Description of a Murine Model of Pneumocystis Pneumonia.

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The onset of clinical signs occurs within 5 weeks following the infectious challenge and immunosuppression can then be withdrawn.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.

IFN-γ Limits Immunopathogenesis but Delays Fungal Clearance during Pneumocystis Pneumonia.

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.

Preclinical and Toxicology Studies of BRD5529, a Selective Inhibitor of CARD9.

BACKGROUND: The caspase recruitment domain-containing protein 9 (CARD9) inhibitor BRD5529 has been shown to be an effective in vitro inhibitor of Pneumocystis ß-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 ß-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.

[Not all cases of groundglas opacity are COVID-19 - Pneumocystis-jirovecii-pneumonia as a differential diagnosis]. / Nicht alles Milchglas ist COVID-19 ­ Pneumocystis-jirovecii-Pneumonie als Differenzialdiagnose.

HISTORY AND CLINICAL FINDINGS: A 68-year-old male patient with psorias and a bullous pemphigoid as an underlying disease developed bilateral groundglass opacities on chest CT under longer-term, higher-dose immunosuppressive therapy with methylprednisolone with clinical symptoms of dry cough, progressive dyspnea and fever. DIAGNOSIS AND THERAPY: After the exclusion of COVID-19, Pneumocystis jirovecii pneumonia (PCP) was detected and a corresponding high-dose therapy with trimethoprim-sulfamethoxazole was initiated promptly. COURSE: Nonetheless, a complicated course with bacterial superinfection and pulmonary aspergillosis as well as ARDS developed. DISCUSSION AND CONCLUSION: In contrast to COVID-19, the typical course, diagnosis and therapy of Pneumocystitis jirovecii pneumonia are discussed. It is particularly emphasized that not all ground glass infiltrates in the CT chest image can be traced back to a COVID-19, even in a pandemic situation. Possible differential diagnoses should always be considered and taken into account in the diagnosis.

Co-infection of Pneumocystis jirovecii pneumonia and pulmonary CMV in a infant with X-linked severe combined immunodeficiency.

We herein report a rare case of co-infection of Pneumocystis jirovecii pneumonia and pulmonary CMV in a 3-month-old infant with X-linked severe combined immunodeficiency, in which diagnostic clues were obtained from the bronchoalveolar lavage fluid. We focus on the value of cytological diagnosis of P. jirovecii pneumonia and pulmonary CMV in the bronchoalveolar lavage fluid. Recognizing morphological characteristics of these pathogenic microorganisms is important to get timely diagnosis and treatment for the patients. Furthermore, repeated severe infections in infants should remind us to screen for immunosuppressed states.

Clinical descriptive analysis of severe Pneumocystis jirovecii pneumonia in renal transplantation recipients.

Pneumocystis jirovecii (P. jirovecii) pneumonia (PJP) is an opportunistic fungal infection after renal transplantation, which is always severe, difficult to diagnose, combined with multiple complications and have poor prognosis. We retrospectively analyzed clinical data, including risk factors, diagnosis, treatment and complications of seven clinical cases suffered with severe PJP after renal transplantation in our department in 2019. All the seven recipients were routinely prescribed with PJP prophylaxis after renal transplantation, and six of them suffered acute graft rejection before the infection. P. jirovecii sequence was identified in blood or broncho-alveolar lavage fluid (BALF) by the metagenomic next-generation sequencing (mNGS) in all patients. All the patients were improved with the therapy trimethoprim-sulfamethoxazole (TMP-SMX) combined with caspofungin for the PJP treatment, but suffered with complications including renal insufficiency, leukopenia, thrombocytopenia, gastrointestinal bleeding, mediastinalemphysema, pulmonary hemorrhage, and hemophagocytic syndrome and other severe infections. Taken together, mNGS is a powerful tool that could be used to diagnose PJP in renal transplantation recipients. And PJP prophylaxis should be prescribed during and after treatment for acute rejection. TMP-SMX is the first-line and effective drug for PJP treatment, but the complications are always life-threatening and lead to poor prognosis. We should pay attention to these life-threatening complications.

COVID-19 and Pneumocystis jirovecii Pulmonary Coinfection-The First Case Confirmed through Autopsy.

Background: Establishing the diagnosis of COVID-19 and Pneumocystisjirovecii pulmonary coinfection is difficult due to clinical and radiological similarities that exist between the two disorders. For the moment, fungal coinfections are underestimated in COVID-19 patients. Case presentation: We report the case of a 52-year-old male patient, who presented to the emergency department for severe dyspnea and died 17 h later. The RT-PCR test performed at his admission was negative for SARS-CoV-2. Retesting of lung fragments collected during autopsy revealed a positive result for SARS-CoV-2. Histopathological examination showed preexisting lesions, due to comorbidities, as well as recent lesions: massive lung thromboses, alveolar exudate rich in foam cells, suprapleural and intra-alveolar Pneumocystisjirovecii cystic forms, and bilateral adrenal hemorrhage. Conclusion: COVID-19 and P.jirovecii coinfection should be considered, particularly in critically ill patients, and we recommend the systematic search for P. jirovecii in respiratory samples.

Aerodigestive adverse effects during intravenous pentamidine infusion for Pneumocystis jirovecii pneumonia prophylaxis.

Aerodigestive adverse effects (AD-AE) during intravenous pentamidine (IV-P) infusion for Pneumocystis jiroveci pneumonia prophylaxis are uncommon in retrospective chart review studies. We conducted a survey in patients on IV-P, which included 31 specific questions. Twenty-five patients were included in the analysis; AD-AE were observed in 22 (88%) with recurrence of symptoms in 88% participants with subsequent infusions. Five leading symptoms were congestion (48%), lip tingling (32%), nausea (28%), tongue tingling (24%), vomiting, and throat swelling (17%); multiple symptoms were reported in 72% of the patients. In conclusion, AD-AE of IV-P infusion are common, self-limited, and tend to be recurrent.

An Usual Presentation of Pneumocystis jirovecii Pneumonia in a Woman Treated With Immune Checkpoint Inhibitor.

We describe a case of Pneumocystis jirovecii pneumonia in an 18-year-old female individual with refractory primary mediastinal B-cell lymphoma treated with the immune checkpoint inhibitor pembrolizumab. She received 11 doses of pembrolizumab without immune-related adverse events (irAEs) before the diagnosis of P. jirovecii pneumonia. However, prophylactic trimethoprim/sulfamethoxazole was discontinued 6 months of postautologous stem cell transplant per standard guidelines. This case report highlights the importance of judicious infectious disease evaluation while on immune checkpoint inhibitor therapy as symptoms can often mimic irAEs. Furthermore, the benefits of immunosuppressive therapy for the treatment of presumptive irAEs must be weighed against the possible increased risk for opportunistic infections.

The risk factor analysis and treatment experience in pneumocystis jirovecii pneumonia after kidney transplantation.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) is an opportunistic infection among solid organ transplantation. The occurrence of PJP is dangerous and fatal if there is no early identification and sufficient treatment. OBJECTIVE: The aim of this study was to evaluate the risk factors and provide appropriate strategies of prophylaxis and treatment for PJP after kidney transplantation in our centre. PATIENTS/METHODS: From January 2009 to December 2018, a total of 167 kidney transplantation recipients with pneumonia were enrolled, including 47 PJP patients as PJP group and 120 non-PJP patients as control group. The clinical characteristics of the two groups were analysed retrospectively. RESULTS: Multivariate analysis showed that high total dosage of ATG [OR, 2.03; 95% CI, 1.12-3.68] and cytomegalovirus (CMV) infection were independent risk factors for PJP. Trimethoprim-sulfamethoxazole (TMP-SMX) (1.44 g q6h)-based treatment was used for 2 weeks, and its dosage and course were adjusted according to the therapeutic effect and side effects. Forty-five cases were recovered after 3 months of follow-up, and two patients died of respiratory failure. TMP-SMX (0.48 g/day) prophylaxis was used for 3-6 months and prolonged to 7-8 months after treatment for acute rejection, which reduced the incidence of PJP compared with those without prophylaxis. CONCLUSION: Our study suggests that the high total dosage of ATG and CMV infection indicate the increased risk of PJP. The strategies of prophylaxis and treatment for PJP after kidney transplantation in our centre were effective.

Of "Cotton Balls" and "Owl's eyes".

Report of a 3-month old girl child who died due to multi-systemic infection of cytomegalovirus (CMV) involving the lungs, liver and kidneys along with pneumocystis jiroveci pneumonia (PJP). The mother of the child tested positive for CMV IgG and HIV with a very low CD4 count (160/ µl). Co-infection of cytomegalovirus and pneumocystis jiroveci always occurs in the setting of immunocompromise. Congenital CMV infection is transmitted through the placenta, especially during the first trimester and causes severe multi-systemic disease whereas perinatal infection is acquired during childbirth/ breastfeeding where the babies have maternal protective antibodies leading to much milder or asymptomatic infection. PJP is more common in infancy and presents as hypoxic pneumonia. CMV causes cyto-nucleomegaly and classic "owl's eye" inclusions on histology while PJP presents with characteristic fluffy "cotton ball" alveolar exudates.

Pneumocystis pneumonia, a COVID-19 mimic, reminds us of the importance of HIV testing in COVID-19.

While clinical environments are highly focused on COVID-19, reports of missed or delayed treatment for conditions that imitate COVID-19, such as pneumonia caused by the fungus Pneumocystis jirovecii, are emerging. Given the uncertain spectrum of COVID-19 presentations and variable sensitivity of laboratory tests for SARS-CoV-2, there is a risk that, without a high index of suspicion, alternative aetiologies may be overlooked while pursuing a diagnosis of COVID-19. The British HIV Association has been calling for the inclusion of HIV testing in all patients admitted to hospital with suspected COVID-19. In this article we reflect on the importance of including HIV testing to prevent avoidable morbidity and mortality in our patients.

Differences and similarities of high-resolution computed tomography features between pneumocystis pneumonia and cytomegalovirus pneumonia in AIDS patients.

BACKGROUND: Accurately differentiating pneumocystis from cytomegalovirus pneumonia is crucial for correct therapy selection in AIDS patients. Hence, the goal of this study was to compare the computerized tomography (CT) features of pneumocystis pneumonia and cytomegalovirus pneumonia in AIDS patients and identify clinical hallmarks to accurately distinguish these two pathologies. METHODS: A total of 112 AIDS patients (78 with pneumocystis pneumonia and 34 cytomegalovirus pneumonia) at Beijing Ditan Hospital from January 2017 to May 2019 were included in this study. Two experienced chest radiologists retrospectively reviewed CT images for 17 features including ground-glass opacity, consolidation, nodules, and halo sign. Binary logistic regression analyses were conducted to identify the significant parameters that distinguished pneumocystis pneumonia from cytomegalovirus pneumonia. Correlations were analyzed by Pearson or Spearman correlation analyses. Result were considered significant if P < 0.05. RESULTS: The presence of consolidation, halo signs, and nodules (all P < 0.05) were significantly more frequent in patients with cytomegalovirus pneumonia than in those with pneumocystis pneumonia. Small nodules (32.5% in cytomegalovirus pneumonia, 6.41% in pneumocystis pneumonia, P < 0.001) without perilymphatic distribution were particularly common in patients with cytomegalovirus pneumonia. Large nodules were not found in any of patients with cytomegalovirus pneumonia. The presence of ground-glass opacity, reticulation, and bronchial wall thickening (all P > 0.05) were common in both groups. CONCLUSIONS: Analysis of consolidation, nodules, and halo signs may contribute to the differential diagnosis of pneumocystis pneumonia or cytomegalovirus pneumonia. However, some CT features considered typical in one or other diseases appear with similar frequency in both cohorts of AIDS patients. CT features are potentially useful for the differential diagnosis of pneumocystis pneumonia and cytomegalovirus pneumonia in AIDS patients.

Radiographic features in investigated for Pneumocystis jirovecii pneumonia: a nested case-control study.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) can be challenging to diagnose, often requiring bronchoscopy. Since most patients suspected of PJP undergo imaging, we hypothesized that the findings of these studies could help estimate the probability of disease prior to invasive testing. METHODS: We created a cohort of patients who underwent bronchoscopy specifically to diagnose PJP and conducted a nested case-control study to compare the radiographic features between patients with (n = 72) and without (n = 288) pathologically proven PJP. We used multivariable logistic regression to identify radiographic features independently associated with PJP. RESULTS: Chest x-ray findings poorly predicted the diagnosis of PJP. However, multivariable analysis of CT scan findings found that "increased interstitial markings" (OR 4.3; 95%CI 2.2-8.2), "ground glass opacities" (OR 3.3; 95%CI 1.2-9.1) and the radiologist's impression of PJP being "possible" (OR 2.0; 95%CI 1.0-4.1) or "likely" (OR 9.3; 95%CI 3.4-25.3) were independently associated with the final diagnosis (c-statistic 0.75). CONCLUSIONS: Where there is clinical suspicion of PJP, the use of CT scan can help determine the probability of PJP. Identifying patients at low risk of PJP may enable better use of non-invasive testing to avoid bronchoscopy while higher probability patients could be prioritized.

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.