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

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.

Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia

2008 ◽  
Vol 75 (2) ◽  
pp. 258-261 ◽  
Author(s):  
Judith Fillaux ◽  
Sonia Malvy ◽  
Muriel Alvarez ◽  
Richard Fabre ◽  
Sophie Cassaing ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pneumocystis Jirovecii ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Pcr Assay

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

2021 ◽  
Vol 156 (Supplement_1) ◽  
pp. S140-S140
Author(s):  
A Kalam
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Low Income ◽  
Water Samples ◽  
Real Time Pcr ◽  
Tap Water ◽  
Watery Diarrhea ◽  
Acid Extraction ◽  
Pcr Assay ◽  
Viral Contamination

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

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