Standardization and Clinical Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis jirovecii Pneumonia

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Its diagnosis remains challenging, especially in microscopy-negative cases. This study aimed to standardize and validate a qPCR assay targeting the mtLSU rRNA gene for detecting P. jirovecii DNA in respiratory samples from patients in Argentina. Materials and Methods The assay was optimized using plasmid dilutions containing the target gene. Analytical specificity was evaluated against 60 fungal, 21 mycobacterial, and 16 bacterial species. Clinical validation included 101 respiratory samples from symptomatic patients and 37 from healthy individuals. An internal positive control (IPC) was included in all reactions to detect inhibitors. Results The qPCR assay showed a detection limit of 8.8 copies/µL and no cross-reactivity. Among microscopy-confirmed cases, 95.5% were qPCR-positive. Notably, 14.9% of microscopy-negative but clinically compatible cases tested positive. ROC analysis yielded an AUC of 0.96, with an optimal Ct cutoff ≤ 36, providing 90.7% sensitivity and 95.8% specificity. No healthy controls tested positive. A “grey zone” (Ct 36–37.8) was observed, requiring clinical correlation. Conclusions This qPCR assay is highly sensitive and specific, offering a valuable diagnostic tool for PjP. Its performance supports implementation in routine diagnostics, especially when microscopy is inconclusive. However, interpretation in the grey zone requires complementary clinical or biomarker data. Pneumocystis jirovecii real-time PCR molecular diagnosis pneumonia immunocompromised patients Figures Figure 1 Figure 2 Introduction Pneumocystis jirovecii is an opportunistic fungus that causes pneumonia (PjP) in immunocompromised individuals. Although the incidence of PjP has significantly declined among people living with HIV (PLHIV) due to effective antiretroviral therapy, the infection remains a major concern in patients with undiagnosed HIV or limited access to treatment. In recent years, there has been a rising incidence of PjP among non-HIV immunocompromised populations, and in these patients, PjP often presents with more severe clinical outcomes and higher mortality, underscoring the need for timely diagnosis and targeted management strategies [1, 2]. In this group, PjP is typically characterized by an acute onset, high fever, severe dyspnea, and marked hypoxemia, in contrast to the slower clinical progression observed in PLHIV [3]. However, a recent meta-analysis estimated the overall prevalence of PLHIV-associated PjP at 35.4%, compared to 10.2% in HIV-negative immunocompromised individuals and PjP continues to exhibit high mortality rates among PLHIV. [4]. Colonization is common across various populations, including immunocompetent individuals, where it does not cause signs or symptoms of PjP, maintaining a low fungal load in the host, with prevalence rates ranging from 10–65% depending on the study group [5, 6 ]. Pneumocystis jirovecii is a species that exclusively infects its host and has not been found in any environmental reservoir outside of human colonization. The transmission of P. jirovecii is airborne, and the amount of Pneumocystis DNA in the air decreases as one moves further away from colonized individuals or those with active PjP [7–9]. Diagnostic challenges arise because P. jirovecii cannot be cultured in conventional microbiology media. The gold standard for diagnosis remains microscopic visualization of the organism in respiratory samples using different stains, with a sensitivity ranging from 49–79% and a specificity of 99% [3]. Immunofluorescence techniques utilizing specific antibodies also offer the ability to visualize both cysts and trophozoites, exhibiting sensitivities of 91% and 95% and specificities of 91% and 95%, respectively [3, 10]. Serum biomarkers have been investigated for the diagnosis of P. jirovecii pneumonia, including 1-3-beta-D-glucan, a fungal cell wall component, as well as markers of inflammation and lung injury such as lactate dehydrogenase and Krebs von den Lungen-6 antigen [11; 12]. In recent years, real-time PCR has emerged as a crucial diagnostic tool for detecting P. jirovecii DNA in clinical samples. It offers high sensitivity and specificity while reducing the risk of amplicon contamination. Furthermore, real-time PCR enables quantitative analysis, which may help differentiate between active PjP and colonization [13]. Various respiratory specimens for PCR analysis have been evaluated, including bronchoalveolar lavage (BAL), bronchial lavage (BL), tracheal aspirates (TA), lung biopsies (LB), induced sputum (IS), oropharyngeal washings (OW), and nasopharyngeal aspirates (NPA). The IS, NPA, and OW samples are important in patients where minimally invasive tissue sampling is required. [14]. While BAL is the preferred sample for diagnosis due to its sensitivity ranging from 96. 8–99.5% [13], its invasiveness may limit its feasibility depending on the patient's condition, requiring less invasive respiratory samples. Consequently, the microscopic diagnosis depends on the type of samples, the quality of the sample, the observer's experience, the fungal load, and the rapid disintegration of fungal cells in fresh samples [10, 15]. This study aims to standardize and validate a qPCR assay targeting the mtLSU rRNA gene for detecting P. jirovecii DNA in respiratory samples from patients in Argentina Materials and Methods Quantitative PCR (qPCR) Cloning the mitochondrial large-subunit rRNA ( mtLSU rRNA ) gene fragment was a critical step in the qPCR standardization assay and a stable control in each assay. A plasmid harboring the target fragment, kindly provided by the Pasteur Institute of Lille, was successfully amplified in Escherichia coli by electroporation and selection on antibiotic-containing agar plates. Plasmid DNA was extracted from multiple transformed colonies. Plasmid concentration was measured in ng/ µL using Qubit® dsDNA HS reagent (high sensitivity) with a Qubit 3.0 fluorometer (Invitrogen- Life Technologies-Malaysia), and the number of copies/ µL was calculated later. Precise quantification of plasmid DNA ensured accurate concentrations for use as quantification standards in subsequent qPCR assays. The real-time PCR assay was standardized based on the protocol by Alanio et al. [16], with modifications to optimize performance under specific laboratory conditions. A 120-bp fragment of the P. jirovecii mtLSU-rRN A gene was amplified using primers PjF1 (5'-CTGTTTCCCTTTCGACTATCTACCTT-3') and PjR1 (5'-CACTGAATATCTCGAGGGAGTATGAA-3'), and the TaqMan probe PjSL (6FAM-5'-TCGCACATAGTCTGATTAT-3'- MGBNFQ). The assay was conducted on a StepOne PCR system (Applied Biosystems) using the TaqMan Universal Master Mix. Optimization involved evaluating probe concentrations (0.10 µM − 0.15 µM − 0.20 µM − 0.30 µM) against plasmid dilutions (10 5 -10 4 -10 3 -10 2 copies /µL) across four primer concentrations (0.3 µM − 0.4 µM − 0.5 µM -0.6 µM), to ensure effective amplification conditions for reliable qPCR performance. The thermal cycling protocol consisted of an initial incubation at 50°C for 2 min, followed by a denaturation step at 95°C for 10 min, and then 45 cycles of 15 s at 95°C and 1 min at 60°C for annealing/extension. Each sample was tested in triplicate, in three different assays. DNA from 60 different fungi was tested to assess analytical specificity, including primary and opportunistic pathogens. It included DNA from 10 Histoplasma capsulatum , 10 Coccidioides spp., 5 Paracoccidioides spp., 10 Aspergillus spp., 5 Penicillium sp., 10 Candida spp., and 10 Cryptococcus spp. Additionally, DNA from 10 Mycobacterium tuberculosis strains, 11 non-tuberculosis mycobacteria, and 16 different bacteria causing respiratory tract infections (2 Legionella spp., 5 Streptococcus pneumoniae , 3 Haemophilus influenzae , 2 Mycoplasma pneumoniae , and 4 Nocardia spp.) provided by the Mycobacteria Service of the Bacteriology Department at INEI-ANLIS "Carlos G. Malbrán" were included. The limit of detection (LOD) was established using a plasmid containing the mtLSU rRNA fragment. Serial 10-fold dilutions of the plasmid were prepared, starting from an initial concentration of 2.2 x 10 8 copies/µL. The minimum amount of plasmid detectable by the previously described qPCR assay was determined in triplicate across three independent experiments. Milli-Q water served as a negative control. Clinical specimens From January 2018 to October 2020, 101 respiratory samples from an equal number of patients presenting with clinical symptoms compatible with PjP were analyzed. The respiratory samples included were: 90 BAL, 2 BL, three induced sputum (IS), one mini-bronchoalveolar lavage (mBAL), four formalin-fixed and paraffin-embedded pulmonary tissue (PT-FFPE), and 1 TA. Samples were collected from four hospitals in the Autonomous City of Buenos Aires (CABA). The participating laboratories belong to high-complexity centers and are part of the National Network of Mycology Laboratories of Argentina, adhering to the National External Program for Quality Control in Mycology (PNCCM for its acronym in Spanish), which periodically offers quality assessments in mycology to them. The patients sought consultation or were admitted to these hospitals, where they underwent clinical and radiological evaluations for protocol inclusion. A portion of each clinical sample was stored at 4°C for later transfer to the National Reference Laboratory in Clinical Mycology (LNRM, for its acronym in Spanish). An ad hoc form that included anonymized patient information accompanied each sample: age, gender, date of admission, specimen type, HIV status, and results from the reference methods for microbiological diagnosis. In addition, OW samples from 37 healthy individuals immunocompetent adults without any known immunosuppressive conditions, chronic lung disease, or recent hospital exposure were analyzed. To obtain these samples, each individual performed a deep oro-pharyngeal lavage (gargle) after tooth brushing, using 15 mL of sterile physiological solution for 30 s and collected the fluid into a sterile tube. Microbiological studies Each sample was analyzed in the hospital laboratory by direct examination using different assays: direct microscopy on wet mount preparations, calcofluor white stain, immunofluorescence, and specific stains (Grocott, Giemsa, Gram-Weighert) according to their protocols. The direct microscopic examinations (DME) results were consigned in the custom form. Molecular Studies The clinical materials received at the LNRM were processed for molecular studies. DNA extraction was performed using the Blood and Tissue Genomic DNA Extraction Kit from Qiagen (DNeasy Blood & Tissue), following the manufacturer's instructions. Subsequently, the extracted DNA was stored at -20°C until further processing. The samples were processed: pure and diluted 1:2 with ultrapure water, using the qPCR method standardized previously. To assess the presence of PCR inhibitors, all clinical samples were systematically tested with an internal positive control (IPC), using the TaqMan® Exogenous Internal Positive Control reagents (Applied Biosystems). Each sample was evaluated in parallel reactions with and without IPC, and no inhibition was detected across the runs. Inclusion of the IPC in every qPCR assay ensured the reliability of negative results and ruled out false negatives due to reaction failure or inhibition. The plasmid, stored at − 20°C, was also used to generate a three-point standard curve in each qPCR assay, and Milli-Q water was used as a negative control. Sample results were expressed in Ct (value of the corresponding curve between cycle and threshold). Statistical analysis A receiver operating characteristic (ROC) curve was generated to determine the potential of standardized qPCR for diagnosing PjP, using the GraphPad Prism program version 8.01 for Windows (GraphPad Software, San Diego, CA, USA). The optimal cut-off was determined using Youden’s index to differentiate active PjP reliably. The diagnostic performance was calculated by comparing it against the direct microscopy diagnosis, and the correlation was calculated with the Kappa index. Cases definitions We applied the consensus definitions established by the EORTC/MSGERC expert group. “Probable PjP” was defined by the presence of clinical and radiological features consistent with P. jirovecii pneumonia, accompanied by either detection of β-D-glucan in serum or identification of P. jirovecii DNA by quantitative real-time PCR in respiratory samples. “Proven PjP” required direct microscopic confirmation of P. jirovecii through conventional or immunofluorescence staining method in respiratory samples [17, 18]. Additionally, cases with detectable P. jirovecii DNA that do not allow differentiation between active infection and colonization were defined as “gray zone”. This corresponds to qPCR results indicating DNA levels that are inconclusive for defining infection status [16]. Results The qPCR protocol was optimized by testing primers, probes, and plasmids at four different concentrations. Analyzing sigmoidal fluorescence variation (ΔRn) throughout the amplification cycles was crucial in stablishing the optimal reaction conditions. Primer concentrations of 0.3 µM paired with a probe concentration of 0.15 µM yielded the most consistent and reproducible Ct values across a wide range of plasmid concentrations. No amplification was observed in DNA samples of 60 fungal species, 21 mycobacteria, and 16 other bacterial species known to cause respiratory infections, confirming the assay’s analytical specificity for P. jirovecii . Using a ΔRn threshold of 0,2, Ct values ranged from 16.69 and 43.02 across serial dilutions from 2.2 x 10 8 to 22 copies of the mtLSU rRNA gene fragment, with an R² of 0.995, demonstrating the linearity of the curve. The Table 1 shows the Ct values corresponding to each gene copy number. The lowest plasmid dilution detected with 100% reproducibility was 220 copies per 25 µL reaction (8.8 copies/µL), with an average Ct = 40.63. Considering that the average mtLSU rRNA gene copy number per P. jirovecii organism is approximately 15, 220 copies/25 µL is equivalent to ≈ 14.7 microorganisms per 25 µL, or approximately 1 microorganism/µL. Table 1 Ct values for different plasmid concentrations in 3 different assays. Copy Number /25 µL Ct values 2.2x10 8 2.2x10 7 2.2x10 6 2.2x10 5 2.2x10 4 2.2x10 3 220 22 2.2 16.91 20.24 24.11 28.98 32.18 36.86 39.91 ND ND 16.69 20.07 24.63 28.25 32.21 36.64 40.42 43.02 ND 17.23 20.01 24.04 28.46 32.20 36.44 41.56 42.13 ND ND: No detected. A total of 101 respiratory samples (90 BAL and 11 other respiratory specimens) from 101 symptomatic patients with clinical suspicion of PjP were analyzed. P. jirovecii cysts and/or trophic forms were identified by direct microscopic examination in 44 of these samples (43.6%), while no fungal elements were observed in the remaining 57 samples (56.4%) or in any of the 37 OW samples obtained from healthy controls. The median age of symptomatic patients was 43 years (range: 19–82); 63 (62%) were male, 31 (31%) were female, and gender data were unavailable for seven patients. Regarding HIV status, 43 patients (43%) were HIV-positive and 49 (49%) had HIV-negative with other immunosuppressive conditions. (Supplementary Table 1). Among the 44 patients with respiratory symptoms and positive microscopy (proven PjP), 42 (95.5%) showed a detectable qPCR signal with Ct values ranging from 11.98 to 37.84. In contrast, two samples (4.5%) were qPCR negative (Ct = ND). Among the 57 microscopy-negative patients with respiratory symptoms and radiological features suggestive of PjP, but negative microscopic results (non- proven PjP), 14 samples showed positive qPCR results (Ct ranging: 24.60–37.80). The remaining 43 samples showed no qPCR amplification, nor did any of the 37 healthy control samples. The overall concordance between the qPCR and the direct microscopy (gold standard) was 88.41%, with a kappa coefficient of 0.75 (95% CI: 0,639-0,864), indicating substantial agreement. The discriminatory capacity of the qPCR assay was further assessed by generating a ROC curve comparing patients with proven PjP (positive microscopy samples) with those without confirmed infection. The area under the ROC curve (AUC) was 0.96 (95% CI: 0.93–0.99), reflecting excellent discriminatory power (Fig. 1 ). The optimal Ct cutoff value was 36.07, corresponding to the highest likelihood ratio (21.54), and associated with sensitivity of 90.70% and specificity of 95.79%. Among the 44 microscopy-confirmed cases, three patients had qPCR with Ct values > 36 (range: 36.53–37.84), and two samples were qPCR no-detectable. Among the 14 qPCR-positive patients with -negative microscopy, four had Ct values < 36 and therefore classified as probable PjP, while 10 had Ct values between 36.23 and 37.80, corresponding to the so-called “gray zone”. The distribution of Ct values for both proven (positive microscopy) and non-proven PjP cases (negative microscopy) with Ct < 40 is shown in Fig. 2 . In the analyzed population, and considering the optimal Ct cutoff value, 44 patients (90.90%) were classified as proven PjP and 4 patients (9.10%) as probable PjP, according to the EORTC/MSGERC criteria [17, 18]. The number of cases increased by 9.1% when probable PjP was included. Among patients with proven/probable PjP, the median age was 46 years. Thirty patients (68.2%) were male, 16 (36.4%) were female, and sex was not recorded in two cases (4.5%). Thirty-nine patients (81.25%) were PLHIV, while the remaining nine (18.75%) were immunocompromised due to other causes and exhibited radiological findings consistent with atypical pneumonia. (Supplementary Table 1). Discussion Pneumocystis jirovecii is a fungal pathogen that causes severe pneumonia in immunocompromised patients, including those with HIV or other immunocompromising conditions. Currently, microscopy- base methods are considered the diagnostic gold standard for PjP; however, their low sensitivity is limited, depending s on the observer's experience, fungal load in clinical specimens, and other technical factors. In this context, molecular diagnostic methods have emerged as valuable tools due to their high sensitivity [16, 19]. In the present study, a highly sensitive and specific qPCR assay targeting the mtLSU rRNA gene of P. jirovecii was successfully optimized. This gene has been previously selected for molecular diagnostics on clinical samples due to its high copy number within the P. jirovecii genome, which enhances detection sensitivity [10, 16, 19, 20]. The assay demonstrated high analytical specificity, with no cross-reactivity observed against a broad panel of fungal and bacterial pathogens associated with respiratory infections. These results are in agree with previous studies using the same target gene across different PCR formats, including assays involving human cytomegalovirus and host DNA controls. [21]. The qPCR assay showed a detection limit of 220 copies per 25 µL (equivalent to 8.8 copies/µL) with 100% reproducibility, consistent with the detection limit reported by Choukri et al. [7], who reported 7 copies/µL using the same target. Although slight differences in detection thresholds may result from sample quality, qPCR conditions, or laboratory equipment, these results underscore the robustness of this molecular target. A Ct ≈ 40.6 was observed for this detection threshold, underscoring the need to establish a reliable cutoff when standardizing in-house assays to reduce false negatives in clinical diagnostics of PjP. We analyzed samples from 101 patients with respiratory symptoms, dividing them into groups based on their direct examination results: 44 positive, 57 negative, and 37 healthy individuals. Among microscopy-positive cases, the qPCR assay showed high sensitivity, detecting P. jirovecii DNA in 95.5% (42/44) with Ct values ≤ 37.8. Notably, 14 out of 57 (14.9%) microscopy-negative but clinically compatible cases also tested positive by qPCR, with Ct values between 36 and 37.8 in 10 patients, and < 36 in the remaining four. This remark the high sensitivity of qPCR and its ability to detect low fungal burdens that might be missed by microscopy. No amplification was observed in healthy controls, indicating the absence of colonization or false positives, and confirming the assay’s high specificity. In our study the overall concordance between direct microscopy and qPCR was 88.4% (kappa = 0.75), indicating substantial agreement between both diagnostic methods. This supports the utility of qPCR as a complementary diagnostic tool, especially in cases where direct examination is inconclusive. However, our concordance rate was slightly lower than that reported by Meliani et al. [22], who reported 91.3% agreement, but higher than the 70% reported by Brancart et al. [23]. The differences likely reflecting inter-study variability in experimental qPCR protocols, sample handling and definitions used for case classification. The inclusion of probable PjP cases in our study, based on clinical and radiological criteria in conjunction with qPCR Ct values, may have influenced the observed concordance values. These findings reinforce the importance of standardizing laboratory methods and clinical algorithms for the diagnosis of PjP across different healthcare centers. The ROC curve analysis showed an area under the curve (AUC) of 0.96 (95% CI, 0.93–0.99), indicating excellent diagnostic performance. A Ct cutoff of ≤ 36 yielded the best balance of sensitivity (90%) and specificity (96%) for distinguishing active infection. This threshold is higher than that reported by Fauchier et al. [19], who proposed Ct < 32, with an AUC of 0.88 (72% sensitivity and 75% specificity), using the same gene. The higher cutoff observed in our study probable reflects a broader patient population, including both HIV-positive individuals, who generally exhibited higher fungal burdens, and non-HIV patients, who typically exhibit lower fungal burdens [19, 24, 25]. Variations in specimen type and immune status influenced the range of Ct values observed, emphasizing the need to adapt diagnostic thresholds accordingly. Gits Musell et al. [26] evaluated a panel of respiratory samples using various qPCR protocols across 16 reference laboratories and reported up to 12-cycle variability for the identical samples. This emphasizes the importance of standardizing in-house qPCR assays and incorporating concentration curves and control materials to ensure consistency across different settings. A critical observation from our data is the identification of a “grey zone” for Ct values between 36 and 37.8, where differentiating between colonization and active infection remains challenging. This overlap was observed in both proven PjP and microscopy negative patients. Similar findings have been reported in previous studies using molecular techniques [16, 19, 27, 28]. In our study, no complementary biomarkers such as serum β-D-glucan or longitudinal follow-up data were available to clarify the clinical significance of theses intermediate Ct values. As such, this range should be interpreted with caution and within the broader clinical and radiological context. All 37 respiratory samples from healthy individuals remained negative, reinforcing the assay’s specificity and reducing concerns about asymptomatic colonization [19, 29]. An aspect that strengthens the technical reliability of this study is the consistent inclusion of an IPC in all qPCR reactions, which allowed us to detect potential PCR inhibitors and ensure the validity of negative results, as recommended in molecular diagnostics protocols [26]. Moreover, the assay’s analytical specificity was evaluated against a wide panel of fungal and bacterial pathogens, helping to reduce the risk of false positives in clinical practice. These measures contribute to the assay’s robustness under routine laboratory conditions. In summary, the standardized qPCR assay developed in this study represents a highly sensitive and specific method for the molecular detection of P. jirovecii . Its ability to detect low fungal burdens enables early diagnosis, particularly in patients with clinical suspicion of PjP but negative microscopic findings. While diagnostic uncertainty persists within the grey Ct zone, the assay remains a valuable complementary tool when interpreted together with clinical, radiological, and immunological data. Overall, these results support its integration into diagnostic algorithms for PjP, especially in situations requiring rapid and accurate detection to guide timely clinical decisions. Declarations Funding This work was supported by the Administración Nacional de Laboratorios e Institutos de Salud (ANLIS) “Dr. Carlos G. Malbrán”. Cristina E. Canteros received support through the VI CONVOCATORIA A FONDOS CONCURSABLES ANLIS, 2015 (Project No. NRU 1524). Competing Interests The authors declare that they have no conflicts of interest. Author contributions Adriana Inés Toranzo and Cristina E. Canteros contributed substantially to the study conception, design, coordination, execution, data analysis, and manuscript preparation. Claudia Frola, Rosana Jordán, Patricia Giorgio, and Ana Ruth Laborde managed patient care, case selection, and clinical interpretation. Roberto Moyano performed statistical analyses, including ROC curve interpretation. Norma Fernández, Agustina Forastiero, Liliana Guelfand, Luciana Farías, Mariana Andreani, Facundo Muise Acevedo, Mariana Viale, and Cecilia López-Joffre contributed to laboratory work, sample processing, and manuscript editing. All authors reviewed and approved the final manuscript. Ethical approval The study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki, and all applicable national regulations. The ANLIS Research Ethics Committee “Dr. Carlos G. Malbrán” granted approval for this study: NRU 1524, VI CONVOCATORIA A FONDOS CONCURSABLES ANLIS, 2015. Additional ethical permission was obtained from the ethics committees of each participating hospitals. Consent to participate Written informed consent was obtained from all individuals enrolled in the study. The consent procedure was reviewed and approved by the same ethics committee. All clinical data were anonymized prior to analysis, and oropharyngeal wash samples from healthy volunteers were obtained in accordance with approved informed consent procedures. Acknowledgements We thank the Institut Pasteur de Lille for kindly providing the cloned fragment of the mitochondrial large-subunit rRNA (mtLSU rRNA) gene. This study was conducted as part of the PhD thesis of Adriana Inés Toranzo, under the direction of Dr. Cristina E. Canteros. References McDonald EG, Afshar A, Assiri B, et al. Pneumocystis jirovecii pneumonia in people living with HIV: a review. Clin Microbiol Rev. 2024;37(1):e0010122. doi:10.1128/cmr.00101-22. Salzer HJF, Schäfer G, Hoenigl M, et al. Clinical, Diagnostic, and Treatment Disparities between HIV-Infected and Non-HIV-Infected Immunocompromised Patients with Pneumocystis jirovecii Pneumonia. Respiration. 2018;96(1):52-65. doi:10.1159/000487713. Krajicek BJ, Thomas CF Jr, Limper AH. Pneumocystis pneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin Chest Med. 2009;30(2):265-vi. doi:10.1016/j.ccm.2009.02.005. Ahmadpour E, Valilou S, Ghanizadegan MA, et al. Global prevalence, mortality, and main characteristics of HIV-associated pneumocystosis: A systematic review and meta-analysis. PLoS One. 2024;19(3):e0297619. Published 2024 Mar 25. doi:10.1371/journal.pone.0297619. Medrano FJ, Montes-Cano M, Conde M, et al. Pneumocystis jirovecii in general population. Emerg Infect Dis. 2005;11(2):245-250. doi:10.3201/eid1102.040487. Ponce CA, Gallo M, Bustamante R, Vargas SL. Pneumocystis colonization is highly prevalent in the autopsied lungs of the general population. Clin Infect Dis. 2010;50(3):347-353. doi:10.1086/649868. Choukri F, Menotti J, Sarfati C, et al. Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia. Clin Infect Dis. 2010;51(3):259-265. doi:10.1086/653933. Fréalle E, Valade S, Guigue N, et al. Diffusion of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis colonization: frequency and putative risk factors. Med Mycol. 2017;55(5):568-572. doi:10.1093/mmy/myw113. Vera C, Rueda ZV. Transmission and Colonization of Pneumocystis jirovecii. J Fungi (Basel). 2021;7(11):979. Published 2021 Nov 18. doi:10.3390/jof7110979. Moodley B, Tempia S, Frean JA. Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii. PLoS One. 2017;12(7):e0180589. Published 2017 Jul 6. doi:10.1371/journal.pone.0180589. Esteves F, Calé SS, Badura R et al. Diagnosis of Pneumocystis pneumonia: evaluation of four serologic biomarkers. Clin Microbiol Infect. 2015;21(4):379.e1-10. doi: 10.1016/j.cmi.2014.11.025. Esteves F, Lee CH, de Sousa B, et al. (1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients. Eur J Clin Microbiol Infect Dis. 2014;33(7):1173-1180. doi:10.1007/s10096-014-2054-6. Brown L, Rautemaa-Richardson R, Mengoli C, et al. Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis. Clin Infect Dis. 2024;79(1):161-168. doi:10.1093/cid/ciae239. Senécal J, Smyth E, Del Corpo O, et al. Non-invasive diagnosis of Pneumocystis jirovecii pneumonia: a systematic review and meta-analysis. Clin Microbiol Infect. 2022;28(1):23-30. doi:10.1016/j.cmi.2021.08.017. Reid AB, Chen SC, Worth LJ. Pneumocystis jirovecii pneumonia in non-HIV-infected patients: new risks and diagnostic tools. Curr Opin Infect Dis. 2011;24(6):534-544. doi:10.1097/QCO.0b013e32834cac17. Alanio A, Desoubeaux G, Sarfati C, et al. Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients. Clin Microbiol Infect. 2011;17(10):1531-1537. doi:10.1111/j.1469-0691.2010.03400.x. Donnelly JP, Chen SC, Kauffman CA, 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(6):1367-1376. doi:10.1093/cid/ciz1008. Lagrou K, Chen S, Masur H, et al. Pneumocystis jirovecii Disease: Basis for the Revised EORTC/MSGERC Invasive Fungal Disease Definitions in Individuals Without Human Immunodeficiency Virus. Clin Infect Dis. 2021;72(Suppl 2):S114-S120. doi:10.1093/cid/ciaa1805. Fauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty PM, 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(6):1487-1495. doi:10.1128/JCM.03174-15. Gupta R, Mirdha BR, Guleria R, et al. Diagnostic significance of nested polymerase chain reaction for sensitive detection of Pneumocystis jirovecii in respiratory clinical specimens. Diagn Microbiol Infect Dis. 2009;64(4):381-388. doi:10.1016/j.diagmicrobio.2009.04.008. Muñoz C, Zuluaga A, Restrepo A, Tobón A, Cano LE, Gonzalez A. Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients. Diagn Microbiol Infect Dis. 2012;72(3):204-213. doi:10.1016/j.diagmicrobio.2011.11.015. Meliani L, Develoux M, Marteau-Miltgen M, et al. Real time quantitative PCR assay for Pneumocystis jirovecii detection. J Eukaryot Microbiol. 2003;50 Suppl:651. doi:10.1111/j.1550-7408.2003.tb00670.x. Brancart F, Rodriguez-Villalobos H, Fonteyne PA, Peres-Bota D, Liesnard C. Quantitative TaqMan PCR for detection of Pneumocystis jiroveci. J Microbiol Methods. 2005;61(3):381-387. doi:10.1016/j.mimet.2005.01.001. Mühlethaler K, Bögli-Stuber K, Wasmer S, et al. Quantitative PCR to diagnose Pneumocystis pneumonia in immunocompromised non-HIV patients. Eur Respir J. 2012;39(4):971-978. doi:10.1183/09031936.00095811 Sarasombath PT, Thongpiya J, Chulanetra M, et al. Quantitative PCR to Discriminate Between Pneumocystis Pneumonia and Colonization in HIV and Non-HIV Immunocompromised Patients. Front Microbiol. 2021;12:729193. Published 2021 Oct 20. doi:10.3389/fmicb.2021.729193. Gits-Muselli M, White PL, Mengoli C, et al. The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay. Med Mycol. 2020;58(6):779-788. doi:10.1093/mmy/myz115. Maillet M, Maubon D, Brion JP, et al. Pneumocystis jirovecii (Pj) quantitative PCR to differentiate Pj pneumonia from Pj colonization in immunocompromised patients. Eur J Clin Microbiol Infect Dis. 2014;33(3):331-336. doi:10.1007/s10096-013-1960-3. Matsumura Y, Ito Y, Iinuma Y, et al. Quantitative real-time PCR and the (1→3)-β-D-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization. Clin Microbiol Infect. 2012;18(6):591-597. doi:10.1111/j.1469-0691.2011.03605.x. Aguilar YA, Rueda ZV, Maya MA, et al. Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?. J Fungi (Basel). 2021;7(12):1036. Published 2021 Dec 2. doi:10.3390/jof7121036. Supplementary Files SupplementaryTable1.docx Cite Share Download PDF Status: Published Journal Publication published 04 Nov, 2025 Read the published version in Mycopathologia → Version 1 posted Reviewers agreed at journal 08 Aug, 2025 Reviewers invited by journal 05 Aug, 2025 Editor invited by journal 18 Jul, 2025 Editor assigned by journal 18 Jul, 2025 First submitted to journal 16 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7142772","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496143461,"identity":"557b6919-2fb3-4c27-bf3d-4957eb3c58b3","order_by":0,"name":"Adriana I Toranzo","email":"","orcid":"","institution":"INEI: Instituto Nacional de Enfermedades Infecciosas","correspondingAuthor":false,"prefix":"","firstName":"Adriana","middleName":"I","lastName":"Toranzo","suffix":""},{"id":496143462,"identity":"36f7f466-99e5-4c5b-af9b-416688056eba","order_by":1,"name":"Norma Fernandez","email":"","orcid":"","institution":"Hospital de Clínicas José de 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18:43:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7142772/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7142772/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11046-025-01016-7","type":"published","date":"2025-11-04T15:57:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88773796,"identity":"7c3147f7-9667-413d-9cb6-acc154973687","added_by":"auto","created_at":"2025-08-11 09:58:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":213499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROC Curve of qPCR Performance Versus Microscopy Detection. \u003c/strong\u003eThe right table shows the area under the curve (AUC), standard error, 95% confidence interval, and p-value, summarizing the diagnostic accuracy of the assay.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142772/v1/222ebe5e9669ab07dfb674cf.jpeg"},{"id":88773804,"identity":"4611949a-82e4-46cc-9bb7-d0ff7d14d685","added_by":"auto","created_at":"2025-08-11 09:58:58","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of qPCR Ct values among positive microscopy samples and negative microscopy.\u003c/strong\u003e The gray line indicates the Ct threshold of 36.07. Horizontal black bars represent the mean Ct value (28.33) and the standard deviation. In the positive microscopy group two samples were undetectable, and data point not displayed in the figure. In the negative microscopy group circle marked samples Ct values \u0026lt;36 (probable cases).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142772/v1/b8c1149433f16ce87bc0425f.jpeg"},{"id":95564160,"identity":"e0694759-73a0-42c9-818e-02b4b2ab72ac","added_by":"auto","created_at":"2025-11-10 16:08:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1033618,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7142772/v1/329104ff-4c45-463a-a149-55e931f3845e.pdf"},{"id":88773799,"identity":"315e4ae9-0459-4492-aec7-50bf024e0235","added_by":"auto","created_at":"2025-08-11 09:58:58","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":24337,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7142772/v1/cc8c929cfbfa54efb41bf80e.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eStandardization and Clinical Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis jirovecii Pneumonia\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003ePneumocystis jirovecii\u003c/em\u003e is an opportunistic fungus that causes pneumonia (PjP) in immunocompromised individuals. Although the incidence of PjP has significantly declined among people living with HIV (PLHIV) due to effective antiretroviral therapy, the infection remains a major concern in patients with undiagnosed HIV or limited access to treatment. In recent years, there has been a rising incidence of PjP among non-HIV immunocompromised populations, and in these patients, PjP often presents with more severe clinical outcomes and higher mortality, underscoring the need for timely diagnosis and targeted management strategies [1, 2]. In this group, PjP is typically characterized by an acute onset, high fever, severe dyspnea, and marked hypoxemia, in contrast to the slower clinical progression observed in PLHIV [3]. However, a recent meta-analysis estimated the overall prevalence of PLHIV-associated PjP at 35.4%, compared to 10.2% in HIV-negative immunocompromised individuals and PjP continues to exhibit high mortality rates among PLHIV. [4].\u003c/p\u003e\u003cp\u003eColonization is common across various populations, including immunocompetent individuals, where it does not cause signs or symptoms of PjP, maintaining a low fungal load in the host, with prevalence rates ranging from 10\u0026ndash;65% depending on the study group [5, 6 ].\u003c/p\u003e\u003cp\u003e\u003cem\u003ePneumocystis jirovecii\u003c/em\u003e is a species that exclusively infects its host and has not been found in any environmental reservoir outside of human colonization. The transmission of \u003cem\u003eP. jirovecii\u003c/em\u003e is airborne, and the amount of \u003cem\u003ePneumocystis\u003c/em\u003e DNA in the air decreases as one moves further away from colonized individuals or those with active PjP [7\u0026ndash;9].\u003c/p\u003e\u003cp\u003eDiagnostic challenges arise because \u003cem\u003eP. jirovecii\u003c/em\u003e cannot be cultured in conventional microbiology media. The gold standard for diagnosis remains microscopic visualization of the organism in respiratory samples using different stains, with a sensitivity ranging from 49\u0026ndash;79% and a specificity of 99% [3]. Immunofluorescence techniques utilizing specific antibodies also offer the ability to visualize both cysts and trophozoites, exhibiting sensitivities of 91% and 95% and specificities of 91% and 95%, respectively [3, 10].\u003c/p\u003e\u003cp\u003eSerum biomarkers have been investigated for the diagnosis of \u003cem\u003eP. jirovecii\u003c/em\u003e pneumonia, including 1-3-beta-D-glucan, a fungal cell wall component, as well as markers of inflammation and lung injury such as lactate dehydrogenase and Krebs von den Lungen-6 antigen [11; 12].\u003c/p\u003e\u003cp\u003eIn recent years, real-time PCR has emerged as a crucial diagnostic tool for detecting \u003cem\u003eP. jirovecii\u003c/em\u003e DNA in clinical samples. It offers high sensitivity and specificity while reducing the risk of amplicon contamination. Furthermore, real-time PCR enables quantitative analysis, which may help differentiate between active PjP and colonization [13]. Various respiratory specimens for PCR analysis have been evaluated, including bronchoalveolar lavage (BAL), bronchial lavage (BL), tracheal aspirates (TA), lung biopsies (LB), induced sputum (IS), oropharyngeal washings (OW), and nasopharyngeal aspirates (NPA). The IS, NPA, and OW samples are important in patients where minimally invasive tissue sampling is required. [14]. While BAL is the preferred sample for diagnosis due to its sensitivity ranging from 96. 8\u0026ndash;99.5% [13], its invasiveness may limit its feasibility depending on the patient's condition, requiring less invasive respiratory samples. Consequently, the microscopic diagnosis depends on the type of samples, the quality of the sample, the observer's experience, the fungal load, and the rapid disintegration of fungal cells in fresh samples [10, 15].\u003c/p\u003e\u003cp\u003eThis study aims to standardize and validate a qPCR assay targeting the \u003cem\u003emtLSU rRNA\u003c/em\u003e gene for detecting \u003cem\u003eP. jirovecii\u003c/em\u003e DNA in respiratory samples from patients in Argentina\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eQuantitative PCR (qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCloning the mitochondrial large-subunit rRNA (\u003cem\u003emtLSU rRNA\u003c/em\u003e) gene fragment was a critical step in the qPCR standardization assay and a stable control in each assay. A plasmid harboring the target fragment, kindly provided by the Pasteur Institute of Lille, was successfully amplified in \u003cem\u003eEscherichia coli\u003c/em\u003e by electroporation and selection on antibiotic-containing agar plates. Plasmid DNA was extracted from multiple transformed colonies. Plasmid concentration was measured in ng/ \u0026micro;L using Qubit\u0026reg; dsDNA HS reagent (high sensitivity) with a Qubit 3.0 fluorometer (Invitrogen- Life Technologies-Malaysia), and the number of copies/ \u0026micro;L was calculated later.\u003c/p\u003e\u003cp\u003ePrecise quantification of plasmid DNA ensured accurate concentrations for use as quantification standards in subsequent qPCR assays.\u003c/p\u003e\u003cp\u003eThe real-time PCR assay was standardized based on the protocol by Alanio \u003cem\u003eet al.\u003c/em\u003e [16], with modifications to optimize performance under specific laboratory conditions. A 120-bp fragment of the \u003cem\u003eP. jirovecii mtLSU-rRN\u003c/em\u003eA gene was amplified using primers PjF1 (5'-CTGTTTCCCTTTCGACTATCTACCTT-3') and PjR1 (5'-CACTGAATATCTCGAGGGAGTATGAA-3'), and the TaqMan probe PjSL (6FAM-5'-TCGCACATAGTCTGATTAT-3'- MGBNFQ).\u003c/p\u003e\u003cp\u003eThe assay was conducted on a StepOne PCR system (Applied Biosystems) using the TaqMan Universal Master Mix. Optimization involved evaluating probe concentrations (0.10 \u0026micro;M \u0026minus;\u0026thinsp;0.15 \u0026micro;M \u0026minus;\u0026thinsp;0.20 \u0026micro;M \u0026minus;\u0026thinsp;0.30 \u0026micro;M) against plasmid dilutions (10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e2\u003c/sup\u003e copies /\u0026micro;L) across four primer concentrations (0.3 \u0026micro;M \u0026minus;\u0026thinsp;0.4 \u0026micro;M \u0026minus;\u0026thinsp;0.5 \u0026micro;M -0.6 \u0026micro;M), to ensure effective amplification conditions for reliable qPCR performance.\u003c/p\u003e\u003cp\u003eThe thermal cycling protocol consisted of an initial incubation at 50\u0026deg;C for 2 min, followed by a denaturation step at 95\u0026deg;C for 10 min, and then 45 cycles of 15 s at 95\u0026deg;C and 1 min at 60\u0026deg;C for annealing/extension. Each sample was tested in triplicate, in three different assays.\u003c/p\u003e\u003cp\u003eDNA from 60 different fungi was tested to assess analytical specificity, including primary and opportunistic pathogens. It included DNA from 10 \u003cem\u003eHistoplasma capsulatum\u003c/em\u003e, 10 \u003cem\u003eCoccidioides\u003c/em\u003e spp., 5 \u003cem\u003eParacoccidioides\u003c/em\u003e spp., 10 \u003cem\u003eAspergillus\u003c/em\u003e spp., 5 \u003cem\u003ePenicillium\u003c/em\u003e sp., 10 \u003cem\u003eCandida\u003c/em\u003e spp., and 10 \u003cem\u003eCryptococcus\u003c/em\u003e spp. Additionally, DNA from 10 \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e strains, 11 non-tuberculosis mycobacteria, and 16 different bacteria causing respiratory tract infections (2 \u003cem\u003eLegionella\u003c/em\u003e spp., 5 \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e, 3 \u003cem\u003eHaemophilus influenzae\u003c/em\u003e, 2 \u003cem\u003eMycoplasma pneumoniae\u003c/em\u003e, and 4 \u003cem\u003eNocardia spp.)\u003c/em\u003e provided by the Mycobacteria Service of the Bacteriology Department at INEI-ANLIS \"Carlos G. Malbr\u0026aacute;n\" were included.\u003c/p\u003e\u003cp\u003eThe limit of detection (LOD) was established using a plasmid containing the \u003cem\u003emtLSU rRNA\u003c/em\u003e fragment. Serial 10-fold dilutions of the plasmid were prepared, starting from an initial concentration of 2.2 x 10\u003csup\u003e8\u003c/sup\u003e copies/\u0026micro;L. The minimum amount of plasmid detectable by the previously described qPCR assay was determined in triplicate across three independent experiments. Milli-Q water served as a negative control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClinical specimens\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFrom January 2018 to October 2020, 101 respiratory samples from an equal number of patients presenting with clinical symptoms compatible with PjP were analyzed. The respiratory samples included were: 90 BAL, 2 BL, three induced sputum (IS), one mini-bronchoalveolar lavage (mBAL), four formalin-fixed and paraffin-embedded pulmonary tissue (PT-FFPE), and 1 TA.\u003c/p\u003e\u003cp\u003eSamples were collected from four hospitals in the Autonomous City of Buenos Aires (CABA). The participating laboratories belong to high-complexity centers and are part of the National Network of Mycology Laboratories of Argentina, adhering to the National External Program for Quality Control in Mycology (PNCCM for its acronym in Spanish), which periodically offers quality assessments in mycology to them.\u003c/p\u003e\u003cp\u003eThe patients sought consultation or were admitted to these hospitals, where they underwent clinical and radiological evaluations for protocol inclusion. A portion of each clinical sample was stored at 4\u0026deg;C for later transfer to the National Reference Laboratory in Clinical Mycology (LNRM, for its acronym in Spanish). An \u003cem\u003ead hoc\u003c/em\u003e form that included anonymized patient information accompanied each sample: age, gender, date of admission, specimen type, HIV status, and results from the reference methods for microbiological diagnosis.\u003c/p\u003e\u003cp\u003eIn addition, OW samples from 37 healthy individuals immunocompetent adults without any known immunosuppressive conditions, chronic lung disease, or recent hospital exposure were analyzed. To obtain these samples, each individual performed a deep oro-pharyngeal lavage (gargle) after tooth brushing, using 15 mL of sterile physiological solution for 30 s and collected the fluid into a sterile tube.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobiological studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEach sample was analyzed in the hospital laboratory by direct examination using different assays: direct microscopy on wet mount preparations, calcofluor white stain, immunofluorescence, and specific stains (Grocott, Giemsa, Gram-Weighert) according to their protocols. The direct microscopic examinations (DME) results were consigned in the custom form.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe clinical materials received at the LNRM were processed for molecular studies. DNA extraction was performed using the Blood and Tissue Genomic DNA Extraction Kit from Qiagen (DNeasy Blood \u0026amp; Tissue), following the manufacturer's instructions. Subsequently, the extracted DNA was stored at -20\u0026deg;C until further processing.\u003c/p\u003e\u003cp\u003eThe samples were processed: pure and diluted 1:2 with ultrapure water, using the qPCR method standardized previously. To assess the presence of PCR inhibitors, all clinical samples were systematically tested with an internal positive control (IPC), using the TaqMan\u0026reg; Exogenous Internal Positive Control reagents (Applied Biosystems). Each sample was evaluated in parallel reactions with and without IPC, and no inhibition was detected across the runs. Inclusion of the IPC in every qPCR assay ensured the reliability of negative results and ruled out false negatives due to reaction failure or inhibition.\u003c/p\u003e\u003cp\u003eThe plasmid, stored at \u0026minus;\u0026thinsp;20\u0026deg;C, was also used to generate a three-point standard curve in each qPCR assay, and Milli-Q water was used as a negative control. Sample results were expressed in Ct (value of the corresponding curve between cycle and threshold).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eA receiver operating characteristic (ROC) curve was generated to determine the potential of standardized qPCR for diagnosing PjP, using the GraphPad Prism program version 8.01 for Windows (GraphPad Software, San Diego, CA, USA). The optimal cut-off was determined using Youden\u0026rsquo;s index to differentiate active PjP reliably. The diagnostic performance was calculated by comparing it against the direct microscopy diagnosis, and the correlation was calculated with the Kappa index.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCases definitions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe applied the consensus definitions established by the EORTC/MSGERC expert group. \u0026ldquo;Probable PjP\u0026rdquo; was defined by the presence of clinical and radiological features consistent with \u003cem\u003eP. jirovecii\u003c/em\u003e pneumonia, accompanied by either detection of β-D-glucan in serum or identification of \u003cem\u003eP. jirovecii\u003c/em\u003e DNA by quantitative real-time PCR in respiratory samples. \u0026ldquo;Proven PjP\u0026rdquo; required direct microscopic confirmation of \u003cem\u003eP. jirovecii\u003c/em\u003e through conventional or immunofluorescence staining method in respiratory samples [17, 18]. Additionally, cases with detectable \u003cem\u003eP. jirovecii\u003c/em\u003e DNA that do not allow differentiation between active infection and colonization were defined as \u0026ldquo;gray zone\u0026rdquo;. This corresponds to qPCR results indicating DNA levels that are inconclusive for defining infection status [16].\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe qPCR protocol was optimized by testing primers, probes, and plasmids at four different concentrations. Analyzing sigmoidal fluorescence variation (\u0026Delta;Rn) throughout the amplification cycles was crucial in stablishing the optimal reaction conditions. Primer concentrations of 0.3 \u0026micro;M paired with a probe concentration of 0.15 \u0026micro;M yielded the most consistent and reproducible Ct values across a wide range of plasmid concentrations.\u003c/p\u003e\n\u003cp\u003eNo amplification was observed in DNA samples of 60 fungal species, 21 mycobacteria, and 16 other bacterial species known to cause respiratory infections, confirming the assay\u0026rsquo;s analytical specificity for \u003cem\u003eP. jirovecii\u003c/em\u003e. Using a \u0026Delta;Rn threshold of 0,2, Ct values ranged from 16.69 and 43.02 across serial dilutions from 2.2 x 10\u003csup\u003e8\u003c/sup\u003e to 22 copies of the \u003cem\u003emtLSU rRNA\u003c/em\u003e gene fragment, with an R\u0026sup2; of 0.995, demonstrating the linearity of the curve. The Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the Ct values corresponding to each gene copy number. The lowest plasmid dilution detected with 100% reproducibility was 220 copies per 25 \u0026micro;L reaction (8.8 copies/\u0026micro;L), with an average Ct\u0026thinsp;=\u0026thinsp;40.63. Considering that the average \u003cem\u003emtLSU rRNA\u003c/em\u003e gene copy number per \u003cem\u003eP. jirovecii\u003c/em\u003e organism is approximately 15, 220 copies/25 \u0026micro;L is equivalent to \u0026asymp;\u0026thinsp;14.7 microorganisms per 25 \u0026micro;L, or approximately 1 microorganism/\u0026micro;L.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCt values for different plasmid concentrations in 3 different assays.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"10\"\u003e\n \u003cp\u003eCopy Number /25 \u0026micro;L\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eCt values\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"background-color: rgb(209, 213, 216);\"\u003e\n \u003cp\u003e\u003cstrong\u003e220\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"background-color: rgb(209, 213, 216);\"\u003e\n \u003cp\u003e\u003cstrong\u003e39.91\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"background-color: rgb(209, 213, 216);\"\u003e\n \u003cp\u003e\u003cstrong\u003e40.42\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"background-color: rgb(209, 213, 216);\"\u003e\n \u003cp\u003e\u003cstrong\u003e41.56\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003eND: No detected.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eA total of 101 respiratory samples (90 BAL and 11 other respiratory specimens) from 101 symptomatic patients with clinical suspicion of PjP were analyzed. \u003cem\u003eP. jirovecii\u003c/em\u003e cysts and/or trophic forms were identified by direct microscopic examination in 44 of these samples (43.6%), while no fungal elements were observed in the remaining 57 samples (56.4%) or in any of the 37 OW samples obtained from healthy controls.\u003c/p\u003e\n\u003cp\u003eThe median age of symptomatic patients was 43 years (range: 19\u0026ndash;82); 63 (62%) were male, 31 (31%) were female, and gender data were unavailable for seven patients. Regarding HIV status, 43 patients (43%) were HIV-positive and 49 (49%) had HIV-negative with other immunosuppressive conditions. (Supplementary Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eAmong the 44 patients with respiratory symptoms and positive microscopy (proven PjP), 42 (95.5%) showed a detectable qPCR signal with Ct values ranging from 11.98 to 37.84. In contrast, two samples (4.5%) were qPCR negative (Ct\u0026thinsp;=\u0026thinsp;ND). Among the 57 microscopy-negative patients with respiratory symptoms and radiological features suggestive of PjP, but negative microscopic results (non- proven PjP), 14 samples showed positive qPCR results (Ct ranging: 24.60\u0026ndash;37.80). The remaining 43 samples showed no qPCR amplification, nor did any of the 37 healthy control samples.\u003c/p\u003e\n\u003cp\u003eThe overall concordance between the qPCR and the direct microscopy (gold standard) was 88.41%, with a kappa coefficient of 0.75 (95% CI: 0,639-0,864), indicating substantial agreement.\u003c/p\u003e\n\u003cp\u003eThe discriminatory capacity of the qPCR assay was further assessed by generating a ROC curve comparing patients with proven PjP (positive microscopy samples) with those without confirmed infection. The area under the ROC curve (AUC) was 0.96 (95% CI: 0.93\u0026ndash;0.99), reflecting excellent discriminatory power (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The optimal Ct cutoff value was 36.07, corresponding to the highest likelihood ratio (21.54), and associated with sensitivity of 90.70% and specificity of 95.79%.\u003c/p\u003e\n\u003cp\u003eAmong the 44 microscopy-confirmed cases, three patients had qPCR with Ct values\u0026thinsp;\u0026gt;\u0026thinsp;36 (range: 36.53\u0026ndash;37.84), and two samples were qPCR no-detectable. Among the 14 qPCR-positive patients with -negative microscopy, four had Ct values\u0026thinsp;\u0026lt;\u0026thinsp;36 and therefore classified as probable PjP, while 10 had Ct values between 36.23 and 37.80, corresponding to the so-called \u0026ldquo;gray zone\u0026rdquo;. The distribution of Ct values for both proven (positive microscopy) and non-proven PjP cases (negative microscopy) with Ct\u0026thinsp;\u0026lt;\u0026thinsp;40 is shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eIn the analyzed population, and considering the optimal Ct cutoff value, 44 patients (90.90%) were classified as proven PjP and 4 patients (9.10%) as probable PjP, according to the EORTC/MSGERC criteria [17, 18]. The number of cases increased by 9.1% when probable PjP was included.\u003c/p\u003e\n\u003cp\u003eAmong patients with proven/probable PjP, the median age was 46 years. Thirty patients (68.2%) were male, 16 (36.4%) were female, and sex was not recorded in two cases (4.5%). Thirty-nine patients (81.25%) were PLHIV, while the remaining nine (18.75%) were immunocompromised due to other causes and exhibited radiological findings consistent with atypical pneumonia. (Supplementary Table\u0026nbsp;1).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003ePneumocystis jirovecii\u003c/em\u003e is a fungal pathogen that causes severe pneumonia in immunocompromised patients, including those with HIV or other immunocompromising conditions. Currently, microscopy- base methods are considered the diagnostic gold standard for PjP; however, their low sensitivity is limited, depending s on the observer's experience, fungal load in clinical specimens, and other technical factors. In this context, molecular diagnostic methods have emerged as valuable tools due to their high sensitivity [16, 19].\u003c/p\u003e\u003cp\u003eIn the present study, a highly sensitive and specific qPCR assay targeting the \u003cem\u003emtLSU rRNA\u003c/em\u003e gene of \u003cem\u003eP. jirovecii\u003c/em\u003e was successfully optimized. This gene has been previously selected for molecular diagnostics on clinical samples due to its high copy number within the \u003cem\u003eP. jirovecii\u003c/em\u003e genome, which enhances detection sensitivity [10, 16, 19, 20]. The assay demonstrated high analytical specificity, with no cross-reactivity observed against a broad panel of fungal and bacterial pathogens associated with respiratory infections. These results are in agree with previous studies using the same target gene across different PCR formats, including assays involving human cytomegalovirus and host DNA controls. [21].\u003c/p\u003e\u003cp\u003eThe qPCR assay showed a detection limit of 220 copies per 25 \u0026micro;L (equivalent to 8.8 copies/\u0026micro;L) with 100% reproducibility, consistent with the detection limit reported by Choukri et al. [7], who reported 7 copies/\u0026micro;L using the same target. Although slight differences in detection thresholds may result from sample quality, qPCR conditions, or laboratory equipment, these results underscore the robustness of this molecular target. A Ct\u0026thinsp;\u0026asymp;\u0026thinsp;40.6 was observed for this detection threshold, underscoring the need to establish a reliable cutoff when standardizing in-house assays to reduce false negatives in clinical diagnostics of PjP.\u003c/p\u003e\u003cp\u003eWe analyzed samples from 101 patients with respiratory symptoms, dividing them into groups based on their direct examination results: 44 positive, 57 negative, and 37 healthy individuals. Among microscopy-positive cases, the qPCR assay showed high sensitivity, detecting \u003cem\u003eP. jirovecii\u003c/em\u003e DNA in 95.5% (42/44) with Ct values\u0026thinsp;\u0026le;\u0026thinsp;37.8. Notably, 14 out of 57 (14.9%) microscopy-negative but clinically compatible cases also tested positive by qPCR, with Ct values between 36 and 37.8 in 10 patients, and \u0026lt;\u0026thinsp;36 in the remaining four. This remark the high sensitivity of qPCR and its ability to detect low fungal burdens that might be missed by microscopy. No amplification was observed in healthy controls, indicating the absence of colonization or false positives, and confirming the assay\u0026rsquo;s high specificity.\u003c/p\u003e\u003cp\u003eIn our study the overall concordance between direct microscopy and qPCR was 88.4% (kappa\u0026thinsp;=\u0026thinsp;0.75), indicating substantial agreement between both diagnostic methods. This supports the utility of qPCR as a complementary diagnostic tool, especially in cases where direct examination is inconclusive. However, our concordance rate was slightly lower than that reported by Meliani et al. [22], who reported 91.3% agreement, but higher than the 70% reported by Brancart \u003cem\u003eet al.\u003c/em\u003e [23]. The differences likely reflecting inter-study variability in experimental qPCR protocols, sample handling and definitions used for case classification. The inclusion of probable PjP cases in our study, based on clinical and radiological criteria in conjunction with qPCR Ct values, may have influenced the observed concordance values. These findings reinforce the importance of standardizing laboratory methods and clinical algorithms for the diagnosis of PjP across different healthcare centers.\u003c/p\u003e\u003cp\u003eThe ROC curve analysis showed an area under the curve (AUC) of 0.96 (95% CI, 0.93\u0026ndash;0.99), indicating excellent diagnostic performance. A Ct cutoff of \u0026le;\u0026thinsp;36 yielded the best balance of sensitivity (90%) and specificity (96%) for distinguishing active infection. This threshold is higher than that reported by Fauchier et al. [19], who proposed Ct\u0026thinsp;\u0026lt;\u0026thinsp;32, with an AUC of 0.88 (72% sensitivity and 75% specificity), using the same gene. The higher cutoff observed in our study probable reflects a broader patient population, including both HIV-positive individuals, who generally exhibited higher fungal burdens, and non-HIV patients, who typically exhibit lower fungal burdens [19, 24, 25]. Variations in specimen type and immune status influenced the range of Ct values observed, emphasizing the need to adapt diagnostic thresholds accordingly.\u003c/p\u003e\u003cp\u003eGits Musell \u003cem\u003eet al.\u003c/em\u003e [26] evaluated a panel of respiratory samples using various qPCR protocols across 16 reference laboratories and reported up to 12-cycle variability for the identical samples. This emphasizes the importance of standardizing in-house qPCR assays and incorporating concentration curves and control materials to ensure consistency across different settings.\u003c/p\u003e\u003cp\u003eA critical observation from our data is the identification of a \u0026ldquo;grey zone\u0026rdquo; for Ct values between 36 and 37.8, where differentiating between colonization and active infection remains challenging. This overlap was observed in both proven PjP and microscopy negative patients. Similar findings have been reported in previous studies using molecular techniques [16, 19, 27, 28]. In our study, no complementary biomarkers such as serum β-D-glucan or longitudinal follow-up data were available to clarify the clinical significance of theses intermediate Ct values. As such, this range should be interpreted with caution and within the broader clinical and radiological context.\u003c/p\u003e\u003cp\u003eAll 37 respiratory samples from healthy individuals remained negative, reinforcing the assay\u0026rsquo;s specificity and reducing concerns about asymptomatic colonization [19, 29].\u003c/p\u003e\u003cp\u003eAn aspect that strengthens the technical reliability of this study is the consistent inclusion of an IPC in all qPCR reactions, which allowed us to detect potential PCR inhibitors and ensure the validity of negative results, as recommended in molecular diagnostics protocols [26]. Moreover, the assay\u0026rsquo;s analytical specificity was evaluated against a wide panel of fungal and bacterial pathogens, helping to reduce the risk of false positives in clinical practice. These measures contribute to the assay\u0026rsquo;s robustness under routine laboratory conditions.\u003c/p\u003e\u003cp\u003eIn summary, the standardized qPCR assay developed in this study represents a highly sensitive and specific method for the molecular detection of \u003cem\u003eP. jirovecii\u003c/em\u003e. Its ability to detect low fungal burdens enables early diagnosis, particularly in patients with clinical suspicion of PjP but negative microscopic findings. While diagnostic uncertainty persists within the grey Ct zone, the assay remains a valuable complementary tool when interpreted together with clinical, radiological, and immunological data. Overall, these results support its integration into diagnostic algorithms for PjP, especially in situations requiring rapid and accurate detection to guide timely clinical decisions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Administraci\u0026oacute;n Nacional de Laboratorios e Institutos de Salud (ANLIS) \u0026ldquo;Dr. Carlos G. Malbr\u0026aacute;n\u0026rdquo;. Cristina E. Canteros received support through the VI CONVOCATORIA A FONDOS CONCURSABLES ANLIS, 2015 (Project No. NRU 1524).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdriana In\u0026eacute;s Toranzo and Cristina E. Canteros contributed substantially to the study conception, design, coordination, execution, data analysis, and manuscript preparation.\u003c/p\u003e\n\u003cp\u003eClaudia Frola, Rosana Jord\u0026aacute;n, Patricia Giorgio, and Ana Ruth Laborde managed patient care, case selection, and clinical interpretation.\u003c/p\u003e\n\u003cp\u003eRoberto Moyano performed statistical analyses, including ROC curve interpretation.\u003c/p\u003e\n\u003cp\u003eNorma Fern\u0026aacute;ndez, Agustina Forastiero, Liliana Guelfand, Luciana Far\u0026iacute;as, Mariana Andreani, Facundo Muise Acevedo, Mariana Viale, and Cecilia L\u0026oacute;pez-Joffre contributed to laboratory work, sample processing, and manuscript editing.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthical approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki, and all applicable national regulations. The ANLIS Research Ethics Committee \u0026ldquo;Dr. Carlos G. Malbr\u0026aacute;n\u0026rdquo; granted approval for this study: NRU 1524, VI CONVOCATORIA A FONDOS CONCURSABLES ANLIS, 2015. Additional ethical permission was obtained from the ethics committees of each participating hospitals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all individuals enrolled in the study. The consent procedure was reviewed and approved by the same ethics committee. All clinical data were anonymized prior to analysis, and oropharyngeal wash samples from healthy volunteers were obtained in accordance with approved informed consent procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Institut Pasteur de Lille for kindly providing the cloned fragment of the mitochondrial large-subunit rRNA (mtLSU rRNA) gene.\u003c/p\u003e\n\u003cp\u003eThis study was conducted as part of the PhD thesis of Adriana In\u0026eacute;s Toranzo, under the direction of Dr. Cristina E. Canteros.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMcDonald EG, Afshar A, Assiri B, et al. Pneumocystis jirovecii pneumonia in people living with HIV: a review. Clin Microbiol Rev. 2024;37(1):e0010122. doi:10.1128/cmr.00101-22.\u003c/li\u003e\n \u003cli\u003eSalzer HJF, Sch\u0026auml;fer G, Hoenigl M, et al. Clinical, Diagnostic, and Treatment Disparities between HIV-Infected and Non-HIV-Infected Immunocompromised Patients with Pneumocystis jirovecii Pneumonia. Respiration. 2018;96(1):52-65. doi:10.1159/000487713.\u003c/li\u003e\n \u003cli\u003eKrajicek BJ, Thomas CF Jr, Limper AH. Pneumocystis pneumonia: current concepts in pathogenesis, diagnosis, and treatment. Clin Chest Med. 2009;30(2):265-vi. doi:10.1016/j.ccm.2009.02.005.\u003c/li\u003e\n \u003cli\u003eAhmadpour E, Valilou S, Ghanizadegan MA, et al. Global prevalence, mortality, and main characteristics of HIV-associated pneumocystosis: A systematic review and meta-analysis. PLoS One. 2024;19(3):e0297619. Published 2024 Mar 25. doi:10.1371/journal.pone.0297619.\u003c/li\u003e\n \u003cli\u003eMedrano FJ, Montes-Cano M, Conde M, et al. Pneumocystis jirovecii in general population. Emerg Infect Dis. 2005;11(2):245-250. doi:10.3201/eid1102.040487.\u003c/li\u003e\n \u003cli\u003ePonce CA, Gallo M, Bustamante R, Vargas SL. Pneumocystis colonization is highly prevalent in the autopsied lungs of the general population. Clin Infect Dis. 2010;50(3):347-353. doi:10.1086/649868.\u003c/li\u003e\n \u003cli\u003eChoukri F, Menotti J, Sarfati C, et al. Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia. Clin Infect Dis. 2010;51(3):259-265. doi:10.1086/653933.\u003c/li\u003e\n \u003cli\u003eFr\u0026eacute;alle E, Valade S, Guigue N, et al. Diffusion of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis colonization: frequency and putative risk factors. Med Mycol. 2017;55(5):568-572. doi:10.1093/mmy/myw113.\u003c/li\u003e\n \u003cli\u003eVera C, Rueda ZV. Transmission and Colonization of Pneumocystis jirovecii. J Fungi (Basel). 2021;7(11):979. Published 2021 Nov 18. doi:10.3390/jof7110979.\u003c/li\u003e\n \u003cli\u003eMoodley B, Tempia S, Frean JA. Comparison of quantitative real-time PCR and direct immunofluorescence for the detection of Pneumocystis jirovecii. PLoS One. 2017;12(7):e0180589. Published 2017 Jul 6. doi:10.1371/journal.pone.0180589.\u003c/li\u003e\n \u003cli\u003eEsteves F, Cal\u0026eacute; SS, Badura R et al. Diagnosis of Pneumocystis pneumonia: evaluation of four serologic biomarkers. Clin Microbiol Infect. 2015;21(4):379.e1-10. doi: 10.1016/j.cmi.2014.11.025.\u003c/li\u003e\n \u003cli\u003eEsteves F, Lee CH, de Sousa B, et al. (1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients. Eur J Clin Microbiol Infect Dis. 2014;33(7):1173-1180. doi:10.1007/s10096-014-2054-6.\u003c/li\u003e\n \u003cli\u003eBrown L, Rautemaa-Richardson R, Mengoli C, et al. Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis. Clin Infect Dis. 2024;79(1):161-168. doi:10.1093/cid/ciae239.\u003c/li\u003e\n \u003cli\u003eSen\u0026eacute;cal J, Smyth E, Del Corpo O, et al. Non-invasive diagnosis of Pneumocystis jirovecii pneumonia: a systematic review and meta-analysis. Clin Microbiol Infect. 2022;28(1):23-30. doi:10.1016/j.cmi.2021.08.017.\u003c/li\u003e\n \u003cli\u003eReid AB, Chen SC, Worth LJ. Pneumocystis jirovecii pneumonia in non-HIV-infected patients: new risks and diagnostic tools. Curr Opin Infect Dis. 2011;24(6):534-544. doi:10.1097/QCO.0b013e32834cac17.\u003c/li\u003e\n \u003cli\u003eAlanio A, Desoubeaux G, Sarfati C, et al. Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients. Clin Microbiol Infect. 2011;17(10):1531-1537. doi:10.1111/j.1469-0691.2010.03400.x.\u003c/li\u003e\n \u003cli\u003eDonnelly JP, Chen SC, Kauffman CA, 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(6):1367-1376. doi:10.1093/cid/ciz1008.\u003c/li\u003e\n \u003cli\u003eLagrou K, Chen S, Masur H, et al. Pneumocystis jirovecii Disease: Basis for the Revised EORTC/MSGERC Invasive Fungal Disease Definitions in Individuals Without Human Immunodeficiency Virus. Clin Infect Dis. 2021;72(Suppl 2):S114-S120. doi:10.1093/cid/ciaa1805.\u003c/li\u003e\n \u003cli\u003eFauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty PM, 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(6):1487-1495. doi:10.1128/JCM.03174-15.\u003c/li\u003e\n \u003cli\u003eGupta R, Mirdha BR, Guleria R, et al. Diagnostic significance of nested polymerase chain reaction for sensitive detection of Pneumocystis jirovecii in respiratory clinical specimens. Diagn Microbiol Infect Dis. 2009;64(4):381-388. doi:10.1016/j.diagmicrobio.2009.04.008.\u003c/li\u003e\n \u003cli\u003eMu\u0026ntilde;oz C, Zuluaga A, Restrepo A, Tob\u0026oacute;n A, Cano LE, Gonzalez A. Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients. Diagn Microbiol Infect Dis. 2012;72(3):204-213. doi:10.1016/j.diagmicrobio.2011.11.015.\u003c/li\u003e\n \u003cli\u003eMeliani L, Develoux M, Marteau-Miltgen M, et al. Real time quantitative PCR assay for Pneumocystis jirovecii detection. J Eukaryot Microbiol. 2003;50 Suppl:651. doi:10.1111/j.1550-7408.2003.tb00670.x.\u003c/li\u003e\n \u003cli\u003eBrancart F, Rodriguez-Villalobos H, Fonteyne PA, Peres-Bota D, Liesnard C. Quantitative TaqMan PCR for detection of Pneumocystis jiroveci. J Microbiol Methods. 2005;61(3):381-387. doi:10.1016/j.mimet.2005.01.001.\u003c/li\u003e\n \u003cli\u003eM\u0026uuml;hlethaler K, B\u0026ouml;gli-Stuber K, Wasmer S, et al. Quantitative PCR to diagnose Pneumocystis pneumonia in immunocompromised non-HIV patients. Eur Respir J. 2012;39(4):971-978. doi:10.1183/09031936.00095811\u003c/li\u003e\n \u003cli\u003eSarasombath PT, Thongpiya J, Chulanetra M, et al. Quantitative PCR to Discriminate Between Pneumocystis Pneumonia and Colonization in HIV and Non-HIV Immunocompromised Patients. Front Microbiol. 2021;12:729193. Published 2021 Oct 20. doi:10.3389/fmicb.2021.729193.\u003c/li\u003e\n \u003cli\u003eGits-Muselli M, White PL, Mengoli C, et al. The Fungal PCR Initiative\u0026apos;s evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay. Med Mycol. 2020;58(6):779-788. doi:10.1093/mmy/myz115.\u003c/li\u003e\n \u003cli\u003eMaillet M, Maubon D, Brion JP, et al. Pneumocystis jirovecii (Pj) quantitative PCR to differentiate Pj pneumonia from Pj colonization in immunocompromised patients. Eur J Clin Microbiol Infect Dis. 2014;33(3):331-336. doi:10.1007/s10096-013-1960-3.\u003c/li\u003e\n \u003cli\u003eMatsumura Y, Ito Y, Iinuma Y, et al. Quantitative real-time PCR and the (1\u0026rarr;3)-\u0026beta;-D-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization. Clin Microbiol Infect. 2012;18(6):591-597. doi:10.1111/j.1469-0691.2011.03605.x.\u003c/li\u003e\n \u003cli\u003eAguilar YA, Rueda ZV, Maya MA, et al. Is It Possible to Differentiate Pneumocystis jirovecii Pneumonia and Colonization in the Immunocompromised Patients with Pneumonia?. J Fungi (Basel). 2021;7(12):1036. Published 2021 Dec 2. doi:10.3390/jof7121036.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pneumocystis jirovecii, real-time PCR, molecular diagnosis, pneumonia, immunocompromised patients","lastPublishedDoi":"10.21203/rs.3.rs-7142772/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7142772/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003ePneumocystis jirovecii\u003c/em\u003e pneumonia (PjP) is a life-threatening infection in immunocompromised individuals. Its diagnosis remains challenging, especially in microscopy-negative cases. This study aimed to standardize and validate a qPCR assay targeting the \u003cem\u003emtLSU rRNA\u003c/em\u003e gene for detecting \u003cem\u003eP. jirovecii\u003c/em\u003e DNA in respiratory samples from patients in Argentina.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003eThe assay was optimized using plasmid dilutions containing the target gene. Analytical specificity was evaluated against 60 fungal, 21 mycobacterial, and 16 bacterial species. Clinical validation included 101 respiratory samples from symptomatic patients and 37 from healthy individuals. An internal positive control (IPC) was included in all reactions to detect inhibitors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe qPCR assay showed a detection limit of 8.8 copies/\u0026micro;L and no cross-reactivity. Among microscopy-confirmed cases, 95.5% were qPCR-positive. Notably, 14.9% of microscopy-negative but clinically compatible cases tested positive. ROC analysis yielded an AUC of 0.96, with an optimal Ct cutoff\u0026thinsp;\u0026le;\u0026thinsp;36, providing 90.7% sensitivity and 95.8% specificity. No healthy controls tested positive. A \u0026ldquo;grey zone\u0026rdquo; (Ct 36\u0026ndash;37.8) was observed, requiring clinical correlation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis qPCR assay is highly sensitive and specific, offering a valuable diagnostic tool for PjP. Its performance supports implementation in routine diagnostics, especially when microscopy is inconclusive. However, interpretation in the grey zone requires complementary clinical or biomarker data.\u003c/p\u003e","manuscriptTitle":"Standardization and Clinical Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis jirovecii Pneumonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 09:58:53","doi":"10.21203/rs.3.rs-7142772/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-08T06:54:45+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-05T16:35:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Mycopathologia","date":"2025-07-18T14:28:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-18T14:25:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Mycopathologia","date":"2025-07-16T14:43:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"mycopathologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"myco","sideBox":"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)","snPcode":"11046","submissionUrl":"https://submission.nature.com/new-submission/11046/3","title":"Mycopathologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d7af3506-1990-4c3f-8c3e-26e583c6f51a","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:05:17+00:00","versionOfRecord":{"articleIdentity":"rs-7142772","link":"https://doi.org/10.1007/s11046-025-01016-7","journal":{"identity":"mycopathologia","isVorOnly":false,"title":"Mycopathologia"},"publishedOn":"2025-11-04 15:57:59","publishedOnDateReadable":"November 4th, 2025"},"versionCreatedAt":"2025-08-11 09:58:53","video":"","vorDoi":"10.1007/s11046-025-01016-7","vorDoiUrl":"https://doi.org/10.1007/s11046-025-01016-7","workflowStages":[]},"version":"v1","identity":"rs-7142772","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7142772","identity":"rs-7142772","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}