Daniel Corcoran

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.

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.

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.

Development of nalidixic acid amphotericin B vancomycin (NAV) medium for the isolation of Campylobacter ureolyticus from the stools of patients presenting with acute gastroenteritis.

Abstract Recently, Campylobacter ureolyticus has been detected for the first time in the faeces of patients with acute gastroenteritis using polymerase chain reaction (PCR) techniques. Cultural isolation of C. ureolyticusis is not possible using the established selective methods for the isolation of thermophilic Campylobacter spp. from faeces. The aim of the current study is to develop a new selective medium capable of isolating C. ureolyticus from faecal samples. The newly-developed medium consists of Anaerobe Basal Agar with 10 g/L additional agar, 2 g/L sodium formate and 3 g/L sodium fumarate dibasic, to which 10 mg/L nalidixic acid, 10 mg/L amphotericin B and 20 mg/L vancomycin (NAV) are added as selective agents. Validation studies have shown that this experimental selective medium completely inhibits growth of Candida spp. and of Enterococcus spp. and permits reduced growth of selected coliforms and Proteus spp. Growth of Campylobacter ureolyticus on NAV medium is optimal in anaerobic and enriched hydrogen atmospheres. Additionally, an overnight enrichment step using Bolton broth to which 2 g/L sodium formate, 3 g/L sodium fumarate dibasic and the NAV supplement are added, in place of the commercial Bolton broth supplement, allows improved recovery of C. ureolyticus from patients’ faeces.

Validation of a norovirus multiplex real-time RT-PCR assay for the detection of norovirus GI and GII from faeces samples.

Abstract Norovirus is a leading cause of infectious non-bacterial gastroenteritis. The virus is highly contagious and has multiple modes of transmission, presenting a growing challenge to hospital-based healthcare. In this study, a total of 120 stool samples are tested for the presence of norovirus GI and GII by the Roche two-step Lightcycler 2.0 assay incorporating primers and probes produced by TIB Molbiol, and the results are compared with results from the National Virus Reference Laboratory. The Roche/TIB Molbiol assay produced 51 positive results and 69 negative results. Discrepancy analysis was performed for six conflicting results using a second real-time polymerase chain reaction (PCR) assay (Roche/TIB Molbiol) and this confirmed that four of the five discrepant positive results were true positives. A single discrepant negative result generated by the Roche assay remained negative using the second assay. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 98%, 98.6%, 98.0% and 98.6 %, respectively. Melting curve analysis was used to differentiate genogroups I and II and this showed that 92% of strains belonged to genogroup II.

Improved Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)-Based Identification of Mycobacterium spp. by Use of a Novel Two-Step Cell Disruption Preparatory Technique

The use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for microbial identification in clinical microbiology in recent years. This development can be attributed to its low operating cost and to its speed and reliability of

Epsilonproteobacteria in humans, New Zealand.

To the Editor: Cornelius et al. (1) addressed the potential of Campylobacter ureolyticus as an emerging pathogen by conducting a molecular study on 128 diarrheal specimens and 49 fecal samples from healthy volunteers. Reporting the identification of C. ureolyticus in 12 (24.5%) of 49 healthy volunteers, a number that they compared with our finding of 349 (23.8%) from Campylobacter spp.–positive samples (2), the authors concluded that C. ureolyticus species “are unlikely causes of diarrhea,” an assertion with which we take issue. This interpretation does not take into account that our screening involved 7,194 symptomatic patients: a sample size 40× greater than that of Cornelius et al. In this context, the likely carriage rate for C. ureolyticus is 1.15%. Also, our assay, which has a limit of detection in the picomolar range, is likely comparable with, if not greater than, that of Cornelius et al. (1). Accounting for variations in geographic location and detection methods, a detection rate of 24.5% in healthy volunteers (overall detection rate 14.7%) is high in contrast to our reported rate of 1.15%. One possible explanation for this discrepancy is that Cornelius et al. “did not specifically exclude volunteers who had had gastrointestinal disturbances in the 10 days before sampling,” Campylobacter can be shed in feces for <4 weeks after infection. Also, Cornelius et al. (1) noted the possibility of “genetically distinct but phenotypically indistinguishable genomospecies differing in their pathogenic potential” to account for the presence of the emerging pathogen C. concisus in healthy volunteers and patients with diarrheal illness. This may also apply for C. ureolyticus. We reported a strong seasonal prevalence of C. ureolytcius and a bimodal age distribution (2). The lack of any related details from Cornelius et al. may undermine their reported detection rates. These factors strongly suggest that the statement, “these species are unlikely causes of diarrhea,” should, at the very least, be taken under advisement.

Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae

Objectives To improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously. Methods PCR primers were designed to detect two N gonorrhoeae genes, namely porA and pgi1. Primers for an internal control targeting the ompW gene of Vibrio cholerae were also designed and incorporated in the assay. The DNA of 45 clinical isolates including 33 N gonorrhoeae isolates, seven non-gonococcal Neisseria species, and five non-Neisseria species was tested using the multiplex PCR assay. Results All 33 N gonorrhoeae isolates were successfully detected by the assay and none of the non-gonococcal isolates was detected. The assay showed a sensitivity and specificity of 100%, and a limit of detection of 1.25 ng of DNA. Conclusion This multiplex PCR assay offers a sensitive and specific assay suitable for the detection of N gonorrhoeae, and offers real potential for diagnostic use.