Search Results (1,032)

Allergic asthma is associated with a reduction in the number of regulatory T cells (Tregs). Although interleukin-27 (IL-27) has been shown to modulate Tregs potentially through the Lymphocyte-activation gene 3 (Lag3) pathway, the underlying mechanism remains incompletely defined. Objective: This study sought to determine whether IL-27 ameliorates airway inflammation in asthma by modulating Tregs in a Lag3-dependent manner. Acute asthma was induced in wild-type (WT) and Lag3 knockout (Lag3^−/−) mice through sensitization and challenge with house dust mite (HDM). A treatment group received intranasal recombinant IL-27 prior to challenges. In WT mice, IL-27 administration significantly attenuated airway inflammation, goblet cell hyperplasia, and total cell counts in bronchoalveolar lavage fluid (BALF), along with reduced levels of Th2 cytokines (IL-4, IL-5). It also upregulated T-bet (Th1) mRNA expression, downregulated GATA-3 (Th2) and RORγt (Th17) levels, and increased the proportions of CD4⁺ Foxp3⁺ Tregs, CTLA4⁺ Tregs, and Lag3⁺ Tregs in lung tissue. Conversely, in Lag3^−/− mice, the protective effects of IL-27 were completely abrogated, with no observed increases in Treg populations or suppression of Th2/Th17 immune responses. The anti-asthmatic effect of exogenous IL-27 is associated with increased Treg frequency and upregulation of inhibitory markers, with Lag3 serving as a pivotal target on Tregs. Full article

Background: Pulmonary aspergillosis remains underdiagnosed in patients with structural lung disease, as conventional methods might miss a part of cases. There are few real-world data on quantitative PCR performance outside immunocompromised populations. We evaluated the Aspergillus ELITe MGB Kit across two years of routine clinical practice at a tertiary pulmonology center. Methods: We retrospectively analyzed 492 consecutive ELITe MGB PCR tests (October 2023–September 2025) at the Marius Nasta Institute of Pneumology, Bucharest, Romania, performed on bronchoalveolar aspirate (n = 219), lavage (BAL; n = 202), and plasma (n = 65). Results were correlated with microscopy, fungal culture, imaging, and host risk factors where available. Results: Of 451 evaluable tests, 140 (31.0%) were significant, 7 (1.6%) low-level detected, and 304 (67.4%) non-significant or negative. Combined positivity was 32.6%. Aspirate outperformed BAL in positivity rate (42.8% vs. 30.8%; p ≈ 0.013); aspirate and BAL had similar median DNA loads (120 copies/mL each) but aspirate showed a longer tail toward very high loads (p < 0.001); raw median DNA loads (67 vs. 120 copies/mL) were not directly comparable between matrices due to differing matrix-specific lower limits of quantification (50 vs. 120 copies/mL, reflecting BAL dilution). Plasma positivity was 1.8%. Positivity peaked in May–June (42.5–45.5%), contrasting with the classical autumn pattern. Chronic obstructive pulmonary disease (COPD) was the dominant risk factor (OR = 3.45). PCR exclusively detected Aspergillus in the majority of cases where microscopy and culture were negative. Conclusions: The ELITe MGB kit demonstrates clinically meaningful diagnostic yield in a real-world pulmonology cohort. Aspirate consistently outperforms BAL, and the low-level detected category identifies a borderline population warranting prospective validation. The unexpected spring–summer positivity peak suggests a greater role for post-viral aspergillosis than previously recognized in this setting. Full article

Background/Objectives: This study is the first to investigate the potential mechanism of star anise extract (SAE) in protecting against cold stress-aggravated acute lung injury (CSALI) in rats. Methods: A rat CSALI model was induced via combined lipopolysaccharide challenge and cold stress exposure. The preventive effects of SAE were evaluated using cytotoxicity assays, quantification of biochemical indices and inflammatory factors, and histopathological examination. Ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC–HRMS)-based serum metabolomics was employed to systematically profile CSALI-associated metabolic alterations and decipher the potential mechanism underlying the preventive effects of SAE. Results: SAE alleviated pathological progression of CSALI, suppressed inflammatory cell migration, markedly reduced pulmonary inflammatory cell infiltration, and ameliorated lung tissue injury in CSALI rats. SAE also improved abnormal liver function indicators and lowered the levels of pro-inflammatory factors in both serum and bronchoalveolar lavage fluid (BALF). Serum metabolomics analysis identified and annotated 24 disease-altered differential metabolites and evaluated the protective effects of SAE on them. These metabolites were significantly enriched in two key metabolic pathways related to the pathogenesis of CSALI, including arachidonic acid metabolism and glycerophospholipid metabolism. Furthermore, based on metabolite changes, phospholipase A2 was hypothesized as a potential key regulatory factor that may cooperate with arachidonic metabolism to suppress the inflammatory cascade. Conclusions: These findings demonstrated that SAE exerted prominent anti-inflammatory activity and effectively protected against lung injury in CSALI rats. Full article

Background: Rare fungal infections may represent under-recognized causes of healthcare-associated sepsis, particularly when caused by emerging or difficult-to-identify pathogens. We aimed to characterize Candida viswanathii isolates recovered in the setting of panel-negative candidemia and to assess the contribution of an integrated diagnostic workflow. Methods: We investigated seven C. viswanathii isolates overall, including three recovered at our institution from blood, urine, and bronchoalveolar lavage of a NICU patient, as well as four bloodstream isolates from a second pediatric center included for comparison. Isolates were analyzed by culture and microscopy, three MALDI-TOF MS platforms, internal transcribed spacer sequencing, Fourier transform infrared (FTIR) spectroscopy and antifungal susceptibility testing. Results: C. viswanathii was repeatedly recovered from blood, urine and bronchoalveolar lavage, while the FilmArray BCID2 panel remained negative. All MALDI-TOF MS systems with updated databases correctly identified the yeast at the species level; identification was confirmed by sequencing. Fourier transform infrared analysis showed clustering of clinical isolates and clearly separated C. viswanathii from related Candida species. All isolates exhibited low MICs to echinocandins and amphotericin B as well as moderately elevated fluconazole MICs (2–4 mg/L). Conclusion: This study supports the use of explicit diagnostic algorithms for rare fungal pathogens in yeast-positive, syndromic panel-negative blood cultures. In this setting, updated MALDI-TOF MS libraries and FTIR spectroscopy may provide useful adjunctive support for the recognition and phenotypic discrimination of atypical yeasts within an integrated laboratory workflow. Full article

Daptomycin is widely used for serious methicillin-resistant Staphylococcus aureus (MRSA) infections, but it can cause eosinophilic pneumonia, an uncommon and potentially severe adverse drug reaction. We report probable daptomycin-induced eosinophilic pneumonia (DIEP) in a 77-year-old woman receiving intravenous daptomycin with rifampin for a postoperative MRSA foot infection with concern for orthopedic-device involvement and osteomyelitis. Daptomycin had been initiated 10 days before admission, when she developed acute hypoxemic respiratory failure with bilateral ground-glass and consolidative opacities on chest computed tomography and a negative evaluation for infection. Daptomycin was discontinued on the day of admission, yet respiratory failure progressed, requiring mechanical ventilation. Bronchoalveolar lavage performed after corticosteroid exposure demonstrated eosinophilia (9%), and she improved with systemic corticosteroids and supportive care. Although she did not meet strict FDA “most likely/definite” criteria, the temporal association, compatible imaging, exclusion of alternative etiologies, abnormal lavage eosinophilia, and clinical response following drug withdrawal support “probable” DIEP criteria. Clinicians managing complex foot infections should consider DIEP when new respiratory symptoms and infiltrates develop during daptomycin therapy, even early in the treatment course and even when peripheral eosinophilia is absent or minimal. Full article

Air pollutants can cause respiratory diseases, highlighting the need for effective preventive and therapeutic strategies. We investigated the protective effects of heat-killed Lacticaseibacillus paracasei ATG-E1 against particulate matter plus diesel exhaust particle (PM₁₀D)-induced airway inflammation. BALB/c mice were intranasally injected with PM₁₀D and treated with heat-killed L. paracasei ATG-E1 via oral gavage for 5 days. In the bronchoalveolar lavage fluid (BALF) and lungs, inflammatory mediators, immune cell subtypes, and histological changes were analyzed, while gut microbiota composition was analyzed in the cecum. Heat-killed L. paracasei ATG-E1 suppressed the infiltration of immune cells, including neutrophils, T cells, and B cells. Furthermore, it decreased various inflammatory mediators, such as C-X-C Motif chemokine ligand (CXCL)-1, macrophage inflammatory protein (MIP)-2, interleukin (IL)-1α, and tumor necrosis factor (TNF)-α, in the BALF and lung tissue, as well as serum symmetric dimethylarginine (SDMA) levels in the PM₁₀D-induced airway inflammation model. Heat-killed L. paracasei ATG-E1 also exhibited a protective effect against lung damage induced by PM₁₀D. Furthermore, heat-killed L. paracasei ATG-E1 treatment shifted the gut microbiota composition, increasing several bacterial genera. The data demonstrate that heat-killed L. paracasei ATG-E1 acts as a protective agent against air pollutant-induced lung injury, suggesting its potential as a candidate adjunctive strategy for prevention. Full article

Background/Objectives: Bulk RNA sequencing (RNA-seq) averages signals across heterogeneous cell populations. Computational deconvolution methods aim to infer cell type composition and cell type-specific gene expression from bulk data, but their performance in equine samples has not been evaluated. In this study, we assessed the ability of computational deconvolution to recover cellular composition and differential expression signals in bronchoalveolar lavage fluid (BALF) from horses with severe equine asthma (SEA) and controls (CTL). Methods: Cryopreserved BALF samples from six SEA and five CTL horses previously analyzed by scRNA-seq were used to generate bulk RNA-seq data. The matched scRNA-seq dataset served as the reference for deconvolution. Performance was evaluated by comparing deconvolution raw and mRNA-corrected estimates with scRNA-seq cell proportions. Differential expression between SEA and CTL was analyzed on bulk RNA-seq, deconvoluted expression profiles, and scRNA-seq pseudobulk data. Results: Deconvolution primarily captured mRNA-derived cell type proportions rather than true cell counts: agreement with scRNA-seq cell counts was moderate (r = 0.62; 95% CI 0.45–0.75) but improved after mRNA content correction (r = 0.83; 95% CI 0.74–0.89). Comparison with mRNA-weighted scRNA-seq proportions showed near-perfect concordance (r = 0.98; 95% CI 0.97–0.99). Cell type-specific performance varied, with stronger correlations for B cells and dendritic cells and weaker performance for neutrophils, T cells and monocytes/macrophages. Recovery of cell type-specific differential expression was inconsistent, frequently showing cross-lineage signal spillover. Although both approaches detected a Th17 signature in SEA, most deconvolution-derived differentially expressed genes overlapped with conventional bulk RNA-seq results. Conclusions: Deconvolution of bulk RNA-seq did not reliably estimate cell counts or provide substantial biological insight beyond conventional bulk analysis, highlighting the value of scRNA-seq for resolving cell type-specific disease mechanisms in equine asthma. Full article

Interleukin-22 binding protein (IL-22BP) is a soluble decoy receptor that competitively inhibits IL-22 by preventing its interaction with the IL-22 receptor. Although the IL-22 receptor is primarily expressed on non-hematopoietic cells, such as airway epithelial cells, the role of IL-22BP in the pathogenesis of asthma remains uncertain. We observed that IL-22BP was upregulated in the airways of wild-type (WT) mice intranasally sensitized and challenged with house dust mite (HDM) extract. To directly elucidate the function of IL-22BP in allergic airway responses, IL-22BP-deficient (IL-22BP−/−) and WT mice were sensitized and challenged with HDM, and airway responses were systematically assessed. IL-22BP−/− mice exhibited significantly lower airway hyperresponsiveness (AHR) compared to WT mice following sensitization and challenge with HDM. In contrast, eosinophil counts in bronchoalveolar lavage (BAL) fluid did not differ significantly between the two groups. Similarly, levels of interleukin (IL)-4, IL-5, IL-6, IL-13, IL-17A, and keratinocyte chemoattractant (KC) in BAL fluid were comparable between WT and IL-22BP−/− mice. Notably, IL-22 levels in lung homogenates were significantly higher in IL-22BP−/− mice than in WT mice after sensitization and challenge with HDM. These findings suggest that inhibition of IL-22BP attenuates the development of allergen-induced AHR, an effect likely mediated through enhanced IL-22 activity rather than alterations in airway inflammation or type 2 cytokine production. Full article

Background: Diphtheria is an acute infectious disease caused by Corynebacterium diphtheriae. Despite several worldwide outbreaks, it is now considered a rare disease by industrialized countries. Clinical manifestations usually account for oropharyngeal lesions, but rare cases of systemic involvement (mainly endocarditis) have been described among non-toxigenic strains. Case description: We report the case of a patient who experienced septic shock, disseminated intravascular coagulation and multiorgan failure due to Corynebacterium diphtheriae infection. The pathogen was further characterized as a highly toxigenic strain. Infective endocarditis with mitral and aortic valve vegetations led to early multiorgan septic embolization. Major stroke, liver function impairment, heart failure and acute kidney injury were the main findings. Unlike the typical forms of infection caused by this pathogen, there was no evidence of airway or skin involvement. Furthermore, apart from hemocultures, none of the other investigations (pharyngeal swabs, bronchoalveolar lavages, urine culture) ever tested positive for the bacteria. Conclusions: The report we present describes a case of C. diphtheriae infection with many atypical characteristics: (i) lack of any pathognomonic signs or symptoms; (ii) extensive endocarditic process (very uncommon for toxigenic strains); (iii) early septic emboli development, with rapid evolution to multiorgan failure; (iv) detection of disseminated intravascular coagulation. Despite disseminated intravascular coagulation being a known complication of septic shock, regardless of the etiological agent, according to our literature research, this is the second known case driven by C. diphtheriae infection in an adult. Full article

The use of inhaled corticosteroids (ICs) could influence the respiratory microbiota. In animals with asthma it is, however, difficult to separate the immunomodulatory effects of ICs from their indirect effects via improvement of ventilation. Our objective was to determine if ICs alter the pulmonary microbiota independently from their effects on lung function, using a blinded, controlled trial in an experimental model of asthma exacerbation in horses. We treated horses with severe asthma with either bronchodilators alone, or in combination with ICs. Twelve horses in exacerbation received long-acting β2-agonist (LABA, salmeterol) or ICs/LABA (fluticasone/salmeterol) by inhalation, for 2 weeks. Lung function and bronchoalveolar lavages (BAL) were performed before and after treatment. 16S rRNA gene quantification and sequencing were performed on BAL fluid, using digital droplet PCR and the Illumina MiSeq platform. Data were processed using the software package mothur v. 1.44.2. In the LABA group, pulmonary bacterial load and the relative abundance of Actinobacteria and Verrucomicrobia phyla decreased with treatment (p < 0.05 for both), and β-diversity differed from baseline (p = 0.007). The relative abundance of families and genera belonging to the Bacteroidetes phylum increased with ICs/LABA (p < 0.05). Lung function significantly improved with both treatments, suggesting that treatment-related differences in pulmonary microbiota could be attributed in part to medication, not solely to change in ventilation. However, it is not clear if these changes are positive or detrimental to the lung environment. Furthermore, lung function following treatment was not perfectly identical between groups. Full article

Objectives: Inflammatory bowel disease (IBD) is associated with extraintestinal comorbidities, and lung diseases are widespread manifestations. Respiratory bacterial insult is a common illness that results in acute lung injury (ALI) in critical patients. IBD concurrence with respiratory infection may further exacerbate lung injury. Tryptophan (Try), an essential amino acid, is processed by gut microbiota and produces aryl hydrocarbon receptor (AhR) ligands. These ligands can activate the AhR pathway that exerts anti-inflammatory properties and provides protection against mucosal barrier injury. This study investigated the effects of dietary Try on lipopolysaccharide (LPS)-stimulated ALI in mice with colitis induced by dextran sodium sulfate (DSS). Methods: Mice with colitis were allocated to four groups: (1) ND-Sal: normal diet + DSS + intratracheal saline injection; (2) ND-LPS: normal diet + DSS + intratracheal LPS injection; (3) TD-Sal: Try diet + DSS + intratracheal saline injection; (4) TD-LPS: Try diet + DSS + intratracheal LPS injection. Mice were sacrificed 24 h after the intratracheal injection. Results: Results showed that colitis resulted in a high disease activity index. Following induction of ALI in colitis mice, neutrophil populations and inflammatory cytokine levels in bronchoalveolar lavage fluid increased. Gene expression levels associated with toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling were upregulated, and tight junction proteins decreased in the lungs. Dietary Try supplementation decreased circulating LPS levels, suppressed pulmonary TLR4/NF-κB signaling, upregulated AhR/interleukin-22 expression, attenuated oxidative stress and improved the capillary–epithelial barrier integrity in DSS-treated mice. Conclusions: These findings imply that Try may have potential therapeutic significance in bacterial-induced ALI in a colitis condition. Full article

Background: Solriamfetol, a dopamine and norepinephrine reuptake inhibitor widely used in narcolepsy management, has not been thoroughly investigated for its anti-fibrotic and anti-inflammatory properties. Herein, we investigated its potential therapeutic applications and underlying mechanisms in both cellular and murine models of pulmonary fibrosis. Methods: To induce fibrosis, C57BL/6 male mice (six-week-old) were administered bleomycin via the intratracheal route. These animals subsequently received solriamfetol orally once per day at dosages of 3 or 10 mg/kg. Histological and immunohistochemical techniques were employed to evaluate inflammatory cell infiltration, collagen accumulation, and α-smooth muscle actin (α-SMA) expression in bronchoalveolar lavage samples and lung tissue sections. Cytokine levels were measured by ELISA, and gene/protein expression of pro-fibrotic markers, A_2A/A_2B adenosine receptors (ARs), adenylate cyclases (ACs), Epac, K_Ca3.1, and adenosine deaminase (ADA) were assessed via quantitative PCR and Western blot. Electrophysiological recordings evaluated K_Ca3.1 channel activity. Purified ADA and normal human lung fibroblasts (NHLFs) were treated with solriamfetol to assess effects on ADA activity and levels of cAMP and adenosine, respectively. Results: Solriamfetol significantly reduced inflammatory cell infiltration, collagen accumulation, and α-SMA expression in fibrotic lungs. Solriamfetol restored downregulated A_2AAR, A_2BAR, ACs, and Epac, while suppressing ADA expression and activity, resulting in elevated extracellular adenosine and intracellular cAMP. The intervention potentiated Epac signaling and inhibited fibroblast activation. Solriamfetol inhibited the K_Ca3.1 current in fibroblasts and reduced K_Ca3.1 protein expression levels in TGFβ-treated fibroblasts and lung tissues from bleomycin-challenged mice. Notably, these effects were abolished by A_2AAR or A_2BAR antagonists, implying that they occur through AR-mediated pathways. Conclusions: Solriamfetol inhibits ADA and reinforces adenosine–cAMP signaling, suppressing pathological fibroblast activation. These findings suggest its therapeutic utility as a novel anti-fibrotic compound for various fibrotic diseases, including pulmonary fibrosis. Full article

Background: Systemic autoimmune rheumatic diseases-associated interstitial lung disease (SARD-ILD) presents with varied disease courses, emphasizing the need for reliable predictors of progression. The prognostic utility of bronchoalveolar lavage (BAL) in SARD-ILD remains underexplored. The objective of this study was to evaluate the role of BAL fluid lymphocyte count in predicting disease progression in patients with SARD-ILD. Methods: This observational study included patients with SARD-ILD undergoing BAL as part of their diagnostic workup. Disease progression was defined as either Forced vital capacity (FVC) decrease >10%, two out of the following three criteria within two years: FVC decrease of 5–10%, worsening symptoms, increased fibrosis on imaging, or any of the following: escalation of treatment, Interstitial lung disease (ILD) exacerbation, lung transplantation, or disease-specific mortality. Logistic regression identified predictors of progression. Time-to-progression was assessed using Kaplan–Meier survival curves. The optimal BAL lymphocyte threshold for predicting progression was identified using the Youden Index and the Wilcoxon method. Results: We identified 89 patients, of whom 30 (33.7%) had progressive disease. Progressors had a significantly higher BAL lymphocyte count compared to non-progressors (31.6 ± 24.8% vs. 14.3 ± 16.5%, p < 0.001). BAL lymphocyte proportion was significantly and independently associated with disease progression (odds ratio, 1.05; 95% confidence interval 1.02–1.07; p < 0.01). A lymphocyte count above 9 percent was associated with a markedly increased risk of disease progression (odds ratio, 13.14; 95% confidence interval, 4.20–51.98; p < 0.01). Conclusions: BAL lymphocyte count was associated with a higher likelihood of progression in SARD-ILD. BAL assessment may help identify patients at increased risk of disease progression. However, these findings should be considered exploratory and require validation in larger prospective studies and across individual SARD-ILD subtypes. Full article

Background: Flexible bronchoscopy (FB) has long been integral to pulmonology, but its bedside role in the intensive care unit (ICU) is expanding. Despite a lack of high-level evidence, FB remains a pivotal tool for airway visualization, sampling, and selected interventions in critically ill patients. Objective: This meta-narrative review critically appraises the clinical use, evolving indications, safety profile, and emerging controversies of FB in ICU settings, particularly regarding the role of non-pulmonologist intensivists. Methods: A structured literature search was conducted using PubMed, Scopus, and Google Scholar for studies published in the past 15 years. Emphasis was placed on observational studies, meta-analyses, and guidelines relevant to FB in ICU patients. Key controversies were grouped under thematic questions based on clinical relevance. Results: A total of 84 articles were retrieved, of which 47 met the predefined inclusion criteria. Seven key thematic domains were synthesized regarding the use of flexible bronchoscopy (FB) in the intensive care unit (ICU) setting. FB performed by trained intensivists was found to be safe and diagnostically effective across a range of ICU populations, including elderly and non-intubated patients. Although procedure-related hypoxemia was reported, it was largely manageable with appropriate precautions. FB demonstrated critical utility in the management of acute respiratory failure (ARF), acute respiratory distress syndrome (ARDS), and sepsis, particularly through bronchoalveolar lavage (BAL), airway secretion clearance, and, selectively, bronchoscopic lung biopsy. The adoption of disposable bronchoscopes may reduce infection risk and economic burden. Furthermore, the integration of advanced techniques such as endobronchial ultrasound (EBUS) and transbronchial cryobiopsy is emerging, although application in the critical care environment remains cautious and selective. Conclusions: With structured training and careful patient selection, FB is an adaptable and often underutilized tool in ICU medicine. Multidisciplinary competency development and institutional protocols can enhance its safe integration. Full article

Pneumocystis is an opportunistic fungal pathogen that causes severe Pneumocystis pneumonia (PCP) in immunocompromised individuals and laboratory animals. Three host-specific species—Pneumocystis murina (P. murina), Pneumocystis carinii (P. carinii), and Pneumocystis jirovecii (P. jirovecii)—are closely associated with infections in humans and laboratory animals. However, the conventional method, microscopic staining, suffers from low sensitivity, operator-dependent subjectivity, and inability to differentiate species, highlighting the urgent need for a multiplex qPCR assay. In this study, we established a multiplex qPCR method targeting the mtLSUrRNA gene of P. murina, the TS gene of P. carinii, and the mtSSUrRNA gene of P. jirovecii. Primers and probes were designed and optimized using a matrix approach. The method was systematically evaluated for sensitivity, specificity, and reproducibility using recombinant plasmid standards and laboratory animal samples. Validation was performed on 260 mouse lung samples, 30 P. murina-positive samples, 25 rat lung samples, 6 rat bronchoalveolar lavage fluid (BALF) samples, and 8 P. carinii-positive samples. Results were compared with single-plex qPCR and staining microscopy (performed on 68 mouse lung samples, 38 Pneumocystis-positive samples). The limits of detection (LOD) were 5 copies/μL for P. murina, 6 copies/μL for P. carinii, and 8 copies/μL for P. jirovecii. Standard curves showed excellent linearity (R² ≥ 0.999) with amplification efficiencies of 90–110%. No non-specific reactions were observed with 22 common pathogens, and intra-/inter-group coefficients of variation (CV%) were below 1%. Moreover, interference testing revealed minimal matrix effects on the amplification system and no mutual interference among the primers and probes. The multiplex qPCR detected all 38 positive samples (100%), showing 100% concordance with single-plex qPCR, whereas Giemsa staining detected none (0%) and toluidine blue staining only 60% (3/5) of the tested positives, suggesting that the multiplex qPCR achieved higher detection rates than staining microscopy. In conclusion, this novel multiplex qPCR method offers high sensitivity, specificity, and reproducibility, providing a sensitive and specific tool for laboratory animal health monitoring and epidemiological surveillance. Its clinical application for human PCP diagnosis requires further validation with authentic human specimens. Full article