Immunocompetent Infants as a Human Reservoir for Pneumocystis jirovecii: Rapid Screening by Non-Invasive Sampling and Real-Time PCR at the Mitochondria1 Large Subunit rRNA Gene

2003 ◽  
Vol 50 (s1) ◽  
pp. 668-669 ◽  
Author(s):  
ANNE TOTET ◽  
LEILA MELIANI; ◽  
PHILIPPE LACUBE; ◽  
JEAN CLAUDE PAUTARD; ◽  
CHRISTIAN RACCURT ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Subunit ◽  
Pneumocystis Jirovecii ◽  
Rapid Screening ◽  
Rrna Gene ◽  
Non Invasive ◽  
Large Subunit Rrna

scholarly journals 744. Clinical Performance of Real-time PCR in the Diagnosis of Pneumocystis jirovecii Pneumonia compared with Immunofluorescence Assay

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S420-S420
Author(s):  
Cristina Veintimilla ◽  
Ana Alvarez-Uria ◽  
Pablo Martin-Rabadan ◽  
Luis Alcala ◽  
Patricia Muñoz ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Subunit ◽  
Immunofluorescence Assay ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Rrna Gene ◽  
Sample Type ◽  
Sensitivity Specificity ◽  
Pcr Targeting

Abstract Background The laboratory diagnosis of Pneumocystis jirovecii pneumonia (PJP) has been traditionally based on microscopy techniques, which have suboptimal sensitivity and depends on the experience and skills of the microbiologist. Molecular detection assays based in PCR (Polymerase chain reaction) could improve sensitivity. Our aim was to evaluate the utility of real-time PCR in the diagnosis of PJP compared with IFA (Immunofluorescence assay) performed in different respiratory samples of patients with PJP suspicion for routine use in a clinical laboratory setting. Methods From September 2015 to April 2018, we studied by a real-time PCR targeting the large subunit of rRNA gene of P. jirovecii (PJ-PCR RealCycler PJIR kit Progenie Molecular) and Immunofluorescence assay (MONOFLUO P. carinii IFA BioRad) in all respiratory samples received for microbiological diagnosis of PJP. The definite clinical diagnosis of PJP was established by infectious disease physicians considering symptoms, radiological and laboratory findings. Results Overall, 302 samples were included (182 bronchoalveolar lavage, 67 sputum, 53 tracheal aspirates). PJ-PCR was positive in 51 (16.9%) and IFA in 11 (3.6%) of the patients with PJP. There were not IFA positive/PCR negative samples. Sensitivity, specificity, PPV and NPV for IFA were 26% (95%CI 15.9-39.6%), 100% (95%CI 98.5-100%), 100% (95% CI 77.2-100%) and 87.2% (95% CI 82.6-90.6%). Whereas, sensitivity, specificity, PPV and NPV for PCR was 92% (95%CI 81.2-96.8%), 98% (95% CI 95.4-99.2%), 90.2% (95% CI 79.0-95.7%) and 98.4% (95% CI 96.0-99.4%). PJ-PCR had sensitivity > 80% and specificity > 90% in all type of samples included. A definitive diagnosis of PJP was considered in 50 (16.6%) patients, including 4 (1.3%) cases with negative PJ-PCR. Five cases (9.8%) with positive PJ-PCR were considered as colonization. Conclusion P. jirovecii PCR improves the sensitivity and NPV of PJP diagnosis respecting to IFA, regardless of respiratory sample type. Our results suggest that Microbiology laboratories should use PCR techniques to diagnose PJP better than IFA. Disclosures All Authors: No reported disclosures

scholarly journals A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

2022 ◽  
Vol 12 ◽  
Author(s):  
Susana Ruiz-Ruiz ◽  
Carolina A. Ponce ◽  
Nicole Pesantes ◽  
Rebeca Bustamante ◽  
Gianna Gatti ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Large Subunit ◽  
Pcr Amplification ◽  
Surface Protein ◽  
Pneumocystis Jirovecii ◽  
Pcr Assay ◽  
Low Levels ◽  
Large Subunit Rrna

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

scholarly journals Sympodiomyces attinorum sp. nov., a yeast species associated with nests of the leaf-cutting ant Atta sexdens

2004 ◽  
Vol 54 (5) ◽  
pp. 1891-1894 ◽  
Author(s):  
Solange C. Carreiro ◽  
Fernando C. Pagnocca ◽  
Maurício Bacci ◽  
Marc-André Lachance ◽  
Odair C. Bueno ◽  
...  
Keyword(s):  
Yeast Species ◽  
Novel Species ◽  
Large Subunit ◽  
Growth Characteristics ◽  
Rrna Gene ◽  
The Novel ◽  
Atta Sexdens ◽  
Leaf Cutting ◽  
Large Subunit Rrna ◽  
Waste Deposit

Four strains of a novel yeast species were isolated from laboratory nests of the leaf-cutting ant Atta sexdens in Brazil. Three strains were found in older sponges and one was in a waste deposit in the ant nests. Sequencing of the D1/D2 region of the large-subunit rRNA gene showed that the novel species, named Sympodiomyces attinorum sp. nov., is phylogenetically related to Sympodiomyces parvus. Unlike Sympodiomyces parvus, Sympodiomyces attinorum can ferment glucose, assimilate methyl α-d-glucoside, salicin and citrate, and grow at 37 °C, thus enabling these two species to be distinguished. Differentiation from other related species is possible on the basis of other growth characteristics. The type strain of Sympodiomyces attinorum is UNESP-S156T (=CBS 9734T=NRRL Y-27639T).

Quantitative Pneumocystis jirovecii real-time PCR to differentiate disease from colonisation

Pathology ◽  
2021 ◽  
Author(s):  
Shu Jin Tan ◽  
Todd Matthew Pryce ◽  
Erin Judith Haygarth ◽  
Peter Alan Boan
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pneumocystis Jirovecii

scholarly journals Detection of Pneumocystis carinii DNA in Blood Specimens from Human Immunodeficiency Virus-Infected Patients by Nested PCR

1999 ◽  
Vol 37 (1) ◽  
pp. 127-131 ◽  
Author(s):  
Meja Rabodonirina ◽  
Laurent Cotte ◽  
André Boibieux ◽  
Karine Kaiser ◽  
Martine Mayençon ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Whole Blood ◽  
Nested Pcr ◽  
Large Subunit ◽  
Pneumocystis Carinii ◽  
Proteinase K ◽  
Rrna Gene ◽  
Immunodeficiency Virus ◽  
Blood Specimens ◽  
Large Subunit Rrna

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp.hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

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