Development of a Real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform

Abstract

Pneumocystis pneumonia (PCP) is an opportunistic infection caused by the fungus Pneumocystis jirovecii. Infection with P. jirovecii can result in serious illness in patients with a weakened immune system, and can lead to death if it is not properly diagnosed and treated. Direct detection of P. jirovecii in lower respiratory tract specimens such as bronchoalveolar lavage (BAL) is preferred for rapid diagnosis, a laboratory service currently not available locally. We report here the development of a diagnostic real-time Polymerase Chain Reaction (PCR) assay using BAL specimens to detect P. jirovecii. By targeting the multi-copy mitochondrial large subunit ribosomal RNA gene (mtLSU rRNA) of P. jirovecii, assay sensitivity is increased. Primer pairs were designed to include a fluorescent reporter dye-labeled primer with a unique MultiCode® base pair isoC on the 5’end and one unlabeled primer. The performance characteristics were determined on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. The cassette contains the reagents needed to perform all of the steps including extraction, purification, amplification, and detection, plus a sample processing control. Accuracy, precision, sensitivity, specificity and stability studies were conducted to validate the assay to meet CLIA requirements. The analytical sensitivity was 89.1%, and the analytical specificity was 100%. The assay could reliably detect 200 organisms/ mL, crossing thresholds (Ct) and melt temperatures (Tm) were consistent, and no cross-reactivity was observed with other pathogens known to cause respiratory infections. The results demonstrated that these primers are specific to Pneumocystis jirovecii. The real-time PCR method using the ARIES® system allowed for rapid and sensitive detection of Pneumocystis pneumonia infections with P. jirovecii using clinical respiratory specimens. DOI: 10.18297/jri/vol3/iss1/5 Received Date: November 26, 2018 Accepted Date: December 20, 2018 https://ir.library.louisville.edu/jri/vol3/iss1/ Affiliations: 1Division of Infectious Diseases, University of Louisville This original article is brought to you for free and open access by ThinkIR: The University of Louisville’s Institutional Repository. It has been accepted for inclusion in The University of Louisville Journal of Respiratory Infections by an authorized editor of ThinkIR. For more information, please contact thinkir@louisville. edu. Recommended Citation: Marimuthu, Subathra; Ghosh, Kuldeep; and Wolf, Leslie A (2019) “Development of a Realtime PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform,” The University of Louisville Journal of Respiratory Infections: Vol. 3 : Iss. 1, Article 5. *Correspondence To: Leslie A. Wolf, PhD Work Address: Infectious Diseases Laboratory, Division of Infectious Diseases, Department of Medicine, University of Louisville, Louisville, Kentucky 40292 USA Work Email: lawolf07@louisville.edu ORIGINAL RESEARCH Copyright: © 2019 The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Materials and Methods Specimens Archived, de-identified PCP positive and negative clinical specimens were provided by Dr. Scott McClellan, University of Michigan, in order to set assay parameters. These specimens were deemed exempt by the University of Michigan Institutional Review Board (S. McClellan, personal communication). Residual BAL specimens from consenting patients were used for analytical sensitivity, accuracy, precision, stability and specificity studies. Residual BAL specimens were determined to be negative for P. jirovecii using this PCR method prior to spiking with known concentrations of P. jirovecii DNA. Briefly, specimen processing was done in Bertin tubes (Precellys Lysis Kit, Reference number KT03961-1-006.2, Bertin Corp, Rockville, MD). First, 400 μl of BAL sample were added to the Bertin tube with 400 μL of AL buffer (QIAGEN Catalog number: 19075, Germantown, MS). The Bertin tube was placed on a vortex adaptor and shaken at 10,000 rpm for 5 minutes to disrupt cells. After incubating at room temperature for 10 minutes, the tubes were centrifuged for 2 minutes at 14,000 rpm. Finally, 400 μL of supernatant was added to tubes containing 5 μL of carrier RNA, vortexed, then 405 μL of sample with carrier RNA was used for the assay. Organisms Fungal isolates were provided by Dr. Alan Junkins, Norton Healthcare, Louisville, KY. Eleven different fungi were spiked into negative BAL specimens and tested to assess cross-reactivity as follows: Candida dubliniensis, Candida parapsilosis, Candida glabrata, Candida krusei, Candida tropicalis, Cryptococcus neoformans, Saccharomyces cerevisiae, Histoplasma capsulatum (yeast form), Aspergillus fumigatus, Aspergillus versicolor and Aspergillus niger. Streptococcus pneumoniae isolates from the University of Louisville Infectious Diseases Laboratory were tested in the same way to assess cross-reactivity. A single colony was selected from freshly subcultured plates using a sterile calibrated loop; the loop was used to inoculate BAL from patient specimens previously determined to be negative for PCP processed as described in the specimen section. College of American Pathologists (CAP) proficiency testing samples (archived IDR-A and IDR-B from 2018 events) containing numerous respiratory viruses and bacteria were used to assess cross-reactivity. For that purpose, 200 μL of proficiency test samples known to contain the following were spiked into negative BAL specimens: Coxsackie virus A9, Chlamydophila pneumoniae strain CWL 029, Influenza A California 07/2009 (H1N1), Bordetella pertussis A639, Influenza B Massachusetts 2/2012, Coronavirus NL63, Parainfluenza Type 2, RSV Type B CH93(18)-18, Bordetella parapertussis A747, Parainfluenza virus Type 4, Echovirus 30 (E-30), Influenza B Strain Brisbane 60/2008, Parainfluenza Type 3, Adenovirus Type 21, Mycoplasma pneumoniae M129, Influenza A strain New York 18/2009 (H1N1), and Human metapneumovirus B2. Also, previously characterized bacterial stocks from the University of Louisville Infectious Diseases Laboratory were tested to assess cross-reactivity [7]. Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae from each bacterial stock were spiked into BAL, using 160 μL of 1x105 CFU/ mL stock as input. Control Materials Due to the limited number of clinical samples available for testing, recombinant P. jirovecii positive controls were purchased from Zeptometrix (NATtrol P. jirovecii Recombinant External Run Control, Catalog number 320679, Buffalo, NY) and EXACT Diagnostics (custom Pneumocystis jirovecii run control, Reference number PCPRC, Fort Worth, TX). Reagents ARIES® MultiCode® DNA ReadyMix® tubes (Part number: 3697, Luminex, Austin, TX) and ARIES® extraction cassettes (Part number: 50-10026, Luminex) were purchased ready to use. Carrier RNA (QIAGEN part number 1017647, Germantown, MD) was prepared as a 1 μg/μL solution in AVE buffer (QIAGEN part number 1026956, Germantown, MD), aliquots were stored at -20°C. Primer pairs, obtained from Integrated DNA Technologies Inc. (IDT, Coralville, IA), were designed to include a FAM reporter-labeled reverse primer with an isodC on the 5’end, and a second unlabeled forward primer for P. jirovecii. The primer set was designed to amplify the mitochondria large subunit (mtLSU) rRNA from P. jirovecii based on a previous study [8]. Primer designs were evaluated by eye, then screened using IDT’s Oligo Analyzer software. Primer sequences are shown in Table 1. Each primer was used at a final concentration of 200 nM. Mouse Hepatitis Virus 2 control primers (MHV2, proprietary, Luminex Part Number 3803, Austin, TX) were used to amplify the Sample Processing Control (SPC; confirms extraction, amplification and detects inhibition). The SPC calibrator function allowed for calibration of detected melting temperatures (Tm) based on MHV2 amplification. Primer Pool Preparation The mtLSU rRNA forward and reverse lyophilized primers were dissolved in Tris-EDTA (TE buffer, Fisher Catalog #BP2473-1, Waltham, MA) to make a 100 μM stock solution of each primer. Further dilution of the 100 μM stock was done with molecular grade water to make 5 μM working stock. To prepare the pooled primer mix, 100 μL of each 5 μM stock primer was combined with 100μL of MHV2 primer stock to make 300 μL total volume. 2 ULJRI Vol 3, (1) 2019 Organism Forward Primer Reverse Primer Gene Target Amplicon Length (bp) Pneumocystis jirovecii 5’-CAG ACT ATG TGC GAT AAG GTA GAT AGT CG-3’ 5’-/56FAM/iMeisodC/GGA GCT TTA ATT ACT GTT CTG GGC-3’ Mitochondrial large subunit ribosomal RNA gene 64 Table 1 Primers used for Luminex ARIES® P. jirovecii PCR assay. PCR Pre-heat Temperature 95C Pre-heat hold time 0 sec Activation Temperature 95C Activation Time 120 sec Denature Temperature 95C Denature Time 5 sec Anneal Temperature 62C Anneal hold Time 7 sec Extension Temperature 72C Extension hold Time 14 sec Number of PCR steps repeat 3 (from Denature to Extend) Optical Read Location Extension Number of PCR cycles 45 Melt Melt Temperature Step 0.5C Melt Start Temperature 60C Melt final Temperature 95C Melt Hold Time 2 sec Optical read location Melt hold time Table 2 PCR Thermal profile used for Luminex ARIES® P. jirovecii PCR assay. Setting Conditions P. jirovecii mtLSU rRNA target FAM channel SPC MHV2 target AP525 channel 1 Amplification curve Cut off (Ct) 1-40 1-45 2 Melt curve (Tm) Cutoff (C) 77.4-78.9 (78.150.75) 74.7-79.7 (77.22.5) 3 Melt curve (Tm) Peak Threshold cutoff (RFU) -700,000 -150,000 SPC= Sample Processing Control; RFU=Change in relative fluorescent units Table 3 P. jirovecii assay Amplification and Melt Curve Settings for ARIES® The complete primer pool