Dynamics of host immune responses and a potential function of Trem2hi interstitial macrophages in Pneumocystis pneumonia.

BACKGROUND: Pneumocystis pneumonia (PCP) is a life-threatening opportunistic fungal infection with a high mortality rate in immunocompromised patients, ranging from 20 to 80%. However, current understanding of the variation in host immune response against Pneumocystis across different timepoints is limited. METHODS: In this study, we conducted a time-resolved single-cell RNA sequencing analysis of CD45+ cells sorted from lung tissues of mice infected with Pneumocystis. The dynamically changes of the number, transcriptome and interaction of multiply immune cell subsets in the process of Pneumocystis pneumonia were identified according to bioinformatic analysis. Then, the accumulation of Trem2hi interstitial macrophages after Pneumocystis infection was verified by flow cytometry and immunofluorescence. We also investigate the role of Trem2 in resolving the Pneumocystis infection by depletion of Trem2 in mouse models. RESULTS: Our results characterized the CD45+ cell composition of lung in mice infected with Pneumocystis from 0 to 5 weeks, which revealed a dramatic reconstitution of myeloid compartments and an emergence of PCP-associated macrophage (PAM) following Pneumocystis infection. PAM was marked by the high expression of Trem2. We also predicted that PAMs were differentiated from Ly6C+ monocytes and interacted with effector CD4+ T cell subsets via multiple ligand and receptor pairs. Furthermore, we determine the surface markers of PAMs and validated the presence and expansion of Trem2hi interstitial macrophages in PCP by flow cytometry. PAMs secreted abundant pro-inflammation cytokines, including IL-6, TNF-α, GM-CSF, and IP-10. Moreover, PAMs inhibited the proliferation of T cells, and depletion of Trem2 in mouse lead to reduced fungal burden and decreased lung injury in PCP. CONCLUSION: Our study delineated the dynamic transcriptional changes in immune cells and suggests a role for PAMs in PCP, providing a framework for further investigation into PCP's cellular and molecular basis, which could provide a resource for further discovery of novel therapeutic targets.

Evaluation of pentamidine tolerability and efficacy between CYP2C19 phenotypes.

Intravenous pentamidine is used for prophylaxis against Pneumocystis jirovecii pneumonia, an infection seen in hematopoietic stem cell transplant recipients. Pentamidine is partially metabolized by CYP2C19, which is vulnerable to pharmacogenetic variation. This retrospective study evaluated allogeneic hematopoietic stem cell transplant patients who received intravenous pentamidine as P. jirovecii pneumonia prophylaxis. The primary objective was the association between CYP2C19 phenotype and discontinuation of pentamidine due to drug-related side effects based on univariate logistic regression (N = 81). Ten patients (12.3%) discontinued pentamidine because of side effects. There was no difference in discontinuation between phenotype groups (p = 0.18) or discontinuation due to side effects (p = 0.76). Overall, no association was seen between phenotypes and pentamidine-related side effects (p = 0.475). Drug discontinuation rates and P. jirovecii pneumonia infection rates were low.

Genetic Mouse Models of Pneumocystis Pneumonia.

Pneumocystis jirovecii causes pneumonia in immunocompromised patients. A major challenge in drug susceptibility testing and in understanding host/pathogen interactions is that Pneumocystis spp. are not viable in vitro. Continuous culture of the organism is not currently available, and therefore, developing new drug targets is very limited. Due to this limitation, mouse models of Pneumocystis pneumonia have proven to be an invaluable resource to researchers. In this chapter, we provide an overview of selected methods used in mouse models of infection including, in vivo Pneumocystis murina propagation, routes of transmission, genetic mouse models available, a P. murina life form-specific model, a mouse model of PCP immune reconstitution inflammatory syndrome (IRIS), and the experimental parameters associated with these models.

Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for Pneumocystis jirovecii Pneumonia.

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.

Case report: Pneumocystis jirovecii pneumonia in a severe case of Aicardi-Goutières syndrome with an IFIH1 gain-of-function mutation mimicking combined immunodeficiency.

Aicardi-Goutières syndrome (AGS) is a genetically determined early-onset progressive encephalopathy caused by mutations leading to overexpression of type I interferon (IFN) and resulting in various clinical phenotypes. A gain-of-function (GOF) mutation in the IFIH1 gene is associated with robust production of type I IFN and activation of the Janus kinase (JAK) signal transducer and activator of the transcription (STAT) pathway, which can cause AGS type 7. We detail the clinical case of an infant who initially presented with Pneumocystis jirovecii pneumonia (PCP), had recurrent respiratory infections, and was later treated with a JAK inhibitor, baricitinib, because of a genetically confirmed GOF mutation in the IFIH1 gene. This spectrum of IFIH1 GOF mutations with overlapping features of hyperinflammation and severe opportunistic infection, which mimics combined immunodeficiency (CID), has not been described before. In this case, therapy with baricitinib effectively blocked IFN-α activation and reduced STAT1 signaling but had no effect on the progression of the neurological disease.

T and B cell abnormalities, pneumocystis pneumonia, and chronic lymphocytic leukemia associated with an AIOLOS defect in patients.

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy.

Toward a humanized mouse model of Pneumocystis pneumonia.

Pneumocystis is an important opportunistic fungus that causes pneumonia in children and immunocompromised individuals. Recent genomic data show that divergence of major surface glycoproteins may confer speciation and host range selectivity. On the other hand, immune clearance between mice and humans is well correlated. Thus, we hypothesized that humanize mice may provide information about human immune responses involved in controlling Pneumocystis infection. CD34-engrafted huNOG-EXL mice controlled fungal burdens to a greater extent than nonengrafted mice. Moreover, engrafted mice generated fungal-specific IgM. Fungal control was associated with a transcriptional signature that was enriched for genes associated with nonopsonic recognition of trophs (CD209) and asci (CLEC7A). These same genes were downregulated in CD4-deficient mice as well as twins with bare lymphocyte syndrome with Pneumocystis pneumonia.

Increased mRNA expression of key cytokines among suspected cases of Pneumocystis jirovecii infection.

BACKGROUND: Pneumocystis pneumonia (PCP) is a fatal infectious disease caused by Pneumocystis jirovecii (PJP). The major factor relevant to morbidity and mortality seems to be the host inflammatory reaction. The objective of this study was to evaluate the role of IL-2, IL-4, IL-10, and IL-13 cytokine mRNA expression among suspected P. jirovecii infection. METHODS: This was a cross-sectional analytical study undertaken in Aseer region, Saudi Arabia. One hundred suspected PCP cases and 100 healthy controls were included in the study. Basic clinical manifestations, radiological findings, microbiological and immunological findings were extracted from the hospital records from January 2019 to August 2019, Pneumocystis detection was done by immune-fluorescent staining (IFAT, Gomorimethanamine silver staining (GMSS), Giemsa staining, Toluidine blue O (TBO), and Pneumocystis RT-PCR. RESULTS: Increased more than 5 fold, 3 fold, 4 fold, and 7 fold of IL-2, IL-4, IL-10, and IL-13 mRNA expression were observed in PCP cases compared to controls. Higher expression of IL-2 mRNA was connected with crept, wheezing and chest X-ray findings like central perihilar infiltrate, patchy infiltrate, consolidation, hilar lymphadenopathy, pneumothorax, pleural effusion which showed higher expression compared to counterpart (p< 0.0001). Higher expression of IL-4 mRNA was found to be significantly associated with weight loss (p=0.002), dyspnea (p=0.003), crept (p=0.01), and chest X-ray findings (p< 0.0001). Significantly increased expression of IL-10 mRNA was observed to be associated with weight loss, dyspnea, night sweats, wheezing, and different findings of chest X-ray compared to their counterparts, whereas, IL-13 mRNA was observed in cases with fever. Suspected cases of PCP confirmed positive by IFTA with higher IL-2, IL-4, and IL-10 mRNA expression compared to negative cases. RT-PCR confirmed PCP cases had significantly higher expression of IL-2, IL-4, and IL-10 as well as IL-13 mRNA compared to negative cases. Positive detected cases by GMSS showed higher IL-2, IL-10 mRNA expression, while Giemsa showed only higher IL-4 mRNA expression compared to negative cases. CONCLUSION: Confirmed cases of P. jirovecii showed higher IL-2, IL-4, IL-10, and IL-13 mRNA expression comparatively to negative cases. Increased expression of cytokines may be indicative of infection severity and could help in patients' management.

Deficiency of mannose-binding lectin is a risk of Pneumocystis jirovecii pneumonia in a natural history cohort of people living with HIV/AIDS in Northern Thailand.

BACKGROUND: Mannose-binding lectin (MBL) plays a pivotal role in innate immunity; however, its impact on susceptibility to opportunistic infections (OIs) has not yet been examined in a natural history cohort of people living with HIV/AIDS. METHODS: We used archived samples to analyze the association between MBL expression types and risk of major OIs including Pneumocystis jirovecii pneumonia (PCP), cryptococcosis, talaromycosis, toxoplasmosis, and tuberculosis in a prospective cohort in Northern Thailand conducted from 1 July 2000 to 15 October 2002 before the national antiretroviral treatment programme was launched. RESULTS: Of 632 patients, PCP was diagnosed in 96 (15.2%) patients, including 45 patients with new episodes during the follow-up period (1006.5 person-years). The total history of PCP was significantly associated with low MBL expression type: high/intermediate (81/587, 13.8%), low (10/33, 30.3%) and deficient (5/12, 41.7%) (p = 0.001), whereas the history of other OIs showed no relation with any MBL expression type. Kaplan-Meier analysis (n = 569; log-rank p = 0.011) and Cox's proportional hazards model revealed that deficient genotype dramatically increased the risk of PCP, which is independent upon sex, age, CD4 count, HIV-1 viral load and hepatitis B and C status (adjusted hazard ratio 7.93, 95% confidence interval 2.19-28.67, p = 0.002). CONCLUSIONS: Deficiency of MBL expression is a strong risk factor determining the incidence of PCP but not other major OIs.

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens.

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)-targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5 hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P. jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1

[Pneumocystis jirovecii and quantitative PCR: Pneumonia or colonization?] / Pneumocytis jirovecii et PCR quantitative : pneumonie ou colonisation à Pneumocystis jirovecii ?

BACKGROUND: Quantitative PCR to detect Pneumocystis jirovecii (Pj) is a new tool for the diagnosis of Pneumocystis jirovecii pneumonia (PJP). The yield of this technique, in cases of low fungal burden, when the standard technique using immunofluorescence (IF) is negative, needs to be evaluated. METHODS: We retrospectively reviewed the charts of all patients with a positive PCR but negative IF test (PCR+/IF-) in bronchoalveolar lavage (BAL) fluid performed over one year. We used an algorithm based on underlying immunosuppression, clinical picture, thoracic CT scan appearances, existence of an alternative diagnosis and the patient's outcome on treatment. Using this, each case was classified as probable PJP, possible PJP or colonization. RESULTS: Among the 416 BAL performed, 48 (12%) were PCR+/IF- and 43 patients were analyzed. Patients were mostly male (56%) with a median age of 60 years. Thirty-five (84%) were immunocompromised: 4 (9%) HIV-infected patients, 26 (60%) with hematologic or solid organ cancer, 3 (7%) were renal transplant recipients. Seven (16%) were classified as probable PPJ and 9 (21%) as possible PJP. Patients with a probable or possible PJP were more frequently admitted to the ICU (P<0.02) and had higher risk of death (P<0.01) when compared to those with colonization. Median PCR levels were very low and were not different between PJP or colonized patients (P=0.23). CONCLUSIONS: Among patients with a positive Pj PCR in BAL but with negative IF, only 37% had probable or possible PJP and PCR could not discriminate PJP from colonization.

PD-1 Deficiency Promotes Macrophage Activation and T-Helper Cell Type 1/T-Helper Cell Type 17 Response in Pneumocystis Pneumonia.

Pneumocystis is an unusual, opportunistic fungal pathogen capable of causing Pneumocystis pneumonia (PCP) in immunocompromised hosts. Although PCP was discovered >100 years ago, its pathogenesis remains unclear. The inhibitory receptor PD-1 (programmed death 1), a negative regulator of activated T cells, has been reported to take part in tumor escape, immune tolerance, and infection immunity. In this study, we examined the role of the PD-1/PD-L1 (programmed death-ligand 1) pathway in patients with PCP and in mice. The expression levels of PD-1/PD-L1 in patients with PCP and in mice were measured by real-time PCR and flow cytometry. The effects of PD-1 deficiency are demonstrated using wild-type and PD-1-/- mice. Our data show that Pneumocystis infection promotes PD-1/PD-L1 expression; PD-1 deficiency enhances the phagocytic function of macrophages and the pulmonary T-helper cell type 1 (Th1)/Th17 response, which might contribute to Pneumocystis clearance; and PD-1 deficiency affects the polarization of macrophages. PCP mice treated with anti-PD-1 antibody showed improved pulmonary clearance of Pneumocystis. Collectively, our results demonstrate that the PD-1/PD-L1 pathway plays a role in regulating the innate and adaptive immune responses, suggesting that manipulation of this pathway may constitute an immunotherapeutic strategy for PCP.

Leishmania donovani and Pneumocystis jirovecii (carinii) diagnosed on bronchoalveolar lavage cytology in a liver transplant recipient with Cytomegalovirus infection.

Liver transplant recipients are prone to several infections, including lung infections, which can lead to substantial morbidity and mortality. Bronchoalveolar lavage (BAL) cytology is a rapid and sensitive diagnostic tool to identify the etiologic agents. We report a rare case of a 24-year-old male, post Live donor liver transplantation for autoimmune chronic liver disease, who presented with cough, fever, weight loss, and cavitatory lesion in lung. BAL cytology revealed Leishmania donovani (LD) and Pneumocystis jirovecii/carinii (PCP). Cytomegalovirus deoxyribonucleic acid polymerase chain reaction (CMV DNA PCR) test showed markedly raised levels. Patient was put on treatment for these multiple infections and showed significant improvement. Thus, rapid diagnosis of infections through BAL cytology is crucial in transplant recipients to institute timely therapy and avoid undesirable empirical treatments. Moreover, this case highlights a rare finding of LD bodies along with PCP in BAL cytology.

IL-4 polymorphism influences susceptibility to Pneumocystis jirovecii pneumonia in HIV-positive patients.

OBJECTIVES: Pneumocystis jirovecii pneumonia (PJP) is an important cause of morbidity and mortality in HIV-positive patients. Polymorphisms in immune genes are increasingly reported to influence susceptibility to fungal infections. We analysed the role of 21 single nucleotide polymorphisms from 19 candidate genes on PJP development in patients from the Swiss HIV Cohort Study. DESIGN AND METHODS: The analysis included patients with a nadir CD4 T-cell count less than 200 cells/µl, divided into a discovery (N = 1645) and a replication (N = 1861) cohort. The associations were analysed by using cumulative incidence curves as well as competing risk regression over 18 years, starting from the estimated date of HIV infection, considering death a competing risk, with censoring at lost follow-up, and assuming the dominant mode of inheritance. RESULTS: The minor allele of rs2243250 in IL-4 was associated with the risk of PJP in the discovery cohort (cumulative incidence 0.18 versus 0.12, P = 0.002). This association was replicated in the validation cohort (0.16 versus 0.12, P = 0.02). It was still significant in multivariate models, adjusted for HIV transmission mode, viral load, CD4 T cells slope, age, antiretroviral therapy, tobacco smoking, hepatitis C virus coinfection, year of cohort entry and PJP prophylaxis (global subhazard ratio 1.42, 95% confidence interval 1.17-1.73, P = 0.0004). CONCLUSION: Our data suggest rs2243250, a single nucleotide polymorphism known to influence IL-4 production, is associated with susceptibility to PJP in HIV-positive patients.

Clinical, genetic and immunological characteristics of 40 Chinese patients with CD40 ligand deficiency.

CD40 ligand (CD40L) deficiency is a rare but life-threatening primary immunodeficiency caused by mutations in the CD40L gene. Here, we investigated a cohort of 40 genetically diagnosed CD40L-deficient patients from the Chinese mainland, analysed their clinical and genetic data, and examined CD40L expression, the proportion of T cell subsets, B cell subsets and T follicular helper (Tfh) cells. The aim was to provide a complete picture of CD40L deficiency. Initial presentations of the patient cohort mainly involved recurrent fever (47.5%) and sinopulmonary infection (42.5%). Life-threatening infections (42.5%), caused by various pathogens, were the most serious threats faced by CD40L-deficient patients, while neutropenia (57.5%) remained the most common complication. Opportunistic infections, including Pneumocystis carinii pneumonia and invasive fungal disease associated with Talaromyces marneffei, were also common in the cohort. In addition, seven patients (17.5%) suffered BCGitis/BCGosis, which is a major problem facing a planned immunization programme in China. It was intriguing that reduced IgM levels were observed in 12.5% of patients, while normal or elevated IgA levels were shown in 47.5% of patients. Thirty-seven unique mutations were identified in 40 patients; of these, 10 were novel. Furthermore, we observed a lower percentage of NK cells, Tfh cells, and central memory CD4+ T cells, and an extremely small class-switched memory B cell population, in CD40L-deficient patients. Patients who underwent hematopoietic stem cell transplantation experienced better disease remission. Taken together, our data establish the largest database about CD40L deficiency in China and provide genetic, immunologic and clinical information about Chinese CD40L-deficient patients.

Genetic polymorphisms associated with treatment failure and mortality in pediatric Pneumocystosis.

Data on the genetic diversity of Pneumocystis jirovecii causing Pneumocystis pneumonia (PCP) among children are still limited, and there are no available data from the Indian subcontinent, particularly associations between genotypes and clinical characteristics. A total of 37 children (62 days-12 years [median 5.5 years]) were included in this study. Pneumocystis was diagnosed by microscopy using Grocott-Gomori methenamine silver stain in 12 cases and by nested PCR using mtLSUrRNA in 25 cases. Genotyping was performed using three different genes, mitochondrial large subunit ribosomal RNA (mtLSUrRNA), dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). mtLSUrRNA genotype 3 and novel mutations at the gene target DHFR (401 T > C) and DHPS 96/98 were frequently observed and clinically associated with severe PCP and treatment failure. Phylogenetic analyses revealed 13 unique sequence types (STs). Two STs (i) 3-DHFR 401 T > C-DHPS 96/98 - PJ1 and (ii) 3-DHFR 401 T > C-DHPS 96- PJ3 were significantly associated with treatment failure and high mortality among PCP-positive patients. In conclusion, the present study strongly suggests the emergence of virulent P. jirovecii strains or genetic polymorphisms, leading to treatment failure and high mortality. Our study is the first of its kind from the Indian subcontinent and has highlighted the genetic diversity of Pneumocystis jirovecii among children and their clinical outcomes. These findings emphasize the need to focus more on genotypes to better understand the epidemiology of Pneumocystis pneumonia.