Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation

Lab on a Chip ◽  
2015 ◽  
Vol 15 (18) ◽  
pp. 3749-3759 ◽  
Author(s):  
G. Czilwik ◽  
T. Messinger ◽  
O. Strohmeier ◽  
S. Wadle ◽  
F. von Stetten ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Nested Pcr ◽  
Pathogen Detection ◽  
Bacterial Pathogen ◽  
Highly Sensitive

Integrated PCR LabDisk and portable LabDisk-Player.

Abstract 11119: Real-Time Nanopore Sequencing for Bacterial Pneumonia After Out-of-Hospital Cardiac Arrest, a Pilot Proof-of-Concept Study

Circulation ◽  
2021 ◽  
Vol 144 (Suppl_2) ◽  
Author(s):  
Alexandra Weissman ◽  
Mariam Bramah Lawani ◽  
Thomas Rohan ◽  
Clifton W CALLAWAY
Keyword(s):  
Patient Care ◽  
Pathogen Detection ◽  
Bacterial Pathogen ◽  
Improve Patient Care ◽  
Antibiotic Administration ◽  
Nanopore Sequencing ◽  
Proof Of Concept ◽  
Chi Square ◽  
Significant Difference ◽  
Improve Patient

Introduction: Pneumonia is common after OHCA but is difficult to diagnose in the first 72 hours following ROSC, this results in early untargeted antibiotic administration based on non-specific imaging and laboratory findings. Antibiotic resistance is rising, is influenced by untargeted antibiotic administration, and can increase patient morbidity and mortality as well as healthcare costs. Precision methods of bacterial pathogen detection in OHCA patients are needed to improve patient care. This proof-of-concept pilot study aimed to assess feasibility of bacterial pathogen sequencing and comparability of sequencing results to clinical culture after OHCA. Methods: Blood and bronchoalveolar lavage (BAL) were obtained from residual clinical specimens collected within 12 hours of ROSC. Bacterial DNA was extracted using the Qiagen PowerLyzer PowerSoil DNA kit, sequenced using the MinION nanopore sequencer, and analyzed with Oxford Nanopore Technologies’ EPI2ME bioinformatics software. Sequencing results were compared to culture results using McNemar’s chi-square statistic. Study-defined pneumonia was based on presence of at least two characteristics within 72 hours of ROSC: fever (temperature ≥38°C); persistent leukocytosis >15,000 or leukopenia <3,500 for 48 hours; persistent chest radiography infiltrates for 48 hours per clinical radiology read; bacterial pathogen cultured. Results: We enrolled 38 consecutive OHCA subjects: mean age 61.8 years (18.0); 16 (42%) female; 25 (66%) White, 7 (18%) Black, 6 (16%) “Other” race; 7 subjects (18%) survived and 31 (82%) died; 16 (42%) subjects had pneumonia. Sequencing results were available in 12 hours while culture results were available in 48-72 hours after collection. There was a non-significant difference in the proportion of the same pathogens identified for each method per McNemar’s chi-square: p = 0.38, difference of 0.095 (-0.095, 0.286). Conclusions: Nanopore sequencing detects pathogenic bacteria comparable to clinical microbiologic culture and in less time. This technology can produce a paradigm shift in early bacterial pathogen detection in OHCA survivors, which can improve patient care. The technology is applicable to other patient populations and for viral and fungal pathogens.

scholarly journals Bacteriophage functional genomics and its role in bacterial pathogen detection

2013 ◽  
Vol 12 (4) ◽  
pp. 354-365 ◽  
Author(s):  
J. Klumpp ◽  
D. E. Fouts ◽  
S. Sozhamannan
Keyword(s):  
Functional Genomics ◽  
Pathogen Detection ◽  
Bacterial Pathogen

scholarly journals A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Bhagya Deepachandi ◽  
Sudath Weerasinghe ◽  
Preethi Soysa ◽  
Nadira Karunaweera ◽  
Yamuna Siriwardana
Keyword(s):  
Nested Pcr ◽  
Case Detection ◽  
Highly Sensitive

scholarly journals Comparative Evaluation of Enteric Bacterial Culture and a Molecular Multiplex Syndromic Panel in Children with Acute Gastroenteritis

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Thomas Kellner ◽  
Brendon Parsons ◽  
Linda Chui ◽  
Byron M. Berenger ◽  
Jianling Xie ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Rectal Swab ◽  
Bacterial Pathogen ◽  
Positive Culture ◽  
Content Type ◽  
Negative Culture ◽  
Percent Agreement ◽  
Stool Samples ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTAlthough enteric multianalyte syndromic panels are increasingly employed, direct comparisons with traditional methods and the inclusion of host phenotype correlations are limited. Luminex xTAG gastrointestinal pathogen panel (GPP) and culture results are highly concordant. However, phenotypic and microbiological confirmatory testing raises concerns regarding the accuracy of the GPP, especially forSalmonellaspp. A total of 3,089 children with gastroenteritis submitted stool specimens, rectal swab specimens, and clinical data. The primary outcome was bacterial pathogen detection agreement for shared targets between culture and the Luminex xTAG GPP. Secondary analyses included phenotype assessment, additional testing of GPP-negative/culture-positive isolate suspensions with the GPP, and in-house and commercial confirmatory nucleic acid testing of GPP-positive/culture-negative extracts. The overall percent agreement between technologies was >99% for each pathogen.Salmonellaspp. were detected in specimens from 64 participants: 12 (19%) by culture only, 9 (14%) by GPP only, and 43 (67%) by both techniques. Positive percent agreement forSalmonellaspp. was 78.2% (95% confidence interval [CI], 64.6%, 87.8%). Isolate suspensions from the 12 participants with specimens GPP negative/culture positive forSalmonellatested positive by GPP. Specimens GPP positive/culture negative forSalmonellaoriginated in younger children with less diarrhea and more vomiting. GPP-positive/culture-negative specimen extracts tested positive using additional assays for 0/2Campylobacter-positive specimens, 0/4Escherichia coliO157-positive specimens, 0/9Salmonella-positive specimens, and 2/3Shigella-positive specimens. For both rectal swab and stool samples, the median cycle threshold (CT) values, determined using quantitative PCR, were higher for GPP-negative/culture-positive samples than for GPP-positive/culture-positive samples (for rectal swabs, 36.9 [interquartile range {IQR}, 33.7, 37.1] versus 30.0 [IQR, 26.2, 33.2], respectively [P = 0.002]; for stool samples, 36.9 [IQR, 33.7, 37.1] versus 29.0 [IQR, 24.8, 30.8], respectively [P = 0.001]). GPP and culture have excellent overall agreement; however, for specific pathogens, GPP is less sensitive than culture and, notably, identifies samples false positive forSalmonellaspp.

A fully integrated bacterial pathogen detection system based on count-on-a-cartridge platform for rapid, ultrasensitive, highly accurate and culture-free assay

2020 ◽  
Vol 152 ◽  
pp. 112007 ◽  
Author(s):  
Won-Il Lee ◽  
Younghyeon Park ◽  
Sajal Shrivastava ◽  
Taekeon Jung ◽  
Montri Meeseepong ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Detection System ◽  
Bacterial Pathogen ◽  
Fully Integrated

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina

2019 ◽  
Vol 67 (S2) ◽  
pp. 159-164 ◽  
Author(s):  
Sabrina Ganzinelli ◽  
Daniel Benitez ◽  
Sambuu Gantuya ◽  
Azirwan Guswanto ◽  
Monica Florin‐Christensen ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Babesia Bovis ◽  
Cattle Herd ◽  
Babesia Bigemina ◽  
Highly Sensitive

scholarly journals Proof of Concept for Recombinant Cellular Controls in Quantitative Molecular Pathogen Detection

2011 ◽  
Vol 77 (7) ◽  
pp. 2531-2533 ◽  
Author(s):  
Peter Rossmanith ◽  
Patrick Mester ◽  
Karin Frühwirth ◽  
Sabine Fuchs ◽  
Martin Wagner
Keyword(s):  
Listeria Monocytogenes ◽  
Sample Preparation ◽  
Quantitative Pcr ◽  
Pathogen Detection ◽  
Single Copy ◽  
Deletion Strain ◽  
Proof Of Concept ◽  
Targeted Deletion ◽  
Process Controls

ABSTRACTIn this study, we present the concept of internal sample process controls (ISPCs) to monitor the efficiency of an analytical chain using sample preparation and quantitative PCR (qPCR). A recombinantListeria monocytogenesΔprfA(targeted deletion) strain containing a competitive artificial single-copy genomic target was applied to naturally contaminated samples to demonstrate its analytical suitability as an ISPC.

scholarly journals The IGHV1-69/IGHJ3 recombinations of unmutated CLL are distinct from those of normal B cells

Blood ◽  
2012 ◽  
Vol 119 (9) ◽  
pp. 2106-2109 ◽  
Author(s):  
Francesco Forconi ◽  
Kathleen N. Potter ◽  
Elisa Sozzi ◽  
Isla Henderson ◽  
Emanuele Cencini ◽  
...  
Keyword(s):  
Amino Acid ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nested Pcr ◽  
Antigen Recognition ◽  
Lymphocytic Leukemia ◽  
Healthy Individuals ◽  
Amino Acid Homology ◽  
Highly Sensitive

Abstract IGHV1-69/51p1 is expressed by ∼ 30% of unmutated chronic lymphocytic leukemia (U-CLL) and combines with selected IGHD and IGHJ genes generating stereotypes if HCDR3 amino acid homology is > 60%. We had previously revealed stereotypic IGHV1-69/IGHJ6 rearrangements in normal naive B cells, thereby identifying potential counterparts of U-CLL. A different stereotypic IGHV1-69/IGHD3-16(RF2)/IGHJ3 rearrangement carrying the CAR(GGx)YD motif in the N1-region, recurrent in 6% IGHV1-69+ve CLL, is exceptionally sequence restricted, strongly suggestive of shared antigen recognition. We have now analyzed IGHV1-69/IGHJ3 rearrangements in circulating B cells of healthy individuals using several PCR-based approaches with IGHV1-69/IGHJ3 CLL sequences for reference. Stereotypes were found, but all were distinct from CLL. Remarkably, even a highly sensitive semi-nested PCR, specific for the CLL-expressed IGHV1-69/IGHD3-16(RF2)/IGHJ3 stereotype, failed to identify the CAR(GGx)YD sequence, although similar motifs were found. These highly specific B cells are not apparent in the accessible normal repertoire and may expand in response to rarely expressed antigens important in the pathogenesis of CLL.

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