scholarly journals Laboratory diagnosis of 37 cases of Bartonella endocarditis based on enzyme immunoassay and real-time PCR

2021 ◽  
Author(s):  
Lev Shapira ◽  
Michal Rasis ◽  
Inbal Binsky Ehrenreich ◽  
Yasmin Maor ◽  
Eugene A. Katchman ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Real Time ◽  
Real Time Pcr ◽  
Heart Valves ◽  
Q Fever ◽  
Laboratory Diagnosis ◽  
Cross Reactivity ◽  
Immunofluorescence Assay ◽  
Complete Agreement ◽  
Pcr Assay

Bartonella spp., mostly B. quintana and B. henselae, are a common cause of culture-negative endocarditis. Serology, using immunofluorescence assay (IFA) and PCR performed on cardiac tissues are the mainstays of diagnosis. We developed an enzyme immunoassay (EIA) and a novel multiplex real-time PCR assay, utilizing Bartonella genus-specific, B. henselae-specific and B. quintana-specific SimpleProbe probes, for diagnosis of Bartonella endocarditis. We aimed to evaluate the performance of these assays. Thirty-seven patients with definite endocarditis, 18 with B. henselae, 18 with B. quintana and one with B. koehlerae were studied. Diagnosis was confirmed by conventional PCR and DNA sequencing of surgical cardiac specimens. Similarly to IFA, anti-Bartonella IgG titers ≥1:800 were found in 94% of patients by EIA; cross-reactivity between B. henselae and B. quintana precluded species-specific serodiagnosis, and frequent (41%) but low-titer cross-reactivity between Coxiella burnetii antibodies and B. henselae antigen was found in patients with Q fever endocarditis. Low-titer (1:100) cross-reactivity was uncommonly found also in patients with brucellosis and culture-positive endocarditis, particularly Enterococcus faecalis endocarditis. Real-time PCR performed on explanted heart valves/vegetations was in complete agreement with results of sequence-based diagnosis with characteristic melting curves. The genus-specific probe identified five additional endocarditis-associated Bartonella spp. at the genus level. In conclusion, EIA coupled with a novel real-time PCR assay can play an important role in Bartonella endocarditis diagnosis and expand the diagnostic arsenal at the disposal of the clinical microbiologist. Since serology remains a major diagnostic tool, recognizing its pitfalls is essential to avoid incorrect diagnosis.

scholarly journals Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay

2009 ◽  
Vol 58 (1) ◽  
pp. 65-68 ◽  
Author(s):  
Abdessalam Cherkaoui ◽  
Dimitri Ceroni ◽  
Stéphane Emonet ◽  
Yan Lefevre ◽  
Jacques Schrenzel
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cell Types ◽  
Cross Reactivity ◽  
Toxin Gene ◽  
Rrna Gene ◽  
Putative Virulence Factor ◽  
Pcr Assay ◽  
Kingella Kingae ◽  
Rtx Toxin

Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

scholarly journals Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR

Foods ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 332
Author(s):  
Jasmin Wrage ◽  
Oxana Kleyner ◽  
Sascha Rohn ◽  
Jürgen Kuballa
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Method ◽  
Immunoglobulin E ◽  
Limit Of Detection ◽  
Cocos Nucifera ◽  
Cross Reactivity ◽  
Pcr Assay ◽  
Probe System ◽  
Ige Mediated

So far, only a few cases of immunoglobulin E (IgE)-mediated coconut allergies have been described in the literature. Due to a growing consumption of coconut-containing foods in occidental countries, the number of coconut allergies may also increase. As there is no causative immunotherapy in clinical routine, appropriate food labelling is particularly important, also with regard to cross-contamination, to prevent serious health consequences. The purpose of this study was to develop a DNA-based detection method for coconut (Cocos nucifera). Initially, three sets of coconut-specific primers were designed and tested. A TaqMan™ probe was then developed to identify and quantify coconut by real-time PCR assay. With 27 other plant and animal species, the specificity of the primer/probe system was tested and cross reactivity was excluded. In a dilution series, a limit of detection of 1 pg/µL was determined. Thus, the developed real-time PCR assay is a suitable method to detect coconut in food.

scholarly journals Real-Time PCR Detection of Parapoxvirus DNA,

2006 ◽  
Vol 52 (2) ◽  
pp. 316-319 ◽  
Author(s):  
Andreas Nitsche ◽  
Mathias Büttner ◽  
Sonja Wilhelm ◽  
Georg Pauli ◽  
Hermann Meyer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Probit Analysis ◽  
Animal Origin ◽  
Pcr Assay ◽  
Medical Practitioners ◽  
B2l Gene ◽  
Zoonotic Infections

Abstract Background: Detection of parapoxviruses is important in various animals as well as in humans as zoonotic infections. Reliable detection of parapoxviruses is fundamental for the exclusion of other rash-causing illnesses, for both veterinarians and medical practitioners. To date, however, no real-time PCR assay for the detection of parapoxviruses has been reported. Methods: A minor groove binder–based quantitative real-time PCR assay targeting the B2L gene of parapoxviruses was developed on the ABI Prism and the LightCycler platforms. Results: The real-time PCR assay successfully amplified DNA fragments from a total of 41 parapoxvirus strains and isolates representing the species orf virus, bovine papular stomatitis virus, pseudocowpoxvirus, and sealpoxvirus. Probit analysis gave a limit of detection of 4.7 copies per assay (95% confidence interval, 3.7–6.8 copies per reaction). Scabs contain a sufficient amount of parapoxvirus DNA and can therefore be used for PCR without any DNA preparation step. No cross-reactivity to human, bovine, or sheep genomic DNA or other DNA viruses, including orthopoxviruses, molluscum contagiosum viruses, and yaba-like disease viruses, was observed. Conclusion: The presented assay is suitable for the detection of parapoxvirus infections in clinical material of human and animal origin.

scholarly journals Development and Validation of aPneumocystis jiroveciiReal-time Polymerase Chain Reaction Assay for Diagnosis ofPneumocystisPneumonia

2015 ◽  
Vol 26 (5) ◽  
pp. 263-267 ◽  
Author(s):  
Deirdre L Church ◽  
Anshula Ambasta ◽  
Amanda Wilmer ◽  
Holly Williscroft ◽  
Gordon Ritchie ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Pathogenic Fungus ◽  
Immunofluorescence Assay ◽  
Immunocompromised Patients ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Predictive Values ◽  
Sensitivity Specificity

BACKGROUND:Pneumocystis jirovecii(PJ), a pathogenic fungus, causes severe interstitialPneumocystispneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP.METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP.RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%.CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients.

scholarly journals A rapid and automated sample-to-result Candida auris real-time PCR assay for high-throughput testing of surveillance samples with BD MAX™ open system

2019 ◽  
Author(s):  
L. Leach ◽  
A. Russell ◽  
Y. Zhu ◽  
S. Chaturvedi ◽  
V. Chaturvedi
Keyword(s):  
New York ◽  
Real Time ◽  
High Throughput ◽  
Open System ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Pcr Assay ◽  
Candida Auris ◽  
Clinical Sensitivity

ABSTRACTThe multidrug-resistant yeast pathogen Candida auris continues to cause outbreaks and clusters of clinical cases worldwide. Previously, we developed a real-time PCR assay for the detection of C. auris from surveillance samples (Leach et al. JCM. 2018: 56, e01223-17). The assay played a crucial role in the ongoing investigations of C. auris outbreak in New York City. To ease the implementation of the assay in other laboratories, we developed an automated sample-to-result real-time C. auris PCR assay using BD MAX™ open system. We optimized sample extraction at three different temperatures and four incubation periods. Sensitivity was determined using eight pools of patient samples, and specificity was calculated using four clades of C. auris, and closely and distantly related yeasts. Three independent extractions and testing of two patient sample pools in the quadruplicate yielded assay precision. BD MAX™ optimum assay conditions were: DNA extraction at 75°C for 20 min, and the use of PerfeCTa Multiplex qPCR ToughMix. The limit of detection (LOD) of the assay was one C. auris CFU/PCR reaction. We detected all four clades of C. auris without cross-reactivity to other yeasts. Of the 110 patient surveillance samples tested, 50 were positive for C. auris using the BD MAX™ System with 96% clinical sensitivity and 94% accuracy compared to the manual assay. BD MAX™ assay allows high-throughput C. auris screening of 180 surveillance samples in a 12-hour workday.

scholarly journals Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

2021 ◽  
Author(s):  
Sudha Chaturvedi ◽  
Tanya R Victor ◽  
Anuradha Marathe ◽  
Ketevan Sidamonidze ◽  
Kelly L Crucillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Coccidioides Immitis ◽  
Coccidioides Posadasii ◽  
Pcr Assay ◽  
Valley Fever ◽  
Clinical Specimens ◽  
Wide Range

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 129 isolates and three primary specimens as C. posadasii and 23 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the surveillance of coccidioidomycosis.

scholarly journals Rapid detection of phenotypes Bombay sedel and nonsecretor rs200157007 SNP (302C > T) by real-time PCR-based methods

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mikiko Soejima ◽  
Yoshiro Koda
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
Association Studies ◽  
Melting Curve ◽  
Curve Analysis ◽  
Complete Agreement ◽  
Melting Curve Analysis ◽  
Duplex Pcr ◽  
Pcr Assay

AbstractThe sedel allele is one of the nonsecretor alleles (se) of FUT2 generated by an Alu-mediated recombination event and was first found in Indian Bombay phenotype individuals who have anti-H, anti-A, and anti-B antibodies in their serum. As well as anti-A, and anti-B antibodies, anti-H is clinically significant because it causes sever hemolytic transfusion reactions. Like sedel, se302 having a missense single nucleotide polymorphism (SNP), 302C > T, is characteristic of South Asians with a frequency of 10–30%. We developed a real-time PCR melting curve analysis for detection of sedel using a 127-bp amplicon encompassing the breakpoint junction. In addition, by performing duplex PCR by amplifying a 65-bp amplicon of the FUT2 coding region at the same time, we could determine the zygosity of sedel in a single tube. We also developed an Eprobe-mediated PCR assay (Eprobe-PCR) for detection of 302C > T of FUT2. These methods were validated by analyzing 58 Tamils and 54 Sinhalese in Sri Lanka. Both the duplex PCR melting curve analysis for determination of sedel zygosity and the Eprobe-PCR assay for detection of 302C > T exactly determined three genotypes. In addition, the results of the present methods were in complete agreement with those obtained by previously established methods. The two present methods were reliable and seem to be advantageous for large-scale association studies of FUT2 polymorphisms in South Asian populations.

scholarly journals Real-time PCR assay for detection and differentiation of Coccidioides immitis and Coccidioides posadasii from culture and clinical specimens

2021 ◽  
Vol 15 (9) ◽  
pp. e0009765
Author(s):  
Sudha Chaturvedi ◽  
Tanya R. Victor ◽  
Anuradha Marathe ◽  
Ketevan Sidamonidze ◽  
Kelly L. Crucillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Coccidioides Immitis ◽  
Coccidioides Posadasii ◽  
Pcr Assay ◽  
Valley Fever ◽  
Clinical Specimens ◽  
Wide Range

Coccidioidomycosis (Valley Fever) is a pulmonary and systemic fungal disease with increasing incidence and expanding endemic areas. The differentiation of etiologic agents Coccidioides immitis and C. posadasii remains problematic in the clinical laboratories as conventional PCR and satellite typing schemes are not facile. Therefore, we developed Cy5- and FAM-labeled TaqMan-probes for duplex real-time PCR assay for rapid differentiation of C. immitis and C. posadasii from culture and clinical specimens. The RRA2 gene encoding proline-rich antigen 2, specific for Coccidioides genus, was the source for the first set of primers and probe. Coccidioides immitis contig 2.2 (GenBank: AAEC02000002.1) was used to design the second set of primers and probe. The second primers/probe did not amplify the corresponding C. posadasii DNA, because of an 86-bp deletion in the contig. The assay was highly sensitive with limit of detection of 0.1 pg gDNA/PCR reaction, which was equivalent to approximately ten genome copies of C. immitis or C. posadasii. The assay was highly specific with no cross-reactivity to the wide range of fungal and bacterial pathogens. Retrospective analysis of fungal isolates and primary specimens submitted from 1995 to 2020 confirmed 168 isolates and four primary specimens as C. posadasii and 30 isolates as C. immitis from human coccidioidomycosis cases, while all eight primary samples from two animals (rhesus monkey and rhinoceros) were confirmed as C. posadasii. A preliminary analysis of cerebrospinal fluid (CSF) and pleural fluid samples showed positive correlation between serology tests and real-time PCR for two of the 15 samples. The Coccidioides spp. duplex real-time PCR will allow rapid differentiation of C. immitis and C. posadasii from clinical specimens and further augment the treatment and surveillance of coccidioidomycosis.

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