Brigid Lucey

Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens.

ABSTRACT The EntericBio system uses a multiplex PCR assay for the simultaneous detection of Campylobacter spp., Salmonella enterica, Shigella spp., and Escherichia coli O157 from feces. It combines overnight broth enrichment with PCR amplification and detection by hybridization. An evaluation of this system was conducted by comparing the results obtained with the system with those obtained by routine culture, supplemented with alternative PCR detection methods. In a study of 773 samples, routine culture and the EntericBio system yielded 94.6 and 92.4% negative results, respectively. Forty-two samples had positive results by culture, and all of these were positive with the EntericBio system. This system detected an additional 17 positive samples (Campylobacter spp., n = 12; Shigella spp., n = 1; E. coli O157, n = 4), but the results for 5 samples (Campylobacter spp., n = 2; Shigella spp., n = 1; E. coli O157, n = 2) could not be confirmed. The target for Shigella spp. detected by the EntericBio system is the ipaH gene, and the molecular indication of the presence of Shigella spp. was investigated by sequence analysis, which confirmed that the ipaH gene was present in a Klebsiella pneumoniae isolate from the patient. The sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 99.3%, 91.5%, and 100%, respectively. Turnaround times were significantly reduced with the EntericBio system, and a result was available between 24 and 32 h after receipt of the sample in the laboratory. In addition, the amount of laboratory waste was significantly reduced by use of this system. In summary, the EntericBio system proved convenient to use, more sensitive than the conventional culture used in this study, and highly specific; and it generated results significantly faster than routine culture for the pathogens tested.

Trends in antimicrobial susceptibility among isolates of Campylobacter species in Ireland and the emergence of resistance to ciprofloxacin

Measurements were made of the susceptibility to six commonly prescribed antibiotics, including erythromycin, tetracycline and ciprofloxacin, of 130 isolates of Campylobacter jejuni and 15 isolates of Campylobacter coli cultured from human and poultry sources during 2000. The results were compared with the results from a collection of strains isolated between 1996 and 1998. The levels of resistance to erythromycin remained low, 2 per cent and 4.4 per cent for the human and poultry isolates, respectively. Resistance to tetracycline had increased to 31 per cent and 24.4 per cent from 13.9 per cent and 18.8 per cent for the human and poultry isolates, respectively. However, the resistance to ciprofloxacin of the strains isolated during 2000 had increased to 30 per cent, whereas between 1996 and 1998 there had been no resistance to this agent among human isolates, and only 3-1 per cent resistance among poultry isolates. The molecular basis for this resistance has been shown to be the result of a single amino acid substitution, Thr-86-lle, in the gyrA subunit of DNA gyrase in C jejuni. A subset of 59 isolates was tested by molecular methods and all of the 25 phenotypically resistant isolates possessed this substitution. None of the human isolates had been treated with ciprofloxacin before their laboratory isolation.

Molecular-based detection of non-culturable and emerging campylobacteria in patients presenting with gastroenteritis.

From January 2009 to May 2010, 436 faecal samples from patients with diarrhoeal illness in Southern Ireland were identified as Campylobacter genus-positive by an automated multiplex PCR; however, 204 (46·8%) of these samples were culture-negative for campylobacters. A combination of Campylobacter-specific uniplex PCR and 16S rRNA sequencing confirmed the presence of Campylobacter DNA in 191 (93·6%) of the culture-negative samples. Species-specific PCR identified C. jejuni (50·7%) C. ureolyticus (41%) and C. coli (5·7%) as the most prevalent species while C. fetus, C. upsaliensis, C. hyointestinalis and C. lari accounted for 10% of culture-negative samples; mixed Campylobacter spp. were detected in 11% of samples. We conclude that non-culturable Campylobacter spp. are responsible for a considerable proportion of human enteritis and the true incidence of infection is likely to be significantly underestimated where conventional Campylobacter culture methods are used in isolation.

Emerging dynamics of human campylobacteriosis in Southern Ireland

Infections with Campylobacter spp. pose a significant health burden worldwide. The significance of Campylobacter jejuni/Campylobacter coli infection is well appreciated but the contribution of non-C. jejuni/C. coli spp. to human gastroenteritis is largely unknown. In this study, we employed a two-tiered molecular study on 7194 patient faecal samples received by the Microbiology Department in Cork University Hospital during 2009. The first step, using EntericBio(®) (Serosep), a multiplex PCR system, detected Campylobacter to the genus level. The second step, utilizing Campylobacter species-specific PCR identified to the species level. A total of 340 samples were confirmed as Campylobacter genus positive, 329 of which were identified to species level with 33 samples containing mixed Campylobacter infections. Campylobacter jejuni, present in 72.4% of samples, was the most common species detected, however, 27.4% of patient samples contained non-C. jejuni/C. coli spp.; Campylobacter fetus (2.4%), Campylobacter upsaliensis (1.2%), Campylobacter hyointestinalis (1.5%), Campylobacter lari (0.6%) and an emerging species, Campylobacter ureolyticus (24.4%). We report a prominent seasonal distribution for campylobacteriosis (Spring), with C. ureolyticus (March) preceeding slightly C. jejuni/C. coli (April/May).

Campylobacter ureolyticus: an emerging gastrointestinal pathogen?

A total of 7194 faecal samples collected over a 1-year period from patients presenting with diarrhoea were screened for Campylobacter spp. using EntericBio(®) , a multiplex-PCR system. Of 349 Campylobacter-positive samples, 23.8% were shown to be Campylobacter ureolyticus, using a combination of 16S rRNA gene analysis and highly specific primers targeting the HSP60 gene of this organism. This is, to the best of our knowledge, the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis and may suggest a role for this organism as an emerging enteric pathogen.

Molecular diagnostics: the changing culture of medical microbiology.

Diagnostic molecular biology is arguably the fastest growing area in current laboratory-based medicine. Growth of the so called ‘omics’ technologies has, over the last decade, led to a gradual migration away from the ‘one test, one pathogen’ paradigm, toward multiplex approaches to infectious disease diagnosis, which have led to significant improvements in clinical diagnostics and ultimately improved patient care.

Comparison of three stool antigen assays with the 13C- urea breath test for the primary diagnosis of Helicobacter pylori infection and monitoring treatment outcome.

Background The urea breath test (UBT) is the gold-standard non-invasive test for the detection of Helicobacter pylori infection, however, the lack of availability of the UBT due to the high cost of the test, and in particular the need for expensive analytical instrumentation, limits the usefulness of this method. Stool antigen assays may offer an alternative non-invasive method for the diagnosis of infection. Objective To compare the accuracy of three stool antigen assays (HpSA, IDEIA HpStAR, and ImmunoCard STAT) against the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome. Methods A total of 102 patients attending two gastroenterology day-case clinics for the investigation of dyspepsia were included. Each patient provided breath and stool samples for analysis. Patients who tested positive for H. pylori by the validated UBT were prescribed triple therapy and invited to return for repeat breath and stool sample analysis 6 weeks post-treatment. Results Of the 102 patients tested, 48 were diagnosed with H. pylori infection by the UBT. The HpSA assay interpreted 38 of these as positive (79% sensitive). Of the 54 UBT-negative patients the HpSA assay interpreted all 54 as negative (100% specific). The IDEIA HpStAR assay correctly identified 44 patients as positive (92% sensitive) and 50 as negative (92.5% specific). The ImmunoCard STAT assay interpreted 38 patients as positive (79% sensitive) and 52 as negative (96.3% specific). Conclusion The findings indicate that the IDEIA HpStAR stool antigen kit is the most accurate assay of the three assays evaluated, and possibly represents a viable alternative to the UBT for the primary diagnosis of H. pylori infection and for monitoring treatment outcome.

Emergence of MRSA clone ST22 in healthy young adults in the community in the absence of risk factors.

One thousand adults aged between 18 and 35 years were investigated for nasal colonization with community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Each volunteer completed a questionnaire to assess the presence or absence of risk factors for hospital-acquired MRSA (HA-MRSA) carriage. All MRSA isolated were characterized by microbiological and molecular methods. A S. aureus carriage rate of 22% and a MRSA carriage rate of 0.7% were observed. Analysis of the questionnaires revealed 121 individuals with HA-MRSA risk factors. Subsequently two MRSA infections with associated risk factors were excluded from calculation of the true carriage rate and an adjusted rate of 0.57% (5/879) was established. All seven MRSA isolates expressed the genotypic profile ST22-MRSA-IV, were PVL negative, agr type 1, and differed only by their antimicrobial susceptibility patterns. ST22-MRSA-IV (EMRSA-15) has shown worldwide spread in the hospital setting but has not been previously documented in isolation in the community.

Improved Detection of Bacterial Pathogens in Patients Presenting with Gastroenteritis by Use of the EntericBio Real-Time Gastro Panel I Assay

ABSTRACT In this study, we evaluated the use of EntericBio real-time Gastro Panel I (Serosep, Limerick, Ireland) for routine use in a clinical microbiology laboratory for simultaneous detection of Campylobacter jejuni, coli, and lari, Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Shigella spp. in feces. This system differs from its predecessor (the EntericBio Panel II system, Serosep) in that it allows real-time detection of pathogens directly from feces, without pre-enrichment. It also specifically detects Campylobacter jejuni, coli, and lari rather than all Campylobacter species, as is the case with the previous system. A total of 528 samples from patients presenting with acute gastroenteritis were screened prospectively with this assay, and results were compared with those of the current method, which combines screening the samples with a molecular assay (the EntericBio Panel II assay) and retrospective culture of the specimens in which the target was detected. Discrepancy analysis was conducted using culture and molecular methods. The real-time assay produced 84 positive results, specifically, Campylobacter spp. (n = 44); Stx1 and/or Stx2 (n = 35); Shigella spp. (n = 3); and Salmonella spp. (n = 6). Of these, 4 samples represented coinfections with Campylobacter spp. and STEC. The real-time assay showed an increased detection rate for pathogens, apart from Salmonella spp. Four Campylobacter-positive and 6 Stx-positive results remained unconfirmed by any other method used. The isolation rates for PCR-positive samples were as follows: Campylobacter spp., 80%; STEC, 45.7%; Salmonella spp., 100%; and Shigella spp., 66.7%. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were 100%, 97.8%, 88.1%, 100%, and 98.1%, respectively.

Genomic investigation into strain heterogeneity and pathogenic potential of the emerging gastrointestinal pathogen Campylobacter ureolyticus.

The recent detection and isolation of C. ureolyticus from patients with diarrhoeal illness and inflammatory bowel diseases warrants further investigation into its role as an emerging pathogen of the human gastrointestinal tract. Regarding the pathogenic mechanisms employed by this species we provide the first whole genome analysis of two C. ureolyticus isolates including the type strain. Comparative analysis, subtractive hybridisation and gene ontology searches against other Campylobacter species identifies the high degree of heterogenicity between C. ureolyticus isolates, in addition to the identification of 106 putative virulence associated factors, 52 of which are predicted to be secreted. Such factors encompass each of the known virulence tactics of pathogenic Campylobacter spp. including adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (ciaB and 16 virB-virD4 genes) and toxin production (S-layer RTX and ZOT). Herein, we provide the first virulence catalogue for C. ureolyticus, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology.