Feasibility of a Molecular Screening Method for Detection of<i>Salmonella enterica</i>and<i>Campylobacter jejuni</i>in a Routine Community-Based Clinical Microbiology Laboratory

Schuurman, T.; Boer, Richard F. de; Zanten, Evert van; Slochteren, K. R. van; Scheper, Henk; Dijk-Alberts, B.; Möller, Anika; Kooistra-Smid, A.M.D.

doi:10.1128/jcm.00896-07

Cited by 42 publications

(33 citation statements)

References 40 publications

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“…irrespective of mPCR outcome, as a delay of more than 1 day (caused by not performing the mPCR during the weekend) would result in a significantly lower potential to obtain a positive result for the MSA guided culture (32).…”

Section: Molecular Screening Approach (I) Specimen Preparationmentioning

confidence: 99%

“…Culturing of S. enterica and Campylobacter spp. was carried out as described previously (32). Briefly, culture of S. enterica consisted of selenite enrichment and selective culturing on salmonellashigella (SS) medium and Hektoen enteric agar (HEA) medium at 35°C, biochemical identification, and group-specific Salmonella serological identification.…”

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confidence: 99%

“…Due to the high throughput of stool screening and the number of possible enteric pathogens, implementation of a molecular approach which uses multiplexing of targets is mandatory (9,10,20,25,26,36,43,44,47). For the detection of enteric pathogens it has been proven feasible to use molec-ular methods with improved performance and turnaround time (TAT) (7,25,32 MATERIALS AND METHODS mPCR technical validation. All primers and probes used for detection of the five enteric pathogens were previously described (4,21,34,43,45), and for some we previously performed extensive clinical validations (32,33,40).…”

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confidence: 99%

“…For the detection of enteric pathogens it has been proven feasible to use molec-ular methods with improved performance and turnaround time (TAT) (7,25,32 MATERIALS AND METHODS mPCR technical validation. All primers and probes used for detection of the five enteric pathogens were previously described (4,21,34,43,45), and for some we previously performed extensive clinical validations (32,33,40). For validation of the mPCR, a total of 147 bacterial/fungal strains were used in selectivity testing.…”

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confidence: 99%

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Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

Boer¹,

et al. 2010

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The detection of bacterial and parasitic gastrointestinal pathogens through culture and microscopy is laborious and time-consuming. We evaluated a molecular screening approach (MSA) for the detection of five major enteric pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC), for use in the daily practice of a clinical microbiology laboratory. The MSA consists of prescreening of stool specimens with two real-time multiplex PCR (mPCR) assays, which give results within a single working day, followed by guided culture/microscopy of the positive or mPCR-inhibited samples. In the present 2-year overview, 28,185 stool specimens were included. The MSA was applied to 13,974 stool samples (49.6%), whereas 14,211 samples were tested by conventional methods only (50.4%). The MSA significantly increased the total detection rate compared to that of conventional methods (19.2% versus 6.4%). The detection of all included pathogens, with the exception of S. enterica, significantly improved. MSA detection frequencies were as follows: C. jejuni, 8.1%; G. lamblia, 4.7%; S. enterica, 3.0%; STEC, 1.9%; and Shigella spp./EIEC, 1.4%. The guided culture/microscopy was positive in 76.8%, 58.1%, 88.9%, 16.8%, and 18.1% of mPCR-positive specimens, respectively. Of all mPCRs, only 1.8% was inhibited. Other findings were that detection of mixed infections was increased (0.9% versus 0.02%) and threshold cycle (C T ) values for MSA guided culture/microscopy-positive samples were significantly lower than those for guided culture/microscopy-negative samples. In conclusion, an MSA for detection of gastrointestinal pathogens resulted in markedly improved detection rates and a substantial decrease in time to reporting of (preliminary) results.

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Section: Molecular Screening Approach (I) Specimen Preparationmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

Boer¹,

et al. 2010

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“…A major advantage of molecular methods is the reduced time to detection (13). Faster diagnostic outputs allow earlier epidemiological investigations and infection control interventions.…”

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confidence: 99%

Comparison of the EntericBio Multiplex PCR System with Routine Culture for Detection of Bacterial Enteric Pathogens

2009

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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.

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Molecular diagnostics of intestinal parasites in returning travellers

Hove

Esbroeck

Vervoort

et al. 2009

Eur J Clin Microbiol Infect Dis

130

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A new diagnostic strategy was assessed for the routine diagnosis of intestinal parasites in returning travellers and immigrants. Over a period of 13 months, unpreserved stool samples, patient characteristics and clinical data were collected from those attending a travel clinic. Stool samples were analysed on a daily basis by microscopic examination and antigen detection (i.e. care as usual), and compared with a weekly performed multiplex real-time polymerase chain reaction (PCR) analysis on Entamoeba histolytica, Giardia lamblia, Cryptosporidium and Strongyloides stercoralis. Microscopy and antigen assays of 2,591 stool samples showed E. histolytica, G. lamblia, Cryptosporidium and S. stercoralis in 0.3, 4.7, 0.5 and 0.1% of the cases, respectively. These detection rates were increased using real-time PCR to 0.5, 6.0, 1.3 and 0.8%, respectively. The prevalence of ten additional pathogenic parasite species identified with microscopy was, at most, 0.5%. A pre-selective decision tree based on travel history or gastro-intestinal complaints could not be made. With increased detection rates at a lower workload and the potential to extend with additional parasite targets combined with fully automated DNA isolation, molecular high-throughput screening could eventually replace microscopy to a large extent.

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Feasibility of a Molecular Screening Method for Detection ofSalmonella entericaandCampylobacter jejuniin a Routine Community-Based Clinical Microbiology Laboratory

Cited by 42 publications

References 40 publications

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

Comparison of the EntericBio Multiplex PCR System with Routine Culture for Detection of Bacterial Enteric Pathogens

Molecular diagnostics of intestinal parasites in returning travellers

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