 43% to 94% and specificities from 95% to 98%, with particularly poor performance for S. enterica. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly for S. enterica, Giardia lamblia and Cryptosporidium spp. As the results indicated that S. enterica detection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag(®) gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). For Campylobacter spp., C. difficile and norovirus, high sensitivities (> 92%) and specificities (> 96%) were observed. For S. enterica, on the original MassCode/Oxford extracts, Luminex sensitivity compared with standard microbiological testing was 84% [95% confidence interval (CI) 73% to 93%], but this dropped to 46% on a fresh extract, very similar to MassCode, with a corresponding increase in specificity from 92% to 99%. Overall agreement on the per-sample diagnosis compared with combined microbiology plus PCR for the main four/all pathogens was 85.6%/64.7%, 87.0%/82.9% and 89.8%/86.8% for the MassCode assay, Luminex assay/MassCode extract and Luminex assay/fresh extract, respectively. Luminex assay results from fresh extracts implied that 5% of samples did not represent infectious diarrhoea, even though enteropathogens were genuinely present. Managing infectious diarrhoea was a significant burden for infection control teams (taking 21% of their time) and better diagnostics were identified as having major potential benefits for patients. CONCLUSIONS: Overall, the Luminex xTag gastrointestinal panel showed similar or superior sensitivity and specificity to the MassCode assay. However, on fresh extracts, this test had low sensitivity to detect a key enteric pathogen, S. enterica; making it an unrealistic option for most microbiology laboratories. Extraction efficiency appears to be a major obstacle for nucleic acid-based tests for this organism, and possibly the whole Enterobacteriaceae family. To improve workflows in service microbiology laboratories, to reduce workload for infection control practitioners, and to improve outcomes for NHS patients, further research on deoxyribonucleic acid-based multiplex gastrointestinal diagnostics is urgently needed. FUNDING: The Health Technology Assessment programme of the National Institute for Health Research.

IS3 profiling identifies the enterohaemorrhagic Escherichia coli O-island 62 in a distinct enteroaggregative E. coli lineage.

BACKGROUND: Enteroaggregative Escherichia coli (EAEC) are important diarrhoeal pathogens that are defined by a HEp-2 adherence assay performed in specialist laboratories. Multilocus sequence typing (MLST) has revealed that aggregative adherence is convergent, providing an explanation for why not all EAEC hybridize with the plasmid-derived probe for this category, designated CVD432. Some EAEC lineages are globally disseminated or more closely associated with disease. RESULTS: To identify genetic loci conserved within significant EAEC lineages, but absent from non-EAEC, IS3-based PCR profiles were generated for 22 well-characterised EAEC strains. Six bands that were conserved among, or missing from, specific EAEC lineages were cloned and sequenced. One band corresponded to the aggR gene, a plasmid-encoded regulator that has been used as a diagnostic target but predominantly detects EAEC bearing the plasmid already marked by CVD432. The sequence from a second band was homologous to an open-reading frame within the cryptic enterohaemorrhagic E. coli (EHEC) O157 genomic island, designated O-island 62. Screening of an additional 46 EAEC strains revealed that the EHEC O-island 62 was only present in those EAEC strains belonging to the ECOR phylogenetic group D, largely comprised of sequence type (ST) complexes 31, 38 and 394. CONCLUSIONS: The EAEC 042 gene orf1600, which lies within the EAEC equivalent of O-island 62 island, can be used as a marker for EAEC strains belonging to the ECOR phylogenetic group D. The discovery of EHEC O-island 62 in EAEC validates the genetic profiling approach for identifying conserved loci among phylogenetically related strains.

The commonly-used DNA probe for diffusely-adherent Escherichia coli cross-reacts with a subset of enteroaggregative E. coli.

BACKGROUND: The roles of diffusely-adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) in disease are not well understood, in part because of the limitations of diagnostic tests for each of these categories of diarrhoea-causing E. coli. A HEp-2 adherence assay is the Gold Standard for detecting both EAEC and DAEC but DNA probes with limited sensitivity are also employed. RESULTS: We demonstrate that the daaC probe, conventionally used to detect DAEC, cross-reacts with a subset of strains belonging to the EAEC category. The cross hybridization is due to 84% identity, at the nucleotide level, between the daaC locus and the aggregative adherence fimbriae II cluster gene, aafC, present in some EAEC strains. Because aaf-positive EAEC show a better association with diarrhoea than other EAEC, this specific cross-hybridization may have contributed to an over-estimation of the association of daaC with disease in some studies. We have developed a discriminatory PCR-RFLP protocol to delineate EAEC strains detected by the daaC probe in molecular epidemiological studies. CONCLUSIONS: A PCR-RFLP protocol described herein can be used to identify aaf-positive EAEC and daaC-positive DAEC and to delineate these two types of diarrhoeagenic E. coli, which both react with the daaC probe. This should help to improve current understanding and future investigations of DAEC and EAEC epidemiology.

Error-prone DNA repair system in enteroaggregative Escherichia coli identified by subtractive hybridization.

Enteroaggregative Escherichia coli (EAEC) are etiologic agents of diarrhea. The EAEC category is heterogeneous, but most in-depth experimentation has focused on prototypical strain, 042. We hypothesized that 60A, another EAEC strain, might posses virulence or fitness genes that 042 does not have. Through subtractive hybridization we identified 60A-specific sequences, including loci present in other E. coli and phage DNA. One locus thus identified was impB, a LexA repressed error-prone DNA repair gene that has been identified in plasmids from other enteric organisms and which we detected in 21 of 34 EAEC strains. An isogenic 60A impB mutant showed decreased survival and mutagenesis after exposure to UV, as well as bile salt exposure, compared to the wild-type strain, and these phenotypes could be complemented in trans. The EAEC strain 60A imp operon differs structurally from previously described homologs. A cryptic gene, impC, present in other imp operons, is absent from 60A. In addition, transcription of impAB in strain 60A occurs from a promoter that is dissimilar to the previously described impC promoter but is still triggered by UV-mediated damage. In strain 60A the impAB and the aggregative adherence fimbriae I (AAF/I)-encoding genes are on the same large plasmid, and the 60A version of the operon is predominantly seen in AAF/I-positive EAEC. Supplementary imp SOS-inducible error-prone repair systems are common among EAEC even though they are absent in prototypical strain 042.

Commensal bacteria do translocate across the intestinal barrier in surgical patients.

BACKGROUND: The "gut origin of sepsis" hypothesis proposes that enteric bacteria may cause sepsis at distant extra-intestinal sites. Whilst there is much circumstantial evidence to support this hypothesis, there is no conclusive proof in humans. The nature of translocating bacteria remains unclear. The aim of this study was to establish the origin of Escherichia coli (E. coli) cultured from mesenteric lymph nodes (MLN) and determine if they belonged to any recognized pathotypes known to cause infections in humans. METHODS: MLN and faecal samples were obtained from 98 patients undergoing colonic resection. E. coli were isolated from 9/98 MLN samples. DNA fingerprints of MLN isolates were compared with faecal isolates from the same patient. MLN isolates were tested for adherence and invasion using HEp-2 epithelial cells, and screened for DNA markers indicative of different pathotypes of E. coli. MLN isolates were also tested for internalisation into Caco-2 cells. RESULTS: All the nine E. coli cultured from MLNs were found to have identical DNA fingerprints to at least one and often several E. coli isolates cultured from faecal samples of the same patient. 8/9 (89%) MLN isolates were weakly adherent and 2/9 (22.2%) were invasive. 8/9 (89%) tested negative for DNA markers. All the nine MLN strains were internalised by Caco-2 cells. CONCLUSION: This study confirms the gut origin of translocating bacteria. Most translocating E. coli do not belong to any recognised pathotype and are therefore normal commensal microflora. Our results suggest that bacterial translocation is more dependent upon the gut epithelium rather than the virulence properties of resident enteric bacteria.