scholarly journals Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients

Thorax ◽  
2007 ◽  
Vol 63 (2) ◽  
pp. 154-159 ◽  
Author(s):  
J F Huggett ◽  
M S Taylor ◽  
G Kocjan ◽  
H E Evans ◽  
S Morris-Jones ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Real Time Pcr ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Pneumocystis Jirovecii ◽  
Pcr Assay

scholarly journals 252. Development and Evaluation of a Novel MultiCode Real-Time PCR Assay for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid and Induced Sputum

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S141-S141
Author(s):  
Baoming Liu ◽  
Karen C Carroll ◽  
Sean Zhang
Keyword(s):  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Induced Sputum ◽  
Pneumocystis Jirovecii ◽  
Mechanical Grinding ◽  
Pcr Assay

Abstract Background Pneumocystis jirovecii is a medically important fungal pathogen responsible for opportunistic infections in immunocompromised hosts with high morbidity and mortality. Compared with standard microscopy based assays, home-brew nucleic acid amplification tests (NAAT) have emerged as sensitive tools for the diagnosis of P. jirovecii pneumonia, but their sensitivities vary depending upon selected genetic targets. Recent studies suggest that the mitochondrial small subunit (mtSSU) is a better NAAT target given its higher copy number and stable expression in the disease process. We aimed to develop and evaluate a mtSSU-targeted MultiCode real-time PCR assay that incorporates a sample processing control (SPC) and enables detection of P. jirovecii in bronchoalveolar lavage fluid (BALF) and induced sputum. Methods Firstly, we compared manual DNA extraction using Zymo Quick DNA kit with automated extraction using the NucliSENS easyMAG system after sample pretreatment with either FastPrep mechanical grinding or vortex-based bead beating. We then determined the mouse hepatitis virus SPC (Luminex) spike-in amount, and optimized the PCR conditions on the ABI 7500 PCR system. A new Pneumocystis mtSSU run control was generated by cloning and transforming mtSSU gene into a genetically engineered E. coli strain, and quantified with a home-brew quantitative TaqMan PCR. Lastly, the performance characteristics of the MultiCode PCR assay were determined. Results Mechanical grinding of BALF or sputum before the easyMAG based extraction was better than the other extraction protocols as evidenced by lower CT of mtSSU or SPC. Diluted SPC added to samples before DNA extraction made its CT within 31–34. With the mtSSU run control, the limit of detection of the new assay was 80 copies/mL. No cross-reactivity was found with 9 respiratory viruses, 8 bacteria or 11 fungi. The assay has high reproducibility for three-day detection of the same sample aliquots for mtSSU (CT: 30.0–30.3; CV%: 0.5–1.6) and SPC (CT: 32.1–32.2; CV%: 0.8–2.4). Conclusion We developed a novel MultiCode real-time PCR assay for detection of P. jirovecii in BALF and sputum, which demonstrated high analytical sensitivity, specificity and reproducibility and warrants further clinical validation. Disclosures All authors: No reported disclosures.

scholarly journals Inter-laboratory comparison of three different real-time PCR assays for the detection of Pneumocystis jiroveci in bronchoalveolar lavage fluid samples

2006 ◽  
Vol 55 (9) ◽  
pp. 1229-1235 ◽  
Author(s):  
Catharina F. M. Linssen ◽  
Jan A. Jacobs ◽  
Pieter Beckers ◽  
Kate E. Templeton ◽  
Judith Bakkers ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Real Time Pcr ◽  
Bronchoalveolar Lavage Fluid ◽  
Medical Center ◽  
Tertiary Care ◽  
Lavage Fluid ◽  
The Other ◽  
Pneumocystis Jiroveci ◽  
Pcr Assays

Pneumocystis jiroveci pneumonia (PCP) is an opportunistic infection affecting immunocompromised patients. While conventional diagnosis of PCP by microscopy is cumbersome, the use of PCR to diagnose PCP has great potential. Nevertheless, inter-laboratory validation and standardization of PCR assays is lacking. The aim of this study was to evaluate the inter-laboratory agreement of three independently developed real-time PCR assays for the detection of P. jiroveci in bronchoalveolar lavage fluid samples. Therefore, 124 samples were collected in three tertiary care laboratories (Leiden University Medical Center, Maastricht Infection Center and Radboud University Nijmegen Medical Centre) and were tested by both microscopy and real-time PCR. Of 41 samples positive for P. jiroveci by microscopy, 40 were positive in all three PCR assays. The remaining sample was positive in a single assay only. Out of 83 microscopy-negative samples, 69 were negative in all three PCR assays. The other 14 samples were found positive, either in all three assays (n=5), in two (n=2) or in one of the assays (n=7). The data demonstrate high inter-laboratory agreement among real-time PCR assays for the detection of P. jiroveci.

Real-time PCR in Bronchoalveolar Lavage Fluid in the Sputum Smear-Negative Patients Suspected of Pulmonary Tuberculosis by Chest CT

CHEST Journal ◽  
2010 ◽  
Vol 138 (4) ◽  
pp. 673A
Author(s):  
Yee Hyung Kim ◽  
Sohee Park ◽  
Cheon Woong Choi ◽  
Jee-Hong Yoo ◽  
Hong Mo Kang ◽  
...  
Keyword(s):  
Pulmonary Tuberculosis ◽  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Real Time Pcr ◽  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Chest Ct ◽  
Sputum Smear ◽  
Smear Negative

scholarly journals 754. Performance Characterization of a Real Time PCR Assay for Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid and Sputum

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S424-S425
Author(s):  
Drew T Bell ◽  
Jeremy Koehlinger ◽  
Bryan H Schmitt
Keyword(s):  
Bronchoalveolar Lavage ◽  
Real Time ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Dna Amplification ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Acid Extraction ◽  
Pcr Assay ◽  
Calcofluor White

Abstract Background Pneumocystis jirovecii pneumonia (PJP) affects immunocompromised patients and contributes significantly to mortality. Outcomes depend on early treatment, making timely and accurate diagnosis critical. Typically, PJP diagnosis is through identification of trophozoite or cyst forms in bronchoalveolar lavage (BAL) fluid or sputum, a labor-intensive and insensitive process. Options for more accessible and sensitive molecular detection are limited. It is known that patients may be colonized, which can cast doubt on the clinical significance of low levels of DNA amplification in qualitative result reporting. In this study, we describe a real time (rt) PCR assay utilizing analyte specific reagent primers targeting the mtLSU gene of P. jirovecii and correlate amplification with morphological PJP identification. Methods IUHPL Clinical Microbiology assessed sputum or BAL fluid from 109 patients with clinical concern for PJP microscopically via fungal stains (GMS, calcofluor white). Comparative rtPCR was conducted as follows. First, 2µL of residual specimen or control were mixed with an 8µL combination of rtPCR mastermix, control DNA, and primer pairs (Simplexa). No nucleic acid extraction was performed. Real time PCR was executed and analyzed on the LIAISON MDX (DiaSorin) platform. Qualitative amplification results and cycle threshold (CT) values were correlated with microscopic methods to establish performance. Chart review was performed to assess the clinical impact of this assay. Results P. jirovecii was microscopically detected in 26% (29/109) of samples, while 31.1% (34/109) exhibited amplification by rtPCR. Agreement between the two methods was 95.4%; rtPCR demonstrated 100% sensitivity and 93.8% specificity in comparison. Conclusion Our results indicate that this assay has exceptional negative predictive value (100%), and therefore may be valuable as a screening test. Considering this data alone, the positive predictive value is lower (85.3%). Further examination of the data, however, revealed that 80% (4/5) of discrepant results demonstrated CT values of >34, while the highest CT for a microscopically positive sample was 31.2. Further clinical correlation may establish a CT cutoff that will reduce false positive and potentially clinically insignificant cases. Disclosures All Authors: No reported disclosures

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