IFN-γ Limits Immunopathogenesis but Delays Fungal Clearance during Pneumocystis Pneumonia.

High levels of IFN-γ are produced in the lung during an adaptive immune response to Pneumocystis, but the effects of this prototypical Th1 cytokine on fungal clearance and immunopathogenesis have not been fully defined. Therefore, Pneumocystis-infected immunodeficient mice were immune reconstituted and administered control or anti-IFN-γ neutralizing Ab to determine how IFN-γ regulates the balance between host defense and immune-mediated lung injury. Mice treated with anti-IFN-γ demonstrated an initial worsening of Pneumocystis pneumonia-related immunopathogenesis, with greater weight loss, heightened lung inflammation, and more severe pulmonary function deficits than control mice. However, IFN-γ neutralization also enhanced macrophage phagocytosis of Pneumocystis and accelerated fungal clearance. When anti-IFN-γ-treated mice were also given IL-4 and IL-13 to promote a Th2-biased lung environment, the accelerated fungal clearance was preserved, but the severity of immunopathogenesis was reduced, and a more rapid recovery was observed. A direct suppressive effect of IFN-γ on macrophages was required but was not solely responsible for delayed fungal clearance, suggesting that IFN-γ acts through multiple mechanisms that likely include modulation of both macrophage and Th polarization. Enhanced Pneumocystis clearance in anti-IFN-γ-treated and IFN-γR-deficient mice was associated with significantly elevated IL-17+ CD4+ T cells and IL-17 protein in the lungs. Furthermore, neutralization of IL-17, but not IL-4, signaling blocked the accelerated fungal clearance observed in anti-IFN-γ-treated mice. Together, these data demonstrate that although IFN-γ delays fungal clearance by suppressing the lung Th17 response, it also serves an important regulatory role that limits immunopathogenesis and preserves pulmonary function.

Protective innate immunity against Pneumocystis does not require Stat6-dependent macrophage polarization.

Pneumocystis species are respiratory fungal pathogens that cause life-threatening opportunistic infections in immunocompromised hosts. Pneumocystis typically evade pulmonary innate immunity but are efficiently eradicated by a functional adaptive immune response. FVB/NJ mice are unique in that they display protective alveolar macrophage-dependent innate immunity against Pneumocystis, and remain resistant to infection even in the absence of CD4+ T lymphocyte function. FVB/NJ alveolar macrophages (AMs) were found to display an M2-biased phenotype at baseline, which was potentiated after stimulation with Pneumocystis, suggesting that macrophage polarization may dictate the outcome of the Pneumocystis-macrophage interaction. To determine whether Stat6, a key global regulator of M2 polarization, was required for FVB/NJ innate immunity, FVB Stat6-/- mice were generated. FVB Stat6-deficient AMs were markedly impaired in their ability to polarize to an M2 phenotype when stimulated with Th2 cytokines. However, FVB Stat6-/- mice remained highly resistant to infection, indicating that Stat6 signaling is dispensable for innate FVB/NJ resistance. Despite the loss of Stat6 signaling, primary AMs from FVB Stat6-/- mice maintained baseline expression of M2 markers, and also strongly upregulated M2-associated genes following direct stimulation with Pneumocystis. Additional FVB/NJ knockout strains were generated, but only FVB MerTK-/- mice showed a marginally increased susceptibility to Pneumocystis infection. Together, these findings demonstrate that effective FVB/NJ innate immunity against Pneumocystis does not require Stat6 signaling and suggest that alternative pathways regulate M2 bias and macrophage-mediated innate resistance in FVB/NJ mice.

Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis.

BACKGROUND: This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations. METHODS: A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined. RESULTS: Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients. CONCLUSIONS: On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested.

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii ß-glucans.

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. ß-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. ß-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae ß-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal ß-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Evaluation of analyte-specific reagents for the direct detection of Pneumocystis jirovecii.

Pneumocystis jirovecii pneumonia (PJP) is a serious and sometimes fatal infection occurring in immunocompromised individuals. High-risk patients include those with low CD4 counts due to human immunodeficiency virus infection and transplant recipients. The incidence of PJP is increasing, and rapid detection of PJP is needed to effectively target treatment and improve patient outcomes. A common method used is an immunofluorescent assay (IFA), which has limitations, including labor costs, low sensitivity, and requirement for expert interpretation. This study evaluates the performance of the DiaSorin Molecular Pneumocystis jirovecii analyte-specific reagent (ASR) in a laboratory-developed test (LDT) for the direct detection of P. jirovecii DNA without prior nucleic acid extraction. Respiratory samples (n = 135) previously tested by IFA from 111 patients were included. Using a composite standard of in-house IFA and reference lab PJP PCR, the percent positive agreement for the LDT using the DiaSorin ASR was 97.8% (90/92). The negative percent agreement was 97.7% (42/43). The lower limit of detection of the assay was determined to be 1,200 copies/mL in bronchoalveolar lavage fluid. Analytical specificity was assessed using cultures of oropharyngeal flora and common respiratory bacterial and fungal pathogens. No cross-reactivity was observed. Our study suggests that the DiaSorin Pneumocystis ASR accurately detects P. jirovecii DNA and demonstrates improved sensitivity compared to the IFA method. IMPORTANCE: Our study is unique compared to other previously published studies on the DiaSorin analyte-specific reagent (ASR) because we focused on microbiological diagnostic methods commonly used (immunofluorescent assay) as opposed to pathology findings or reference PCR. In addition, in our materials and methods, we describe the protocol for the use of the DiaSorin ASR as a singleplex assay, which will allow other users to evaluate the ASR for clinical use in their lab.

Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii.

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within  30-40 min, comprising a 25-30 min RPA reaction and a 5-10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

MiR-150 levels are related to in-hospital mortality in non-HIV Pneumocystis pneumonia patients.

Pneumocystis pneumonia (PCP) is a common opportunistic infection that occurs in immunocompromised patients. Compared with HIV patients, PCP in non-HIV patients tends to follow up a more urgent course and poorer prognosis. Therefore, markers that could predict survival of PCP patients in non-HIV population are of great value. MiRNA-150 has been widely studied in many diseases since it has been identified as a vital regulator of immune cell differentiation and activation. We thus conduct this study aiming to evaluate the prognostic value of miR-150 level in non-HIV PCP. First, the expression levels of miR-150 were compared between PCP patients and healthy volunteers. The miR-150 levels in immune cells were also detected in PCP mouse models. Then the prognostic value of miR-150 was further assessed in another PCP population (n = 72). The expression levels of miR-150 were measured by reverse transcription real-time PCR (RT-PCR) technique. Our data demonstrated significantly decreased miR-150 expression levels in PCP patients and mouse models compared to controls. The miR-150 levels also decreased in various immune cells of PCP mouse models. With a cut-off value of 3.48, the area under the curve, sensitivity, specificity of miR-150 to predicate PCP mortality were 0.845, 68.2% and 96.0%, respectively. In conclusion, miR-150 expression value might serve as a potential biomarker to identify PCP patients at high risk of death.

Pneumocystis pneumonia (PCP) remains a fatal risk for immunosuppressed patients. MiR-150 takes part in immune regulation, and thus is involved in infection control. Our study indicated that the miR-150 expression may act as a potential biomarker for predicting mortality of PCP patients.

Features and global impact of invasive fungal infections caused by Pneumocystis jirovecii: A systematic review to inform the World Health Organization fungal priority pathogens list.

This systematic review evaluates the current global impact of invasive infections caused by Pneumocystis jirovecii (principally pneumonia: PJP), and was carried out to inform the World Health Organization Fungal Priority Pathogens List. PubMed and Web of Science were used to find studies reporting mortality, inpatient care, complications/sequelae, antifungal susceptibility/resistance, preventability, annual incidence, global distribution, and emergence in the past 10 years, published from January 2011 to February 2021. Reported mortality is highly variable, depending on the patient population: In studies of persons with HIV, mortality was reported at 5%-30%, while in studies of persons without HIV, mortality ranged from 4% to 76%. Risk factors for disease principally include immunosuppression from HIV, but other types of immunosuppression are increasingly recognised, including solid organ and haematopoietic stem cell transplantation, autoimmune and inflammatory disease, and chemotherapy for cancer. Although prophylaxis is available and generally effective, burdensome side effects may lead to discontinuation. After a period of decline associated with improvement in access to HIV treatment, new risk groups of immunosuppressed patients with PJP are increasingly identified, including solid organ transplant patients.

Prognostic analysis of concurrent Pneumocystis jirovecii pneumonia in patients with systemic lupus erythematosus: a retrospective study.

BACKGROUND: Systemic lupus erythematosus (SLE) has been less deadly since the advent of corticosteroid-sparing medications. SLE patients still have a higher mortality rate than the general population. Infectious disease is reported as one of the major causes of death in patients with SLE. Although bacteria are the most often isolated pathogens from patients with SLE, Pneumocystis jirovecii pneumonia (PJP) is more deadly than bacterial infection. METHODS: We retrospectively enrolled consecutive patients with SLE concurrent with PJP (SLE-PJP) in our center between January 2014 and December 2022. The participants were classified into two groups: survivors and non-survivors. Cox regression models and Kaplan‒Meier survival analyses were conducted to explore prognostic factors for survival. RESULTS: There were 57 patients with SLE (42.0 ± 15.8 years old, 78.9% female) complicated with PJP, 22 (38.6%) of whom died. Compared with the survival group, the non-survival group had more patients with hyperglycemia or diabetes mellitus, invasive ventilation (p < 0.01), respiratory failure, intensive care unit admission, non-invasive ventilation, and hospital-acquired pneumonia (p < 0.05). The non-survival group showed a higher neutrophil percentage, lactate dehydrogenase, D-dimer (p < 0.001), urea, high-sensitivity C-reactive protein (hsCRP), erythrocyte sedimentation rate (ESR), and ferritin (p < 0.05). It also had lower minimal albumin, hemoglobin (p < 0.001), immunoglobulin G, complement 3, peripheral lymphocyte count, platelet, NK cell count, and CD4+ T-cell count (p < 0.05). Multivariate analysis indicated that hyperglycemia or diabetes mellitus (HR = 4.25, p < 0.01, 95% CI: 1.51-11.97), thrombocytopenia (HR = 4.22, p < 0.01, 95% CI: 1.63-10.91) and lower complement 3 (C3) (HR = 4.06, p < 0.01, 95% CI: 1.60-10.33) were independent risk factors for the survival of SLE-PJP patients. CONCLUSIONS: The mortality rate of patients with SLE-PJP is still high. Hyperglycemia, decreased C3, and thrombocytopenia are independent survival risk factors.

Development of a clinical risk score for the prediction of Pneumocystis jirovecii pneumonia in hospitalised patients.

BACKGROUND: The performance and availability of invasive and non-invasive investigations for the diagnosis of Pneumocystis jirovecii pneumonia (PCP) vary across clinical settings. Estimating the pre-test probability of PCP is essential to the optimal selection and interpretation of diagnostic tests, such as the 1,3-ß-D-glucan assay (BDG), for the prioritization of bronchoscopy, and to guide empiric treatment decisions. We aimed to develop a multivariable risk score to estimate the pre-test probability of PCP. METHODS: The score was developed from a cohort of 626 individuals who underwent bronchoscopy for the purposes of identifying PCP in a Canadian tertiary-care centre, between 2015 and 2018. We conducted a nested case-control study of 57 cases and 228 unmatched controls. Demographic, clinical, laboratory, and radiological data were included in a multivariable logistic regression model to estimate adjusted odds ratios for PCP diagnosis. A clinical risk score was derived from the multivariable model and discrimination was assessed by estimating the score's receiver operating characteristic curve. RESULTS: Participants had a median age of 60 years (interquartile range [IQR] 49-68) and 115 (40%) were female; 40 (14%) had HIV and 49 (17%) had a solid organ transplant (SOT). The risk score included prior SOT or HIV with CD4 ≤ 200/µL (+ 2), serum lactate dehydrogenase ≥ 265.5 IU/mL (+ 2), radiological pattern typical of PCP on chest x-ray (+ 2) or CT scan (+ 2.5), and PCP prophylaxis with trimethoprim-sulfamethoxazole (-3) or other antimicrobials (-2). The median score was 4 points (IQR, 2-4.5) corresponding to a 28% probability of PCP. The risk prediction model had good discrimination with a c-statistic of 0.79 (0.71-0.84). Given the operating characteristics of the BDG assay, scores ≤ 3 in patients without HIV, and ≤ 5.5 in those with HIV, paired with a negative BDG, would be expected to rule out PCP with 95% certainty. CONCLUSION: We propose the PCP Score to estimate pre-test probability of PCP. Once validated, it should help clinicians determine which patients to refer for invasive investigations, when to rely on serological testing, and in whom to consider pre-emptive treatment.

Pneumocystis jirovecii and Nocardia pneumonia in a middle-aged male with Nephrotic syndrome: a case report and literature review.

INTRODUCTION: Nephrotic syndrome (NS) is a common chronic kidney disease that is often accompanied by a state of immunodeficiency. Immunosuppression increases the risk of infections, with Pneumocystis jirovecii and Nocardia brasiliensis being two opportunistic pathogens that can cause severe infections in patients with compromised immune function. This study presents a case of a middle-aged male patient with NS concurrently infected with Pneumocystis jirovecii and Nocardia brasiliensis. It aims to synthesize the pertinent diagnostic approaches and treatment experiences. Notably, there have been no reported cases of NS occurring simultaneously with both Pneumocystis jirovecii pneumonia and Nocardia pneumonia. CASE PRESENTATION: A 58-year-old male farmer presented to the hospital with a one-week history of persistent fever, cough, and sputum production. His maximum body temperature was recorded at 39 °C, and he produced yellow viscous sputum. This patient had a one-year history of NS, managed with long-term oral corticosteroid and cyclophosphamide therapy. Admission chest computed tomography displayed interstitial changes in both lungs. After failing to detect any pathogens through routine etiological tests, we successfully identified Nocardia brasiliensis, Pneumocystis jirovecii, and Lodderomyces elongisporus using bronchoscopy-guided sputum samples through metagenomic next-generation sequencing (mNGS) technology. Subsequently, we initiated a combined treatment regimen for the patient using trimethoprim-sulfamethoxazole, meropenem, and moxifloxacin, which yielded remarkable therapeutic outcomes. CONCLUSION: The adoption and promotion of mNGS technologies have significantly resolved the difficulty in early pathogen detection, guiding clinicians from empirical to genomic diagnosis, achieving prevention before treatment, and thereby enhancing patient survival rates.

Outcome of Pneumocystis pneumonia in transplant and non-transplant HIV-negative immunocompromised patients.

BACKGROUND: Previous studies showed HIV-negative immunocompromised patients are susceptible to Pneumocystis pneumonia (PCP). However, the PCP outcome has not been compared among HIV-negative immunocompromised patients. METHODS: In this retrospective cohort study at the University Health Network, we included all HIV-negative immunocompromised patients who fulfilled the European Organization for Research and Treatment of Cancer (EORTC) PCP diagnosis criteria from December 2018 to December 2019. We compared the demographics, comorbidities, course of illness, and PCP outcome (28-day mortality and composite outcome [i.e., death or intensive care unit (ICU) admission]) between solid organ transplant (SOT) and non-SOT patients. RESULTS: Of 160 non-HIV patients with PCP diagnoses, 118 patients fulfilled EORTC criteria (76 males [64.4%], median [range] age: 65.5 [21-87] years). PCP presentation in SOT recipients (n = 14) was more severe than non-SOT patients (n = 104): acute presentation (onset <7 days before admission: 11/14 [78.6%] vs. 51/104 [56%], p = .037), shortness of breath (100% vs. 75/104 [74.3%], p = .037), median [range] O2 saturation (88% [75%, 99%] vs. 92%[70%, 99%], p = .040), and supplemental O2 requirement (12/14 [85.7%] vs. 59/104 [56.7%], p = .044). The mortality [4/14, (28.6%) vs. 15/104 (14.4%), p = .176], ICU admission (10/14 [71.4%] vs. 18/104 [17.3%], p < .0001), and mechanical ventilation (8/14 [57.1%] vs. 18/104 [17.3%], p = .0007) in SOT patients was different from non-SOT patients. In multivariable analysis, SOT recipients were at greater risk of composite outcome than non-SOT patients (aOR [CI95%]: 12.25 [3.08-48.62], p < .001). CONCLUSION: PCP presentation and outcomes in SOT recipients are more severe than in non-SOT patients. Further studies are required to explore the biological reasons for this difference.

Interstitial Lesions in Both Lungs Finally Diagnosed as a Rare Coinfection of Influenza A Virus and Pneumocystis Jiroveci.

BACKGROUND: Influenza is an acute respiratory infection caused by influenza viruses, with influenza A virus (IAV) being the most common and most likely to progress to critically ill cases leading to death. Pneumocystis jiroveci is an opportunistic lung-causing fungus that occurs most often in immunocompromised individuals and can cause Pneumocystis jiroveci pneumonia (PJP). It is rare for both diseases to occur in the same patient. METHODS: Appropriate laboratory tests, chest computed tomography (CT), bronchoalveolar lavage fluid, second-generation macro gene sequencing, and pathogenetic tests to clarify the diagnosis. RESULTS: G test and LDH were high, and chest CT showed rapidly progressive interstitial pneumonia, which was confirmed by bronchoalveolar lavage fluid and macrogenomic second-generation sequencing (mNGS) to be a mixed infection of H. influenzae type A virus and Pneumocystis jiroveci. CONCLUSIONS: In rapidly progressive interstitial pneumonia, bronchoalveolar lavage and mNGS should be done early to clarify the presence of infection with specific pathogenic organisms.

Seronegative HIV-1 infection in a Japanese man presenting with Pneumocystis pneumonia: Analysis of long-term antibody response and literature review.

Seronegative human immunodeficiency virus (HIV) infection, where an HIV-specific antibody response is lacking even in chronic or late-stage HIV infections, is extremely rare. Here, we report the case of a 50-year-old Japanese man presenting with Pneumocystis pneumonia who did not produce antibodies against HIV-1 until the initiation of antiretroviral therapy (ART). Fourth-generation antigen-antibody testing temporarily reverted from weakly positive to negative soon after initiating ART, likely due to a reduction in viral load (assessed by p24 antigen levels). His HIV-1 antibody titers remained low or indeterminate even after four years of ART. A literature review suggested that the absence of HIV-1-specific antibody production may be associated with unimpeded HIV replication and rapid CD4+ T cell decline. Seronegative HIV infection can lead to deferred diagnosis and treatment, thereby increasing the risk of transmitting the virus to others or developing opportunistic illnesses. It is important to combine multiple tests for diagnosis, depending on the medical condition. Further studies are required to investigate the host factors involved in the production of HIV-1-specific antibodies.

Description of a Murine Model of Pneumocystis Pneumonia.

Pneumocystis pneumonia is a serious lung infection caused by an original ubiquitous fungus with opportunistic behavior, referred to as Pneumocystis jirovecii. P. jirovecii is the second most common fungal agent among invasive fungal infections after Candida spp. Unfortunately, there is still an inability to culture P. jirovecii in vitro, and so a great impairment to improve knowledge on the pathogenesis of Pneumocystis pneumonia. In this context, animal models have a high value to address complex interplay between Pneumocystis and the components of the host immune system. Here, we propose a protocol for a murine model of Pneumocystis pneumonia. Animals become susceptible to Pneumocystis by acquiring an immunocompromised status induced by iterative administration of steroids within drinking water. Thereafter, the experimental infection is completed by an intranasal challenge with homogenates of mouse lungs containing Pneumocystis murina. The onset of clinical signs occurs within 5 weeks following the infectious challenge and immunosuppression can then be withdrawn. At termination, lungs and bronchoalveolar lavage (BAL) fluids from infected mice are analyzed for fungal load (qPCR) and immune response (flow cytometry and biochemical assays). The model is a useful tool in studies focusing on immune responses initiated after the establishment of Pneumocystis pneumonia.

Rapid Diagnosis of Pneumocystis jirovecii Pneumonia and Respiratory Tract Colonization by Next-Generation Sequencing.

OBJECTIVES: To describe the epidemiology of Pneumocystis jirovecii pneumonia and colonization diagnosed by next-generation sequencing (NGS) and explore the usefulness of the number of P. jirovecii sequence reads for the diagnosis of P. jirovecii pneumonia. METHODS: We examined the NGS results for P. jirovecii in respiratory samples collected from patients and analysed their clinical, radiological and microbiological characteristics. RESULTS: Among 285 respiratory samples collected over a 12-month period (January to December 2022), P. jirovecii sequences were detected in 56 samples from 53 patients. Fifty (94.3%) of the 53 patients were HIV-negative. Following our case definitions, 37 (69.8%) and 16 (30.2%) of the 53 patients had P. jirovecii infection and colonization respectively. P. jirovecii infection was associated with presence of underlying disease with immunosuppression (94.6% vs 18.8%, P < 0.05), positive serum 1,3-ß-D-glucan (41.2% vs 0%, P < 0.01) and higher number of P. jirovecii sequence reads (P < 0.005). In contrast, P. jirovecii colonization was associated with the male sex (93.8% vs 54.1%, P < 0.01), another definitive infectious disease diagnosis of the respiratory tract (43.8% vs 2.7%, P < 0.001) and higher survival (100% vs 67.6%, P < 0.01). Although P. jirovecii pneumonia was associated with higher number of P. jirovecii reads in respiratory samples, only a sensitivity of 82.14% and a specificity of 68.75% could be achieved. CONCLUSION: Detection of P. jirovecii sequences in respiratory samples has to be interpreted discreetly. A combination of clinical, radiological and laboratory findings is still the most crucial in determining whether a particular case is genuine P. jirovecii pneumonia.

Microbiological characterization of Pneumocystis jirovecii pneumonia using quantitative PCR from nasopharyngeal specimens: a retrospective study in a Canadian province from 2019 to 2023.

Bronchoalveolar lavage is usually employed for molecular diagnosis of Pneumocystis jirovecii but requires a specialized procedure. By contrast, nasopharyngeal (NP) specimens are easily obtained. In this retrospective study of 35 patients with paired NP and bronchoscopy specimens, NP specimens had a 100% negative percent agreement (95% CI 80.5-100) but only 72.2% positive percent agreement (95% CI 46.5-90.3).

Clinical characteristics and risk factor analysis of Pneumocystis jirovecii pneumonia in patients with CKD: a machine learning-based approach.

Patients with chronic kidney disease (CKD) who are being treated with immunosuppressive medications are at risk for developing Pneumocystis jirovecii pneumonia (PCP). We attempted to characterize the clinical aspects of PCP in CKD patients in order to alert high-risk patients with bad prognosis. A retrospective study of CKD patients was conducted from June 2018 to June 2022. Based on PCP diagnostic criteria, these patients were divided into PCP and non-PCP groups. Using univariate and multivariate logistic regression analysis, risk indicators were evaluated, and nomogram and decision tree were developed. Of the CKD patients screened for Pneumocystis carinii nucleic acid, 1512 were included. Two-hundred forty four (16.14%) were diagnosed with PCP. Of the PCP, 88.5% was receiving glucocorticoid (GC) therapy, of which 66.3% received more than 0.5 mg/kg GC. Multivariate analysis showed that membranous nephropathy (OR 2.35, 95% CI 1.45-3.80), immunosuppressive therapy (OR 1.94, 95% CI 1.06-3.69), and ground glass opacity of CT scanning (OR 1.71, 95% CI 1.10-2.65) were associated with increased risk of Pneumocystis carinii infection. The AUC of nomogram based on logistics regression was 0.78 (0.75-0.81). The mortality in patients with PCP was 32.40%. Univariate analysis and decision tree showed that pulmonary insufficiency (PO2: OR 0.98, 95% CI 0.96-1.00), elevated APTT (OR 1.07, 95% CI 1.04-1.11), and reduced hemoglobin (OR 0.97, 95% CI 0.96-0.98) were associated with poor prognosis. PCP is not rare in CKD patients, particularly in those treated with immunosuppressive therapy. Considering the high mortality of the cases, further studies on the prevention and management of these patients are needed.

Bronchial aspirate obtained during bronchoscopy yields increased fungal load compared to bronchoalveolar lavage fluid in patients at risk of invasive aspergillosis and Pneumocystis pneumonia.

Bronchoalveolar lavage fluid (BALF) is a standard respiratory sample for diagnosing invasive fungal diseases like Pneumocystis pneumonia (PCP) and invasive pulmonary aspergillosis (IPA). However, procedural variations exist across medical centers and wards. This study aimed to compare the diagnostic potential of BALF and bronchial aspirate (BA) obtained during bronchoscopy in 173 patients suspected of fungal infections. A prospective observational study was conducted from April 2020 to November 2021. BALF and BA were collected during bronchoscopy and subjected to direct examination, fungal culture, Aspergillus fumigatus qPCR (AfqPCR), and Pneumocystis jirovecii qPCR (PjqPCR). Galactomannan detection was performed on BALF. Patients were classified based on established European Organization for Research and Treatment of Cancer (EORTC) criteria. Out of 173 patients, 75 tested positive for at least one test in BA or BALF. For Aspergillus, proportion of positive AfqPCR (14.5% vs. 9.2%; P < 0.0001) and fungal loads (Cq of 31.3 vs. 32.8; P = 0.0018) were significantly higher in BA compared to BALF. For Pneumocystis, fungal loads by PjqPCR was also higher in BA compared to BALF (Cq of 34.2 vs. 35.7; P = 0.003). BA only detected A. fumigatus and P. jirovecii in 12 (42.9%) and 8 (19.5%) patients, respectively. BA obtained during a BAL procedure can be a suitable sample type for increased detection of P. jirovecii and A. fumigatus by qPCR. The use of BA in diagnostic algorithms requires further investigation in prospective studies.

Bronchoalveolar lavage fluid (BALF) vs. bronchial aspirate (BA) for fungal diagnosis in 173 patients suspected of invasive fungal infection: BA showed higher fungal loads than in BALF by qPCR for the detection of Aspergillus fumigatus and Pneumocystis jirovecii.

The effect of COVID-19 on the frequency of Pneumocystis jirovecii pneumonia: a monocentric, retrospective, and observational study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) and SARS-CoV2 share some similarities in their effects on the respiratory system, clinical presentation, and management. The COVID-19 pandemic required rapid action to curb transmission and mitigate its lethiferous impact. Non-pharmaceutical interventions (NPIs) were globally adopted. We hypothesized that these measures reduced the transmission and acquisition of P. jirovecii in both hospital and community settings. METHODS: We conducted a retrospective observational study on 2950 respiratory specimens from patients with suspected pulmonary infection, analyzed at the Laboratory of Parasitology Unit of the Policlinico Tor Vergata of Rome, Italy, from January 2014 to December 2022. RESULTS: We show a significant reduction in the frequency of PCP in the COVID-19 pandemic era compared to the previous period. Among the four sequence types of P. jirovecii identified, genotype 1 was the most prevalent (37%). We observed a non-significant trend of decreasing cases with genotype 1 and increasing cases with genotype 3 over the study period. CONCLUSIONS: The nationwide implementation of NPIs against COVID-19 may have changed the microbiological landscape of exposure, thereby decreasing the exposure to P. jirovecii and consequently reducing the incidence of PCP.