Development and Evaluation of a Real-Time PCR Assay for Detection of Pneumocystis jirovecii on the Fully Automated BD MAX Platform

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.

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

Thorax ◽

10.1136/thx.2007.081687 ◽

2007 ◽

Vol 63 (2) ◽

pp. 154-159 ◽

Cited By ~ 78

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

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

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.944 ◽

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

Frontiers in Microbiology ◽

10.3389/fmicb.2021.787554 ◽

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.

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