B.C. Millar

PCR-IMS detection and molecular typing of Cryptosporidium parvum recovered from a recreational river source and an associated mussel (Mytilus edulis) bed in Northern Ireland

PCR-IMS was used to detect Cryptosporidium spp. in environmental water samples in Northern Ireland which had previously tested negative by a conventional IFA staining method. Oocysts of C. parvum detected in river water and final treated sewage effluent collected from various sites along the river Lagan were identified as genotype 2 (animal origin) based on polymorphisms observed at the thrombospondin related adhesion protein gene locus. Similarly, genotype 1 (human origin) oocysts of C. parvum were detected in the marine filter feeder mussel, Mytilus edulis, collected from the shores of Belfast Lough. Detection of the human genotype of Cryptosporidium in mussels destined for human consumption identifies the organisms serious potential as a foodborne pathogen. This work highlights the possible value of monitoring filter feeder systems, in conjunction with specific molecular epidemiological tools, as an alternative monitoring system for the parasite within the aquatic environment.

Prevalence of bacterial faecal pathogens in separated and unseparated stored pig slurry

To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm.

Genotypic subgrouping of clinical isolates of Candida albicans and Candida dubliniensis by 25S intron analysis

Aims: To determine the frequency, distribution and association of genotypes of Candida albicans and C. dubliniensis in invasive and noninvasive clinical isolates.

Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern Ireland

Aims: To investigate the incidence and genotype of Cryptosporidium parvum oocysts in drinking water sources in Northern Ireland for the period 1996–1999, and to compare conventional and molecular methods of detection.

A rapid molecular assay for the detection of antibiotic resistance determinants in causal agents of infective endocarditis

Aims: To develop and employ a PCR amplification system, directly from clinical specimens, for the rapid molecular detection of common antimicrobial resistance genes for streptococci, staphylococci and enterococci organisms causing infective endocarditis (IE).

Molecular genotyping of human cryptosporidiosis in Northern Ireland: epidemiological aspects and review

BackgroundCryptosporidium parvum is the most common of the protozoal pathogens associated with gastrointestinal disease in Northern Ireland. Genotyping techniques are valuable in helping to elucidate sources and modes of transmission of this parasite. There have been no reports on the prevalence of genotypes in Northern Ireland, mainly due to a lack of discriminatory genotyping techniques, which recently have become available.AimTo investigate the genotype ofC. parvum oocysts isolated from human faeces in sporadic cases of cryptosporidiosis in Northern Ireland.MethodsThirty-nine isolates ofC. parvum, representing 79.6% of the total 1998 laboratory reports for the Eastern Health and Social Services Board, were investigated. Following DNA extraction from oocysts the thrombospondinrelated adhesive protein 2 (TRAP-C2) locus was amplified by polymerase chain reaction (PCR) and subsequently sequenced.ResultsThe majority of isolates (87.2%) were classified as bovine genotype II with the remainder (12.8%) being the human genotype I.ConclusionsThere is a high prevalence of the bovine genotype II parasite in sporadic cases around the greater Belfast area. Epidemiologically, this suggests that the most frequent mode of transmission may be from animals to humans, but does not suggest a high proportion of human to human spread.

Determination of verocytotoxin and eae gene loci by multiplex PCR in Escherichia coli O157:H7 isolated from human faeces in Northern Ireland: a four-year study of trends, 1997-2000.

Abstract This study aims to determine the distribution and frequency of verocytotoxin genes in human faecal clinical isolates of Escherichia coli O157 in Northern Ireland during the period 1997–2000, using a special four-target multiplex polymerase chain reaction (PCR) assay. One hundred and thirty two isolates of E. coli O157:H7 cultured during the four-year period (1997 [n=28]; 1998 [n=25]); 1999 (n=43); 2000 [n=36]), representing approximately 79% of total E. coli O157 laboratory isolations throughout N. Ireland, are examined for the presence of verocytotoxin gene loci (VT1, VT2 and eae) using a multiplex PCR assay. These isolates originate from the four Regional Area Health Boards that constitute the healthcare system in N. Ireland as follows: Eastern (53.8%; n=71), Northern (34.1%; n=45), Western (6.8%; n=9) and Southern (5.3%; n=7). Results showed that over 80% of these isolates possessed the VT2 and eae gene loci, with the remainder being predominantly VT1-, VT2 and eae-positive. None possessed the VT1 gene locus alone. Development and adoption of this simple four-target (three virulence and one control gene loci) multiplex PCR assay and subsequent recording of resulting verocytotoxin-typing data in a database, permitted local, rapid determination of carriage of known molecular virulence determinants of E. coli O157 isolates, which may aid in outbreak-related epidemiological investigations or other longitudinal studies.

Atypical mycobacterial infection in patients with cystic fibrosis: update on clinical microbiology methods

While patients with cystic fibrosis (CF) have had dramatic improvement in their survival rates, this has been accompanied by the emergence of more virulent pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex organisms. In addition, there has been emergence of organisms of increasing clinical significance such as the nontuberculous mycobacterial (NTM). Although TB infection in patients with CF is extremely uncommon, there is growing concern with regard to atypical Mycobacterium spp, in particular Mycobacterium abscessus. Many methods of decontamination of sputum, which have been adapted from TB methodologies, are ineffective; as shown by the overgrowth of P. aeruginosa, it is essential that decontamination methods are optimized to overcome this. Establishing optimal methods of isolation and determining accurate levels of prevalence is of importance as, although NTM may be isolated relatively infrequently in CF populations, their clinical status in pulmonary disease is now beginning to emerge.

Development of a genus-specific PCR assay for the molecular detection, confirmation and identification of Fusobacterium spp.

Abstract A genus-specific polymerase chain reaction (PCR)-based assay is developed for the detection and identification of clinically relevant Fusobacterium species, including F. nucleatum and F. necrophorum. Two 16S ribosomal DNA (rDNA) primers, FUSO1 (forward primer: 5’-GAG AGA GCT TTG CGT CC-3’ [17-mer]) and FUSO 2 (reverse primer: 5’-TGG GCG CTG AGG TTC GAC -3’ [18-mer]) are designed to target conserved regions of the 16S rDNA gene for Fusobacterium spp. Subsequent proof-of-principle studies employing this assay detected Fusobacterium spp. in the faeces of eight (10%) out of 80 patients with suspected gastrointestinal infection. This assay may be used for the genus-specific detection of Fusobacterium spp. from clinical specimens and for subsequent species identification.