Applicability of a rapid duplex real-time PCR assay for speciation of<i>Campylobacter jejuni</i>and<i>Campylobacter coli</i>directly from culture plates

Best, Emma L.; Powell, Ella J; Swift, Craig; Grant, Kathleen A.; Frost, J. A.

doi:10.1016/s0378-1097(03)00845-0

Cited by 123 publications

(107 citation statements)

References 17 publications

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“…Given that gastroenteritis due to C. jejuni infections are hyperendemic among young children (< 5 years) in tropical resource-poor settings, it is expected that the prevalence of C. jejuni-induced GBS would be prevalent among individuals of < 5 years of age. Other researchers, using a broader case definition that includes all age groups for GBS surveillance, have published reports of bimodal distribution of GBS cases by age, with more cases identified in young adults (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and the elderly (significantly higher) [4,16,29,30].…”

Section: Discussionmentioning

confidence: 99%

“…(Table 1) was used in our current study [25][26][27]. Multiplex qPCRs were performed in 50 µL reactions and included the following components: 25 µL TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, USA); 12 µL internal positive control (IPC) mix (Applied Biosystems, Foster City, USA; 7.5 µM mapA primer with 1.5 µM mapA probe; 15 µM ceuE primer with 3 µM ceuE probe; and 15 µM 16SrRNA primer with 3 µM 16SrRNA probe (Inqaba Technologies, Pretoria, South Africa, (primers); and Roche, Johannesburg, South Africa, probes); 7 µL template DNA; and 3 µL of deionized water.…”

Section: Multiplex Qpcrmentioning

confidence: 99%

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Detection of Campylobacter species in stool specimens from patients with symptoms of acute flaccid paralysis in South Africa

Thobela¹,

Smith²,

Moonsamy³

et al. 2018

J Infect Dev Ctries

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Introduction: Guillain-Barré Syndrome (GBS) is an autoimmune disease characterized by acute or subacute symmetrical ascending motor weakness, areflexia, and mild-to-moderate sensory abnormalities. Campylobacter jejuni is reported to be the most common bacterium associated with GBS cases. Despite the eradication of polio, the number of reported GBS cases remains considerably high in South Africa with the causative agents not being well described. Methodology: The aim of the study was to investigate the proportion of Campylobacter spp. detected in stool specimens from patients with symptoms of acute flaccid paralysis (AFP). Stool specimens from patients presenting with AFP, that were negative for polio and non-polio enteroviruses (NPENT), were processed and screened for the presence of Campylobacter spp. using quantitative PCR (qPCR). Results: Of the 512 stool specimens screened between October 2014 to December 2015, 12% (62/512) were positive for Campylobacter spp. Of these 62 Campylobacter infections: 77.4% (48/62) was C. jejuni; 19.4% (12/62) was Campylobacter coli; 3.2% (2/62) was mixed infections of C. jejuni and C. coli. Conclusions: True association of the disease with Campylobacter spp. will enable the proportion of Campylobacter-induced GBS to be better described in South Africa; this can only be done through systematic studies that include bacterial culture and serology together with molecular methodologies.

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Section: Discussionmentioning

confidence: 99%

Section: Multiplex Qpcrmentioning

confidence: 99%

Detection of Campylobacter species in stool specimens from patients with symptoms of acute flaccid paralysis in South Africa

Thobela¹,

Smith²,

Moonsamy³

et al. 2018

J Infect Dev Ctries

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“…Real-time amplification was carried out on an ABI PRISM 7700 sequence detection system (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) by using the TaqMan Universal PCR master mix (Applied Biosystems). The primers and probes have been described previously for S. enterica (16), C. jejuni (5), and PhHV (32) and were purchased from Applied Biosystems. Probes were labeled (at the 5Ј end) with 6-carboxyfluorescein (S. enterica), VIC (C. jejuni), or 2Ј-chloro-5Ј-fluoro-7Ј,8Ј-fused phenyl-1,4-dichloro-6-carboxyfluorescein (NED) (PhHV).…”

Section: Methodsmentioning

confidence: 99%

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

Schuurman¹,

Boer²,

Zanten³

et al. 2007

J Clin Microbiol

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Conventional diagnostic methods for the detection of Salmonella enterica and Campylobacter jejuni are laborious and time-consuming procedures, resulting in final results, for the majority of specimens, only after 3 to 4 days. Molecular detection can improve the time to reporting of the final results from several days to the next day. However, molecular assays for the detection of gastrointestinal pathogens directly from stool specimens have not made it into the routine clinical microbiology laboratory. In this study we have assessed the feasibility of a real-time PCR-based molecular screening method (MSM), aimed at S. enterica and C. jejuni, in the daily practice of a routine clinical microbiology laboratory. We have prospectively analyzed 2,067 stool specimens submitted for routine detection of gastrointestinal bacterial pathogens over a 7-month period. The MSM showed 98 to 100% sensitivity but routine culture showed only 77.8 to 86.8% sensitivity when an extended "gold standard" that included all culture-positive and all MSM-positive specimens, as confirmed by an independent secondary PCR of a different target gene, was used. An overall improvement in the rate of detection of both pathogens of 15 to 18% was observed. Both approaches performed nearly identically with regard to the specificity, positive predictive value, and negative predictive value, with the values for MSM being 99.7%, 93.1 to 96.6%, and 99.8 to 100%, respectively, and those for routine culture being 100%, 100%, and 97.6 to 99.5%, respectively. Finally, the final results were reported between 3 and 4 days earlier for negative specimens compared to the time of reporting of the results of routine culture. Positive specimens, on the other hand, required an additional 2 days to obtain a final result compared to the time required for routine culture, although preliminary MSM PCR-positive results were reported, on average, 2.9 to 3.8 days before the final routine culture results were reported. In conclusion, MSM can be incorporated into the daily practice of a routine clinical microbiology laboratory with ease. Furthermore, it provides an improvement in the screening for S. enterica and C. jejuni and substantially improves the time to the reporting of negative results.

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“…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.…”

mentioning

confidence: 99%

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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Applicability of a rapid duplex real-time PCR assay for speciation ofCampylobacter jejuniandCampylobacter colidirectly from culture plates

Cited by 123 publications

References 17 publications

Detection of Campylobacter species in stool specimens from patients with symptoms of acute flaccid paralysis in South Africa

Detection of Campylobacter species in stool specimens from patients with symptoms of acute flaccid paralysis in South Africa

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

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

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