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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 pneumonia (PCP) is an important cause of morbidity in immunocompromised patients, with a higher mortality in non-HIV than in HIV patients. P. jirovecii is one of the rare transmissible pathogenic fungi and the only one that depends fully on the host to survive and proliferate. Transmissibility among humans is one of the main specificities of P. jirovecii . Hence, the description of multiple outbreaks raises questions regarding preventive care management of the disease, especially in the non-HIV population. Indeed, chemoprophylaxis is well codified in HIV patients but there is a trend for modifications of the recommendations in the non-HIV population. In this review, we aim to discuss the mode of transmission of P. jirovecii , identify published outbreaks of PCP and describe molecular tools available to study these outbreaks. Finally, we discuss public health and infection control implications of PCP outbreaks in hospital setting for in- and outpatients.

Background. Increasing reports of Pneumocystis DNA in noninvasive respiratory specimens from immunocompetent asymptomatic adults and the characteristic lung tropism of Pneumocystis suggest that asymptomatic pulmonary infections with Pneumocystis occur after primary infection. However, studies searching for Pneumocystis in the autopsied lungs of healthy immunocompetent adults have not met with success. Methods. Lungs of people who died of violent causes (accidents, homicide, and suicide) and of nonviolent causes (diseases causing a rapid demise in the street) in Santiago, Chile—for whom an autopsy was legally required—were examined for Pneumocystis by nested polymerase chain reaction (PCR) DNA amplification of the mitochondrial large subunit ribosomal RNA–specific P. jirovecii gene and immunofluorescent microscopic analysis. Lung tissue concentration methods and analysis of ∼3% of the weight of the right upper lobe (RUL) were needed to reach the sensitivity threshold of the assays. Individuals determined to be P. jirovecii negative after analysis of 3% of the RUL weight in the violent death group were confirmed to be negative by analyzing additional tissue, totaling 6%–7% of the RUL weight. Results. P. jirovecii was identified by nested PCR in 50 (64.9%) of 77 individuals (34 [61.8%] of 55 in the violent death group and 15 [78.9%] of 19 in the nonviolent death group; P>.05) and additionally by microscopic analysis in all individuals who tested positive for P. jirovecii DNA in the violent death group. Analysis of tissue beyond 3.0% of the RUL weight for the individuals who tested negative yielded consistently negative results. Conclusions. A mild P. jirovecii pulmonary infection is prevalent in more than half of the general adult population. Our results strengthen the concept that immunocompetent adults develop frequent self-limited reinfections throughout life and participate in the circulation of P. jirovecii as an infective reservoir for susceptible individuals.

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.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii . This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens. Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens.

Diagnosis of Pneumocystis jirovecii (PJ) pneumonia ordinarily requires invasive procedures that could be avoided by PCR methodologies, if these could be designed with adequate cut-off values for confounding background carriage. We designed a novel quantitative real-time PCR assay to detect the mitochondrial large subunit rRNA gene of PJ in oral washes. To benchmark levels of PJ carriage versus infection, we tested asymptomatic immunosuppressed patients including Danish (n = 88) and West African HIV-infected (n = 142) patients, renal transplant recipients (n = 51), rheumatologic patients (n = 102), patients with inflammatory bowel diseases (n = 98), and healthy blood donors (controls, n = 50). The fungal burden in patients with PJ pneumonia (PCP, n = 7) was also investigated. Danish HIV-infected patients (with viremia/low CD4) and recent transplant recipients were at most risk of being carriers (prevalence of 23% and 16.7% respectively), whereas PJ was rarely detected among rheumatologic patients, patients with inflammatory bowel diseases, and untreated West African HIV patients. PJ was not detected among healthy controls. The fungal burden in patients with PCP fell rapidly on treatment. The quantitative PCR method described could conceivably discriminate between carriage and disease, given suitable threshold values for the former, and predict treatment efficacy by measures of the fungal burden in daily oral washes.

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.

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.

Background Idiopathic inflammatory myopathies (IIM) are associated with a significantly higher risk of opportunistic infections including Pneumocystis jirovecii pneumonia (PJP), a potentially fatal opportunistic infection. However, no prior studies have evaluated PJP infection in subtypes of IIM. Objectives To investigate the prevalence and mortality rate of PJP infection in subgroups of IIM patients stratified according to myopathy-specific antibodies. Methods In the first part of the study, 463 consecutive patients with IIM were prospectively followed for a period of at least 1 year to analyze the incidence of PJP. In the second part of the study, we enrolled 30 consecutive PJP patients with any rheumatic disease in order to identify the mortality rate and risk factors by Cox regression analysis. The Kaplan-Meier method with log-rank testing was used to assess differences in survival. Results The prevalence of PJP in IIM patients was found to be 3.0/100 person-years, while in MDA5 + DM patients it was 7.5/100 person-years and in MDA5 − IIM patients 0.7/100 person-years ( P < 0.05). PJP typically occurred in the first 2 months in the case of MDA5 + DM patients who had a significant decrease in their CD4 + T cell counts and lymphocyte counts ( P < 0.05). In PJP patients, 3-month mortality was higher for MDA5 + DM patients than in those with other rheumatic diseases (83.3% vs 38.9%, P < 0.05). Alarmingly, MDA5 + DM patients seemed not to benefit from prompt anti-PJP treatment, unlike patients with other rheumatic diseases whose survival improved when anti-PJP treatment was started within 6 days ( P < 0.05). Conclusion PJP has an alarming high incidence and mortality in MDA5 + DM patients. Timely treatment for PJP seems not to improve the prognosis of patients with this particular subtype. Hence, there remains a crucial unmet need to develop PJP prophylaxis for MDA5 + DM patients.