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The Cd40l -/- mouse is a well-established model of X-linked hyper-immunoglobulin M (IgM) syndrome, an immunodeficiency disorder of human beings characterized by the lack of expression of the CD40 ligand (CD40L) on activated T-cells, predisposing to infections with opportunistic pathogens like Pneumocystis jirovecii . The aim of our study was to describe the pulmonary lesions in Cd40l -/- mice experimentally infected with Pneumocystis murina , in comparison with naturally infected severe combined immunodeficient (SCID) mice. Formalin-fixed paraffin-embedded lungs from 26 Cd40l -/- , 11 SCID, and 5 uninfected Cd40l -/- mice were examined by histology and immunohistochemistry for the presence of the pathogen and for leukocyte populations (CD3, CD4, CD45R/B220, CD8a, Iba-1, Ly-6G, CD206, MHC II, and NKp46/NCR1). Infection was confirmed by immunohistochemistry in 18/26 (69%) Cd40l -/- mice and in 11/11 (100%) SCID mice. Fourteen out of 26 (54%) Cd40l -/- mice had interstitial pneumonia. Twenty-three out of 26 (88%) Cd40l -/- mice had peribronchiolar/perivascular lymphoplasmacytic infiltrates, rich in B-cells and Mott cells. Acidophilic macrophage pneumonia was additionally found in 20/26 (77%) Cd40l -/- mice. Only 4/11 (36%) SCID mice had interstitial pneumonia, but no peribronchiolar/perivascular infiltrates or acidophilic macrophage pneumonia were observed in this strain. This study represents the first description of pulmonary histopathological lesions in Cd40l -/- mice infected with P. murina . We speculate that the singular characteristics of the inflammatory infiltrates observed in Cd40l -/- mice could be explained by the specific immune phenotype of the model., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., Competing Interests: The authors declare no conflict of interest.

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.

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] O 2 saturation (88% [75%, 99%] vs. 92%[70%, 99%], p = .040), and supplemental O 2 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., (© 2024 Wiley Periodicals LLC.)

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., (© 2024. The Author(s).)

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-β-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., (© 2024. The Author(s).)

Background: Identification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency., Methods: To set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)-rRNA gene., Results: Based on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probe-based qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: ≤32 as infection, >35 as colonization, and a grey zone between these values (32 < X ≤ 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR)., Conclusions: This study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis -related conditions., Competing Interests: The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rouhi, Erami, Rastgufar, Jahani, Aboutalebian, Soltani, Fakhim and Mirhendi.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

Pneumocystis jirovecii pneumonia (PjP) poses a serious risk to individuals with compromised immune systems, such as individuals with HIV/AIDS or undergoing immunosuppressive therapies for cancer or solid organ transplants. Severe PjP triggers excessive lung inflammation, resulting in lung function decline and consequential alveolar damage, potentially culminating in acute respiratory distress syndrome. Non-HIV patients face a 30%-60% mortality rate, emphasizing the need for a deeper understanding of inflammatory responses in PjP. Prior research emphasized macrophages in Pneumocystis infections, neglecting neutrophils' role in tissue damage. Consequently, the overemphasis on macrophages led to an incomplete understanding of the role of neutrophils and inflammatory responses. In the current investigation, our RNAseq studies on a murine surrogate model of PjP revealed heightened activation of the NLRP3 inflammasome and NETosis cell death pathways in their lungs. Immunofluorescence staining confirmed neutrophil extracellular trap (NET) presence in the lungs of the P. murina -infected mice, validating our findings. Moreover, isolated neutrophils exhibited NETosis when directly stimulated with P. murina . Isolated NETs compromised P. murina viability in vitro , highlighting the potential role of neutrophils in controlling fungal growth and promoting inflammation during P. murina pneumonia through NLRP3 inflammasome assembly and NETosis. These pathways, essential for inflammation and pathogen elimination, bear the risk of uncontrolled activation leading to excessive tissue damage and persistent inflammation. This pioneering study is the first to identify the formation of NETs and inflammasomes during Pneumocystis infection, paving the way for comprehensive investigations into treatments aimed at mitigating lung damage and augmenting survival rates for individuals with PjP .IMPORTANCE Pneumocystis jirovecii pneumonia (PjP) affects individuals with weakened immunity, such as HIV/AIDS, cancer, and organ transplant patients. Severe PjP triggers lung inflammation, impairing function and potentially causing acute respiratory distress syndrome. Non-HIV individuals face a 30%-60% mortality rate, underscoring the need for deeper insight into PjP's inflammatory responses. Past research focused on macrophages in managing Pneumocystis infection and its inflammation, while the role of neutrophils was generally overlooked. In contrast, our findings in P. murina -infected mouse lungs showed neutrophil involvement during inflammation and increased expression of NLRP3 inflammasome and NETosis pathways. Detection of neutrophil extracellular traps further indicated their involvement in the inflammatory process. Although beneficial in combating infection, unregulated neutrophil activation poses a potential threat to lung tissues. Understanding the behavior of neutrophils in Pneumocystis infections is crucial for controlling detrimental reactions and formulating treatments to reduce lung damage, ultimately improving the survival rates of individuals with PjP., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Potential conflicts of interest. A. A. has received honoraria from Gilead Sciences. S. C.-A. A. has received united educational grants from F2G Ltd and consulted for MSD Australia. T. R. R. has received grants from Pfizer and the Irish Department of Agriculture, Food and the Marine and honoraria from Gilead and Menarini Pharma. P. L. W. has performed diagnostic evaluations and received meeting sponsorship from Associates of Cape Cod, Bruker, Dynamiker, and Launch Diagnostics; speaker fees, expert advice fees, and meeting sponsorship from Gilead; and speaker and expert advice fees from F2G. J. P. D. has received personal fees from F2G, Gilead Sciences, and Pfizer. W. J. H. has received lecture honoraria from AbbVie, Amgen, AstraZeneca, Celgene/BMS, Gilead Sciences, Janssen, MSD, and Pfizer; has received support to attend meetings and travel support from AbbVie, Alexion, Astellas, Janssen, Lilly, MSD, Novartis, and Pfizer; and has participated on advisory boards for AbbVie, Amgen, AstraZeneca, Basilea, Celgene/BMS, Gilead Sciences, Janssen, and Sanofi-Aventis. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Competing Interests: The authors declare no conflict of interest.

Background: Pneumocystis jirovecii is the most emerging life-threating health problem that causes acute and fatal pneumonia infection. It is rare and more contagious for patients with leukemia and immune-deficiency disorders. Until now there is no treatment available for this infection therefore, it is needed to develop any treatment against this pathogen., Methods: In this work, we used comparative proteomics, robust immune-informatics, and reverse vaccinology to create an mRNA vaccine against Pneumocystis jirovecii by targeting outer and transmembrane proteins. Using a comparative subtractive proteomic analysis of two Pneumocystis jirovecii proteomes, a distinct non-redundant Pneumocystis jirovecii (strain SE8) proteome was chosen. Seven Pneumocystis jirovecii transmembrane proteins were chosen from this proteome based on hydrophilicity, essentiality, virulence, antigenicity, pathway interaction, protein-protein network analysis, and allergenicity., Objective: The reverse vaccinology approach was used to predict the immunogenic and antigenic epitopes of major histocompatibility complex (MHC) I, II and B-cells from the selected proteins on the basis of their antigenicity, toxicity and allergenicity. These immunogenic epitopes were linked together to construct the mRNA-based vaccine. To enhance the immunogenicity, suitable adjuvant, linkers (GPGPG, KK, and CYY), and PRDRE sequences were used., Results: Through predictive modeling and confirmation via the Ramachandran plot, we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: -0.271, instability index: 39.53, antigenicity: 1.0428). The physiochemical profiling of vaccine construct was predicted it an antigenic, efficient, and potential vaccine. Notably, strong interactions were observed between the vaccine construct and TLR-3/TLR-4 (-1301.7 kcal/mol-1 and -1374.7 kcal/mol-1)., Conclusions: The results predicted that mRNA-based vaccines trigger a cellular and humoral immune response, making the vaccine potential candidate against Pneumocystis jirovecii and it is more suitable for in-vitro analysis and validation to prove its effectiveness., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

SUMMARYEvery human being is presumed to be infected by the fungus Pneumocystis jirovecii at least once in his or her lifetime. This fungus belongs to a large group of species that appear to exclusively infect mammals, with P. jirovecii being the only one known to cause disease in humans. The mystery of P. jirovecii origin and speciation is just beginning to unravel. Here, we provide a review of the major steps of P. jirovecii evolution. The Pneumocystis genus likely originated from soil or plant-associated organisms during the period of Cretaceous ~165 million years ago and successfully shifted to mammals. The transition coincided with a substantial loss of genes, many of which are related to the synthesis of nutrients that can be scavenged from hosts or cell wall components that could be targeted by the mammalian immune system. Following the transition, the Pneumocystis genus cospeciated with mammals. Each species specialized at infecting its own host. Host specialization is presumably built at least partially upon surface glycoproteins, whose protogene was acquired prior to the genus formation. P. jirovecii appeared at ~65 million years ago, overlapping with the emergence of the first primates. P. jirovecii and its sister species P. macacae , which infects macaques nowadays, may have had overlapping host ranges in the distant past. Clues from molecular clocks suggest that P. jirovecii did not cospeciate with humans. Molecular evidence suggests that Pneumocystis speciation involved chromosomal rearrangements and the mounting of genetic barriers that inhibit gene flow among species., Competing Interests: The authors declare no conflict of interest.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Introduction: Pneumocystis jirovecii pneumonia (PJP) is a well-known and frequent opportunistic infection in HIV patients. However, there has been an increase in the number of reports of PJP in other immunosuppressed patients with autoimmune inflammatory disorders or because of chemotherapy and high doses of steroids, especially when used in combination as part of immunosuppressive therapy., Objective: Despite the increasing importance of PJP in non-HIV patients, there is a lack of comprehensive and updated information on the epidemiology, pathogenesis, diagnosis, microbiology, treatments, and prophylaxis of this infection in this population. Therefore, the objective of this systematic review is to synthesize information on these aspects, from a perspective of evidence-based medicine., Methods: The protocol is prepared following the preferred reporting items for systematic reviews and meta-analyses (PRISMA-P) guidelines. We will perform a systematic review of literature published between January 2010 and July 2023, using the databases PubMed, Google Scholar, ScienceDirect, and Web of Science. In addition, manual searches will be carried out through related articles, and references to included articles. The main findings and clinical outcomes were extracted from all the eligible studies with a standardized instrument. Two authors will independently screen titles and abstracts, review full texts, and collect data. Disagreements will be resolved by discussion, and a third reviewer will decide if there is no consensus. We will synthesize the results using a narrative or a meta-analytic approach, depending on the heterogeneity of the studies., Expected Results: It is expected that this systematic review will provide a comprehensive and up-to-date overview of the state-of-the-art of PJP in non-HIV patients. Furthermore, the study will highlight possible gaps in knowledge that should be addressed through new research., Conclusions: Here, we present the protocol for a systematic review which will consider all existing evidence from peer-reviewed publication sources relevant to the primary and secondary outcomes related to diagnosing and managing PJP in non-HIV patients., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Orozco-Ugarriza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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-β-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., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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., Competing Interests: Melissa Miller is a member of the DiaSorin Molecular Scientific Advisory Board

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. β-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. β-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 β-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 β-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., Competing Interests: The authors declare no conflict of interest.

Importance: Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs., Competing Interests: The authors declare no conflict of interest.

Atovaquone is an FDA-approved antiparasitic and antifungal therapeutic that is currently used as a prophylactic agent to prevent Pneumocystis carinii pneumonia (PCP) infections in acute myeloid leukemia (AML) patients after receiving hematopoietic stem cell transplantation (HSCT). Recent studies have shown that atovaquone has shown potential as an anticancer agent. The high variability in atovaquone bioavailability prompts the need for therapeutic drug monitoring, especially in pediatric patients. The goal of our study was to develop and validate the performance of an assay to quantify atovaquone plasma concentrations collected from pediatric cancer patients. Briefly, an organic-based solvent system is used to precipitate protein and extract the atovaquone content from each patient-derived plasma sample. After completing a second stage of sample dilution (5000-fold overall), a 2 μL volume of the plasma extract is analyzed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based bioanalytical method described., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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)., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Pneumocystis , an important opportunistic fungal pathogen that parasitizes in multiple mammalian lungs, may cause life-threatening Pneumocystis pneumonia (PCP) and even death among immunocompromised individuals. With the rapid development of high-throughput sequencing and multi-omics technologies, systematic comparative analyses of genome, transcriptome, and whole-genome sequencing results demonstrate that Pneumocystis is a type of obligate biotrophic fungi, and requires obtaining nutrition from hosts. In addition, sexual reproduction is an essential process for Pneumocystis survival, production and transmission, and asexual reproduction facilitates Pneumocystis survival, which provides new insights into understanding of the whole developmental process of Pneumocystis in the host lung and inter-host transmission of Pneumocystis . This review summarizes the advances in the reproduction mode of Pneumocystis and underlying mechanisms, which provides insights into prevention and treatment of PCP, notably for the prophylaxis against nosocomial transmission of PCP.

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Introduction: Invasive and superficial fungal infections are increasingly reported in Algeria, testifying to the increase in their frequency in parallel with the increase in risk factors and the availability of diagnostic means, at least in university hospitals (CHU). The latter, located in the major northern cities, are equipped with high-performance diagnostic tools compared to hospitals in the interior of the country., Methods: A comprehensive search of published and grey literature was undertaken. Prevalence and incidence of discrete fungal diseases were estimated using a deterministic modelling approach based on populations at risk. Population (2021) and major underlying disease risk groups were obtained from UNAIDS, WHO Tuberculosis and the international transplant registries as well as published data for asthma and COPD. The health service profile was summarised from national documentation., Results: Among the 43.6 million, including 12.9 million children, living in Algeria, the most prevalent fungal diseases are tinea capitis (>1.5 million), recurrent vaginal candidiasis (>500,000) and allergic fungal lung and sinus disorders (>110,000) and chronic pulmonary aspergillosis (>10,000). Life-threatening invasive fungal infection incidence includes 774 Pneumocystis pneumonia in AIDS, 361 cryptococcal meningitis, 2272 candidaemia and 2639 invasive aspergillosis cases. Fungal keratitis probably affects >6000 eyes each year., Conclusions: Fungal infections are underestimated in Algeria because they are sought in patients with risk factors only after bacterial infections when they should be sought in parallel. The diagnosis is only accessible in hospitals in large cities and the work carried out in mycology is rarely published, making the estimation of the burden of these conditions difficult., (© 2023 The Authors. Mycoses published by Wiley-VCH GmbH.)

Introduction: With the extensive use of immunosuppressants, immunosuppression-associated pneumonitis including Pneumocystis jirovecii pneumonia (PCP) has received increasing attention. Though aberrant adaptive immunity has been considered as a key reason for opportunistic infections, the characteristics of innate immunity in these immunocompromised hosts remain unclear., Methods: In this study, wild type C57BL/6 mice or dexamethasone-treated mice were injected with or without Pneumocystis . Bronchoalveolar lavage fluids (BALFs) were harvested for the multiplex cytokine and metabolomics analysis. The single-cell RNA sequencing (scRNA-seq) of indicated lung tissues or BALFs was performed to decipher the macrophages heterogeneity. Mice lung tissues were further analyzed via quantitative polymerase chain reaction (qPCR) or immunohistochemical staining., Results: We found that the secretion of both pro-inflammatory cytokines and metabolites in the Pneumocystis -infected mice are impaired by glucocorticoids. By scRNA-seq, we identified seven subpopulations of macrophages in mice lung tissues. Among them, a group of Mmp12 + macrophages is enriched in the immunocompetent mice with Pneumocystis infection. Pseudotime trajectory showed that these Mmp12 + macrophages are differentiated from Ly6c + classical monocytes, and highly express pro-inflammatory cytokines elevated in BALFs of Pneumocystis -infected mice. In vitro , we confirmed that dexamethasone impairs the expression of Lif , Il1b , Il6 and Tnf , as well as the fungal killing capacity of alveolar macrophage (AM)-like cells. Moreover, in patients with PCP, we found a group of macrophages resembled the aforementioned Mmp12 + macrophages, and these macrophages are inhibited in the patient receiving glucocorticoid treatment. Additionally, dexamethasone simultaneously impaired the functional integrity of resident AMs and downregulated the level of lysophosphatidylcholine, leading to the suppressed antifungal capacities., Conclusion: We reported a group of Mmp12 + macrophages conferring protection during Pneumocystis infection, which can be dampened by glucocorticoids. This study provides multiple resources for understanding the heterogeneity and metabolic changes of innate immunity in immunocompromised hosts, and also suggests that the loss of Mmp12 + macrophages population contributes to the pathogenesis of immunosuppression-associated pneumonitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Li, Zhao, Liu, Li, Yang, Tong and Song.)

Aim: In the past, Pneumocystis jirovecii belonged to the Protozoa group, but is currently taxonomically included in the kingdom Fungi. P. jirovecii is an opportunistic pathogen, responsible for pneumocystis pneumonia with frequent complications of immunocompromised patients. Delayed initiation of appropriate therapy increases the risk of death in immunocompromised patient. The aim of this work was to determine and evaluate the reliability of methods of laboratory diagnosis of pneumocystosis used in routine laboratories as well as the occurrence of this disease in patients from Slovakia during 19 years., Material and Methods: The diagnosis is based on microscopic examination (Giemsa- and Gram-Weigert-staining) and detection of parasite DNA by classical or real-time PCR in bronchoalveolar lavage and sputum., Results: Pneumocysts were detected in 190 persons (5.7%) from the whole group of patients. Cancer patients represented the riskiest group in terms of pneumocystosis, which was confirmed by the highest percentage (57.9%) of individuals infected with P. jirovecii. Compared with the PCR, 33.7% sensitivity and 100% specificity of microscopy was calculated by using a binary classification test. Molecular methods are more sensitive in the detection of P. jirovecii compared to microscopic evidence and currently represent a reliable detection system in the diagnosis of pneumocystosis., Conclusion: In view of the increasing number of immunocompromised persons, diagnostics of P. jirovecii in patients with pulmonary complications is essential. This was also confirmed in our study, where the number of examinations and detection of this opportunistic pathogen increased over the years.

Background: Anecdotally, the burden of fungal diseases in Vietnam is rapidly rising, but there has been no updated estimate on this issue since a previous report in 2015., Objectives: In this study, we aimed at estimating the incidence and prevalence of serious fungal infections for the year 2020., Methods: We made estimates with a previously described methodology, using reports on the incidence and prevalence of various established risk factors for fungal infections from local, regional or global sources., Results: We estimated 2,389,661 cases of serious fungal infection occurred in Vietnam in 2020. The most common condition was recurrent vaginal candidiasis (4047/100,000 women annually). Among people living with HIV, we estimated 451 cases of cryptococcal meningitis, 1030 of pneumocystis pneumonia, 166 of histoplasmosis and 1612 of talaromycosis annually. Candidaemia incidence was estimated at 12/100,000 population each year. Owing to its high burden of tuberculosis and respiratory diseases, Vietnam had high rates of severe infections caused by Aspergillus species. Incidence of invasive aspergillosis is 24/100,000 population, allergic bronchopulmonary aspergillosis 78/100,000 and severe asthma with fungal sensitisation 102/100,000. Five-year period prevalence of chronic pulmonary aspergillosis is 120/100,000 population /5-year period. Mucormycosis, fungal keratitis and tinea capitis were estimated at 192, 14,431 and 201 episodes each year, respectively., Conclusions: The number of patients with mycoses in Vietnam is likely underestimated due to a lack of local data and limited diagnostic capacity, but at least 2.5% of the population might have some form of serious fungal disease., (© 2022 The Authors. Mycoses published by Wiley-VCH GmbH.)

Objectives: Pneumocystis pneumonia (PCP) is an opportunistic infection that causes significant morbidity and mortality in the immunocompromised population. This population is growing and diversifying, yet contemporary epidemiology is lacking. We investigated the population-level incidence of PCP over the past decade., Methods: We conducted a descriptive study of all hospital admissions in England from April 2012 to March 2022. PCP episodes, age, median length of stay, gender and episodes of other respiratory fungal infections were collected. Consumption of Trimethoprim-Sulfamethoxazole was obtained between January 2019 and May 2022., Results: The incidence of PCP increased from 2·2-4·5/100,000 population between 2012/2013 and 2019/2020 (p < 0·0001). There was a drop in 2020/2021 to 2·7/100,000 before returning to 3.9/100,000 in 2021/2022. PCP episodes rose as a proportion of all-cause admissions as well as a proportion of episodes due to other fungal infections. The proportion of PCP patients aged 75+ increased from 14% to 26%. The median length of stay was 13.5 days. Consumption of intravenous Trimethoprim-Sulfamethoxazole increased from 0.24 × 100,000 to 0.30 × 100,000 defined daily doses., Conclusions: The incidence of PCP is rising rapidly and represents a significant burden to the healthcare system. Further study into who is at risk of PCP is needed to better determine who should be given prophylaxis., (Copyright © 2023 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Background: Pneumocystis jirovecii (PJ) is an opportunistic pathogenic fungus, and PJ pneumonia (PJP) is a commonly problem in HIV-positive patients. While PJP is not caused by HIV, it generally advances rapidly and can quickly lead to severe respiratory failure. To improve pediatricians' understanding of the condition and aid early accurate diagnoses and therapy, we examined the clinical characteristics of five instances of non-HIV related PJP (NH-PJP) in children and the efficacy of metagenomic next-generation sequencing (mNGS) in its diagnosis., Methods: From January 2020 to June 2022, five children with NH-PJP were admitted to the PICU of the First Affiliated Hospital of Zhengzhou University. We retrospectively summarize the clinical presentation, previous histories, routine laboratory findings, treatment, outcome of regression, and results of mNGS in these five children., Results: Five male children between the ages of 11 months and 14 years had an acute onset on NH-PJP, three of the children had chest tightness after activity, shortness of breath and paroxysmal dry cough, - and two had high fever and dry cough. All five of the children had several flocculent high-density pictures in both lungs at the beginning of the disease, and lung auscultation revealed coarse breath sounds in both lungs, one of which was accompanied by a modest quantity of dry rales. PJ nuclear sequences were found in one patient and four patients' blood and alveolar lavage fluid. All five children were treated with Trimethoprim-sulfamethoxazole (TMP-SMX) in combination with Caspofungin and corresponding symptomatic treatment. Four patients were cured and one patient died., Conclusion: Children commonly encounter an initial exposure to NH-PJP, which manifests as a high fever, dry cough, chest discomfort, dyspnea that worsens over time, fast disease progression, and a high death rate. The clinical presentation of children with PJ infection should be taken into consideration along with the results for diagnose. mNGS has higher sensitivity and a shorter detection period compared to identification of PJP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Niu, Wang, Jia, Zhang and Wei.)

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 (PO 2 : 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pneumocystis jirovecii kills hundreds of thousands of immunocompromised patients each year. Yet many aspects of the biology of this obligate pathogen remain obscure because it is not possible to culture the fungus in vitro independently of its host. Consequently, our understanding of Pneumocystis pathobiology is heavily reliant upon bioinformatic inferences. We have exploited a powerful combination of genomic and phylogenetic approaches to examine the evolution of transcription factors in Pneumocystis species. We selected protein families (Pfam families) that correspond to transcriptional regulators and used bioinformatic approaches to compare these families in the seven Pneumocystis species that have been sequenced to date with those from other yeasts, other human and plant pathogens, and other obligate parasites. Some Pfam families of transcription factors have undergone significant reduction during their evolution in the Pneumocystis genus, and other Pfam families have been lost or appear to be in the process of being lost. Meanwhile, other transcription factor families have been retained in Pneumocystis species, and some even appear to have undergone expansion. On this basis, Pneumocystis species seem to have retained transcriptional regulators that control chromosome maintenance, ribosomal gene regulation, RNA processing and modification, and respiration. Meanwhile, regulators that promote the assimilation of alternative carbon sources, amino acid, lipid, and sterol biosynthesis, and iron sensing and homeostasis appear to have been lost. Our analyses of transcription factor retention, loss, and gain provide important insights into the biology and lifestyle of Pneumocystis. IMPORTANCE Pneumocystis jirovecii is a major fungal pathogen of humans that infects healthy individuals, colonizing the lungs of infants. In immunocompromised and transplant patients, this fungus causes life-threatening pneumonia, and these Pneumocystis infections remain among the most common and serious infections in HIV/AIDS patients. Yet we remain remarkably ignorant about the biology and epidemiology of Pneumocystis due to the inability to culture this fungus in vitro . Our analyses of transcription factor retentions, losses, and gains in sequenced Pneumocystis species provide valuable new views of their specialized biology, suggesting the retention of many metabolic and stress regulators and the loss of others that are essential in free-living fungi. Given the lack of in vitro culture methods for Pneumocystis, this powerful bioinformatic approach has advanced our understanding of the lifestyle of P. jirovecii and the nature of its dependence on the host for survival.

Background: Pneumocystis jirovecii pneumonia (PJP) remains an important cause of morbidity and mortality in non-HIV immunocompromised patients especially in transplant recipients. But its diagnosis remains challenging due to the insuffificient performance of conventional methods for diagnosing Pneumocystis jirovecii(P. jirovecii) infection. Therefore, the auxiliary diagnostic function of metagenomics next-generation sequencing (mNGS) in clinical practice is worth of exploring., Method: 34 non-HIV immunocompromised patients who were diagnosed as PJP by clinical manifestations, imaging findings, immune status of the host, and Methenamine silver staining were tested by mNGS from October 2018 to December 2020 in Sichuan Provincial People's Hospital. The clinical performances of mNGS for P. jirovecii infection diagnosis were also evaluated with genome reads abundance and comparing with other traditional diagnostic methods., Results: We diagnosed a total of 34 non-HIV PJP patients by the clinical composite diagnosis. Our data shows that, compared with the clinical microbiological test, the detection rate of mNGS for P. jirovecii in non-HIV infected PJP patients is significantly higher than that of Methenamine silver staining and serum 1-3-β-D-glucan. mNGS can be used as an auxiliary diagnostic tool to help diagnosis. The number of reads mapped to the genome of P. jirovecii and the duration of patients from onset to sampling collection were statistically significant between the two groups (Reads>100 and Reads ≤ 100) (8days vs . 23days, p =0.020). In addition, univariate analysis showed that C-reactive protein (15.8mg/L vs .79.56mg/L, p =0.016), lactate dehydrogenase (696U/l vs . 494U/l, p =0.030) and procalcitonin (0.09ng/ml vs . 0.59ng/ml, p =0.028) was also statistically significant between the two groups., Conclusions: An effective detection rate was achieved in PJP patients using mNGS testing of bronchoalveolar lavage fluid (BALF) or blood. The study also confirmed that the abundance of reads of P. jirovecii is related to the interval between the onset and sample collection. And the inflammation status during simultaneous mNGS detection might determine the abundance of pathogens. Hence, we conclude that the mNGS strategy could benefit disease diagnosis as well as treatment when complicated clinical infections appeared., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Yue, Wu, Gao, He and Pan.)

Background: Due to the inability to be cultured in vitro , the biological characteristics and pathogenicity of Pneumocystis jirovecii remain unclear. Intestinal microflora disorder is related to the occurrence and development of various pulmonary diseases. This work explores the pathogenesis of pneumocystis pneumonia (PCP) in acquired immune deficiency syndrome (AIDS) patients from a microbiome perspective, to provide better strategies for the diagnosis, treatment, and prevention of PCP., Methods: Subjects were divided into three groups: human immunodeficiency virus (HIV)-infected patients combined with PCP, HIV-infected patients without PCP, and HIV-negative. Stool and bronchoalveolar lavage fluid (BALF) samples were collected, total DNA was extracted, and 16S rRNA high-throughput sequencing was performed using an Illumina MiSeq platform. PICRUSt and BugBase were used to predict microflora functions, and correlation analysis of intestinal and lung bacterial flora was conducted., Results: Compared with the HIV- group, prevotella and another 21 genera in the intestinal microbiome were statistically different in the HIV+ group; 25 genera including Escherichia-Shigella from HIV+PCP+ group were statistically different from HIV+PCP- group. The abundance of Genera such as Porphyromonas was positively or negatively correlated with CD16/CD56+ (μL). Compared with the HIV- group, identification efficiency based on area under the curve (AUC) >0.7 for the HIV+ group identified seven genera in the gut microbiota, including Enterococcus (total AUC = 0.9519). Compared with the HIV+PCP- group, there were no bacteria with AUC >0.7 in the lung or intestine of the HIV+PCP+ group. The number of shared bacteria between BALF and fecal samples was eight species in the HIV- group, 109 species in PCP- patients, and 228 species in PCP+ patients, according to Venn diagram analysis. Changes in various clinical indicators and blood parameters were also closely related to the increase or decrease in the abundance of intestinal and pulmonary bacteria, respectively., Conclusions: HIV infection and PCP significantly altered the species composition of lung and intestinal microbiomes, HIV infection also significantly affected intestinal microbiome gene functions, and PCP exacerbated the changes. The classification model can be used to distinguish HIV+ from HIV- patients, but the efficiency of bacterial classification was poor between PCP+ and PCP- groups. The microbiomes in the lung and gut were correlated to some extent, providing evidence for the existence of a lung-gut axis, revealing a potential therapeutic target in patients with HIV and PCP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhu, Liu, Zhao, Shi, Li, Ling, Cheng, Dong and Xu.)

Pneumocystis jirovecii pneumonia (PCP), caused by fungal species named Pneumocystis jirovecii, is a frequent opportunistic infection in those with human immunodeficiency virus (HIV) infection. However, PCP has been documented in immunocompetent patients. This study aims to determine if P. jirovecii detection occurs in asthma patients following coronavirus disease 2019 (COVID-19) in a Jordanian cohort. Also, to evaluate a method of TaqMan quantitative polymerase chain reaction (qPCR) assay to detect P. jirovecii, from sputum samples. The nasopharyngeal swabs were used to detect SARS-CoV-2 and sputum samples were tested for P. jirovecii using real time qPCR assay. Beta-tubulin (BT) and Dihydrofolate reductase (DHFR) genes were the directed targets of P. jirovecii. The results showed that the mean qPCR efficiencies of BT and DHFR were 96.37% and 100.13%, respectively. Three out of 31 included patients (9.7%) had a positive P. jirovecii. All of the three patients had used oral corticosteroids (OCS) in the last two months due asthma exacerbation and were treated with OCS for COVID-19. This is the first study based in Jordan to demonstrate that P. jirovecii and COVID-19 can co-exist and that it is important to maintain a broad differential diagnosis, especially in immunocompromised patients. Chronic lung disease can be a risk factor for the P. jirovecii colonization possibly due to corticosteroid's immunosuppression.

Background: Pneumocystis jirovecii can result in a serious pulmonary infection, Pneumocystis jirovecii pneumonia, in immunocompetent hosts. The diagnosis of Pneumocystis jirovecii pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies ( e.g. , insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific transcription mediated amplification and CRISPR/Cas13a-based diagnostics targeted P. jirovecii -mitochondrial large subunit ribosomal RNA., Methods: The procedure of the diagnostics included amplification of the extracted RNA samples by transcription mediated amplification, followed by CRISPR/Cas13 detection, and ultimately, the judgment of the results after 30 minutes of fluorescence signal. Later, the diagnostic performance of the CRISPR/Cas13-based diagnostics were tested on the 62 surplus clinical samples., Results: This CRISPR/Cas13-based diagnostics achieved limits of detection of approximately 2 copies/µL transcribed RNA templates, with no cross reaction to other respiratory pathogens, including bacteria and fungi. Similar to in-house quantitative real-time polymerase chain reaction, CRISPR/Cas13-based diagnostics was still positive in 243-fold diluted bronchial alveolar lavage fluid. A preliminary evaluation of 62 surplus bronchial alveolar lavage fluid samples from patients suspected of Pneumocystis jirovecii pneumonia showed that CRISPR/Cas13-based diagnostics achieved a 78.9% sensitivity and a 97.7% specificity in the diagnosis of Pneumocystis jirovecii pneumonia., Conclusion: Our study demonstrates that the CRISPR/Cas13-based diagnostics technique has good performance for the accurate and specific diagnosis of Pneumocystis jirovecii pneumonia., Competing Interests: Authors XG, YS, and XH are employed by Hangzhou MatriDx Biotechnology Co., Ltd. China. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhan, Gao, Li, Si, Li, Han, Sun, Li and Ye.)

Background: The caspase recruitment domain-containing protein 9 (CARD9) inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis β-glucan-induced proinflammatory signaling, suggesting its viability as a candidate for preliminary anti-Pneumocystis drug testing in the rodent Pneumocystis pneumonia (PCP) model., Methods: Mice were injected intraperitoneally (IP) daily with either vehicle or BRD5529 at 0.1 or 1.0 mg/kg for 2 weeks. Mouse weights were taken daily. At day 14, mice were euthanized, weighed, and analyzed by flexiVent™ for lung stiffness. Lungs, liver, and kidney were then harvested for hematoxylin and eosin (H&E) staining and pathology scoring. Lung samples were further analyzed for proinflammatory cytokines via enzyme-linked immunosorbent assay (ELISA) and extracellular matrix generation via quantitative polymerase chain reaction (qPCR). Blood collection postmortem was performed for blood chemistry analysis. Furthermore, administration of BRD5529 prior to the intratracheal inoculation of fungal β-glucans, which are known proinflammatory mediators via the Dectin-1-CARD9 pathway, resulted in significant reductions in lung tissue interleukin-6 and tumor necrosis factor-α, suggesting the exciting possibility of the use of this CARD9 inhibitor as an additional therapeutic tool in fungal infections., Results: BRD5529 at both IP doses resulted in no significant changes in daily or final weight gain, and analysis of lung stiffness by flexiVent™ showed no significant differences between the groups. Furthermore, ELISA results of proinflammatory cytokines showed no major differences in the respective groups. qPCR analysis of extracellular matrix transcripts were statistically similar. Examination and pathology scoring of H&E slides from lung, liver, and kidney in all groups, as well as subsequent pathology scoring, showed no significant change. Blood chemistry analysis revealed similar, non-significant patterns., Conclusions: In our initial general safety and toxicology assessments, BRD5529 displayed no inherent safety concerns in the analyzed parameters. These data support broader in vivo testing of the inhibitor as a timed adjunct therapy to the deleterious proinflammatory host immune response often associated with anti-Pneumocystis therapy., (© 2022. The Author(s).)

Pneumocystis jirovecii pneumonia (PJP) is an opportunistic fungal infection that may affect patients with immunosuppression. In order to improve the diagnosis accuracy for PJP, facilitating the collection of data across Europe to reliably assess the performance of diagnostic tests for PJP is essential to improve the care of critically ill patients developing this severe condition. Such large data can be collected thanks to the contribution of several European hospitals in the compilation of a dedicated electronic Case Report Form (eCRF). The main focus of this work is to create an interface with high ergonomics both in the compilation and in the subsequent validation of the records.

Background: The current diagnostic gold standard for Pneumocystis jirovecii is represented by microscopic visualization of the fungus from clinical respiratory samples, as bronchoalveolar-lavage fluid, defining "proven" P. jirovecii pneumonia, whereas qPCR allows defining "probable" diagnosis, as it is unable to discriminate infection from colonization. However, molecular methods, such as end-point PCR and qPCR, are faster, easier to perform and interpret, thus allowing the laboratory to give back the clinician useful microbiological data in a shorter time. The present study aims at comparing microscopy with molecular assays and beta-D-glucan diagnostic performance on bronchoalveolar-lavage fluids from patients with suspected Pneumocystis jirovecii pneumonia. Bronchoalveolar-lavage fluid from eighteen high-risk and four negative control subjects underwent Grocott-Gomori's methenamine silver-staining, end-point PCR, RT-PCR, and beta-D-glucan assay., Results: All the microscopically positive bronchoalveolar-lavage samples (50%) also resulted positive by end-point and real time PCR and all, but two, resulted positive also by beta-D-glucan quantification. End-point PCR and RT-PCR detected 10 (55%) and 11 (61%) out of the 18 samples, respectively, thus showing an enhanced sensitivity in comparison to microscopy. All RT-PCR with a Ct < 27 were confirmed microscopically, whereas samples with a Ct ≥ 27 were not., Conclusions: Our work highlights the need of reshaping and redefining the role of molecular diagnostics in a peculiar clinical setting, like P. jirovecii infection, which is a rare but also severe and rapidly progressive clinical condition affecting immunocompromised hosts that would largely benefit from a faster diagnosis. Strictly selected patients, according to the inclusion criteria, resulting negative by molecular methods could be ruled out for P. jirovecii pneumonia., (© 2022. The Author(s).)

Pneumocystis jirovecii pneumonia (PCP) is a life-threatening fungal infection in immunocompromised patients. Traditionally, the laboratory diagnosis of PCP relied on the visualization of organisms by microscopy as Pneumocystis cannot be readily cultured in the laboratory. The polymerase chain reaction (PCR) method is preferred over the conventional microscopic methods as PCR is rapid and found to have higher sensitivity. This retrospective study aimed to analyze the diagnostic value of a real-time PCR (qPCR) for routine diagnosis of PCP in immunocompromised patients with various underlying conditions. The qPCR targets a 121 bp fragment of P.jirovecii mitochondrial large subunit rRNA gene. The study was conducted in a 2600-bed tertiary care hospital between January and December 2019. All patients whose respiratory samples were tested for PCP by qPCR were included. The clinical diagnosis was made for each patient and categorized into PCP and non-PCP based on multi-component clinical criteria by a multi-disciplinary team. The performance characteristics of qPCR were analyzed using clinical diagnosis as the reference. A total of 339 respiratory samples from 289 patients were tested for PCP by qPCR during the study period. The overall sensitivity and specificity of qPCR were 84.75% (95% CI, 73.01% to 92.78%) and 96.1% (95% CI, 92.7 to 98.2), respectively. The sensitivity was slightly higher among HIV-infected patients (91%) than the non- HIV group (81%). The PCR exhibited higher sensitivity in bronchoalveolar lavage (BAL) (94%) than in sputum samples (81%). The colonization can be ruled out with the cycle threshold (C T ) value of below 34 with a sensitivity and specificity of 100% and 78%, respectively. The real-time PCR showed good sensitivity and specificity for routine diagnosis of PCP in patients with various underlying conditions. In addition, a cut-off C T value (≤ 34) was determined to exclude colonization from active pneumonia., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 SFMM. Published by Elsevier Masson SAS. All rights reserved.)

Background: To evaluate the diagnostic value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in immunocompromised patients for the diagnosis of suspected pneumonia in comparison with that of conventional microbiological tests (CMTs)., Methods: Sixty-nine immunocompromised patients with suspected pneumonia received both CMTs and mNGS of BALF were analyzed retrospectively. The diagnostic value was compared between CMTs and mNGS, using the clinical composite diagnosis as the reference standard., Results: Sixty patients were diagnosed of pneumonia including fifty-two patients with identified pathogens and eight patients with probable pathogens. Taking the composite reference standard as a gold standard, 42 pathogens were identified by CMTs including nine bacteria, 17 fungi, 8 virus, 6 Mycobacterium Tuberculosis, and two Legionella and 19(45%) of which were detected by BALF culture. As for mNGS, it identified 76 pathogens including 20 bacteria, 31 fungi, 14 virus, 5 Mycobacterium Tuberculosis, four Legionella and two Chlamydia psittaci. The overall detection rate of mNGS for pathogens were higher than that of CMTs. However, a comparable diagnostic accuracy of mNGS and CMTs were found for bacterial and viral infections. mNGS exhibited a higher diagnostic accuracy for fungal detection than CMTs (78% vs. 57%, P < 0.05), which mainly because of the high sensitivity of mNGS in patients with Pneumocystis jirovecii pneumonia (PJP) (100% vs. 28%, P < 0.05). Nineteen patients were identified as pulmonary co-infection, mNGS test showed a higher detection rate and broader spectrum for pathogen detection than that of CMTs in co-infection. Moreover, Pneumocystis jirovecii was the most common pathogen in co-infection and mNGS have identified much more co-pathogens of PJP than CMTs., Conclusions: mNGS of BALF improved the microbial detection rate of pathogens and exhibited remarkable advantages in detecting PJP and identifying co-infection in immunocompromised patients., (© 2022. The Author(s).)

Introduction: This study aims to assess the value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) in the diagnosis of Pneumocystis jirovecii pneumonia (PJP) and its mixed infection in non-human immunodeficiency virus (HIV) immunosuppressed patients., Methods: A total of 198 non-HIV immunosuppressed patients with severe pneumonia were enrolled, including 77 PJP patients and 121 patients infected by other pathogens. BALF-mNGS and traditional detection methods were used., Results: The positive detection rate of various pathogens of BALF-mNGS was higher than that of the conventional methods, especially for mixed pathogens. The sensitivity and specificity of BALF-mNGS for the diagnosis of PJP were 97.40% and 85.12%, respectively. Compared with traditional methods, the sensitivity of BALF-mNGS was significantly higher than that of blood fungal G (BG)/lactate dehydrogenase (LDH) and BALF-microscopy (p<0.05), and its specificity was significantly higher than that of BG/LDH (p<0.05). In addition, the average detection time of BALF-mNGS (32.76 ± 10.32 h) was also significantly shorter than conventional methods (p<0.01), especially for mixed infections that were common in non-HIV immunosuppressed patients. In patients only detected as positive by BALF-mNGS, the underlying diseases mainly manifested as hematological malignancies with agranulocytosis and within 8 months after hematopoietic stem cell or solid organ transplantation., Conclusions: BALF-mNGS technology is faster, more sensitive, and more comprehensive in detecting P. jirovecii and its mixed infection in immunosuppressed patients., Competing Interests: ZL and HX are employed by Hugobiotech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Wang, Zhang, Xu, Li, Gao, Wu, Han, Wang, Yao, Lou, Xia, Shi and Li.)

Background: Pneumocystis jirovecii is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised hosts. Over an 11-month period, we observed a rise in cases of PCP among kidney-transplant recipients (KTR), prompting an outbreak investigation., Methods: Clinical and epidemiologic data were collected for KTR diagnosed with PCP between July 2019 and May 2020. Pneumocystis strain typing was performed using restriction fragment length polymorphism analyses and multilocus sequence typing in combination with next-generation sequencing. A transmission map was drawn, and a case-control analysis was performed to determine risk factors associated with PCP., Results: Nineteen cases of PCP in KTR were diagnosed at a median of 79 months post-transplantation; 8 received monthly belatacept infusions. Baseline characteristics were similar for KTR on belatacept versus other regimens; the number of clinic visits was numerically higher for the belatacept group during the study period (median 7.5 vs 3). Molecular typing of respiratory specimens from 9 patients revealed coinfection with up to 7 P. jirovecii strains per patient. A transmission map suggested multiple clusters of interhuman transmission. In a case-control univariate analysis, belatacept, lower absolute lymphocyte count, non-White race, and more transplant clinic visits were associated with an increased risk of PCP. In multivariate and prediction power estimate analyses, frequent clinic visits was the strongest risk factor for PCP., Conclusions: Increased clinic exposure appeared to facilitate multiple clusters of nosocomial PCP transmission among KTR. Belatacept was a risk factor for PCP, possibly by increasing clinic exposure through the need for frequent visits for monthly infusions., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod , dhps , dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb ) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes ( dhfr, dhps and cyb ) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China., (© 2022 Ting Xue et al., published by Sciendo.)