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The burden of nosocomial Pneumocystis infections in transplantation units in France was evaluated through a retrospective survey. Over 12 years, 16 outbreaks occurred, including 13 among renal transplant recipients (RTRs). We performed Pneumocystis jirovecii genotyping in 5 outbreaks, which suggested that specific strains may have been selected by RTRs.

Background Pneumocystis pneumonia (PCP) is a common acquired immune deficiency syndrome (AIDS)-related opportunistic infection. Recent reports estimate that more than 400,000 patients with human immunodeficiency virus (HIV) develop PCP each year globally. However, the timing of antiretroviral therapy (ART) initiation for HIV-infected patients with PCP is still controversial, and the benefits and risks of early initiation of ART are not completely clear. We thus designed this study in order to determine the optimal timing for ART initiation for HIV-positive patients with moderate to severe PCP. Methods This study will be an open-label, multi-centre, prospective randomised controlled trial. A total of 200 subjects will be randomly assigned to an early ART initiation group (≤14 days after PCP diagnosis) and a deferred ART initiation group (>14 days after PCP diagnosis) at a 1:1 ratio. All subjects will be followed up for 48 weeks after starting ART. The primary endpoint is incidence of disease progression (including new or relapsing opportunistic infections and death) at week 48. The secondary endpoints are the changes in CD4 counts from baseline at weeks 12, 24 and 48; the degree of virological suppression (HIV RNA < 50 copies/mL) at weeks 24 and 48; the rate of development of PCP-associated immune reconstitution inflammatory syndrome; and adverse events over 48 weeks. Discussion We hope that the results of this study will reveal the optimal timing for initiation of ART in HIV-infected patients with moderate to severe PCP. Trial registration This trial was registered as one of the 12 trials under the name of a general project at chictr.org.cn on February 1, 2019. The registration number of the general project is ChiCTR1900021195 .

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro . One of the main drug targets in P . jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker’s yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses. Pneumocystis jirovecii is a fungus that can cause life-threatening pneumonia in immunocompromised patients. Here, the authors sequence the genomes of P. jirovecii and two other Pneumocystis species, and show the unexpected absence of chitin (a near universal fungal cell wall component).

Background and objective Pneumocystis jirovecii ( Pj ) pneumonia (PJP) is a life-threatening opportunistic infection primarily affecting immunocompromised individuals. Detecting Pj is challenging, particularly in distinguishing between Pj colonization (PJC) and infection. We aimed to systematically evaluate the diagnostic accuracy of various tests in differentiating Pj colonization from infection. Methods Systematic reviews and meta-analyses were performed. Searches were conducted in PubMed, Embase, and Web of Science. Original clinical studies reporting sensitivity and specificity data for diagnostic tests such as quantitative polymerase chain reaction (qPCR), nested PCR, (1,3)-Beta-D glucan (BDG), metagenomic next-generation sequencing (mNGS), and digital PCR (ddPCR) to differentiate PJC from PJP were included. Quality assessment was performed using QUADAS-2 tool, and data processing followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Diagnostic performance was evaluated using either a random-effects or fixed-effects model. Results Twenty-eight studies (2,550 patients, 1,445 with PJP) were included, with moderate methodological quality. The pooled sensitivity of these diagnostic tests was 0.80 (95% CI 0.77–0.82) and specificity was 0.83 (95% CI 0.81–0.85), with a diagnostic odds ratio (DOR) of 23.12. Among the individual tests, BDG (5 studies) showed high pooled sensitivity (0.83, 95% CI 0.77–0.88) but lower specificity (0.78, 95% CI 0.69–0.85). mNGS (3 studies) had the highest performance, with pooled sensitivity and specificity both at 0.87 (95% CI 0.80–0.92 and 95% CI 0.77–0.94, respectively), and the highest DOR of 41.57. qPCR (19 studies) demonstrated adequate pooled sensitivity (0.78, 95% CI 0.76–0.81) and high specificity (0.83, 95% CI 0.81–0.86), with a DOR of 20.44. Conclusion While BDG has low specificity and mNGS is costly with no standardized interpretation, along with the limited number of relevant studies in BDG and mNGS, this meta-analysis concluded that qPCR remains valuable for distinguishing P. jirovecii infection from colonization. A well-designed randomized clinical trial that standardizes the technical aspects of the qPCR protocol is needed to assess its effectiveness and provide a solid basis for clinical diagnosis.

Pneumocystis pneumonia is an opportunistic disease caused by invasion of unicellular fungus Pneumocystis jirovecii. Initially, it was responsible for majority of morbidity and mortality cases among HIV-infected patients, which later have been reduced due to the introduction of anti-retroviral therapy, as well as anti-Pneumocystis prophylaxis among these patients. Pneumocystis pneumonia, however, is still a significant cause of mortality among HIV-negative patients being under immunosuppression caused by different factors, such as transplant recipients as well as oncologically treated ones. The issue of pneumocystosis among these people is particularly emphasized in the article, since rapid onset and fast progression of severe symptoms result in high mortality rate among these patients, who thereby represent the group of highest risk of developing Pneumocystis pneumonia. In contrast, fungal invasion in immunocompetent people usually leads to asymptomatic colonization, which frequent incidence among healthy infants has even suggested the possibility of its association with sudden unexpected infant death syndrome. In the face of emerging strains with different epidemiological profiles resulting from genetic diversity, including drug-resistant genotypes, the colonization phenomenon desires particular attention, discussed in this article. We also summarize specific and sensitive methods, required for detection of Pneumocystis invasion and for distinguish colonization from the disease.

Pneumocystis jirovecii causes severe pneumonia in immunocompromised individuals, leading to high mortality and an economic burden. There is a need for early detection methods suitable for low-resource settings and rapid point-of-care diagnostics. This study developed a detection method using Recombinase Polymerase Amplification (RPA) followed by CRISPR/Cas12a with fluorescence detection. The RPA primers and CRISPR-derived RNAs (crRNAs) were specifically designed to target the mitochondrial small subunit rRNA (mtSSU rRNA) gene of P. jirovecii . A total of 83 clinical samples were tested using this method, including 39 confirmed and 44 suspected cases of P. jirovecii infection. The combination of crRNA5 and crRNA6 demonstrated higher sensitivity compared to the current real-time PCR detection method, with a limit of detection (LOD) of 1 copy per reaction and showed no cross-reactions with other respiratory pathogens. The concordance of this method was validated with both infected and non-infected patients. In conclusion, the method developed in this study potentially provides a highly sensitive and rapid tool suitable for the early and on-site detection of P. jirovecii pneumonia. Furthermore, this method holds potential applications for the detection of other human pathogens, representing a significant advancement in diagnostic capabilities for low-resource settings.

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.

Objective To delineate the clinical differences between Pneumocystis jirovecii pneumonia (PJP) and colonization, identify independent risk factors associated with PJP development, and construct a multidimensional diagnostic model to address the ongoing clinical challenge of accurately distinguishing P. jirovecii infection status in practice. Materials and methods This retrospective study analyzed the clinical characteristics, imaging findings, and laboratory parameters of patients who tested positive for P. jirovecii by next-generation sequencing (NGS) at the First Hospital of Jilin University between January 2014 and October 2024. Multivariable logistic regression was performed to determine independent predictors of PJP. Results Of the 292 patients included in the analysis (210 diagnosed with PJP and 82 classified as colonized), those with PJP had significantly higher rates of immunosuppression (64.4% vs. 9.9%, P <  0.001) and markedly increased P. jirovecii sequence counts from NGS (median: 1,686 vs. 4, P <  0.001).Human immunodeficiency virus coinfection, decreased lymphocyte count, elevated BDG levels, and increased LDH levels were identified as independent risk factors for PJP. A diagnostic model incorporating these four variables demonstrated excellent predictive capability, yielding an area under the receiver operating characteristic curve of 0.892 ( P <  0.001; 95% confidence interval: 0.855–0.929). The optimal NGS sequence count threshold for differentiating PJP from colonization was determined to be 37, achieving a sensitivity of 91% and a specificity of 87.8% (area under the receiver operating characteristic curve: 0.964). Conclusions The developed risk prediction model—comprising lymphocyte count, BDG, and LDH levels—facilitates rapid, pre-NGS clinical risk stratification for PJP, enabling prompt and informed therapeutic decision-making. When NGS results yield a P. jirovecii -specific sequence reads below the cutoff value of 37, a definitive diagnosis of PJP is unlikely. However, such findings should be interpreted in the context of the patient’s clinical presentation and assessed using the diagnostic model to ensure an accurate evaluation of infection status.

The fungus Pneumocystis causes severe pneumonia in patients with weakened immune systems. It possesses a genetic system to vary the antigens at the surface of its cells that are presented to the immune system of the patient. We report for the first time that this system may have been implicated in the infections of renal transplant recipients involved in a suspected outbreak. Our observations suggest that the antigens presented might be selected to avoid the elimination of the fungus by the immune response specific to each patient. The resistance of the fungus to the immunosuppressant mycophenolate administered to these patients to prevent organ rejection probably also played a role in the infections during the suspected outbreak.