Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens

2020; Elsevier BV; Volume: 22; Issue: 12 Linguagem: Inglês

10.1016/j.jmoldx.2020.10.003

ISSN

1943-7811

Autores

Baoming Liu, Marissa Totten, Saman Nematollahi, Kausik Datta, Warda Memon, Subathra Marimuthu, Leslie A. Wolf, Karen C. Carroll, Sean X. Zhang,

Tópico(s)

Pneumonia and Respiratory Infections

Resumo

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)–targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5 hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P. jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1 < CT < 40.0 indicates the presence of a low load of the organism and needs further determination of either colonization or probable/possible Pneumocystis pneumonia. Overall, the new assay demonstrates excellent analytical and clinical performance and may be more sensitive than mtLSU PCR target for the detection of P. jirovecii. The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)–targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5 hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P. jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1 < CT < 40.0 indicates the presence of a low load of the organism and needs further determination of either colonization or probable/possible Pneumocystis pneumonia. Overall, the new assay demonstrates excellent analytical and clinical performance and may be more sensitive than mtLSU PCR target for the detection of P. jirovecii. Pneumocystis jirovecii is an opportunistic fungal pathogen causing Pneumocystis pneumonia (PCP), with high morbidity and mortality in HIV-infected as well as non–HIV-immunocompromised patient populations.1Ma L. Cissé O.H. Kovacs J.A. A molecular window into the biology and epidemiology of Pneumocystis spp.Clin Microbiol Rev. 2018; 31: e00009-e00018Crossref PubMed Scopus (25) Google Scholar, 2Finn K.M. Ginns L.C. Robbins G.K. Wu C.C. Branda J.A. Case records of the Massachusetts General Hospital: case 20-2014: a 65-year-old man with dyspnea and progressively worsening lung disease.N Engl J Med. 2014; 370: 2521-2530Crossref PubMed Scopus (3) Google Scholar, 3Cushion M.T. Pneumocystis: mycology, p 2015–2029.in: Jorgensen J.H. Pfaller M.A. Carroll K.C. Funke G. Landry M.L. Richter S.S. Warnock D.W. Manual of Clinical Microbiology 2015. ed 11, vol 2. ASM Press, Washington, DC2015Crossref Google Scholar Coincident with increasing adoption of transplantation and broad application of immunosuppressant agents, PCP is increasingly diagnosed among oncology patients, solid organ and stem cell transplants, as well as those receiving corticosteroids and other immune-modulating agents.1Ma L. Cissé O.H. Kovacs J.A. A molecular window into the biology and epidemiology of Pneumocystis spp.Clin Microbiol Rev. 2018; 31: e00009-e00018Crossref PubMed Scopus (25) Google Scholar,4Doyle L. Vogel S. Procop G.W. Pneumocystis PCR: it is time to make PCR the test of choice.Open Forum Infect Dis. 2017; 4: ofx193Crossref PubMed Scopus (14) Google Scholar, 5White P.L. Price J.S. Backx M. Therapy and management of Pneumocystis jirovecii infection.J Fungi (Basel). 2018; 4: E127Crossref PubMed Google Scholar, 6Vindrios W. Argy N. Le Gal S. Lescure F.X. Massias L. Le M.P. Wolff M. Yazdanpanah Y. Nevez G. Houze S. Dorent R. Lucet J.C. Outbreak of Pneumocystis jirovecii infection among heart transplant recipients: molecular investigation and management of an interhuman transmission.Clin Infect Dis. 2017; 65: 1120-1126Crossref PubMed Scopus (17) Google Scholar, 7Enoch D.A. Yang H. Aliyu S.H. Micallef C. The changing epidemiology of invasive fungal infections.Methods Mol Biol. 2017; 1508: 17-65Crossref PubMed Scopus (147) Google Scholar Pneumocystis jirovecii has been traditionally identified by microscopic examination using a variety of non-Pneumocystis specific chemical stains.3Cushion M.T. Pneumocystis: mycology, p 2015–2029.in: Jorgensen J.H. Pfaller M.A. Carroll K.C. Funke G. Landry M.L. Richter S.S. Warnock D.W. Manual of Clinical Microbiology 2015. ed 11, vol 2. ASM Press, Washington, DC2015Crossref Google Scholar,8Procop G.W. Haddad S. Quinn J. Wilson M.L. Henshaw N.G. Reller L.B. Artymyshyn R.L. Katanik M.T. Weinstein M.P. Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods.J Clin Microbiol. 2004; 42: 3333-3335Crossref PubMed Scopus (108) Google Scholar The advent of anti-Pneumocystis monoclonal antibodies made direct fluorescent antibody assays (DFAs) a reliable and specific method for Pneumocystis identification. A positive DFA is an accepted microbiological diagnostic criterion for proven PCP, as recommended by the European Organization for Research and Treatment of Cancer/Mycoses Study Group.9Donnelly J.P. Chen S.C. Kauffman C.A. Steinbach W.J. Baddley J.W. Verweij P.E. et al.Revision and update of the consensus definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.Clin Infect Dis. 2020; 71: 1367-1376Crossref PubMed Scopus (355) Google Scholar However, it still requires microscopic examination under a fluorescence microscope and, therefore, is often subjective and less sensitive than molecular methods.3Cushion M.T. Pneumocystis: mycology, p 2015–2029.in: Jorgensen J.H. Pfaller M.A. Carroll K.C. Funke G. Landry M.L. Richter S.S. Warnock D.W. Manual of Clinical Microbiology 2015. ed 11, vol 2. ASM Press, Washington, DC2015Crossref Google Scholar,4Doyle L. Vogel S. Procop G.W. Pneumocystis PCR: it is time to make PCR the test of choice.Open Forum Infect Dis. 2017; 4: ofx193Crossref PubMed Scopus (14) Google Scholar Nucleic acid amplification tests have been increasingly recognized as useful tools for detection of P. jirovecii with higher sensitivity than microscopy-based assays.3Cushion M.T. Pneumocystis: mycology, p 2015–2029.in: Jorgensen J.H. Pfaller M.A. Carroll K.C. Funke G. Landry M.L. Richter S.S. Warnock D.W. Manual of Clinical Microbiology 2015. ed 11, vol 2. ASM Press, Washington, DC2015Crossref Google Scholar,4Doyle L. Vogel S. Procop G.W. Pneumocystis PCR: it is time to make PCR the test of choice.Open Forum Infect Dis. 2017; 4: ofx193Crossref PubMed Scopus (14) Google Scholar,8Procop G.W. Haddad S. Quinn J. Wilson M.L. Henshaw N.G. Reller L.B. Artymyshyn R.L. Katanik M.T. Weinstein M.P. Detection of Pneumocystis jiroveci in respiratory specimens by four staining methods.J Clin Microbiol. 2004; 42: 3333-3335Crossref PubMed Scopus (108) Google Scholar,10Lackner M. Lass-Flörl C. Commercial molecular tests for fungal diagnosis from a practical point of view.Methods Mol Biol. 2017; 1508: 85-105Crossref PubMed Scopus (10) Google Scholar, 11Botterel F. Cabaret O. Foulet F. Cordonnier C. Costa J.M. Bretagne S. Clinical significance of quantifying Pneumocystis jirovecii DNA by using real-time PCR in bronchoalveolar lavage fluid from immunocompromised patients.J Clin Microbiol. 2012; 50: 227-231Crossref PubMed Scopus (67) Google Scholar, 12Guegan H. Robert-Gangneux F. Molecular diagnosis of Pneumocystis pneumonia in immunocompromised patients.Curr Opin Infect Dis. 2019; 32: 314-321Crossref PubMed Scopus (10) Google Scholar, 13Dalpke A.H. Hofko M. Zimmermann S. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii on the fully automated BD MAX platform.J Clin Microbiol. 2013; 51: 2337-2343Crossref PubMed Scopus (18) Google Scholar, 14Fauchier T. Hasseine L. Gari-Toussaint M. Casanova V. Marty P.M. Pomares C. Detection of Pneumocystis jirovecii by quantitative PCR to differentiate colonization and pneumonia in immunocompromised HIV-positive and HIV-negative patients.J Clin Microbiol. 2016; 54: 1487-1495Crossref PubMed Scopus (74) Google Scholar Although various P. jirovecii gene targets, such as unicopy CDC2 and multicopy major surface glycoprotein (MSG), have been chosen for development of TaqMan or LightCycler real-time PCR methods, the mitochondrial large subunit rRNA (mtLSU) gene is one of the most widely used molecular targets for real-time PCR assays.12Guegan H. Robert-Gangneux F. Molecular diagnosis of Pneumocystis pneumonia in immunocompromised patients.Curr Opin Infect Dis. 2019; 32: 314-321Crossref PubMed Scopus (10) Google Scholar, 13Dalpke A.H. Hofko M. Zimmermann S. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii on the fully automated BD MAX platform.J Clin Microbiol. 2013; 51: 2337-2343Crossref PubMed Scopus (18) Google Scholar, 14Fauchier T. Hasseine L. Gari-Toussaint M. Casanova V. Marty P.M. Pomares C. Detection of Pneumocystis jirovecii by quantitative PCR to differentiate colonization and pneumonia in immunocompromised HIV-positive and HIV-negative patients.J Clin Microbiol. 2016; 54: 1487-1495Crossref PubMed Scopus (74) Google Scholar, 15Arcenas R.C. Uhl J.R. Buckwalter S.P. Limper A.H. Crino D. Roberts G.D. Wengenack N.L. A real-time polymerase chain reaction assay for detection of Pneumocystis from bronchoalveolar lavage fluid.Diagn Microbiol Infect Dis. 2006; 54: 169-175Crossref PubMed Scopus (62) Google Scholar, 16Marimuthu S. Ghosh K. Wolf L.A. Development of a real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform.Univ Louisville J Res Infect. 2019; 3: 1-6Google Scholar However, several pitfalls of using the mtLSU PCR target have been reported. In 2017, Le Gal et al17Le Gal S. Robert-Gangneux F. Pépino Y. Belaz S. Damiani C. Guéguen P. Pitous M. Virmaux M. Lissillour E. Pougnet L. Guillaud-Saumur T. Toubas D. Valot S. Hennequin C. Morio F. Hasseine L. Bouchara J.P. Totet A. Nevez G. A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: a French multicenter study.Med Mycol. 2017; 55: 180-184Crossref PubMed Scopus (15) Google Scholar reported a false-negative result when using an mtLSU-targeted real-time PCR assay because of a rare point mutation (C210T) in the probe binding region. Recently, Valero et al18Valero C. Buitrago M.J. Gits-Muselli M. Benazra M. Sturny-Leclère A. Hamane S. Guigue N. Bretagne S. Alanio A. Copy number variation of mitochondrial DNA genes in Pneumocystis jirovecii according to the fungal load in BAL specimens.Front Microbiol. 2016; 7: 1413Crossref PubMed Scopus (18) Google Scholar have demonstrated that the copy number of mtLSU varies with infectious stages and decreases when the fungal load is low. Such a fungal load–dependent copy number variation of mtLSU may compromise the sensitivity of mtLSU-targeted PCRs.18Valero C. Buitrago M.J. Gits-Muselli M. Benazra M. Sturny-Leclère A. Hamane S. Guigue N. Bretagne S. Alanio A. Copy number variation of mitochondrial DNA genes in Pneumocystis jirovecii according to the fungal load in BAL specimens.Front Microbiol. 2016; 7: 1413Crossref PubMed Scopus (18) Google Scholar On the other hand, mitochondrial small subunit rRNA (mtSSU) gene was identified as the most stable mitochondrial marker and may be a better nucleic acid amplification test target given its constant presence with higher copy numbers during infections.18Valero C. Buitrago M.J. Gits-Muselli M. Benazra M. Sturny-Leclère A. Hamane S. Guigue N. Bretagne S. Alanio A. Copy number variation of mitochondrial DNA genes in Pneumocystis jirovecii according to the fungal load in BAL specimens.Front Microbiol. 2016; 7: 1413Crossref PubMed Scopus (18) Google Scholar In this study, a MultiCode real-time PCR assay targeting mtSSU (Pneumocystis mtSSU ARIES PCR assay) for the detection of P. jirovecii in bronchoalveolar lavage fluid (BALF) was developed and evaluated on the fully automated ARIES platform (Luminex Corp., Austin, TX). MultiCode technology is characterized by utilization of synthesized, isomer cytosine (isoC) and isomer guanine, which pair with each other, but not with natural bases (Figure 1A). Unlike TaqMan real-time PCR using a labeled probe and a set of regular primers, MultiCode real-time PCR does not use probes (Figure 1A). Rather, this technology employs the dynamic interaction between isomer guanine–labeled free quencher and isoC-labeled primer to allow efficient detection of decrease in fluorescence, which is directly proportional to the initial target numbers (Figure 1, B and C). The analytical characteristics of the new assay were determined, and its clinical testing performance was evaluated in 180 retrospectively collected BALF specimens. A total of 180 BALF specimens, collected from 180 unique subjects at the Johns Hopkins Hospital between April 2018 and July 2019, were included in the study. Among all 180 BALF specimens, 20 were positive for P. jirovecii by DFA; the remaining 160 specimens were randomly chosen from archived DFA-negative samples within the same collection time frame. Among the 180 patients, 48.3% (87/180) were male. The age of the patient population ranged from 4 months to 93 years old, with a median of 57 years old. Except for 6 patients without any medical history and 3 patients without any underlying conditions, the remaining 171 patients had at least one of the underlying conditions: transplantation (50); HIV positive (20); cancer (63: lymphoma, 11; leukemia, 14; myeloma, 5; lung cancer, 5; prostate cancer, 3; melanoma, 2; and other cancers, 23); cystic fibrosis (22); interstitial lung disease (10); chronic obstructive pulmonary disease (18); diabetes mellitus (6); and common variable immunodeficiency (10). A total of 76 patients received PCP prophylaxis: sulfamethoxazole/trimethoprim (55); atovaquone (17); pentamidine (1); and dapsone (3). A total of 77 patients received steroid treatment. The study was approved by the Johns Hopkins Institutional Review Board. Pneumocystis jirovecii mtSSU and mtLSU plasmids were each constructed by conventional PCR cloning techniques.19Liu B. Panda D. Mendez-Rios J.D. Ganesan S. Wyatt L.S. Moss B. Identification of poxvirus genome uncoating and DNA replication factors with mutually redundant roles.J Virol. 2018; 92: e02152-17Crossref PubMed Scopus (12) Google Scholar, 20Yang D. Li N.L. Wei D. Liu B. Guo F. Elbahesh H. Zhang Y. Zhou Z. Chen G.Y. Li K. The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function.PLoS Negl Trop Dis. 2019; 13: e0007537Crossref PubMed Scopus (13) Google Scholar, 21Liu B. Li N.L. Shen Y. Bao X. Fabrizio T. Elbahesh H. Webby R.J. Li K. The C-terminal tail of TRIM56 dictates antiviral restriction of influenza A and B viruses by impeding viral RNA synthesis.J Virol. 2016; 90: 4369-4382Crossref PubMed Scopus (53) Google Scholar, 22Liu B. Li N.L. Wang J. Shi P.Y. Wang T. Miller M.A. Li K. Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus.J Virol. 2014; 88: 13821-13835Crossref PubMed Scopus (48) Google Scholar, 23Wei D. Li N.L. Zeng Y. Liu B. Kumthip K. Wang T.T. Huo D. Ingels J.F. Lu L. Shang J. Li K. The molecular chaperone GRP78 contributes to toll-like receptor 3-mediated innate immune response to hepatitis C virus in hepatocytes.J Biol Chem. 2016; 291: 12294-12309Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Briefly, partial mtSSU gene sequence (nucleotides 965 to 1350, according to reference strain sequence JX499143) or mtLSU gene sequence (nucleotides 732 to 1078 of JX499143) were amplified from BALF specimens of P. jirovecii DFA-positive samples. Then, the amplicons were inserted into pCR2.1 TOPO vector (Thermo Fisher, Foster City, CA) via rapid cloning technology to generate plasmid pCR2.1-mtSSU or pCR2.1-mtLSU. The identities of plasmids were confirmed by the conventional PCR and DNA sequencing in both directions by Sanger sequencing techniques.24Ding H. Liu B. Zhao C. Yang J. Yan C. Yan L. Zhuang H. Li T. Amino acid similarities and divergences in the small surface proteins of genotype C hepatitis B viruses between nucleos(t)ide analogue-naïve and lamivudine-treated patients with chronic hepatitis B.Antivir Res. 2014; 102: 29-34Crossref PubMed Scopus (17) Google Scholar, 25Peng Y. Liu B. Hou J. Sun J. Hao R. Xiang K. Yan L. Zhang J. Zhuang H. Li T. Naturally occurring deletions/insertions in HBV core promoter tend to decrease in hepatitis B e antigen-positive chronic hepatitis B patients during antiviral therapy.Antivir Ther. 2015; 20: 623-632Crossref PubMed Scopus (8) Google Scholar, 26Liu B. Yang J.X. Yan L. Zhuang H. Li T. Novel HBV recombinants between genotypes B and C in 3'-terminal reverse transcriptase (RT) sequences are associated with enhanced viral DNA load, higher RT point mutation rates and place of birth among Chinese patients.Infect Genet Evol. 2018; 57: 26-35Crossref PubMed Scopus (4) Google Scholar PCR and sequencing primers were mtSSU-forward (5′-GTGTTGCATGGCTGTCTTTAGTTCG-3′) and mtSSU-reverse (5′-CAAGAAATGGACGTAAGAACTTGAATTTC-3′) for mtSSU gene and pAZ102-H (forward; 5′-GTGTACGTTGCAAAGTACTC-3′) and pAZ102-E (reverse; 5′-GATGGCTGTTTCCAAGCCCA-3′) for mtLSU gene.27Wakefield A.E. Pixley F.J. Banerji S. Sinclair K. Miller R.F. Moxon E.R. Hopkin J.M. Detection of Pneumocystis carinii with DNA amplification.Lancet. 1990; 336: 451-453Abstract PubMed Scopus (380) Google Scholar Thermal cycling conditions were as follows: 95°C for 2 minutes; 95°C for 20 seconds, 50°C for 20 seconds, and 72°C for 1 minute, 35 cycles; and 72°C for 10 minutes. Pneumocystis jirovecii mtSSU run control was generated by transforming pCR2.1-mtSSU plasmids into One Shot TOP10 chemically competent Escherichia coli (Thermo Fisher) and grown on ampicillin-containing Luria-Bertani agar plate or in Luria-Bertani broth (Thermo Fisher) for 18 hours. Blue-white colony screening and colony PCR with primers of mtSSU-forward and mtSSU-reverse were performed to confirm the presence of mtSSU gene insert. The P. jirovecii mtSSU run control was quantified with a laboratory-developed quantitative TaqMan real-time PCR on an ABI 7500 real-time PCR system with the primers Pj1098F (5′-TCATGACCCTTATGAAGTGGGC-3′) and Pj1173R (5′-GCTCCGACTTCCATCATTGC-3′) as well as probe P1125P (5′-FAM-ACGTGCTGCAAAATTTTCTACAATGGG-BHQ1-3′).18Valero C. Buitrago M.J. Gits-Muselli M. Benazra M. Sturny-Leclère A. Hamane S. Guigue N. Bretagne S. Alanio A. Copy number variation of mitochondrial DNA genes in Pneumocystis jirovecii according to the fungal load in BAL specimens.Front Microbiol. 2016; 7: 1413Crossref PubMed Scopus (18) Google Scholar Thermal cycling conditions were as follows: 50°C for 2 minutes; 95°C for 10 minutes; and 95°C for 15 seconds and 60°C for 1 minute, 45 cycles. Copy numbers of mtSSU DNA were calculated on the basis of a standard curve generated using serially diluted pCR2.1-mtSSU plasmid. To facilitate developing the Pneumocystis mtSSU ARIES PCR assay, MultiCode PCR conditions were first optimized by running P. jirovecii MultiCode real-time PCRs targeting mtSSU or mtLSU on an ABI 7500 system (Applied Biosystems, Foster City, CA). Briefly, 400 μL BALF was added into ceramic beads containing Lysing Matrix D tubes (MP Biomedicals, Irvine, CA) and subjected to mechanical grinding in a FastPrep 24 high-power mechanical grinder (MP Biomedicals) with the recommended program for fungi (speed, 6.0 m/second; time, 30 seconds).28Jeddi F. Piarroux R. Mary C. Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases.Parasite. 2013; 20: 52Crossref PubMed Scopus (20) Google Scholar After centrifugation, 250 μL supernatant was used for total nucleic acid extraction on the NucliSENS easyMAG automated extraction system (bioMérieux Inc., Durham, NC),29Liu B. Forman M. Valsamakis A. Optimization and evaluation of a novel real-time RT-PCR test for detection of parechovirus in cerebrospinal fluid.J Virol Methods. 2019; 272: 113690Crossref PubMed Scopus (2) Google Scholar during which mouse hepatitis virus DNA sample processing control (SPC; Luminex, Austin, TX) was added to lysed samples. Then, 25 μL eluate was obtained and 5 μL was used for MultiCode PCR following the manufacturer's recommendation. MultiCode real-time PCR primers for mtLSU included unlabeled forward primer PCW3A-for (5′-CAGACTATGTGCGATAAG GTAGATAGTCG-3′) and FAM/isoC-labeled reverse primer PCW4-rev (5’-/56-FAM/iMe-isodC/GGAGCTTTAATTACTGTTCTGGGC-3′).14Fauchier T. Hasseine L. Gari-Toussaint M. Casanova V. Marty P.M. Pomares C. Detection of Pneumocystis jirovecii by quantitative PCR to differentiate colonization and pneumonia in immunocompromised HIV-positive and HIV-negative patients.J Clin Microbiol. 2016; 54: 1487-1495Crossref PubMed Scopus (74) Google Scholar,16Marimuthu S. Ghosh K. Wolf L.A. Development of a real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform.Univ Louisville J Res Infect. 2019; 3: 1-6Google Scholar The primers for mtSSU included FAM/isoC-labeled forward primer Pj1098F (5’-/56-FAM/iMe-isodC/TCATGACCCTTATGAAGTGGGC-3′) and unlabeled reverse primer Pj1173R (5′-GCTCCGACTTCCATCATTGC-3′).18Valero C. Buitrago M.J. Gits-Muselli M. Benazra M. Sturny-Leclère A. Hamane S. Guigue N. Bretagne S. Alanio A. Copy number variation of mitochondrial DNA genes in Pneumocystis jirovecii according to the fungal load in BAL specimens.Front Microbiol. 2016; 7: 1413Crossref PubMed Scopus (18) Google Scholar The optimized thermal cycling conditions were as follows: 50°C for 7 minutes; 95°C for 2 minutes; and 95°C for 5 seconds, 62°C for 7 seconds, and 72°C for 14 seconds, 45 cycles. Of note, unlike the above-mentioned mtSSU TaqMan real-time PCR, which used 60°C as annealing temperature, 62°C was chosen in this MultiCode PCR and the Pneumocystis mtSSU ARIES PCR assay to ensure a high specificity. The principle and workflow of the Luminex ARIES platform are illustrated in Figure 1. The Pneumocystis MultiCode real-time PCR assay targeting mtSSU was transitioned onto the Luminex ARIES platform with some modifications and optimizations. Briefly, 400 μL BALF was mixed with 400 μL AL lysis buffer (Qiagen, Germantown, MD) in a Bertin tube with beads of different sizes (Bertin Corp., Rockville, MD), followed by shaking the Bertin tube on a vortex adaptor at 14,000 × g for 5 minutes to disrupt cells. After 10-minute incubation and centrifugation (14,000 × g, 2 minutes) both at room temperature, 400 μL of supernatant was mixed with 5 μL carrier RNA (1 μg/μL; Qiagen) and 10 μL PCR-grade proteinase K (25 mg/mL; Sigma, St. Louis, MO), and then added into an ARIES Extraction Cassette (Luminex) (Figure 1C). On the other end of the cassette was mounted a MultiCode DNA Ready Mix tube (Luminex) containing FAM/isoC-labeled primer Pj1098F, unlabeled primer Pj1173R, and mouse hepatitis virus control primer 2 (working concentration, 100 to 200 nmol/L). Subsequently, the cassette was loaded into the ARIES platform (Luminex) to perform DNA extraction, MultiCode real-time PCR, melting curve analysis, data analysis, and reporting (Figure 1C). Thermal cycling conditions were as follows: 50°C for 7 minutes; 95°C for 2 minutes; and 95°C for 5 seconds, 62°C for 7 seconds, and 72°C for 14 seconds, 45 cycles. After PCR, melting curve analysis was performed by increasing the temperature from 60°C to 95°C, with 0.5°C melting step, and the melting temperatures were held for 2 seconds for optical reading. Pneumocystis jirovecii mtSSU and SPC signals were obtained at FAM and AP525 channels, respectively. The entire procedure took 2.5 hours from sample to answer. Stringent call thresholds were set up for amplification and melting curves of mtSSU and SPC to ensure high analytical specificity (Supplemental Figure S1). Specifically, threshold cycle (CT) = 40.0 was established as the positive amplification call threshold for mtSSU. Temperatures of 78.8°C to 82.5°C and 74.7°C to 79.7°C were set up as melting temperature windows for mtSSU and SPC, respectively. Relative fluorescent units of 700,000 and 150,000 were chosen as melting curve peak (-Δrelative fluorescent unit) cutoffs for mtSSU and SPC, respectively. If needed, additional data analysis can be performed using the User Defined Protocol module in the SYNCT software 1.1u2(build 349) (Luminex). The Pneumocystis mtSSU ARIES PCR assay was evaluated for the following analytical characteristics: i) The limit of detection (LOD): P. jirovecii mtSSU run control was serially diluted in negative BALF to 1,600, 800, and 40 copies/mL and tested (10 replicates per dilution). LOD was defined as the lowest concentration with a detection rate ≥90%. ii) Analytical specificity: 1-mL aliquots of negative BALF samples confirmed by DFA and Pneumocystis mtSSU ARIES PCR assay were spiked individually with a broad range of respiratory viruses, bacteria, and fungi (a loop of bacteria or fungi for inoculation; stock virus, 1:10 dilution). Using four Candida species (Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis) as examples, the amount of DNA added into ARIES reactions was estimated to range from 256 to 360 ng. These contrived BALF specimens were tested by the Pneumocystis mtSSU ARIES PCR assay to examine any cross-reactivity with the spiked pathogens. iii) Reproducibility: three replicates of P. jirovecii mtSSU run control at 8000 copies/mL were tested over 3 consecutive days. iv) Specimen stability: aliquots of pooled, DFA-positive BALF specimens were detected with the Pneumocystis mtSSU ARIES PCR assay immediately or after storage at room temperature for 24 or 48 hours, 4°C for 24 hours or 7 days, or −25°C for 7 or 32 days. A total of 180 residual BALF specimens after DFA analysis were de-identified and split into two aliquots for parallel testing of either mtSSU or mtLSU target PCRs at two laboratories in a blinded manner. One set of aliquots was tested by the Pneumocystis mtSSU ARIES PCR assay performed at Johns Hopkins Hospital (B.L.). The other set was shipped on dry ice to the Division of Infectious Diseases, University of Louisville, to be tested by the in-house developed Pneumocystis mtLSU ARIES PCR assay (S.M.),16Marimuthu S. Ghosh K. Wolf L.A. Development of a real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform.Univ Louisville J Res Infect. 2019; 3: 1-6Google Scholar which used the same sample pretreatment and extraction protocol as that for the mtSSU ARIES PCR assay, except for an additional proteinase K digestion step added to the sample pretreatment step for the mtSSU ARIES PCR assay. Statistical differences were determined by t-test, Fisher exact χ2 test, McNemar test for analysis of variance, proportional difference, or exact probabilities with SPSS software version 11.5 (SPSS Inc., Chicago, IL) and GraphPad QuickCalcs (GraphPad Software, San Diego, CA) where appropriate. P < 0.05 was considered statistically significant. Receiver operating characteristic curve was generated by using GraphPad Prism software version 8.2 for Windows (GraphPad Software). mtSSU and mtLSU targets for P. jirovecii detection in MultiCode or TaqMan real-time PCR were compared on ABI 7500 system using plasmid dilutions and clinical samples. When testing the same molar concentration of mtSSU and mtLSU plasmid dilutions, ranging from 107 to 102 copies/mL, MultiCode real-time PCR targeting mtSSU yielded larger changes in relative fluorescent units and lower CT values than its counterpart targeting mtLSU (Figure 2A), suggesting the mtSSU target can lead to a higher analytical sensitivity for the detection of a broad concentration range of P. jirovecii DNA. When testing a BALF specimen positive for P. jirovecii by DFA, MultiCode PCR targeting mtSSU yielded 4.2 lower CT values than the counterpart targeting mtLSU, whereas the SPC for both PCRs yielded similar CT values (Figure 2B). Similar results were obtained when two other P. jirovecii DFA-positive samples were tested for mtSSU and mtLSU by TaqMan real-time PCRs (data not shown). These results suggested that mtSSU is a more sensitive target than mtLSU for ARIES assay development. Given the fact that P. jirovecii is uncultivable in vitro, a P. jirovecii mtSSU run control was generated, whose molar concentration was determined to be 4 × 1010 copies/mL using a quantitative TaqMan real-time PCR (Figure 3A). Pneumocyst

Referência(s)