Bronchial aspirate obtained during bronchoscopy yields increased fungal load compared to bronchoalveolar lavage fluid in patients at risk of invasive aspergillosis and Pneumocystis pneumonia.

Bronchoalveolar lavage fluid (BALF) is a standard respiratory sample for diagnosing invasive fungal diseases like Pneumocystis pneumonia (PCP) and invasive pulmonary aspergillosis (IPA). However, procedural variations exist across medical centers and wards. This study aimed to compare the diagnostic potential of BALF and bronchial aspirate (BA) obtained during bronchoscopy in 173 patients suspected of fungal infections. A prospective observational study was conducted from April 2020 to November 2021. BALF and BA were collected during bronchoscopy and subjected to direct examination, fungal culture, Aspergillus fumigatus qPCR (AfqPCR), and Pneumocystis jirovecii qPCR (PjqPCR). Galactomannan detection was performed on BALF. Patients were classified based on established European Organization for Research and Treatment of Cancer (EORTC) criteria. Out of 173 patients, 75 tested positive for at least one test in BA or BALF. For Aspergillus, proportion of positive AfqPCR (14.5% vs. 9.2%; P < 0.0001) and fungal loads (Cq of 31.3 vs. 32.8; P = 0.0018) were significantly higher in BA compared to BALF. For Pneumocystis, fungal loads by PjqPCR was also higher in BA compared to BALF (Cq of 34.2 vs. 35.7; P = 0.003). BA only detected A. fumigatus and P. jirovecii in 12 (42.9%) and 8 (19.5%) patients, respectively. BA obtained during a BAL procedure can be a suitable sample type for increased detection of P. jirovecii and A. fumigatus by qPCR. The use of BA in diagnostic algorithms requires further investigation in prospective studies.

Bronchoalveolar lavage fluid (BALF) vs. bronchial aspirate (BA) for fungal diagnosis in 173 patients suspected of invasive fungal infection: BA showed higher fungal loads than in BALF by qPCR for the detection of Aspergillus fumigatus and Pneumocystis jirovecii.

Oculosystemic pneumocystosis in 2 sibling Chihuahuas.

Sibling female and male Chihuahuas were evaluated for a 9-month history of tachypnea that failed to respond to fenbendazole, doxycycline, amoxicillin-clavulanate, and prednisone. Physical examination identified tachypnea, hyperpnea, and harsh bronchovesicular lung sounds. Fundic examination disclosed diffuse chorioretinitis, manifested as multifocal chorioretinal granulomas in the female dog and occasional chorioretinal scars in the male dog. Thoracic radiographs indicated moderate to severe interstitial to broncho-interstitial infiltrates in both dogs. Serum and urine antigen and antibody testing in the female dog failed to identify infectious agents, but cytologic assessment of hepatic lymph node, liver, and splenic aspirates identified Pneumocystis trophozoites. Infection was confirmed in both dogs by 28S rRNA PCR sequencing from multiple tissue samples. The female dog responded well to trimethoprim-sulfamethoxazole, but the male dog was euthanized because of liver failure, presumably related to antimicrobial treatment.

Evaluation of the PneumoGenius® PCR assay for the diagnosis of Pneumocystis pneumonia and the detection of Pneumocystis dihydropteroate synthase mutations in respiratory samples.

Pneumocystis pneumonia (PCP) is the most frequent fungal opportunistic infection defining AIDS in HIV-infected patients, and is of growing importance in HIV-negative patients. In this latter category of patients, the diagnosis mainly relies on real-time polymerase chain reaction (qPCR) detection of Pneumocystis jirovecii (Pj) on respiratory samples. The PneumoGenius® kit (PathoNostics) allows the simultaneous detection of Pj mitochondrial large subunit (mtLSU) and dihydropteroate synthase (DHPS) polymorphisms, which could be of interest to anticipate therapeutic failure. This study aimed at evaluating its clinical performance on 251 respiratory specimens (239 patients), (i) for P. jirovecii detection in clinical samples, and (ii) for DHPS polymorphisms detection in circulating strains. Patients were classified according to modified European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, as having proven PCP (n = 62), probable PCP (n = 87), Pneumocystis colonization (n = 37), and no PCP (n = 53). Compared with in-house qPCR, the sensitivity of PneumoGenius® assay for P. jirovecii detection reached 91.9% (182/198), the specificity was excellent (100%, 53/53) and the global concordance was 93.6% (235/253). A total of four diagnoses of proven/probable PCP were missed by the PneumoGenius® assay, reaching a 97.5% sensitivity (157/161) in this sub-group. The 12 other 'false-negative' results were obtained in patients diagnosed as colonized using the in-house PCR. DHPS genotyping was successful for 147/182 samples with PneumoGenius® and revealed dhps mutation in 8 samples, which were all confirmed by sequencing. In conclusion, PneumoGenius® assay missed the detection of low-burden PCP. This lower sensitivity for PCP diagnosis can be balanced by a higher specificity (P. jirovecii colonization less frequently detected) and the efficient detection of DHPS hot spot mutations.

The diagnosis of Pneumocystis pneumonia (PCP) relies on DNA detection of P. jirovecii in respiratory samples. In this study, we show that the commercial assay PneumoGenius® has a lower sensitivity than our in-house qPCR for PCP diagnosis, but provides accurate results for DHPS genotyping.

Real-life epidemiology and current outcomes of hospitalized adults with invasive fungal infections.

We aimed to describe the current epidemiology of both hosts with invasive fungal infections (IFIs) and causative fungi. And, detail outcomes of these infections at 12 weeks in a real-life cohort of hospitalized patients. The study was retrospective and observational to describe IFI diagnosed in a tertiary hospital (February 2017-December 2021). We included all consecutive patients meeting criteria for proven or probable IFI according to EORTC-MSG and other criteria. A total of 367 IFIs were diagnosed. 11.7% were breakthrough infections, and 56.4% were diagnosed in the intensive care unit. Corticosteroid use (41.4%) and prior viral infection (31.3%) were the most common risk factors for IFI. Lymphoma and pneumocystis pneumonia were the most common baseline and fungal diseases. Only 12% of IFI occurred in patients with neutropenia. Fungal cultures were the most important diagnostic tests (85.8%). The most frequent IFIs were candidemia (42.2%) and invasive aspergillosis (26.7%). Azole-resistant Candida strains and non-fumigatus Aspergillus infections represented 36.1% and 44.5% of the cases, respectively. Pneumocystosis (16.9%), cryptococcosis (4.6%), and mucormycosis (2.7%) were also frequent, as well as mixed infections (3.4%). Rare fungi accounted for 9.5% of infections. Overall, IFI mortality at 12 weeks was 32.2%; higher rates were observed for Mucorales (55.6%), Fusarium (50%), and mixed infections (60%). We documented emerging changes in both hosts and real-life IFI epidemiology. Physicians should be aware of these changes to suspect infections and be aggressive in diagnoses and treatments. Currently, outcomes for such clinical scenarios remain extremely poor.

Current epidemiology of the host and fungi and IFI treatments are changing. Real-life data on this subject are scarce. We present our most recent evidence to highlight the importance of the ongoing challenges that require further investigation and clinical adjustments.

Pneumocystis Colonization in Dogs Is as in Humans.

Pneumocystis is an atypical fungus that resides in the pulmonary parenchyma of many mammals, including humans and dogs. Immunocompetent human hosts are usually asymptomatically colonised or show subtle clinical signs, but some immunocompromised people can develop florid life-threatening Pneumocystis pneumonia (PCP). Since much less is known concerning Pneumocystis in dogs, we posit the question: can Pneumocystis colonization be present in dogs with inflammatory airway or lung disease caused by other pathogens or disease processes? In this study, Pneumocystis DNA was detected in bronchoalveolar lavage fluid (BALF) of 22/255 dogs (9%) with respiratory distress and/or chronic cough. Although young dogs (<1 year-of-age) and pedigree breeds were more often Pneumocystis-qPCR positive than older dogs and crossbreds, adult dogs with other infectious conditions and/or a history of therapy-resistant pulmonary disease could also be qPCR-positive, including two patients with suppression of the immune system. Absence of pathognomonic clinical or radiographic signs render it impossible to convincingly discriminate between overt PCP versus other lung/airway disease processes colonised by P. canis. It is possible that colonisation with P. canis might play a certain role as a co-pathogen in some canine patients with lower respiratory disease.

Pneumocystis jirovecii genetic diversity in a Spanish tertiary hospital.

Pneumocystis jirovecii is associated with non-noxious colonization or severe pneumonia in immunocompromised hosts. Epidemiological investigations have been hampered by the lack of a standardized typing scheme. Thus, only partial molecular data on Spanish P. jirovecii cases are available. Recently, a new ISHAM consensus multilocus sequence typing scheme (MLST) targeting ß-TUB, mt26S, CYB, and SOD with a publicly accessible database has been launched to overcome this problem. The molecular epidemiology of P. jirovecii from immunocompromised patients either colonized (n = 50) or having pneumonia (n = 36) seen between 2014 and 2018 at a single center in Barcelona, Spain, was studied. The new ISHAM consensus MSLT scheme was used to investigate the local epidemiology and identify possible unnoticed outbreaks. Mutations in the DHPS gene, not included in the scheme but giving information about potential sulfa treatment failure, were also studied. The study assigned 32 sequence types (ST) to 72.2% pneumonia and 56% colonization cases. The most frequent STs were ST21 (18.5%), ST22 (14.8%), and ST37(14.8%). For non-unique STs, ST3, ST30 and ST31 were found only in pneumonia cases, whereas ST27 was associated exclusively to colonizations. Despite 38 patients sharing similar STs, only two were involved in a potential cross transmission event. No DHPS mutations were identified. The new consensus typing scheme was useful to ascertain the molecular epidemiology of P. jirovecii in our center revealing a high genetic diversity and the potential association of specific STs to colonization and pneumonia cases. LAY SUMMARY: A newly described MLST scheme aims at providing a standardized tool to study and compare Pneumocystis jirovecii epidemiology. A high diversity among P. jirovecii isolates from patients in Barcelona, Spain, and a potential association between specific STs and infection/colonization were identified.

X-linked CD40 ligand deficiency in a 1-year-old male Shih Tzu with secondary Pneumocystis pneumonia.

An approximately 1-year-old male intact Shih Tzu dog was referred to a tertiary facility with a history of progressive tachypnea, increased respiratory effort, and weight loss over a 3-month period that failed to improve with empirical antimicrobial treatment. Upon completion of a comprehensive respiratory evaluation, the dog was diagnosed with severe Pneumocystis pneumonia and secondary pulmonary hypertension. Clinical signs resolved and disease resolution was confirmed after completion of an 8-week course of trimethoprim-sulfonamide, 4-week tapering dose of prednisone to decrease an inflammatory response secondary to acute die-off of organisms, a 2-week course of clopidogrel to prevent clot formation, and a 2-week course of a phosphodiesterase-5 inhibitor to treat pulmonary hypertension. Immunodiagnostic testing and genetic sequencing were performed to evaluate for potential immunodeficiency as an underlying cause for the development Pneumocystis pneumonia, and identified an X-linked CD40 ligand deficiency.

First molecular detection of Pneumocystis spp. in red foxes (Vulpes vulpeslinnaeus, 1758) and raccoon dogs (Nyctereutes procyonoidesgray, 1834).

Fungal organisms of the genus Pneumocystis may cause Pneumocystis pneumonia (PCP) in humans, but also domestic and wild mammals. Almost every animal species hosts its own genetically distinct Pneumocystis species, however information is sparse. In this study, 62 red foxes (Vulpes vulpes) and 37 raccoon dogs (Nyctereutes procyonoides) were collected in North-East Germany. The lung tissues of the animals were analysed by a new designed specific pan-Pneumocystis mtLSU rRNA gene PCR and sequencing. With this PCR, detection and discrimination of all known Pneumocystis spp. in a single step should be possible. This first detection of Pneumocystis spp. in 29/62 (46.8%) red foxes and 29/37 (78.4%) raccoon dogs indicated, that they harbour two dissimilar strains, as seen by specific single nucleotide position changes (SNPs). Nevertheless, five samples with contrary SNPs showed a probable inter-species transmission.

Clinical and pathological findings of Rhodococcus equi infection in foals / Achados clínicos e patológicos da infecção por Rhodococcus equi em potros

Infection by Rhodococcus equi is considered one of the major health concerns for foals worldwide. In order to better understand the disease's clinical and pathological features, we studied twenty cases of natural infection by R. equi in foals. These cases are characterized according to their clinical and pathological findings and immunohistochemical aspects. Necropsy, histologic examination, bacterial culture, R. equi and Pneumocystis spp. immunohistochemistry were performed. The foals had a mean age of 60 days and presented respiratory signs (11/20), hyperthermia (10/20), articular swelling (6/20), prostration (4/20), locomotor impairment (3/20) and diarrhea (3/20), among others. The main lesions were of pyogranulomatous pneumonia, seen in 19 foals, accompanied or not by pyogranulomatous lymphadenitis (10/20) and pyogranulomatous and ulcerative enterocolitis (5/20). Pyogranulomatous osteomyelitis was seen in 3 foals, one of which did not have pulmonary involvement. There was lymphoplasmacytic (4/20), lymphoplasmacytic and neutrophilic (1/20) or pyogranulomatous arthritis (1/20), affecting multiple or singular joints. Immunohistochemistry revealed to be a valuable tool for the detection of R. equi, confirming the diagnosis in all cases. Furthermore, pulmonary immunostaining for Pneumocystis spp. demonstrates that a coinfection with R. equi and this fungal agent is a common event in foals, seen in 13 cases.(AU)

Infecção por Rhodococcus equi é considerado um dos maiores problemas sanitários para potros em todo o mundo. Para melhor compreender a apresentação clínica e patológica da enfermidade, foram avaliados vinte casos de infecção natural por R. equi em potros. Os casos são caracterizados de acordo com seus achados clínicos e patológicos e aspectos imuno-histoquímicos. Foram realizados exames de necropsia, histologia, bacteriologia e imuno-histoquímica para R. equi e Pneumocystis spp. Os potros tinham idade media de 60 dias e apresentaram sinais respiratórios (11/20), hipertermia (10/20), aumento de volume articular (6/20), prostração (4/20), distúrbios locomotores (3/20) e diarreia (3/20), entre outros. As lesões mais importantes eram pneumonia piogranulomatosa, vista em 19 potros, acompanhada ou não por linfadenite piogranulomatosa (10/20) e enterocolite ulcerativa (5/20). Osteomielite piogranulomatosa foi constatada em três potros, um dos quais não apresentava envolvimento pulmonar. Artrites afetando uma ou múltiplas articulações eram caracterizadas por infiltrado linfoplasmocítico (4/20), linfoplasmocítico e neutrofílico (1/20) e piogranulomatoso (1/20). A imuno-histoquímica demonstrou ser uma ferramenta valiosa na detecção de R. equi, permitindo confirmar o diagnóstico em todos os casos avaliados. Além disso, a imuno-histoquímica para Pneumocystis spp. demonstra que a coinfecção por R. equi e o agente fúngico é um evento frequente em potros, constatado em 13 casos.(AU)

Confirmed case of Pneumocystis pneumonia in a Maltese Terrier × Papillon dog being treated with toceranib phosphate.

CASE REPORT: A 7-year-old female-neutered Maltese Terrier × Papillon dog was presented with tachypnoea and weight loss following 12 months of therapy with toceranib phosphate for a metastatic, histologically-low-grade mast cell tumour. The dog was diagnosed with Pneumocystis canis based on PCR with supportive clinical, radiographic and cytological findings. No other clinical evidence of immunocompromise was identified through assessment of haematology and immunoglobulin quantification. Clinical signs completely resolved with a short course of potentiated sulfonamides and discontinuation of the toceranib. CONCLUSION: To the authors' knowledge this represents the first case of Pneumocystis in a dog secondary to immunomodulatory drug therapy. It is also the first case of opportunist infection secondary to a tyrosine kinase inhibitor in dogs.

Molecular detection of Pneumocystis in the lungs of cats.

The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats' lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.

Comparative Population Genomics Analysis of the Mammalian Fungal Pathogen Pneumocystis.

Pneumocystis species are opportunistic mammalian pathogens that cause severe pneumonia in immunocompromised individuals. These fungi are highly host specific and uncultivable in vitro Human Pneumocystis infections present major challenges because of a limited therapeutic arsenal and the rise of drug resistance. To investigate the diversity and demographic history of natural populations of Pneumocystis infecting humans, rats, and mice, we performed whole-genome and large-scale multilocus sequencing of infected tissues collected in various geographic locations. Here, we detected reduced levels of recombination and variations in historical demography, which shape the global population structures. We report estimates of evolutionary rates, levels of genetic diversity, and population sizes. Molecular clock estimates indicate that Pneumocystis species diverged before their hosts, while the asynchronous timing of population declines suggests host shifts. Our results have uncovered complex patterns of genetic variation influenced by multiple factors that shaped the adaptation of Pneumocystis populations during their spread across mammals.IMPORTANCE Understanding how natural pathogen populations evolve and identifying the determinants of genetic variation are central issues in evolutionary biology. Pneumocystis, a fungal pathogen which infects mammals exclusively, provides opportunities to explore these issues. In humans, Pneumocystis can cause a life-threatening pneumonia in immunosuppressed individuals. In analysis of different Pneumocystis species infecting humans, rats, and mice, we found that there are high infection rates and that natural populations maintain a high level of genetic variation despite low levels of recombination. We found no evidence of population structuring by geography. Our comparisons of the times of divergence of these species to their respective hosts suggest that Pneumocystis may have undergone recent host shifts. The results demonstrate that Pneumocystis strains are widely disseminated geographically and provide a new understanding of the evolution of these pathogens.

Diagnosis of pneumocystis pneumonia in a 2-year-old King Charles Cavalier Spaniel using the polymerase chain reaction.

A 2-year-old, female spayed, King Charles Cavalier Spaniel was presented for evaluation of dyspnea, inappetence, and lethargy. Thoracic radiographs revealed a moderate diffuse interstitial lung pattern affecting the perihilar and caudodorsal lung fields, and an echocardiogram revealed severe pulmonary hypertension. A bronchoalveolar lavage (BAL) was performed, and cytology revealed mixed inflammation with cysts and trophozoites consistent with Pneumocystis. Pneumocystis infection was later confirmed with PCR. To the author's knowledge, this report represents the first case of canine pneumocystis pneumonia diagnosed antemortem with PCR from a BAL sample. Pneumocystis represents an important, but uncommon cause of afebrile pneumonia in immunosuppressed dogs.

Extreme Tetralogy of Fallot With Polycythemia in a Ferret (Mustela putorius furo).

A 5-month-old, intact male ferret (Mustela putorius furo) was presented with apathy, tachypnea and exercise intolerance. On initial physical examination, tachypnea and cyanosis were the two primary clinical signs detected. The complete blood count also revealed a severe polycythemia, with lymphopenia, neutrophilia and eosinophilia. Further diagnostic imaging tests were performed. The thoracic radiographs revealed cardiomegaly and a diffuse alveolar pulmonary pattern. Consequently, an echocardiography was performed and showed an interventricular septal defect, pulmonic artery occlusion, overriding of the aorta and right ventricle hypertrophy. All abnormalities were compatible with a tetralogy of Fallot (TOF). As a treatment plan, a phlebotomy was performed and the ferret was posteriorly maintained with oxygen and fluid therapy. The following day, the hematocrit decreased 11% and the respiratory distress improved. Four days later, the ferret got clinically worse and was euthanized. A necropsy was performed and confirmed the presence of an extreme TOF with a diffused interstitial pneumonia secondary to Pneumocystis carinii. To the author's knowledge, this is the first report of an extreme tetralogy of Fallot with polycythemia in a ferret.

Comparison of Pneumocystis nucleic acid and antibody profiles and their associations with other respiratory pathogens in two Austrian pig herds.

Pneumocystis carinii f. sp. suis (PCS) nucleic acid and antibody profiles on two Austrian-farrow-to-finish farms were investigated. Furthermore, associations with other respiratory pathogens were evaluated. Respiratory specimen and sera from pigs of five age classes between the 1st week and the 3rd month of life as well as samples from sows were analyzed. On Farm A, PCS infection occurred early in life. The suckling piglets were already infected in the 1st week of life and the pigs remained positive until the 3rd month of life. On Farm B, pigs were infected later, between 3 and 4 months of age. The maximum PCS nucleic acid load on Farm A was 8.3 log10 genome copies/mL BALF, whereas on Farm B the PCS burden was significantly lower, with 4.0 log10 genome copies/mL BALF. Anti-PCS antibodies were detected in sows, as maternal antibodies in suckling piglets and as an immunological reaction to infection. On both farms, PCS infection was accompanied by several co-infections. On Farm A, there were concurrent infections with PRRSV, a virulent strain of Haemophilus parasuis, and Mycoplasma hyopneumoniae. On Farm B, PCS was accompanied by infections with swine influenza virus, Mycoplasma hyopneumoniae, and a non-virulent strain of Haemophilus parasuis. The results clearly show that the PCS profiles can vary between farms. Younger pigs may be more susceptible as they had higher PCS burdens. It is possible that PCS may contribute to a respiratory disease in pigs and further investigation of its potential role is warranted.

Pneumocystis carinii infection with severe pneumomediastinum and lymph node involvement in a Whippet mixed-breed dog.

A 3.5-y-old Whippet mixed-breed dog was presented with a history of respiratory distress, exercise intolerance, and generalized demodicosis. Hematologic alterations included marked leukocytosis and neutrophilia. Radiographic examination showed a diffuse interstitial and mild peripheral alveolar lung pattern and pneumomediastinum. Because the cytologic examination of the bronchoalveolar aspirate was not diagnostic and a persistent perforation of the upper respiratory tract could not be ruled out, the dog was submitted to thoracoscopy, and subsequently the left cranial lung lobe as well as mediastinal and sternal lymph nodes were resected. Pulmonary pneumocystosis with spread to the thoracic lymph nodes was suspected after histologic investigation of lung and lymph nodes, which was confirmed by in situ hybridization, PCR, and subsequent Sanger sequencing. We document a rare, simultaneous occurrence of severe pulmonary and thoracic lymph node pneumocystosis with spontaneous pneumomediastinum in a dog. Definitive diagnosis was achieved through the use of Grocott methenamine silver staining, in situ hybridization, and PCR.

Molecular diagnosis of Pneumocystis pneumonia in dogs.

Pneumocystis pneumonia (PCP) is a life-threatening fungal disease that can occur in dogs. The aim of this study was to provide a preliminary genetic characterisation of Pneumocystis carinii f.sp.'canis' (P. canis) in dogs and thereby develop a reliable molecular protocol to definitively diagnose canine PCP. We investigated P. canis in a variety of lung specimens from dogs with confirmed or strongly suspected PCP (Group 1, n = 16), dogs with non-PCP lower respiratory tract problems (Group 2, n = 65) and dogs not suspected of having PCP or other lower respiratory diseases (Group 3, n = 11). Presence of Pneumocystis DNA was determined by nested PCR of the large and small mitochondrial subunit rRNA loci and by a real-time quantitative polymerase chain reaction (qPCR) assay developed using a new set of primers. Molecular results were correlated with the presence of Pneumocystis morphotypes detected in cytological/histological preparations. Pneumocystis DNA was amplified from 13/16 PCP-suspected dogs (Group 1) and from 4/76 dogs of control Groups 2 and 3 (combined). The latter four dogs were thought to have been colonized by P. canis. Comparison of CT values in 'infected' versus 'colonized' dogs was consistent with this notion, with a distinct difference in molecular burden between groups (CT ≤ 26 versus CT range (26

Establishment of a quantitative real-time PCR for the detection of Pneumocystis carinii f. sp. suis in bronchoalveolar lavage samples from pigs.

Respiratory diseases in pigs are mostly polymicrobial, and the involved pathogens can vary from farm to farm. The impact of Pneumocystis carinii (P. c.) f. sp. suis on respiratory disorders has not been comprehensively appraised because tests were limited to lung tissue samples and, for this reason, it was not possible to detect the fungus in living animals. In the present study, bronchoalveolar lavage fluid (BALF) from 12 pigs and oral fluid samples from 9 pigs were tested for the presence of Pneumocystis by quantitative real-time PCR. The results from these 2 clinical specimens were compared with Pneumocystis quantities in lung tissue samples. Pneumocystis quantities in BALF correlated significantly to those in lung tissue. BALF has proved to be an adequate specimen for detection of various respiratory pathogens in pigs, and the collection procedure directly on farms is also well established. In contrast to the BALF results, all oral fluid samples were negative. Thus, specimens from the lower respiratory tract should generally be preferred for the detection of Pneumocystis Additionally, under farm conditions, oral fluid is mainly collected in the form of collective samples per pen. In the present study, oral swab sampling of individual pigs was intended but failed in 3 of 12 pigs because they did not salivate sufficiently. As a conclusion, only BALF can be recommended as a useful tool for Pneumocystis herd monitoring in vivo.

Barcoding markers for Pneumocystis species in wildlife.

Lung specimens (n = 216) from six wildlife species were examined for occurrence of Pneumocystis species in pulmonary tissues. Among small mammals the shrew Sorex antinorii (80 %) were most frequently colonized. In contrast, foxes and badgers did not yield positive amplification. Host-specificity was noted, at least at the level of the host genus. Phylogenetic trees based on partial mtLSU and mtSSU showed high diversity of species corresponding to animal host diversity. Nuclear rDNA ITS data confirmed unambiguous separation of species. In conclusion, ITS is an excellent marker to distinguish species of the genus Pneumocystis.

Common variable immune deficiency in a Pomeranian with Pneumocystis carinii pneumonia.

A Pomeranian dog, 1 year- and 8 month-old neutered female, was presented with persistent respiratory distress and recurrent generalized demodicosis. Physical examination revealed cyanosis, rough respiratory sounds, multifocal alopecia and dermal erosions on the dorsal side of the forelimbs, perineal area and skin around the eyes. A severe diffuse interstitial lung pattern was observed on thoracic radiographs. The blood examination revealed neutrophilia and hypoglobulinemia. Serum immunoglobulin concentrations of IgG and IgA were low. Histopathological examination revealed severe diffuse interstitial pneumonia with Pneumocystis carinii infection. Severe lymphoid depletion was observed in the spleen and other organs with lymphoid follicles consisted mainly of CD3-positive T cells and few cells of B-cell lineage. B-cell hypoplasia with subsequent antibody deficiency was suspected.