Semiquantitative Real-Time PCR to Distinguish Pneumocystis Pneumonia from Colonization in a Heterogeneous Population of HIV-Negative Immunocompromised Patients.

Pneumocystis jirovecii is a threat to iatrogenically immunosuppressed individuals, a heterogeneous population at rapid growth. We assessed the ability of an in-house semiquantitative real-time PCR assay to discriminate Pneumocystis pneumonia (PCP) from colonization and identified risk factors for infection in these patients. Retrospectively, 242 PCR-positive patients were compared according to PCP status, including strata by immunosuppressive conditions, human immunodeficiency virus (HIV) infection excluded. Associations between host characteristics and cycle threshold (CT) values, semiquantitative real-time PCR correlates of fungal loads in lower respiratory tract specimens, were investigated. CT values differed significantly according to PCP status. Overall, a CT value of 36 allowed differentiation between PCP and colonization with sensitivity and specificity of 71.3% and 77.1%, respectively. A CT value of less than 31 confirmed PCP, whereas no CT value permitted exclusion. A considerable diversity was uncovered; solid organ transplant (SOT) recipients had significantly higher fungal loads than patients with hematological malignancies. In SOT recipients, a CT cutoff value of 36 resulted in sensitivity and specificity of 95.0% and 83.3%, respectively. In patients with hematological malignancies, a higher CT cutoff value of 37 improved sensitivity to 88.5% but reduced specificity to 66.7%. For other conditions, assay validity appeared inferior. Corticosteroid usage was an independent predictor of PCP in a multivariable analysis and was associated with higher fungal loads at PCP expression. Semiquantitative real-time PCR improves differentiation between PCP and colonization in immunocompromised HIV-negative individuals with acute respiratory syndromes. However, heterogeneity in disease evolution requires separate cutoff values across intrinsic and iatrogenic predisposition for predicting non-HIV PCP. IMPORTANCE Pneumocystis jirovecii is potentially life threatening to an increasing number of individuals with compromised immune systems. This microorganism can cause severe pneumonia in susceptible hosts, including patients with cancer and autoimmune diseases and people undergoing solid organ transplantation. Together, these patients constitute an ever-diverse population. In this paper, we demonstrate that the heterogeneity herein has important implications for how we diagnose and assess the risk of Pneumocystis pneumonia (PCP). Specifically, low loads of microorganisms are sufficient to cause infection in patients with blood cancer compared to those in solid organ recipients. With this new insight into host versus P. jirovecii biology, clinicians can manage patients at risk of PCP more accurately. As a result, we take a significant step toward offering precision medicine to a vulnerable patient population. One the one hand, these patients have propensity for adverse effects from antimicrobial treatment. On the other hand, this population is susceptible to life-threatening infections, including PCP.

Effect of Subcutaneous Anti-CD20 Antibody-Mediated B Cell Depletion on Susceptibility to Pneumocystis Infection in Mice.

Prior work has shown that parenterally administered anti-CD20 (5D2) inhibits CD4+ T cell priming in response to challenge with Pneumocystis murina and predisposes to pneumonia. In this study, we investigated the effect of subcutaneous anti-CD20 antibody and Pneumocystis infection. In mice with primary infection, anti-CD20 antibody treatment depleted both CD19+ and CD27+ CD19+ cells but not T cells in the lung at days 14 and 28 after Pneumocystis inoculation. Although anti-CD20 antibody treatment impaired fungal clearance at day 14 postinfection, fungal burden in the lungs was substantially reduced at day 28 in both depleted and control mice in the low-dose group. Subcutaneous anti-CD20 antibody treatment did not alter antigen-specific serum immunoglobulin levels in mice compared with control mice, and there were no significant differences in the numbers of lung gamma interferon-positive (IFN-γ+) CD4+, interleukin 4-positive (IL-4+) CD4+, IL-5+ CD4+, and IL-17A+ CD4+ cells between depleted and control mice after infection. In mice with secondary infection, the lung fungal burden was comparable between depleted and control mice 14 days after reinfection. Low-dose subcutaneous anti-CD20 antibody treatment may delay fungal clearance, but it did not impair the ability of the host to clear Pneumocystis infection, irrespective of primary or secondary infection.IMPORTANCE Anti-CD20 antibody therapy is used for both cancer and autoimmune disease but has been shown to be associated with Pneumocystis pneumonia in humans. This study shows that low-dose subcutaneous anti-CD20 can modulate B cell populations without grossly perturbing fungal immunity against Pneumocystis lung infection.

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.

Risk factors associated with Pneumocystis jirovecii pneumonia in juvenile myositis in North America.

OBJECTIVES: Pneumocystis jirovecii pneumonia (PJP) is associated with significant morbidity and mortality in adult myositis patients; however, there are few studies examining PJP in juvenile myositis [juvenile idiopathic inflammatory myopathy (JIIM)]. The purpose of this study was to determine the risk factors and clinical phenotypes associated with PJP in JIIM. METHODS: An research electronic data capture (REDCap) questionnaire regarding myositis features, disease course, medications and PJP infection characteristics was completed by treating physicians for 13 JIIM patients who developed PJP (PJP+) from the USA and Canada. Myositis features and medications were compared with 147 JIIM patients without PJP (PJP-) from similar geographic regions who enrolled in National Institutes of Health natural history studies. RESULTS: PJP+ patients were more often of Asian ancestry than PJP- patients [odds ratio (OR) 8.7; 95% CI 1.3, 57.9]. Anti- melanoma differentiation associated protein 5 (MDA5) autoantibodies (OR 12.5; 95% CI 3.0, 52.4), digital infarcts (OR 43.8; 95% CI 4.2, 460.2), skin ulcerations (OR 12.0; 95% CI 3.5, 41.2) and interstitial lung disease (OR 10.6; 95% CI 2.1, 53.9) were more frequent in PJP+ patients. Before PJP diagnosis, patients more frequently received pulse steroids, rituximab and more immunosuppressive therapy compared with PJP- patients. Seven PJP+ patients were admitted to the intensive care unit and four patients died due to PJP or its complications. CONCLUSIONS: PJP is a severe infection in JIIM that can be associated with mortality. Having PJP was associated with more immunosuppressive therapy, anti-MDA5 autoantibodies, Asian race and certain clinical features, including digital infarcts, cutaneous ulcerations and interstitial lung disease. Prophylaxis for PJP should be considered in juvenile myositis patients with these features.

Fatal outcome of anti-MDA5 juvenile dermatomyositis in a paediatric COVID-19 patient: a case report.

Anti-melanoma differentiation-associated gene 5 juvenile dermatomyositis (anti-MDA5 JDM) is associated with high risk of developing rapidly progressive interstitial lung disease (RP-ILD). Here we report an 11-year-old girl with anti-MDA5 JDM and RP-ILD which led to a fatal outcome, further aggravated by SARS-CoV-2 infection. She was referred to our hospital after being diagnosed with anti-MDA5 JDM and respiratory failure due to RP-ILD. On admission, fibrobronchoscopy with bronchoalveolar lavage (BAL) revealed Pneumocystis jirovecii infection so treatment with intravenous trimethoprim-sulfamethoxazole was initiated. Due to RP-ILD worsening, immunosuppressive therapy was intensified using methylprednisolone pulses, cyclophosphamide, tofacitinib and intravenous immunoglobulin without response. She developed severe hypoxemic respiratory failure, pneumomediastinum and pneumothorax, further complicated with severe RP-ILD and cervical subcutaneous emphysema. Three real-time RT-PCR for SARS-CoV-2 were made with a negative result. In addition, she was complicated with a secondary hemophagocytic lymphohistiocytosis and a fourth real-time PCR for SARS-CoV-2 performed in BAS sample was positive. Despite aggressive treatment of RP-ILD due to anti-MDA5 JDM, there was no improvement of respiratory failure in the following days and patient developed refractory septic shock and died. Anti-MDA5 JDM patients with RP-ILD have a poor prognosis with a high mortality rate. For this reason, intensive immunosuppressive therapy is essential including the use of promising drugs such as tofacitinib. COVID-19 in children with underlying health conditions like anti-MDA5 JDM may still be at risk for disease and severe complications.

A Comprehensive Evaluation of Risk Factors for Pneumocystis jirovecii Pneumonia in Adult Solid Organ Transplant Recipients: A Systematic Review and Meta-analysis.

BACKGROUND: There is no consensus guidance on when to reinitiate Pneumocystis jirovecii pneumonia (PJP) prophylaxis in solid organ transplant (SOT) recipients at increased risk. The 2019 American Society of Transplantation Infectious Diseases Community of Practice (AST IDCOP) guidelines suggested to continue or reinstitute PJP prophylaxis in those receiving intensified immunosuppression for graft rejection, cytomegalovirus (CMV) infection, higher dose of corticosteroids, or prolonged neutropenia. METHODS: A literature search was conducted evaluating all literature from existence through April 22, 2020, using MEDLINE and EMBASE. (The International Prospective Register of Systematic Reviews registration number: CRD42019134204). RESULTS: A total of 30 studies with 413 276 SOT recipients were included. The following factors were associated with PJP development: acute rejection (pooled odds ratio [pOR], 2.35; 95% confidence interval [CI], 1.69-3.26); study heterogeneity index [I2] = 23.4%), CMV-related illnesses (pOR, 3.14; 95% CI, 2.30-4.29; I2 = 48%), absolute lymphocyte count <500 cells/mm3 (pOR, 6.29; 95% CI, 3.56-11.13; I2 = 0%), BK polyomavirus-related diseases (pOR, 2.59; 95% CI, 1.22-5.49; I2 = 0%), HLA mismatch ≥3 (pOR, 1.83; 95% CI, 1.06-3.17; I2 = 0%), rituximab use (pOR, 3.03; 95% CI, 1.82-5.04; I2 = 0%), and polyclonal antibodies use for rejection (pOR, 3.92; 95% CI, 1.87-8.19; I2 = 0%). On the other hand, sex, CMV mismatch, interleukin-2 inhibitors, corticosteroids for rejection, and plasmapheresis were not associated with developing PJP. CONCLUSIONS: PJP prophylaxis should be considered in SOT recipients with lymphopenia, BK polyomavirus-related infections, and rituximab exposure in addition to the previously mentioned risk factors in the American Society of Transplantation Infectious Diseases Community of Practice guidelines.

A complex COVID-19 case with rheumatoid arthritis treated with tocilizumab.

Recurrences of COVID-19 were observed in a patient with long-term usage of hydroxychloroquine, leflunomide, and glucocorticoids due to her 30-year history of rheumatoid arthritis (RA). Tocilizumab was applied and intended to target both COVID-19 and RA. However, disease of this patient aggravated after usage of tocilizumab. After the discussion of a multiple disciplinary team (MDT) including rheumatologists, antimicrobial treatments were applied to target the potential opportunistic infections (Pneumocystis jirovecii and Aspergillus fumigatus), which were authenticated several days later via high throughput sequencing. As an important cytokine in immune responses, IL-6 can be a double-edged sword: interference in the IL-6-IL-6 receptor signaling may save patients from cytokine release storm (CRS), but can also weaken the anti-infectious immunity, particularly in rheumatic patients, who may have received a long-term treatment with immunosuppressive/modulatory agents. Thus, we suggest careful considerations before and close monitoring in the administration of tocilizumab in rheumatic patients with COVID-19. Besides tocilizumab, several disease-modifying antirheumatic drugs (DMARDs) can also be applied in the treatment of COVID-19. Therefore, we also reviewed and discussed the application of these DMARDs in COVID-19 condition.

Clinical Characteristics and Risk Factors for Pneumocystis Jirovecii Pneumonia during Immunosuppressive Treatment in Patients with Ulcerative Colitis: A Retrospective Study.

AIMS: This study aimed to clarify the clinical characteristics of Pneumocystis jirovecii pneumonia (PJP) infection in patients with ulcerative colitis (UC) and to identify risk factors for PJP using a retrospective case-control study. METHODS: Of 4,525 patients with UC treated between 2007 and 2019, we identified those who satisfied the criteria for PJP. The Lichtiger clinical activity index (LCI) was compared between the initiation of immunosuppressive drug treatment and the onset of PJP. A retrospective case-control study was conducted using a PJP group and a non-PJP group. RESULTS: Nine patients experienced PJP, of whom two died. Since October 2014, there were no cases of PJP among UC patients aged ≥50 years who were prescribed three or more immunosuppressive agents given prophylactic sulfamethoxazole-trimethoprim (TPM-SMX). The median LCI (range) was 13 (8-17) at the initiation of treatment versus 2 (1-8) at PJP onset (p = 0.016). The median time to PJP onset was 83 days after treatment initiation. In the PJP group the median age was significantly greater (p = 0.022), three immunosuppressants were used significantly more frequently (p = 0.004), and the lymphocyte counts during treatment were significantly lower (p < 0.01) than in the non-PJP group. The cut-off lymphocyte count that distinguished PJP patients from non-PJP patients was 570/µL according to a receiver-operating curve analysis. CONCLUSIONS: Prophylactic administration of TPM-SMX prevented further cases of PJP. The onset of PJP occurred at the same time as the symptoms of UC were stabilizing and the immunosuppressive drugs were being reduced. Greater age, lower lymphocyte count, and treatment with three immunosuppressive drugs were risk factors for PJP.

A critical role for CARD9 in pneumocystis pneumonia host defence.

Caspase recruitment domains-containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C-type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4-depleted CARD9-/- and immunocompetent hosts. Card9 gene-disrupted (CARD9-/- ) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild-type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9-/- macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin-1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9-/- animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9-/- animals during PCP, T-helper cell cytokines were normal in immunocompetent CARD9-/- animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.

Pneumocystis carinii Major Surface Glycoprotein Dampens Macrophage Inflammatory Responses to Fungal ß-Glucan.

BACKGROUND: Pneumocystis major surface glycoprotein (Msg) is a 120-kD surface protein complex on the organism with importance in adhesion and immune recognition. In this study, we show that Msg significantly impairs tumor necrosis factor (TNF)-α secretion by macrophages induced by Saccharomyces cerevisiae and Pneumocystis carinii (Pc) ß-glucans. METHODS: Major surface glycoprotein was shown to greatly reduce ß-glucan-induced Dectin-1 immunoreceptor tyrosine-based activating motif (ITAM) phosphorylation. Major surface glycoprotein also down regulated Dectin-1 receptor messenger ribonucleic acid (mRNA) expression in the macrophages. It is interesting that Msg incubation with macrophages resulted in significant mRNA upregulation of both C-type lectin receptors (CLR) Mincle and MCL in Msg protein presence alone but to even greater amounts in the presence of Pc ß-glucan. RESULTS: The silencing of MCL and Mincle resulted in TNF-α secretions similar to that of macrophages treated with Pneumocystis ß-glucan alone, which is suggestive of an inhibitory role for these 2 CLRs in Msg-suppressive effects on host cell immune response. CONCLUSIONS: Taken together, these data indicate that the Pneumocystis Msg surface protein complex can act to suppress host macrophage inflammatory responses to the proinflammatory ß -glucan components of the organisms.

Pneumocystis Jirovecii Pneumonia in Newly Diagnosed HIV Infection: A Challenging Case Report.

Pneumocystis jirovecii is a potentially life-threatening opportunistic pathogen particularly affecting the lungs, mainly in immunosuppressed individuals and HIV-infected patients with a low CD4 cell count. A 50-year-old man presented with a 1-week history of pleuritic chest pain and fever. He was also hypoxic with oxygen saturation of 86% on room air. Detailed clinical history revealed that he had fatigue, dyspnea, night sweats, generalized bone pain and a loss of about 10 kg in weight over the past six months without intention. Chest imaging showed diffuse bilateral infiltrates. Diagnostic bronchoscopy was performed. Transbronchial biopsy and bronchoalveolar lavage were received simultaneously. The presence of P. jirovecii was suspected in hematoxylin-eosin-stained slides, and Gomori's methenamine silver stain was used to confirm the diagnosis. A blood test revealed dyslipidemia, hypothyroidism, increased plasma levels of the gonadotropins and positive HIV antibodies with a CD4+ cell count of 48/µL. CMV co-infection was found with CMV viral load of 6738 copies/ml in plasma. Herein, we present a case with Pneumocystis jirovecii pneumonia (PCP) that led to a new diagnosis of Human immonudeficiency virus. As in our case, diagnosis of disease through the pathological examination of tissues (biopsy samples) or bodily fluids could lead to the recognition of an unrevealed HIV-infection.

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.

Transcriptomic and Proteomic Approaches to Finding Novel Diagnostic and Immunogenic Candidates in Pneumocystis.

Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and posttransplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome by comparisons to proteins encoded within the genomes of other fungi using NCBI BLAST. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form (the ascus) and the replicative form (the trophozoite [troph]) reside within the alveolar space of the host. To that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivoGsc1, encoding a ß-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis GSC-1 ectodomain immunization generated a strong antibody response that demonstrated the ability to recognize the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina Finally, mice immunized with the GSC-1 ectodomain had limited fungal burden following natural transmission of Pneumocystis using a cohousing model.IMPORTANCE The current report enhances our understanding of Pneumocystis biology in a number of ways. First, the current study provided a preliminary annotation of the Pneumocystis murina genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of Pneumocystis These findings allow better characterization of the Pneumocystis life cycle in vivo and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against Pneumocystis.

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.

Characterization of Pneumocystis murina Bgl2, an Endo-ß-1,3-Glucanase and Glucanosyltransferase.

Glucan is the major cell wall component of Pneumocystis cysts. In the current study, we have characterized Pneumocystis Bgl2 (EC 3.2.1.58), an enzyme with glucanosyltransferase and ß-1,3 endoglucanase activity in other fungi. Pneumocystis murina, Pneumocystis carinii, and Pneumocystis jirovecii bgl2 complementary DNA sequences encode proteins of 437, 447, and 408 amino acids, respectively. Recombinant P. murina Bgl2 expressed in COS-1 cells demonstrated ß-glucanase activity, as shown by degradation of the cell wall of Pneumocystis cysts. It also cleaved reduced laminaripentaose and transferred oligosaccharides, resulting in polymers of 6 and 7 glucan residues, demonstrating glucanosyltransferase activity. Surprisingly, confocal immunofluorescence analysis of P. murina-infected mouse lung sections using an antibody against recombinant Bgl2 showed that the native protein is localized primarily to the trophic form of Pneumocystis in both untreated mice and mice treated with caspofungin, an antifungal drug that inhibits ß-1,3-glucan synthase. Thus, like other fungi, Bgl2 of Pneumocystis has both endoglucanase and glucanosyltransferase activities. Given that it is expressed primarily in trophic forms, further studies are needed to better understand its role in the biology of Pneumocystis.