scholarly journals Direct Molecular Detection and Genotyping ofBorrelia burgdorferi Sensu Latoin Cerebrospinal Fluid of Children with Lyme Neuroborreliosis

2018 ◽  
Vol 56 (5) ◽  
Author(s):  
Bjørn Barstad ◽  
Hanne Quarsten ◽  
Dag Tveitnes ◽  
Sølvi Noraas ◽  
Ingvild S. Ask ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Real Time ◽  
Short Duration ◽  
Real Time Pcr ◽  
Diagnostic Value ◽  
Lyme Neuroborreliosis ◽  
Content Type ◽  
Causative Agents ◽  
Csf Pleocytosis ◽  
Pcr Assays

ABSTRACTThe current diagnostic marker of Lyme neuroborreliosis (LNB), theBorrelia burgdorferisensu latoantibody index (AI) in the cerebrospinal fluid (CSF), has insufficient sensitivity in the early phase of LNB. We aimed to elucidate the diagnostic value of PCR forB. burgdorferisensu latoin CSF from children with symptoms suggestive of LNB and to exploreB. burgdorferisensu latogenotypes associated with LNB in children. Children were prospectively included in predefined groups with a high or low likelihood of LNB based on diagnostic guidelines (LNB symptoms, CSF pleocytosis, andB. burgdorferisensu latoantibodies) or the detection of other causative agents. CSF samples were analyzed by twoB. burgdorferisensu lato-specific real-time PCR assays and, ifB. burgdorferisensu latoDNA was detected, were further analyzed by five singleplex real-time PCR assays for genotype determination. For children diagnosed as LNB patients (58 confirmed and 18 probable) (n= 76) or non-LNB controls (n= 28), the sensitivity and specificity of PCR forB. burgdorferisensu latoin CSF were 46% and 100%, respectively.B. burgdorferisensu latoDNA was detected in 26/58 (45%) children with AI-positive LNB and in 7/12 (58%) children with AI-negative LNB and symptoms of short duration. Among 36 children with detectableB. burgdorferisensu latoDNA, genotyping indicatedBorrelia garinii(n= 27) and non-B. garinii(n= 1) genotypes, while 8 samples remained untyped. Children with LNB caused byB. gariniidid not have a distinct clinical picture. The rate of detection ofB. burgdorferisensu latoDNA in the CSF of children with LNB was higher than that reported previously. PCR forB. burgdorferisensu latocould be a useful supplemental diagnostic tool in unconfirmed LNB cases with symptoms of short duration.B. gariniiwas the predominant genotype in children with LNB.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

2012 ◽  
Vol 78 (10) ◽  
pp. 3630-3637 ◽  
Author(s):  
Karol Krak ◽  
Martina Janoušková ◽  
Petra Caklová ◽  
Miroslav Vosátka ◽  
Helena Štorchová
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Glomus Intraradices ◽  
Large Subunit ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Content Type ◽  
Copy Numbers ◽  
Pcr Assays

ABSTRACTReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates ofGlomus intraradicessensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

scholarly journals Triplex Real-Time PCR without DNA Extraction for the Monitoring of Meningococcal Disease

Diagnostics ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 58 ◽  
Author(s):  
Melissa Whaley ◽  
Laurel Jenkins ◽  
Fang Hu ◽  
Alexander Chen ◽  
Seydou Diarra ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Real Time ◽  
Dna Extraction ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection Range ◽  
Clinical Specimens ◽  
Large Numbers ◽  
Pcr Assays

Detection of Neisseria meningitidis has become less time- and resource-intensive with a monoplex direct real-time PCR (drt-PCR) to amplify genes from clinical specimens without DNA extraction. To further improve efficiency, we evaluated two triplex drt-PCR assays for the detection of meningococcal serogroups AWX and BCY. The sensitivity and specificity of the triplex assays were assessed using 228 cerebrospinal fluid (CSF) specimens from meningitis patients and compared to the monoplex for six serogroups. The lower limit of detection range for six serogroup-specific drt-PCR assays was 178–5264 CFU/mL by monoplex and 68–2221 CFU/mL by triplex. The triplex and monoplex showed 100% agreement for six serogroups and the triplex assays achieved similar sensitivity and specificity estimates as the monoplex drt-PCR assays. Our triplex method reduces the time and cost of processing CSF specimens by characterizing six serogroups with only two assays, which is particularly important for testing large numbers of specimens for N. meningitidis surveillance.

scholarly journals Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris

2017 ◽  
Vol 55 (8) ◽  
pp. 2445-2452 ◽  
Author(s):  
Milena Kordalewska ◽  
Yanan Zhao ◽  
Shawn R. Lockhart ◽  
Anuradha Chowdhary ◽  
Indira Berrio ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Related Species ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Invasive Infections ◽  
Candida Lusitaniae ◽  
Pcr Assays ◽  
Phenotypic Tests

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae . Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species.

scholarly journals Quantification of Human Fecal Bifidobacterium Species by Use of Quantitative Real-Time PCR Analysis Targeting thegroELGene

2012 ◽  
Vol 78 (8) ◽  
pp. 2613-2622 ◽  
Author(s):  
Jana Junick ◽  
Michael Blaut
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Cell Number ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Content Type ◽  
Pcr Assays

ABSTRACTQuantitative real-time PCR assays targeting thegroELgene for the specific enumeration of 12 human fecalBifidobacteriumspecies were developed. The housekeeping genegroEL(HSP60in eukaryotes) was used as a discriminative marker for the differentiation ofBifidobacterium adolescentis,B. angulatum,B. animalis,B. bifidum,B. breve,B. catenulatum,B. dentium,B. gallicum,B. longum,B. pseudocatenulatum,B. pseudolongum, andB. thermophilum. The bifidobacterial chromosome contains a single copy of thegroELgene, allowing the determination of the cell number by quantification of thegroELcopy number. Real-time PCR assays were validated by comparing fecal samples spiked with known numbers of a givenBifidobacteriumspecies. Independent of theBifidobacteriumspecies tested, the proportion ofgroELcopies recovered from fecal samples spiked with 5 to 9 log10cells/g feces was approximately 50%. The quantification limit was 5 to 6 log10groELcopies/g feces. The interassay variability was less than 10%, and variability between different DNA extractions was less than 23%. The method developed was applied to fecal samples from healthy adults and full-term breast-fed infants. Bifidobacterial diversity in both adults and infants was low, with mostly ≤3Bifidobacteriumspecies andB. longumfrequently detected. The predominant species in infant and adult fecal samples wereB. breveandB. adolescentis, respectively. It was possible to distinguishB. catenulatumandB. pseudocatenulatum. We conclude that thegroELgene is a suitable molecular marker for the specific and accurate quantification of human fecalBifidobacteriumspecies by real-time PCR.

scholarly journals Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

2013 ◽  
Vol 80 (3) ◽  
pp. 1177-1184 ◽  
Author(s):  
Delphine Bibbal ◽  
Estelle Loukiadis ◽  
Monique Kérourédan ◽  
Carine Peytavin de Garam ◽  
Franck Ferré ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Specific Detection ◽  
Content Type ◽  
E Coli ◽  
Cattle Feces ◽  
Pcr Assays

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targetedeaesubtypes. The simultaneous presence ofstx,eae, and one of the five O group markers was found in 58.0% of the samples, and the five targetedstxpluseaeplus O genetic combinations were detected 143 times. However, taking into consideration the association betweeneaesubtypes and O group markers, the resultingstxpluseaesubtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22E. colistrains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive forstx,eaeand an O group marker, but that were negative for the correspondingeaesubtype, were successful. Characterization of the 24E. coliisolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenicE. coli(aEPEC). Finally, the more discriminatingeaesubtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

scholarly journals Detection and Tracking of a Novel Genetically Tagged Biological Simulant in the Environment

2012 ◽  
Vol 78 (23) ◽  
pp. 8281-8288 ◽  
Author(s):  
Peter A. Emanuel ◽  
Patricia E. Buckley ◽  
Tiffany A. Sutton ◽  
Jason M. Edmonds ◽  
Andrew M. Bailey ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Test Material ◽  
Wild Type ◽  
Lidar System ◽  
Content Type ◽  
Detection And Tracking ◽  
Background Material ◽  
Pcr Assays ◽  
Aberdeen Proving Ground

ABSTRACTA variant ofBacillus thuringiensissubsp.kurstakicontaining a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material. In this study, we demonstrate the utility of the genetically “barcoded” simulant in a controlled indoor setting and in an outdoor release. In an ambient breeze tunnel test, spores deposited on tiles were reaerosolized and detected by real-time PCR at distances of 30 m from the point of deposition. Real-time PCR signals were inversely correlated with distance from the seeded tiles. An outdoor release of powdered spore simulant at Aberdeen Proving Ground, Edgewood, MD, was monitored from a distance by a light detection and ranging (LIDAR) laser. Over a 2-week period, an array of air sampling units collected samples were analyzed for the presence of viable spores and using barcode-specific real-time PCR assays. BarcodedB. thuringiensissubsp.kurstakispores were unambiguously identified on the day of the release, and viable material was recovered in a pattern consistent with the cloud track predicted by prevailing winds and by data tracks provided by the LIDAR system. Finally, the real-time PCR assays successfully differentiated barcodedB. thuringiensissubsp.kurstakispores from wild-type spores under field conditions.

scholarly journals Pathogen Identification by Multiplex LightMix Real-Time PCR Assay in Patients with Meningitis and Culture-Negative Cerebrospinal Fluid Specimens

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Karoline Wagner ◽  
Burkard Springer ◽  
Valeria P. Pires ◽  
Peter M. Keller
Keyword(s):  
Cerebrospinal Fluid ◽  
Bacterial Meningitis ◽  
Real Time ◽  
Antimicrobial Therapy ◽  
Real Time Pcr ◽  
Bacterial Identification ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Culture Negative

ABSTRACT Acute bacterial meningitis is a medical emergency, and delays in initiating effective antimicrobial therapy result in increased morbidity and mortality. Culture-based methods, thus far considered the “gold standard” for identifying bacterial microorganisms, require 24 to 48 h to provide a diagnosis. In addition, antimicrobial therapy is often started prior to clinical sample collection, thereby decreasing the probability of confirming the bacterial pathogen by culture-based methods. To enable a fast and accurate detection of the most important bacterial pathogens causing meningitis, namely, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Streptococcus agalactiae, and Listeria monocytogenes, we evaluated a commercially available multiplex LightMix real-time PCR (RT-PCR) in 220 cerebrospinal fluid (CSF) specimens. The majority of CSF samples were collected by lumbar puncture, but we also included some CSF samples from patients with symptoms of meningitis from the neurology department that were recovered from shunts. CSF samples were analyzed by multiplex RT-PCR enabling a first diagnosis within a few hours after sample arrival at our institute. In contrast, bacterial identification took between 24 and 48 h by culture. Overall, a high agreement of bacterial identification between culture and multiplex RT-PCR was observed (99%). Moreover, multiplex RT-PCR enabled the detection of pathogens, S. pneumoniae (n = 2), S. agalactiae (n = 1), and N. meningitidis (n = 1), in four culture-negative samples. As a complement to classical bacteriological CSF culture, the LightMix RT-PCR assay proved to be valuable by improving the rapidity and accuracy of the diagnosis of bacterial meningitis.

scholarly journals Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia

2015 ◽  
Vol 54 (3) ◽  
pp. 625-630 ◽  
Author(s):  
Milène Sasso ◽  
Elsa Chastang-Dumas ◽  
Sophie Bastide ◽  
Sandrine Alonso ◽  
Catherine Lechiche ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Essential Element ◽  
Laboratory Diagnosis ◽  
Final Diagnosis ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Content Type ◽  
Two Samples ◽  
Pcr Assays

Pneumonia due toPneumocystis jirovecii(PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258–261, doi:http://dx.doi.org/10.1016/j.mimet.2008.06.009), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category,Pneumocystis jiroveciiDNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detectPneumocystis jiroveciiDNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden.

scholarly journals Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

2014 ◽  
Vol 53 (2) ◽  
pp. 618-625 ◽  
Author(s):  
Lalitha Gade ◽  
Dale E. Grgurich ◽  
Thomas M. Kerkering ◽  
Mary E. Brandt ◽  
Anastasia P. Litvintseva
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Response To Treatment ◽  
Pcr Assay ◽  
Content Type ◽  
The Real ◽  
Fungal Meningitis ◽  
Exserohilum Rostratum ◽  
Fungal Dna ◽  
Pcr Assays

Exserohilum rostratumwas the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range andE. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection ofE. rostratumin body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive forE. rostratumto 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data.

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