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Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived Pneumocystis. Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.

Pneumocystis jirovecii colonization is frequent during chronic obstructive pulmonary disease (COPD) and patients constitute potential contributors to its interhuman circulation. However, the existence of an environmental reservoir cannot be excluded. We assessed the prevalence and factors associated with Pneumocystis colonization during COPD, and studied circulation between patients and their domestic environment. Pneumocystis molecular detection and mtLSU genotyping were performed in oro-pharyngeal washes (OPW) sampled in 58 patients with COPD acute exacerbation, and in indoor dust, sampled in patients' homes using electrostatic dust collectors (EDCs). Lung and systemic inflammation was assessed. Pneumocystis carriage was evaluated in 28 patients after 18 months at stable state. Pneumocystis was detected in 11/58 OPWs during exacerbation (19.0%). Colonized patients presented a significantly lower body mass index, and higher serum IL-17 and CD62P. One patient presented positive detection of typable isolates in both OPW and EDC, with both isolates harboring mtLSU genotype 3. Pneumocystis genotype 1 was further detected in EDCs from three non-colonized patients and one colonized patient with non-typable isolate. Genotypes 1 and 2 were predominant in clinical isolates (both 42%), with genotype 3 representing 16% of isolates. Pneumocystis was detected in 3/28 patients at stable state (10.7%). These data suggest that Pneumocystis colonization could be facilitated by a lower BMI and be related to acute alteration of lung function during COPD exacerbation. It also suggests Th17 pathway and platelet activation could be involved in the anti-Pneumocystis response during colonization. Last, Pneumocystis detection in EDCs supports its potential persistence in indoor dust., Lay Summary: Chronic obstructive pulmonary disease patients tend to be more frequently colonized by Pneumocystis during exacerbation (19.0%) than at stable state (10.7%). Factors associated with colonization include lower BMI, higher IL-17, and CD62P. Pneumocystis detection in patients' dwellings suggests potential persistence in indoor dust., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

In the non-AIDS group, several underlying conditions and immune defects could lead to different PCP presentations. This study compared PCP presentation and outcome according to the underlying disease. A secondary analysis of a previously published prospective observational study including 544 PCP patients was done. Only non-AIDS patients were included. Underlying disease was defined as chronic lymphocytic leukemia (CLL), organ transplantation, solid cancer, allogeneic hematopoietic stem cell transplant (AHSCT), other hematological diseases, and immunosuppressive treatment. Clinical characteristics and outcomes were compared between groups. Multiple correspondent analyses compared clinical characteristics at diagnosis. Day 30 mortality was analyzed. Three hundred and twenty-one patients were included in the study. The underlying diseases were hematological malignancy (n = 75), AHSCT (n = 14), CLL (n = 19), solid organ transplant (n = 94), solid tumor (n = 39), and immunosuppressive treatment (n = 57). Compared with other underlying diseases, PCP related to CLL was closer to PCP related to AIDS presentation (long duration of symptoms before diagnosis, high level of dyspnea, and low oxygen saturation at diagnosis). Day 30 mortality was associated with underlying disease, oxygen flow, and shock at ICU admission. PCP presentations may vary according to the underlying reason for immunosuppression. Response to treatment and adjuvant steroid therapy should be analyzed regarding this result.

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: "MucoFong" program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran-rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37 °C and on Sabouraud-chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20-27 °C. Each plate was checked twice a week during 3 weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16 days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

Pneumocystis jirovecii pneumonia (PCP) in patients without AIDS is increasingly common. We conducted a prospective cohort study of consecutive patients with proven PCP; of 544 patients, 223 (41%) had AIDS (AIDS patients) and 321 (59%) had other immunosuppressive disorders (non-AIDS patients). Fewer AIDS than non-AIDS patients required intensive care or ventilation, and the rate of hospital deaths--17.4% overall--was significantly lower for AIDS versus non-AIDS patients (4% vs. 27%; p<0.0001). Multivariable analysis showed the odds of hospital death increased with older age, receipt of allogeneic bone marrow transplant, immediate use of oxygen, need for mechanical ventilation, and longer time to treatment; HIV-positive status or receipt of a solid organ transplant decreased odds for death. PCP is more often fatal in non-AIDS patients, but time to diagnosis affects survival and is longer for non-AIDS patients. Clinicians must maintain a high index of suspicion for PCP in immunocompromised patients who do not have AIDS.

Background: Pneumocystis without obvious accompanying pathology is occasionally reported in autopsied infant lungs. Its prevalence and significance are unknown. Interestingly, this mild infection induces a strong activation of mucus secretion-related genes in young immunocompetent rodents that has not been explored in infants. Excess mucus is induced by multiple airway offenders through nonspecific pathways and would explain a cofactor role of Pneumocystis in respiratory disease. We undertook characterization of the prevalence of Pneumocystis and associated mucus in infant lungs., Methods: Samples from 128 infants (mean age, 101 days) who died suddenly and unexpectedly in Santiago during 1999-2004 were examined for Pneumocystis using nested polymerase chain reaction (nPCR) amplification of the P. jirovecii mtLSU ribosomal RNA gene and immunofluorescence microscopy (IF). Pneumocystis-negative infants 28 days and older and their age-closest positives were studied for MUC5AC expression and Pneumocystis burden by Western blot and quantitative PCR, respectively., Results: Pneumocystis DNA was detected by nPCR in 105 of the 128 infants (82.0%) and Pneumocystis organisms were visualized by IF in 99 (94.3%) of the DNA-positive infants. The infection was commonest at 3-4 months with 40 of 41 (97.6%) infants of that age testing positive. MUC5AC was significantly increased in Pneumocystis-positive tissue specimens (P = .013). Death was unexplained in 113 (88.3%) infants; Pneumocystis was detected in 95 (84.0%) of them vs 10 of 15 (66.7%) with explained death (P = .28)., Conclusions: A highly focal Pneumocystis infection associated to increased mucus expression is almost universally present in the lungs of infants dying unexpectedly in the community regardless of autopsy diagnosis.

Pneumocystis carriage was detected in 12.5% of 104 cystic fibrosis (CF) patients during a prospective multicenter French study, with a prevalence of genotype 85C/248C and geographic variations. It was significantly associated with the absence of Pseudomonas aeruginosa colonization and a greater forced expiratory volume in 1 s. Results are discussed considering the natural history of CF.

Is Pneumocystis pneumonia (PcP) a transmissible fungal disease? Does nosocomial PcP occur? Is there Pneumocystis transmission in the community? These questions, which could not be tackled before the 2000s, may at present be approached using either noninvasive detection methods or experimental transmission models. Represented by a unique entity (P. carinii) for almost one century, the Pneumocystis genus was shown to contain several species, being P. jirovecii the sole species identified in humans hitherto. Molecular methods combined with cross infection experiments revealed strong host specificity that precludes Pneumocystis inter-species transmission. In contrast, respiratory transmission between mammals of a same species is usually highly active, even between immunocompetent hosts. Other transmission ways could also exist. New data show that human being is the unique P. jirovecii reservoir; it would constitute the sole infection source in both hospital and community., (© 2012 médecine/sciences – Inserm / SRMS.)

Genotypes of two different loci of the Pneumocystis jirovecii mitochondrial gene were studied in specimens from a total of 75 Pneumocystis pneumonia patients in Spain, France and Cuba. A new genotype of the mitochondrial small subunit rRNA gene of P. jirovecii (160A/196T) was identified, which was revealed to be the most common in these three countries, especially in Cuba where its proportion reached 93.8%. Our data imply that the new genotype might be circulating worldwide and also suggests that the distribution of P. jirovecii genotypes could be narrower in islands such as Cuba.

Immunosuppressed patients are at high risk of acquiring airborne fungal infections, mainly caused by Aspergillus species. Although HEPA filters are recommended to prevent environmental exposure, mobile air-treatment units can be an alternative. However, many different models of mobile units are available but there are few data on their fungal aero-decontamination efficacy and usefulness in the prevention of Aspergillus infections. Thus, we developed a challenge test, based on the aerosolization of 10(6) Aspergillus niger conidia, in order to compare the particle and fungal decontamination efficacy of the following four mobile air-treatment systems; Plasmair T2006, Mobil'Air 1200 (MA1200), Mobil'Air 600 (MA600) combined with Compact AirPur Mobile C250 (C250), and the prototype unit Compact AirPur Mobile 1800 (C1800). The use of all these air-treatment systems was able to significantly decrease the concentration of particles or fungal viable conidia. ISO7 was the maximum particle class reached within 20 min with the Plasmair T2006 and MA1200, 1 h by the combined MA600/C250, and 1 h and 30 min with the C1800. After 2 h, fungal counts were significantly lower with Plasmair T2006, MA1200 and the combined MA600/C250 (2.2 ± 1.9 to 5.0 ± 3.7 CFU/m(3)) than achieved with the C1800 (23.8 ± 12.8 CFU/m(3); P ≤ 6.0E-3). All the air-treatment systems were able to decrease aerial particle and fungal counts, but their efficacy was variable, depending on the units' air-treatment modalities and rates of air volume that was processed. This comparative study could be helpful in making an informed choice of mobile units, and in improving the prevention of air-transmitted fungal infections in non-protected areas.

Objectives: To determine the rate and identify risk factors of Pneumocystis jirovecii (P. jirovecii) colonization among patients with systemic autoimmune diseases., Methods: We conducted an observational study in patients with systemic autoimmune diseases in an internal medicine department. Each week, five patients with systemic diseases were randomly selected for colonization screening. Patients complaining of recent respiratory symptoms were excluded. P. jirovecii PCR was performed on induced sputum samples. Univariate and multivariate logistic regression analyses of clinical and biological data were performed to determine predictors of Pneumocystis colonization. Pneumocystis pneumonia occurrence in P. jirovecii-positive PCR patients was recorded during a 1-year follow-up., Results: P. jirovecii was detected in 11/67 (16%) subjects. Comparing the features in P. jirovecii-positive and P. jirovecii-negative PCR patients, only male gender was significantly associated with Pneumocystis colonization. In multivariate analysis with regard to gender, the higher prevalence of P. jirovecii colonization in men was largely explained by higher daily CSs [odds ratio (OR) = 1.6; 95% CI 1.1, 2.3] and lower total lymphocyte level (OR = 0.9; 95% CI 0.8, 0.99). No P. jirovecii-positive PCR patient developed Pneumocystis pneumonia during the 1-year follow-up, but corticosteroid amounts were significantly lower at the end of follow-up than on inclusion., Conclusion: This is the first study on P. jirovecii colonization in patients with systemic autoimmune diseases. We found a high prevalence of colonization and identified CS therapy and lymphocyte counts as risk factors for colonization. We recommend screening for P. jirovecii colonization in patients with systemic autoimmune diseases receiving immunosuppressant treatment. Further studies are needed to determine the role of subclinical colonization in disease transmission and the persistence of Pneumocystis colonization.

Pneumocystis jirovecii is an atypical fungus exhibiting pulmonary tropism and a highly defined host specificity. It is generally regarded as an opportunistic microorganism causing serious pneumonia in AIDS patients. However, with the currently rising number of patients receiving immunosuppressive therapies for malignancies, allogeneic organ transplantations and autoimmune diseases, Pneumocystis pneumonia is becoming more and more recognized in non-HIV immunosuppressed individuals. The clinical presentation in HIV-infected patients may differ from that in other immunocompromised patients and its diagnosis continues to be challenging as there are no specific symptoms or signs. Cotrimoxazole is the drug of choice for prophylaxis and therapy of any form or severity of Pneumocystis pneumonia, but there are only a few options for other alternative treatments. The management of this pneumonia remains a major challenge for all physicians caring for immunosuppressed patients.

Pneumocystis sp. is transmitted through the airborne route and presents a high host-species-specificity. Occasional reports of Pneumocystis pneumonia in still births and newborn infants suggest that other routes of transmission, e.g. transplacental might occur. The latter has been reported in rabbits but available data indicate that transplacental transmission of Pneumocystis seems not to occur in corticosteroid-treated rats and in SCID mice. The present study was undertaken to evaluate transplacental transmission of Pneumocystis oryctolagi. The spontaneously-acquired pneumocystosis rabbit model using hybrid California/New Zealand white female rabbits was selected because of similarities among rabbit and human placentas. Three different experiments were conducted in France and Chile. Pneumocystis organisms were detected by microscopy in the lungs of pregnant does and Pneumocystis DNA was found in the lungs of fetuses from the multiparous does from the second week to the end of gestation. Pneumocystis DNA was not detected in fetuses from primiparous does. Detection of Pneumocystis oryctolagi--DNA in fetuses of multiparous does and not in those of primiparous ones, suggests that transplacental transmission may be favored by multiple gestations. Whether Pneumocystis-DNA in fetal tissues from multiparous does resulted from transplacental passage of viable transmissible forms requires further investigation.

In addition to the serological systematic screening tests, kits to measure the avidity of toxoplasma IgG antibodies are currently available. Since high-avidity IgG toxoplasma antibodies have been shown to exclude recent infection, IgG avidity determination is especially useful in ruling out acute infection having occurred in the 3-4 prior months of pregnancy. We therefore compared the efficacy of two toxoplasma IgG avidity ELISA kits: SFRI (SFRI Laboratoire) and VIDAS Toxo-IgG avidity kit (bioMérieux). The agreement of the results from the 2 commercial assays were analysed using 55 serum samples, in terms of global mother-child Toxoplasma results and outcome, specially with light of the results of Toxoplasma antenatal, postnatal assays and of clinical follow up of children.

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.

The detection of Pneumocystis DNA in clinical specimens by using PCR assays is leading to important advances in Pneumocystis pneumonia (PcP) clinical diagnosis, therapy and epidemiology. Highly sensitive and specific PCR tools improved the clinical diagnosis of PcP allowing an accurate, early diagnosis of Pneumocystis infection, which should lead to a decreased duration from onset of symptoms to treatment, a period with recognized impact on prognosis. This aspect has marked importance in HIV-negative immunocompromised patients, who develop often PcP with lower parasite rates than AIDS patients. The specific amplification of selected polymorphous sequences of Pneumocystis jirovecii genome, especially of internal transcribed spacer regions of the nuclear rRNA operon, has led to the identification of specific parasite genotypes which might be associated with PcP severity. Moreover, multi-locus genotyping revealed to be a useful tool to explore person-to-person transmission. Furthermore, PCR was recently used for detecting P. jirovecii dihydropteroate synthase gene mutations, which are apparently associated with sulfa drug resistance. PCR assays detected Pneumocystis-DNA in bronchoalveolar lavage fluid or biopsy specimens, but also in oropharyngeal washings obtained by rinsing of the mouth. This non-invasive procedure may reach 90%-sensitivity and has been used for monitoring the response to treatment in AIDS patients and for typing Pneumocystis isolates.

Simian populations represent valuable models for understanding the epidemiology of human pneumocystosis. The present study aims to describe the circulation of Pneumocystis organisms within a social organization of healthy crab-eating macaques (Macaca fascicularis) living in a natural setting in France. Animals were followed for up to 2 years. Deep nasal swab and blood samples were collected monthly from each animal under general anaesthesia. Environmental air was sampled for a 1 week period every month in the park where the macaques dwelt. Pneumocystis DNA was detected by nested-PCR of mitochondrial large subunit rRNA (mtLSU) gene in nasal swab and air samples. Anti-Pneumocystis IgG antibodies were detected in serum samples by indirect immuno-fluorescence assay. Pneumocystis DNA was detected in 168 of 500 swab samples examined (33.6 %). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. Positive detection of Pneumocystis DNA was not related to the detection of serum anti-Pneumocystis antibody. During the second year of the study, Pneumocystis DNA was amplified more frequently from unweaned macaques than from adults or subadults. The mtLSU sequence showed marked polymorphism with eight Pneumocystis sequence types representing two distinct groups. On the whole, a constant and intensive circulation of Pneumocystis organisms within the community was observed. However, the implication of the various members of the colony was probably different and several levels of colonization by Pneumocystis may occur in immunocompetent macaques.

Objective: To investigate the incidence of the association of Trichomonas and Pneumocystis in the lung., Study Design: Sixty-six bronchoalveolar lavage fluid (BALF) samples from immunocompromised patients with pneumocystosis were retrospectively examined microscopically., Results: Trichomonads were found as coinfecting agents in 60% of BALF samples. The frequency and abundance of trichomonads was increased, up to 100%, in cases rich in Pneumocystis., Conclusion: The data suggest that pulmonary Trichomonas infection occurs frequently in the course of Pneumocystis pneumonia. The role of trichomonads in causing alveolar damage during Pneumocystis pneumonia is hypothetical.

A disseminated Fusarium oxysporum infection with skin localization was diagnosed in a woman with a relapse of B-acute leukemia during induction chemotherapy. The infection was refractory to amphotericin B-lipid complex alone but responded successfully when voriconazole was added.

A 41-year-old man was hospitalized, presenting increasing dyspnea and extensive ground-glass opacities on chest X-ray. Infection by human immunodeficiency virus was confirmed. Cytologic examination of bronchoalveolar lavage fluid revealed numerous trichomonads and aggregates of Pneumocystis sp. Treatment was followed by rapid improvement of respiratory symptoms and chest X-ray. The trichomonad species found in the lungs was identified as Trichomonas vaginalis by small-subunit rRNA gene amplification and sequencing. With the exception of rare cases of contamination of newborn babies during delivery, T. vaginalis has never been found in lungs in healthy or immunocompromised adults. In the present case, T. vaginalis is found as coinfecting agent. Our data, like those found in the literature, suggest that trichomonads are overlooked parasites that may be regularly implicated in diverse human pathologies.

As monkeys-derived Pneumocystis is closely related to P. jirovecii, simian populations should be considered as valuable models for the understanding of the epidemiology of human pneumocystosis. In the present study, the impact of environmental factors on the carriage of Pneumocystis was evaluated in socially organized group of immunocompetent macaques (Macaca fascicularis). The tribe, maintained in partial release at the Primatology Center of Strasbourg in France, comprised 29 animals at the end of the study. From December 2000 to November 2002, deep nasal swab samples were collected monthly from each animal under general anaesthesia. The presence of Pneumocystis DNA was assessed by nested PCR of mtLSU rRNA gene. No case of pneumocystosis was reported during the study. Pneumocystis DNA was detected in 166 out of 481 swab samples examined (34.5%). The number of macaques with detectable Pneumocystis DNA was highly variable from one month to another. However, Pnemocystis carriage was clearly correlated to the mean precipitation rates.

The infectious power of Pneumocystis carinii f. sp. hominis was explored by inoculating SCID mice intranasally with either P. carinii f. sp. hominis or P. carinii f. sp. muris isolates. Only mice inoculated with mouse parasites developed Pneumocystis pneumonia, as assessed by microscopy and PCR. These results suggest that humans do not contract pneumocystosis from animals.