scholarly journals Pre-earthquake non-epidemic Vibrio cholerae in Haiti

2014 ◽  
Vol 8 (01) ◽  
pp. 120-122 ◽  
Author(s):  
Jie Liu ◽  
Christopher Winstead-Derlega ◽  
Eric Houpt ◽  
Rebecca Heidkamp ◽  
Jean Pape ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Sybr Green ◽  
Molecular Assays ◽  
Nutrition Study ◽  
Stool Samples ◽  
Pediatric Nutrition ◽  
Mgb Probe ◽  
Taqman Mgb Probe

Introduction: To our knowledge, there was no record of Vibrio cholerae in Haiti until the 2010 post earthquake outbreak. Methodology: This study describes the analysis of 301 stool samples from 117 infants in Port-au-Prince, Haiti, who participated in a pediatric nutrition study between July 2008 and October 2009. Results: Nine samples were identified positive with both SYBR Green and Taqman-MGB probe based molecular assays targeting V. cholerae hlyA and toxR, respectively (Ct = 33 – 40), but none were O1 or O139. Conclusions: Our results from multiple molecular assays demonstrate the presence of non-O1/O139 V. cholerae DNA in stools collected from nine asymptomatic Haitian infants two years prior to the 2010 earthquake.

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real‐time quantitative PCR

2018 ◽  
Vol 42 (1) ◽  
pp. 109-117 ◽  
Author(s):  
Chun Liu ◽  
Yanming M. Guo ◽  
Jizhen Z. Cao ◽  
De‐Feng Zhang ◽  
Ou‐Qin Chang ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Quantitative Pcr ◽  
Mgb Probe ◽  
Taqman Mgb Probe ◽  
Detection And Quantification

scholarly journals Development of Rapid Extraction Method of Mycobacterium avium Subspecies paratuberculosis DNA from Bovine Stool Samples

Diagnostics ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 36 ◽  
Author(s):  
Sören Hansen ◽  
Marco Roller ◽  
Lamia Alslim ◽  
Susanne Böhlken-Fascher ◽  
Kim Fechner ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Extraction Method ◽  
Magnetic Beads ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Rapid Identification ◽  
Cold Chain ◽  
Silica Membrane ◽  
Molecular Assays ◽  
Stool Samples

The rapid identification of Mycobacterium avium subspecies paratuberculosis (MAP) infected animals within the herd is essential for preventing the spread of the disease as well as avoiding human exposure. Although culture is seen as the gold standard, there are various molecular assays available i.e., polymerase chain reaction (PCR) or isothermal amplification technique (recombinase polymerase amplification (RPA)) for the detection of MAP. The accuracy of the molecular assays is highly dependent on the DNA extraction method. In order to establish a rapid point of need system for the detection of MAP DNA from stool samples, we developed a rapid DNA extraction protocol (MAP DNA SpeedXtract) specified for use in combination with the RPA. The whole procedure from “sample in” to “result out” was conducted in a mobile suitcase laboratory. The DNA extraction is based on reverse purification by magnetic beads, which reduces the required technical demand. The MAP DNA SpeedXtract was performed within 25 min and only three pipetting steps were needed. The amplification and detection time were 20 min in RPA. The sensitivity and specificity of the developed protocol in comparison with the lab-based silica membrane column extraction and real-time PCR were 90.9% (n = 22) and 100% (n = 23), respectively. In conclusion, we established a rapid and reliable protocol for the extraction and detection of MAP DNA. All reagents are cold chain independent. The entire setup is ideal for point of need identification of MAP infected cases.

scholarly journals Evaluation of Copan FecalSwab as Specimen Type for Use in Xpert C. difficile Assay

2017 ◽  
Vol 55 (10) ◽  
pp. 3123-3129 ◽  
Author(s):  
Michael J. Mashock ◽  
Matthew L. Faron ◽  
Blake W. Buchan ◽  
Nathan A. Ledeboer
Keyword(s):  
Test Performance ◽  
Pcr Assay ◽  
Content Type ◽  
Molecular Assays ◽  
Specificity And Sensitivity ◽  
Collection Device ◽  
Stool Samples ◽  
Stool Specimens ◽  
Xpert Assay ◽  
Preservation Medium

ABSTRACT Liquid-based microbiology (LBM) devices incorporating flocked swabs and preservation medium ease transport of specimens and improve specimen yield compared to traditional fiber wound swabs; however, the performance of LBM collection devices has not been evaluated in many molecular assays. It is unclear how the differences in matrix and specimen loading with an LBM device will affect test performance compared to traditional collection devices. The purpose of this study was to evaluate the performance of specimens collected in FecalSwab transport medium (Copan Diagnostics, Murrieta, CA) compared to unpreserved stool using the Cepheid Xpert C. difficile assay (Cepheid, Sunnyvale, CA). Results equivalent to unpreserved stool samples were obtained when 400 μl of FecalSwab-preserved stool was employed in the Xpert assay. The positive and negative percent agreement of specimens inoculated with FecalSwab medium ( n = 281) was 97.0% (95% confidence interval [CI], 90.9 to 96.4%) and 99.4% (95% CI, 96.4 to 99.9%), respectively, compared to reference results obtained using unpreserved stool. Throughout this study, only four discrepant results occurred when comparing preserved specimens to unpreserved stool specimens in the Xpert C. difficile PCR assay. Post discrepant analysis, using the BD MAX Cdiff assay, the specificity and sensitivity both increased to 100%. The high positive and negative percent agreements observed in this study suggest that stool preserved in FecalSwab media yields equivalent results to using unpreserved stool when tested on the Xpert C. difficile assay, allowing laboratories to adopt this liquid-based microbiology collection device.

scholarly journals Quantitative Thresholds Enable Accurate Identification ofClostridium difficileInfection by the Luminex xTAG Gastrointestinal Pathogen Panel

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Sixto M. Leal ◽  
Elena B. Popowitch ◽  
Kara J. Levinson ◽  
Teny M. John ◽  
Bethany Lehman ◽  
...  
Keyword(s):  
Test Performance ◽  
Predictive Value ◽  
High Specificity ◽  
Clinical Suspicion ◽  
Performance Characteristics ◽  
Accurate Identification ◽  
Data Set ◽  
Content Type ◽  
Molecular Assays ◽  
Stool Samples

ABSTRACTClostridium difficilecolonizes the gastrointestinal (GI) tract, resulting in either asymptomatic carriage or a spectrum of diarrheal illness. If clinical suspicion forC. difficileis low, stool samples are often submitted for analysis by multiplex molecular assays capable of detecting multiple GI pathogens, and some institutions do not report this organism due to concerns for high false-positive rates. Since clinical disease correlates with organism burden and molecular assays yield quantitative data, we hypothesized that numerical cutoffs could be utilized to improve the specificity of the Luminex xTAG GI pathogen panel (GPP) forC. difficileinfection. Analysis of cotested liquid stool samples (n= 1,105) identified a GPP median fluorescence intensity (MFI) value cutoff of ≥1,200 to be predictive of two-step algorithm (2-SA; 96.4% concordance) and toxin enzyme immunoassay (EIA) positivity. Application of this cutoff to a second cotested data set (n= 1,428) yielded 96.5% concordance. To determine test performance characteristics, concordant results were deemed positive or negative, and discordant results were adjudicated via chart review. Test performance characteristics for the MFI cutoff of ≥150 (standard), MFI cutoff of ≥1,200, and 2-SA were as follows (respectively): concordance, 95, 96, and 97%; sensitivity, 93, 78, and 90%; specificity, 95, 98, and 98%; positive predictive value, 67, 82, and 81%;, and negative predictive value, 99, 98, and 99%. To capture the high sensitivity for organism detection (MFI of ≥150) and high specificity for active infection (MFI of ≥1,200), we developed and applied a reporting algorithm to interpret GPP data from patients (n= 563) with clinician orders only for syndromic panel testing, thus enabling accurate reporting ofC. difficilefor 95% of samples (514 negative and 5 true positives) irrespective of initial clinical suspicion and without the need for additional testing.

scholarly journals Detection and species identification of microsporidial infections using SYBR Green real-time PCR

2011 ◽  
Vol 60 (4) ◽  
pp. 459-466 ◽  
Author(s):  
Spencer D. Polley ◽  
Samuel Boadi ◽  
Julie Watson ◽  
Alan Curry ◽  
Peter L. Chiodini
Keyword(s):  
Real Time ◽  
Species Identification ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Enterocytozoon Bieneusi ◽  
Sybr Green ◽  
Pcr Assay ◽  
Real Time Analysis ◽  
Highly Sensitive ◽  
Stool Samples

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

scholarly journals A Tail Fiber Protein and a Receptor-Binding Protein Mediate ICP2 Bacteriophage Interactions with Vibrio cholerae OmpU

2021 ◽  
Author(s):  
Andrea N.W. Lim ◽  
Minmin Yen ◽  
Kimberley D. Seed ◽  
David W. Lazinski ◽  
Andrew Camilli
Keyword(s):  
Host Range ◽  
Vibrio Cholerae ◽  
Receptor Binding ◽  
Mutant Phenotype ◽  
Tail Fiber ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Strains ◽  
Stool Samples ◽  
Receptor Binding Protein

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host-range mutants within infant rabbits infected with a mixture of wild type and OmpU mutant strains. ICP2 host-range mutants, that can now infect OmpU mutant strains, had missense mutations in putative tail fiber gene gp25 and putative adhesin gp23. Using site-specific mutagenesis we show that single or double mutations in gp25 are sufficient to generate the host-range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to give a host-range mutant phenotype. All ICP2 host-range mutants retained the ability to plaque on wild type V. cholerae cells. The strength of binding of host-range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host-range mutants evolve by a two-step process where, first, gp25 mutations are selected for their broad host-range, albeit accompanied by low level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near wild type efficiencies of adsorption and subsequent phage multiplication. Importance Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to a renewed interest in phage biology and their potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular co-evolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

Cholera outbreak investigation, Bhadola, Delhi, India, April–May 2018

2020 ◽  
Vol 114 (10) ◽  
pp. 762-769
Author(s):  
Akhileshwar Singh ◽  
Rakesh Gupta ◽  
Tanzin Dikid ◽  
Ekta Saroha ◽  
Naresh Chand Sharma ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Fecal Coliforms ◽  
Prevention Measures ◽  
Cholera Outbreak ◽  
Public Health Action ◽  
The North ◽  
Municipal Water ◽  
Health Action ◽  
Vibrio Cholerae O1 ◽  
Stool Samples

Abstract Background In the Gangetic plains of India, including Delhi, cholera is endemic. On 10 May 2018, staff at the north Delhi district surveillance unit identified a laboratory-confirmed cholera outbreak when five people tested positive for Vibrio cholerae O1 Ogawa serotype in Bhadola. We investigated to identify risk factors and recommend prevention measures. Methods We defined a case as ≥3 loose stools within 24 h in a Bhadola resident during 1 April–29 May 2018. We searched for cases house-to-house. In a 1 : 1 unmatched case control study, a control was defined as an absence of loose stools in a Bhadola resident during 1 April–29 May 2018. We selected cases and controls randomly. We tested stool samples for Vibrio cholerae by culture. We tested drinking water for fecal contamination. Using multivariable logistic regression we calculated adjusted ORs (aORs) with 95% CIs. Results We identified 129 cases; the median age was 14.5 y, 52% were females, 27% were hospitalized and there were no deaths. Symptoms were abdominal pain (54%), vomiting (44%) and fever (29%). Among 90 cases and controls, the odds of illness were higher for drinking untreated municipal water (aOR=2.3; 95% CI 1.0 to 6.2) and not knowing about diarrhea transmission (aOR=4.9; 95% CI 1.0 to 21.1). Of 12 stool samples, 6 (50%) tested positive for Vibrio cholerae O1 Ogawa serotype. Of 15 water samples, 8 (53%) showed growth of fecal coliforms. Conclusions This laboratory-confirmed cholera outbreak associated with drinking untreated municipal water and lack of knowledge of diarrhea transmission triggered public health action in Bhadola, Delhi.

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