Real-Time PCR for Detection of NDM-1 Carbapenemase Genes from Spiked Stool Samples

ABSTRACTAn in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification ofblaNDM-1DNA was linear over 10 log dilutions (r2= 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other β-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 101to 3 × 101CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 101to 4 × 103CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 101CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of theblaNDM-1qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time forblaNDM-1detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

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Real-time sampling of travelers shows intestinal colonization by multidrug-resistant bacteria to be a dynamic process with multiple transient acquisitions

10.1101/827915 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anu Kantele ◽

Esther Kuenzli ◽

Steven J Dunn ◽

David AB Dance ◽

Paul N Newton ◽

...

Keyword(s):

Real Time ◽

Multidrug Resistant ◽

Daily Basis ◽

International Travel ◽

Resistant Bacteria ◽

Middle Income ◽

Gram Negative ◽

Extended Spectrum Beta Lactamase ◽

Daily Sampling ◽

Stool Samples

AbstractBackgroundAntimicrobial resistance (AMR) is highly prevalent in low- and middle-income countries (LMIC). International travel contributes substantially to the global spread of intestinal multidrug-resistant gram-negative (MDR-GN) bacteria. Of the 100 million annual visitors to LMIC, 30–70% become colonized by MDR-GN bacteria. The phenomenon has been well documented, but since sampling has only been conducted after travelers’ return home, data on the actual colonization process are scarce. We aimed to characterize colonization dynamics by exploring stool samples abroad on a daily basis while visiting LMIC.MethodsA group of 20 European volunteers visiting Lao People’s Democratic Republic for three weeks provided daily stool samples and filled in daily questionnaires. Acquisition of extended-spectrum beta-lactamase-producing gram-negative bacteria (ESBL-GN) was examined by selective stool cultures followed by whole-genome sequencing (WGS) of isolates.ResultsWhile colonization rates were 70% at the end of the study, daily sampling revealed that all participants had acquired ESBL-GN at some time point during their overseas stay, the colonization status varying day by day. WGS analysis ascribed the transient pattern of colonization to sequential acquisition of new strains, resulting in a loss of detectable colonization by the initial MDR-GN strains. All but one participant acquired multiple strains (n=2–7). Of the total of 83 unique strains identified (53 E. coli, 10 Klebsiella, 20 other ESBL-GN species), some were shared by as many as four subjects.ConclusionsThis is the first study to characterize in real time the dynamics of acquiring MDR-GN during travel. Our data show multiple transient colonization events indicative of constant microbial competition.

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Development of a Rapid and Fully Automated Multiplex Real-Time PCR Assay for Identification and Differentiation of Vibrio cholerae and Vibrio parahaemolyticus on the BD MAX Platform

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.639473 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhenpeng Li ◽

Hongxia Guan ◽

Wei Wang ◽

He Gao ◽

Weihong Feng ◽

...

Keyword(s):

Vibrio Cholerae ◽

Real Time ◽

Real Time Pcr ◽

Vibrio Parahaemolyticus ◽

Health Concern ◽

Specific Gene ◽

Amplification Efficiency ◽

Pcr Assay ◽

Percent Agreement ◽

Stool Samples

Vibrio cholerae and Vibrio parahaemolyticus are common diarrheal pathogens of great public health concern. A multiplex TaqMan-based real-time PCR assay was developed on the BD MAX platform; this assay can simultaneously detect and differentiate V. cholerae and V. parahaemolyticus directly from human fecal specimens. The assay includes two reactions. One reaction, BDM-VC, targets the gene ompW, the cholera toxin (CT) coding gene ctxA, the O1 serogroup specific gene rfbN, and the O139 serogroup specific gene wbfR of V. cholerae. The other, BDM-VP, targets the gene toxR and the toxin coding genes tdh and trh of V. parahaemolyticus. In addition, each reaction contains a sample process control. When evaluated with spiked stool samples, the detection limit of the BD MAX assay was 195–780 CFU/ml for V. cholerae and 46–184 CFU/ml for V. parahaemolyticus, and the amplification efficiency of all genes was between 95 and 115%. The assay showed 100% analytical specificity, using 63 isolates. The BD MAX assay was evaluated for its performance compared with conventional real-time PCR after automated DNA extraction steps, using 164 retrospective stool samples. The overall percent agreement between the BD MAX assay and real-time PCR was ≥ 98.8%; the positive percent agreement was 85.7% for ompW, 100% for toxR/tdh, and lower (66.7%) for trh because of a false negative. This is the first report to evaluate the usage of the BD MAX open system in detection and differentiation of V. cholerae and V. parahaemolyticus directly from human samples.

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Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

Microorganisms ◽

10.3390/microorganisms8111801 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1801

Author(s):

Michael Bording-Jorgensen ◽

Brendon D. Parsons ◽

Gillian A.M. Tarr ◽

Binal Shah-Gandhi ◽

Colin Lloyd ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Gene Detection ◽

Culture Positivity ◽

Hands On ◽

Culture Independent ◽

Chromogenic Agar ◽

Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Pathogens ◽

10.3390/pathogens10060656 ◽

2021 ◽

Vol 10 (6) ◽

pp. 656

Author(s):

Konstantin Tanida ◽

Andreas Hahn ◽

Kirsten Alexandra Eberhardt ◽

Egbert Tannich ◽

Olfert Landt ◽

...

Keyword(s):

Real Time ◽

Indigenous People ◽

Real Time Pcr ◽

Latent Class ◽

Enterocytozoon Bieneusi ◽

Enteric Disease ◽

Immunosuppressed Patients ◽

Study Population ◽

Stool Samples ◽

Pcr Assays

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

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Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

Virology Journal ◽

10.1186/s12985-021-01541-z ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Ellen Kathrin Link ◽

Matthias Eddicks ◽

Liangliang Nan ◽

Mathias Ritzmann ◽

Gerd Sutter ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Diagnostic Sensitivity ◽

Locked Nucleic Acid ◽

Porcine Circovirus ◽

Amplification Efficiency ◽

Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

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Key parameters for genomics-based real-time detection and tracking of multidrug-resistant bacteria: a systematic analysis

The Lancet Microbe ◽

10.1016/s2666-5247(21)00149-x ◽

2021 ◽

Author(s):

Claire L Gorrie ◽

Anders Gonçalves Da Silva ◽

Danielle J Ingle ◽

Charlie Higgs ◽

Torsten Seemann ◽

...

Keyword(s):

Real Time ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Multidrug Resistant Bacteria ◽

Systematic Analysis ◽

Detection And Tracking ◽

Real Time Detection

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Prevalence of Mastitis and Antibiotic Resistance of Bacterial Isolates from CMT Positive Milk Samples Obtained from Dairy Cows, Camels, and Goats in Two Pastoral Districts in Southern Ethiopia

Animals ◽

10.3390/ani11061530 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1530

Author(s):

Amanuel Balemi ◽

Balako Gumi ◽

Kebede Amenu ◽

Sisay Girma ◽

Muuz Gebru ◽

...

Keyword(s):

Antibiotic Resistance ◽

Dairy Cows ◽

Raw Milk ◽

Multidrug Resistant ◽

Southern Ethiopia ◽

Resistant Bacteria ◽

Coagulase Negative Staphylococcus ◽

Bacterial Isolates ◽

E Coli ◽

Milk Samples

A study was carried out from August 2017 to February 2018 on lactating dairy cows, one-humped dromedary camels, and goats to determine mastitis in the Bule Hora and Dugda Dawa districts of in Southern Ethiopia. Milk samples from 564 udder quarters and udder halves from 171 animals consisting of 60 dairy cows, 51 camels, and 60 goats were tested for mastitis. Sixty-four positive udder milk samples were cultured, and bacterial mastitis pathogens were isolated and identified. The antibiotic resistance of bacterial isolates from milk with mastitis was tested against nine antimicrobials commonly used in the study area. Cow- and quarter-level prevalence of mastitis in dairy cows, camels, and goats was 33.3%, 26.3%, and 25% and 17.6%, 14.5%, and 20%, respectively. In cattle, the prevalence was significantly higher in Dugda Dawa than in Bule Hora. Major bacterial isolates were coagulase-negative Staphylococcus species (39.1%), S. aureus (17.2%), S. hyicus (14.1%), and S. intermedius and Escherichia coli (9.4% each). In camels, udder abnormality and mastitis were significantly higher in late lactation than in early lactation. Mastitis tends to increase with parity in camels. E. coli isolates were highly resistant to spectinomycin, vancomycin, and doxycycline, whereas most S. aureus isolates were multidrug-resistant. Most of the rural and periurban communities in this area consume raw milk, which indicates a high risk of infection with multidrug-resistant bacteria. We recommend a community-focused training program to improve community awareness of the need to boil milk and the risk of raw milk consumption.

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