scholarly journals Detection of Aspergillus DNA by a nested PCR assay is able to improve the diagnosis of invasive aspergillosis in paediatric patients

2009 ◽  
Vol 58 (10) ◽  
pp. 1291-1297 ◽  
Author(s):  
Margit Hummel ◽  
Birgit Spiess ◽  
Julia Roder ◽  
Gregor von Komorowski ◽  
Matthias Dürken ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Invasive Aspergillosis ◽  
Nested Pcr ◽  
Fungal Infections ◽  
High Sensitivity ◽  
Clinical Findings ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Imaging Results ◽  
Adolescent Patients

Fungal infections are a leading cause of morbidity and mortality in severely immunocompromised patients and have been increasing in incidence in recent years. Invasive aspergillosis (IA) is the most common filamentous fungal infection and is, in adults as well as in children, difficult to diagnose. Several PCR assays to detect Aspergillus DNA have been established, but so far, studies on molecular tools for the diagnosis of IA in children are few. We evaluated the results of a nested PCR assay to detect Aspergillus DNA in clinical samples from paediatric and adolescent patients with suspected IA. Blood and non-blood samples from immunocompromised paediatric and adolescent patients with suspected invasive fungal infection were sent for processing Aspergillus PCR to our laboratory. PCR results from consecutive patients from three university children's hospitals investigated between November 2000 and January 2007 were evaluated. Fungal infections were classified according to the EORTC classification on the grounds of clinical findings, microbiology and radio-imaging results. Two hundred and ninety-one samples from 71 patients were investigated for the presence of Aspergillus DNA by our previously described nested PCR assay. Two, 3 and 34 patients had proven, probable and possible IA, respectively. Sensitivity (calculated from proven and probable patients, n=5) and specificity (calculated from patients without IA, n=32) rates of the PCR assay were 80 and 81 %, respectively. Our nested PCR assay was able to detect Aspergillus DNA in blood, cerebrospinal fluid and bronchoalveolar lavage samples from paediatric and adolescent patients with IA with high sensitivity and specificity rates.

scholarly journals Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Jeremy Ratcliff ◽  
Dung Nguyen ◽  
Monique Andersson ◽  
Peter Simmonds
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Nested Pcr ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Copy Detection ◽  
Accurate Identification ◽  
Laboratory Equipment ◽  
Rna Transcripts

ABSTRACTAccurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

Development of a high-sensitivity nested PCR assay for the detection of Clostridium piliforme in clinical samples

2010 ◽  
Vol 185 (2) ◽  
pp. 222-224 ◽  
Author(s):  
Alisson Niepceron ◽  
Dominique Licois
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Clostridium Piliforme

scholarly journals Itraconazole Oral Solution for Primary Prophylaxis of Fungal Infections in Patients with Hematological Malignancy and Profound Neutropenia: a Randomized, Double-Blind, Double-Placebo, Multicenter Trial Comparing Itraconazole and Amphotericin B

2000 ◽  
Vol 44 (7) ◽  
pp. 1887-1893 ◽  
Author(s):  
J. L. Harousseau ◽  
A. W. Dekker ◽  
A. Stamatoullas-Bastard ◽  
A. Fassas ◽  
W. Linkesch ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Invasive Aspergillosis ◽  
Amphotericin B ◽  
Hematological Malignancy ◽  
Fungal Infections ◽  
Treated Group ◽  
Multicenter Trial ◽  
Oral Solution ◽  
Double Blind ◽  
Systemic Fungal Infection

ABSTRACT Systemic and superficial fungal infections are a major problem among immunocompromised patients with hematological malignancy. A double-blind, double-placebo, randomized, multicenter trial was performed to compare the efficacy and safety of itraconazole oral solution (2.5 mg/kg of body weight twice a day) with amphotericin B capsules (500 mg orally four times a day) for prophylaxis of systemic and superficial fungal infection. Prophylactic treatment was initiated on the first day of chemotherapy and was continued until the end of the neutropenic period (>0.5 × 109 neutrophils/liter) or up to a maximum of 3 days following the end of neutropenia, unless a systemic fungal infection was documented or suspected. The maximum treatment duration was 56 days. In the intent-to-treat population, invasive aspergillosis was noted in 5 (1.8%) of the 281 patients assigned to itraconazole oral solution and in 9 (3.3%) of the 276 patients assigned to oral amphotericin B; of these, 1 and 4 patients died, respectively. Proven systemic fungal infection (including invasive aspergillosis) occurred in 8 patients (2.8%) who received itraconazole, compared with 13 (4.7%) who received oral amphotericin B. Itraconazole significantly reduced the incidence of superficial fungal infections as compared to oral amphotericin B (2 [1%] versus 13 [5%]; P = 0.004). Although the incidences of suspected fungal infection (including fever of unknown origin) were not different between the groups, fewer patients were administered intravenous systemic antifungals (mainly intravenous amphotericin B) in the group receiving itraconazole than in the group receiving oral amphotericin B (114 [41%] versus 132 [48%];P = 0.066). Adequate plasma itraconazole levels were achieved in about 80% of the patients from 1 week after the start of treatment. In both groups, the trial medication was safe and well tolerated. Prophylactic administration of itraconazole oral solution significantly reduces superficial fungal infection in patients with hematological malignancies and neutropenia. The incidence of proven systemic fungal infections, the number of deaths due to deep fungal infections, and the use of systemic antifungals tended to be lower in the itraconazole-treated group than in the amphotericin B-treated group, without statistical significance. Itraconazole oral solution is a broad-spectrum systemic antifungal agent with prophylactic activity in neutropenic patients, especially for those at high risk of prolonged neutropenia.

scholarly journals Validation and Clinical Application of a Molecular Method for Identification of Histoplasma capsulatum in Human Specimens in Colombia, South America

2009 ◽  
Vol 17 (1) ◽  
pp. 62-67 ◽  
Author(s):  
Cesar Muñoz ◽  
Beatriz L. Gómez ◽  
Angela Tobón ◽  
Karen Arango ◽  
Angela Restrepo ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Predictive Value ◽  
Histoplasma Capsulatum ◽  
Clinical Samples ◽  
Healthy Individuals ◽  
Molecular Method ◽  
Pcr Assay ◽  
Molecular Assays ◽  
Panfungal Pcr ◽  
Negative Controls

ABSTRACT The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the H istoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented ≥98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.

scholarly journals Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens

1998 ◽  
Vol 36 (5) ◽  
pp. 1169-1175 ◽  
Author(s):  
Jo-Anne Van Burik ◽  
David Myerson ◽  
Randall W. Schreckhise ◽  
Raleigh A. Bowden
Keyword(s):  
Fungal Infection ◽  
Whole Blood ◽  
Limit Of Detection ◽  
Fungal Infections ◽  
Small Subunit ◽  
Rrna Gene ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Blood Specimens ◽  
Panfungal Pcr

A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient’s blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection.

scholarly journals Diagnostic potential of nested PCR, galactomannan EIA, and Beta-D-glucan for invasive aspergillosis in pediatric patients

2012 ◽  
Vol 6 (04) ◽  
pp. 352-357 ◽  
Author(s):  
Parisa Badiee ◽  
Abdolvahab Alborzi ◽  
Mahammad Karimi ◽  
Bahman Pourabbas ◽  
Pedram Haddadi ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Nested Pcr ◽  
Pediatric Patients ◽  
Clinical Samples ◽  
Predictive Values ◽  
Blood Samples ◽  
Diagnostic Potential ◽  
Increased Risk ◽  
Galactomannan Antigen ◽  
Noninvasive Tests

Introduction: Limited specific data and investigations are available for invasive aspergillosis (IA) in pediatric patients. We evaluated the diagnostic potential of three noninvasive tests including the Platelia Aspergillus EIA kit for using galactomannan antigen, (1,3)-β-D-glucan Detection Reagent Kit, and nested-PCR for Aspergillus DNA in sera. We evaluated the diagnostic potential of three noninvasive tests including EIA for galactomannan antigen  (Platelia Aspergillus), nested  PCR assay for Aspergillus DNA and test for  (1→3)-β-D-glucan (Glucatell assay Kit). Methodology: All pediatric patients treated at the hematology/oncology unit who were at increased risk of developing invasive aspergillosis were enrolled. Clinical samples were examined for Aspergillus infections by mycological methods. Serial blood samples were collected twice weekly and evaluated by noninvasive tests. Results: We analyzed 230 consecutive blood samples from 62 pediatric patients. The incidence rate of invasive aspergillosis in the patients was found to be 27.4%, and the etiologic agents were Aspergillus flavus, Aspergillus fumigatus, and Aspergillus spp.  The sensitivity, specificity, positive and negative predictive values, and likelihood ratios for positive and negative results of galactomannan in patients with proven and probable IA were 90%, 92%, 81.8%, 96%, 11.25, and 0.1; for beta-D-glucan they were 50%, 46%, 26%, 70.6%, 0.9, 0.9; and for nested-PCR they were 80%, 96.2%, 88.9%, 92.6%, 21, and 0.2, respectively. Conclusions: The conventional methods are not able to detect IA, due to the lack of valid and proper sampling. Galactomannan and nested-PCR tests in serum, with enough accuracy and reliability, can serve as noninvasive methods for the detection of IA in pediatric patients. However, the beta-D-glucan test cannot serve as an efficient diagnostic tool in those with hematologic disorders. 

scholarly journals Toward point-of-care diagnostics of Candida auris

2020 ◽  
Author(s):  
Geoffrey Mulberry ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi ◽  
Brian N. Kim
Keyword(s):  
New York ◽  
Real Time ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

Whole-genome sequence-guided PCR for the rapid identification of thePseudomonas aeruginosa ST175 high-risk clone directly from clinical samples

2020 ◽  
Author(s):  
Gabriel Cabot ◽  
Paula Lara-Esbrí ◽  
Xavier Mulet ◽  
Antonio Oliver
Keyword(s):  
High Risk ◽  
Sensitivity And Specificity ◽  
Genome Sequence ◽  
Treatment Strategies ◽  
High Sensitivity ◽  
Whole Genome Sequence ◽  
Clinical Samples ◽  
Whole Genome ◽  
Pcr Assay ◽  
Pcr Targeting

Abstract Objectives Pseudomonas aeruginosa frequently show MDR/XDR profiles, which are associated with worldwide-disseminated high-risk clones (HRCs). We developed a PCR assay for the detection in clinical samples of ST175, an HRC that is widespread in European countries. Methods The whole-genome sequence was obtained for one ST175 isolate using a PacBio RSII sequencer. Reads from multiple isolates belonging to ST175 and the PAO1 reference strain were mapped against the ST175 genome to identify potentially specific regions. Once curated, using the BLAST database to search for the presence of those regions in any other organism, we designed a specific PCR for the detection of ST175. Results Assembly of the ST175 PacBio-sequenced genome resulted in three contigs with a total length of 7 087 985 bases, encoding 6566 coding sequences. Specific regions for ST175 genomes were detected and a PCR targeting a 318 bp fragment located within a 3177 bp ORF coding for a putative reverse transcriptase was designed. The PCR test was first evaluatedin silico against 229 XDRP. aeruginosa genomes (73 ST175) from two multicentre studies, yielding 100% sensitivity and specificity. Then, the PCR was evaluatedin vitro in 25 isolates (12 ST175) and in 120 clinical samples (30 urine samples, 30 blood cultures, 30 sputum samples and 30 rectal swabs) of which 10% contained ST175, yielding again 100% sensitivity and specificity. Conclusions The PCR assay developed, showing high sensitivity and specificity for the detection of the ST175 HRC directly from clinical samples, could become a useful tool for guiding infection control and treatment strategies in areas with a high prevalence of this clone.

scholarly journals Detection of Sporothrix schenckii in Clinical Samples by a Nested PCR Assay

2003 ◽  
Vol 41 (4) ◽  
pp. 1414-1418 ◽  
Author(s):  
S. Hu ◽  
W.-H. Chung ◽  
S.-I. Hung ◽  
H.-C. Ho ◽  
Z.-W. Wang ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Sporothrix Schenckii ◽  
Clinical Samples ◽  
Pcr Assay
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