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

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 


Introduction
Pneumocystis pneumonia (PCP) is a major health concern for immunocompromised individuals, whether due to HIV infection, chemotherapy or solid organ transplantation [1].PCP is caused by the opportunistic fungal pathogen Pneumocystis jirovecii, a pathogen specific to humans, having no other reservoir [2].Individuals suffering from PCP usually present with non-specific symptoms such as fever, cough, shortness of breath, and fatigue.Sulfa drugs are the most common treatment for infection [3].
A rapid and accurate laboratory test is important for early diagnosis and treatment since any delay in treatment can significantly increase the risk of mortality.This organism has not been successfully cultured to date, so diagnosis depends on other laboratory methods.Traditionally, PCP has been diagnosed with the help of numerous staining methods or fluorescent antibody assays.Detection of nucleic acids from P. jirovecii in clinical samples has been evaluated numerous times, with certain authors making the case that molecular detection, such as quantitative PCR, should be the test of choice for PCP [4,5].While concerns about colonized versus infected patients have been raised, combining quantitative molecular detection with clinical judgment should reduce false positive results.
We developed and evaluated a real-time PCR assay to detect P. jirovecii targeting mitochondrial large subunit ribosomal RNA gene (mtLSU rRNA) in lower respiratory tract specimens using the Luminex ARIES ® instrument.The objective was to provide a rapid and sensitive test to health care practitioners serving immunocompromised patients in our area, since sending these specimens to an out of state reference laboratory is expensive and takes at least one week for results.The mtLSU rRNA target was chosen because of the copy number and previous comparative studies [6].The Luminex ARIES ® is an open-platform instrument which uses MultiCode-RTx ® base pairing (isoC:isoG) technology.This instrument was selected because it simplifies the extraction, amplification and detection process without affecting the robustness of diagnostic testing.Selective primers, labeled with fluorescent dyes, facilitate real time detection of both the mtLSU rRNA gene target for PCP and an internal control in a single-step.This assay enables rapid diagnosis with great sensitivity and specificity with little handson time required.

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 1x10 5 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.The complete primer pool was then mixed well by vortexing, spun down and stored in 60 μL aliquots at -20°C.

ARIES ® Cassette and Instrument Preparation
Six (6) µL of the mtLSU rRNA pooled primer mix were added to each MultiCode ® DNA ReadyMix ® tube, then attached to the ARIES ® cassette.ARIES ® Cassettes (Part Number: 50-10026, Luminex, Austin, TX) contained the extraction reagents.Four hundred (400) µL of processed BAL sample were added to a 1.5 mL micro centrifuge tube along with 5 µL of a 1 µg/mL carrier RNA stock.The tube was vortexed for five seconds prior to transferring the entire volume to the cassette and loaded onto the ARIES ® instrument.The extraction, PCR reaction and result generation then proceeded automatically (PCR thermal cycle profile in Table 2).

Sensitivity
For analytical sensitivity, the LOD was determined for the mtLSU rRNA target in BAL specimens that were negative by the Luminex ARIES ® PCR assay described here.The Zeptometrix PCP positive control (400 µL, 2x10 5 organisms per mL) was used as the initial dilution, followed by 6 ten-fold dilutions in the negative BAL specimen, then tested as described above.The LOD assay was conducted three times independently (Table 4).

Accuracy
A panel of 24 (18 BAL, five sputum and one induced sputum) previously known positive and ten (eight BAL, one bronchial wash, and one sputum) known negative patient specimens provided by Dr. Scott McClellan (University of Michigan) were tested.Additionally, 17 spiked positive samples [PCP controls at varying dilutions: nine Zeptometrix controls at 2x10 5 , 2x10 4 , 2x10 3 , 2x10 2 organisms per mL, and eight Exact Diagnostics controls at 125,000 copies/mL, 12,500 copies/mL, 1250 copies/ mL and 125 copies/mL in BAL (Table 5)] and 14 negative blinded BAL samples were also tested by different technologists.

Precision
Reproducibility studies to determine precision were conducted using the Zeptometrix PCP control diluted 1:100 (2x10 3 organisms/mL) in negative BAL samples.To demonstrate intra-assay and inter-assay precision, positive controls were tested for seven days and negative controls were tested for five days in duplicate, then for 10-15 days in singlet using different technologists.Average and standard deviations were calculated using MS Excel (Table 6, A&B).

Stability
Specimen stability studies were conducted by incubation of negative BAL samples spiked with known PCP Zeptometrix positive control diluted 1:100 (2x10 3 organisms/mL) and stored at 4°C, then assayed daily for 8 days (Table 7A).In addition, nine different known P. jirovecii positive clinical BAL samples were pooled and stored at 4°C, then assayed daily for 8 days (Table 7B).

Specificity
Eleven different fungi, eight different bacteria and 13 different viruses were spiked into negative BAL and tested in to assess cross-reactivity (Table 8).

Data Analysis
Microsoft Excel ® was used to calculate standard deviations for replicate experiments, while MedCalc Diagnostic Test Evaluation was used to determine analytic sensitivity and specificity [9].The User Defined Protocol (UDP) module in the SYNCT ® software was used to set assay parameters.The SYNCT ® software was installed on an external computer for 3    After completing data analysis and PCR assay validation, the Ct and Tm cut off values were set and locked in the SYNCT ® software assay file.Next, the assay file was loaded on the ARIES ® instrument for subsequent diagnostic laboratory testing (Table 3).In order for a specimen to be called positive for P. jirovecii, the following conditions had to be met: 1.
The SPC for any negative specimen must have a valid Ct value and Tm to exclude sample inhibition.

The specimen must have Ct and Tm values in the
established range for the target organism P. jirovecii.

Results
Using a combination of positive and negative patient specimens, and spiked known concentrations of recombinant external positive controls, the performance characteristics of the PCR assay on the ARIES ® system were established.The results of this study were highly reproducible, as demonstrated by testing positive and negative controls with different technologists on different days.We obtained consistent results with low standard deviations (Tables 4, 6A&B and 7A&B).Analytical sensitivity for the P. jirovecii assay was 89.1% (95% CI 76.43-96.38%),while analytical specificity was 100% (95% CI 87.23 -100), (Table 5).Stability studies were performed using positive control material spiked into negative BAL and pooled P. jirovecii clinical positive BAL samples, stored at 2-8°C for eight days and tested daily for 8 days.Results showed that DNA was detected by the PCR assay under these storage conditions (Table 7A and B).Finally, a variety of fungi, bacteria, and viruses that can cause respiratory infections were tested to establish specificity.There was no cross-reactivity observed with the P. jirovecii mtLSU rRNA primers using 32 different organisms (Table 8).By including the SPC, any inhibition of PCR would have been detected, and none was observed during these studies (Table 8).
The assay could reliably detect 3000 copies of mtLSU rRNA that equated to 200 organisms/mL of P. jirovecii (Table 4) in respiratory specimens.It is important to note that P. jirovecii is estimated to have 15 copies of the mtLSU rRNA gene/organism [10], allowing for more sensitive detection by PCR.

Discussion
Quality laboratory results are dependent on proper specimen collection, transport, storage and processing procedures, as well as adherence to good molecular laboratory techniques to avoid false positive and false negative results.As with any laboratory test, regardless of the method used to diagnose Pneumocystis pneumonia, clinical judgment in light of patient presentation and history are the ultimate deciding factors for patient treatment and management [11].
The strengths of this study include the demonstration of the precision, limit of detection using quantitated target DNA and demonstration of specificity.In addition, the ability to adapt the PCR assay to the Luminex ARIES ® platform so that extraction, amplification and detection occur in a closed system reduces potential cross-contamination.Some limitations of this study include the small number of clinical samples available for testing and the use of spiked BAL samples to mimic clinical samples.Due to a limited number of available clinical specimens, stability studies were conducted with positive control material in the form of plasmids as well as with pooled positive clinical   specimens.Comparison to a "gold standard" assay was also problematic.Numerous publications regarding PCR detection of P. jirovecii have used a wide variety of gene targets, methods, instruments, and limits of detection reporting units (i.e.trophic form equivalents [12], plasmid copy/uL [13], DNA copy/uL [11] or cysts/amplification [14], making direct comparisons challenging [15]).
A valid concern with PCP molecular detection assays is that this PCR assay could detect colonized patients as well as patients with infections.While this PCR assay was used in a qualitative manner, the Ct values provided some indication of organism load when P. jirovecii was detected [12,13].Example instrument results are provided in Figures 1A and 1B.Based on a likely human-to-human transmission route [16][17][18], detecting colonization may be relevant in certain situations.Setting a cutoff for colonization may not be practical for all situations, however, such as fungal load variability in HIV-infected versus non-infected immunosuppressed patients, or pediatric patients versus adult patients [8,11,19].While microscopic analysis remains a valid laboratory method for determining infection with these organisms, it requires a high level of technical expertise.It also lacks the sensitivity of molecular diagnostics by an estimated factor of 100 [14], thus supporting the need for molecular diagnostic assays for detection of P. jirovecii.
Exploration of quantitative PCR assays for P. jirovecii, adding other targets (e.g.drug resistance markers or virulence factors), and identifying other high copy number genes such as the mitochondrial smaller subunit (mtSSU) rRNA [10] are future goals to make this assay more sensitive and useful to clinicians.
In particular, drug resistance markers are of great interest in an era of antibiotic stewardship initiatives.A study conducted in Chile observed a high prevalence of mutations in the dihydropteroate synthase (DHPS) gene in P. jirovecii infected patients, despite a lack of previous exposure to trimethoprimsulfamethoxazole or dapsone [18].This study suggested a role for person-to-person transmission.The presence of the DHPS mutation correlated with twice longer duration of mechanical ventilation in this hospitalized group of 56 patients [18], indicating an important area for future study.
The benefits of the ARIES ® P. jirovecii assay include minimal hands-on time for technologists, one step specimen processing with a robust internal sample processing control, all within a closed system.Results are available within two hours while providing rapid, specific and sensitive results for health care providers.The ability to tailor PCR assays to the local health care community, and to add new targets with guidance from Luminex technical support, adds a high degree of future P. jirovecii assay improvements.Based on the performance characteristics established by our laboratory, the ARIES ® real-time PCR assay meets the CLIA requirements for diagnostic testing using BAL specimens from patients suspected of having PCP.Having the real-time PCR assay available to health care providers managing immunocompromised patients in our community will provide results within 24 hours of receipt, leading to better patient management.

Figure 1A
Figure 1ANegative Control Result: Sample data from ARIES ® instrument with a negative control illustrating the amplification curve (Ct) and melt temperature (Tm) for the internal control (SPC).

Figure 1B
Figure 1BPositive Control Result: Sample data from ARIES ® instrument with a positive control illustrating the amplification curve (Ct) and melt temperature (Tm) for P. jirovecii.

Table 2
PCR Thermal profile used for Luminex ARIES ® P. jirovecii PCR assay.

Table 3
P. jirovecii assay Amplification and Melt Curve Settings for ARIES ®

Table 4
Assay Sensitivity for P. jirovecii in BAL Specimens

Table 5
Accuracy Results for the P. jirovecii PCR Assay

Table 6A
Intra-assay Precision Studies for the P. jirovecii PCR Assay Table 6B Inter-assay Precision Studies for the P. jirovecii PCR Assay

Table 7A P
. jirovecii Stability Studies using positive control spiked into negative BAL Ct=Cycle threshold; Tm=Melt temperature; ND=Not Detected; SPC=Sample Processing Control detailed data analysis and editing of Ct and Tm cutoff values.

Table 8
Specificity Studies for the P. jirovecii PCR assay