Revisiting the Pneumocystis host specificity paradigm and transmission ecology in wild Southeast Asian rodents.

Pneumocystis fungi are opportunistic parasites of mammalian lungs whose evolution, ecology and host specificity in natural host populations remain poorly understood and controversial. Using an extensive collection of 731 lung samples from 27 rodent species sampled in five Southeast Asian countries, and nested PCR amplification of mitochondrial and nuclear genes, we investigated the host specificity and genetic structure of Pneumocystis lineages infecting wild rodents. We also identified the rodent species playing a central role in the transmission of these parasites using network analysis and centrality measurement and we characterized the environmental conditions allowing Pneumocystis infection in Southeast Asia using generalized linear mixed models. Building upon an unprecedented Pneumocystis sampling from numerous rodent species belonging to closely related genera, our findings provide compelling evidence that the host specificity of Pneumocystis lineages infecting rodents is not restricted to a single host species or genus as often presented in the literature but it encompasses much higher taxonomic levels and more distantly related rodent host species. The phylogenetic species status at both mitochondrial and nuclear genetic markers of at least three new Pneumocystis lineages, highly divergent from Pneumocystis species currently described, is also suggested by our data. Our models show that the probability of Pneumocystis infection in rodent hosts is positively correlated to environmental variables reflecting habitat fragmentation and landscape patchiness. Synanthropic and habitat-generalist rodents belonging to the Rattus, Sundamys and Bandicota genera played a role of bridge host species for Pneumocystis spreading in these heterogeneous habitats, where they can reach high population densities. These are critical findings improving our understanding of the ecology of these enigmatic parasites and the role played by cospeciation and host switches in their evolution. Our results also confirmed the role of land-use change and habitat fragmentation in parasite amplification and spillover in rodents.

Colonización por Pneumocystis jirovecii en mujeres gestantes y recién nacidos en Lima, Perú / neumocystis jirovecii colonisation in pregnant women and newborns in Lima, Peru

ANTECEDENTES: La primoinfección por Pneumocystis jirovecii ocurre de forma asintomática antes de los 6 meses de edad, lo que sugiere que la infección se adquiere muy precozmente en la vida. Se ha descrito también la presencia de neumonía por Pneumocystis en recién nacidos, lo que indica la necesidad de estudiar la colonización en el binomio madre-hijo. OBJETIVOS: Evaluar la prevalencia de colonización de Pneumocystis en gestantes y explorar la potencial transmisión transplacentaria. MÉTODOS: Estudio transversal que incluyó a mujeres gestantes mayores de 18 años con 37 o más semanas de gestación y negativas para el VIH que acudieron al Hospital Cayetano Heredia en los años 2016-2017. Se obtuvo información clínica y demográfica de la gestante y del recién nacido. Se tomaron muestras de lavado orofaríngeo/hisopado nasal de la gestante, de placenta y de aspirado nasofaríngeo/hisopado nasal del recién nacido. Todas las muestras respiratorias fueron analizadas mediante PCR anidada. En el caso de las muestras de placenta solo fueron analizadas aquellas procedentes de mujeres con resultados positivos de PCR para Pneumocystis en las muestras respiratorias. RESULTADOS: De las 92 gestantes incluidas en el estudio cinco presentaban colonización por Pneumocystis (5,43%). Se evaluaron las muestras de 87 recién nacidos y las placentas de las cinco madres con PCR positiva, no encontrándose ADN de Pneumocystis en ninguna de ellas. CONCLUSIONES: Aunque el 5,43% de las mujeres gestantes estuvieran colonizadas por Pneumocystis no pudo determinarse el rol de esta colonización en la transmisión a sus recién nacidos, ya que en ninguno de ellos se demostró la presencia de Pneumocystis

BACKGROUND: Pneumocystisjirovecii primary infection occurs asymptomatically before 6 months of age, suggesting that the infection is acquired very early in life. Furthermore, Pneumocystis pneumonia has been described in newborns, which emphasizes the importance of studying Pneumocystis colonization in mother-infant pairs. AIMS: To evaluate the prevalence of Pneumocystis colonization among pregnant women and to determine the potential transplacental transmission. METHODS: A cross-sectional study was carried out on HIV-negative women over 18 years-old, and 37 or more weeks of pregnancy attending Hospital Cayetano Heredia Maternity unit during 2016-2017. Clinical and demographical information was collected on them and their newborns. Oropharyngeal washes, nasal swabs, and placenta samples were collected from women, as well as a nasopharyngeal aspirate and nasal swab from newborns. All respiratory samples were analysed by nested-PCR for the detection of Pneumocystis. Placenta samples from women with a positive PCR result in their respiratory samples were also analysed by nested-PCR. RESULTS: Of the 92 pregnant women included, five of them (5.43%) were colonized by Pneumocystis. Pneumocystis DNA was not found in any of the 87 available newborn samples or in the placentas of the five women who had a positive result by PCR in their upper respiratory samples. CONCLUSIONS: It was found that 5.43% of the pregnant women were colonized by Pneumocystis, there was no evidence of any role of this colonization in the transmission to their newborns, since none of them tested positive for Pneumocystis

Investigation of nosocomial pneumocystis infections: usefulness of longitudinal screening of epidemic and post-epidemic pneumocystis genotypes.

BACKGROUND: Twenty-five patients, of whom 22 were renal transplant recipients, developed Pneumocystis jirovecii infections at the nephrology department of Reims University Hospital (France) from September 2008 to October 2009, whereas only four sporadic cases had been diagnosed in this department over the 14 previous years. AIM: This outbreak was investigated by analysing patient encounters and P. jirovecii types. METHODS: A transmission map was drawn up. P. jirovecii typing at DHPS, ITS and mtLSU rRNA sequences was performed in the patients of the cluster (18 patients with Pneumocystis pneumonia (PCP) and seven colonized patients), 10 unlinked control patients (six PCP patients and four colonized patients), as well as 23 other patients diagnosed with P. jirovecii (nine PCP patients and 14 colonized patients) in the same department over a three-year post-epidemic period. FINDINGS: Eleven encounters between patients harbouring the same types were observed. Three PCP patients and one colonized patient were considered as possible index cases. The most frequent types in the cluster group and the control group were identical. However, their frequency was significantly higher in the first than in the second group (P < 0.01). Identical types were also identified in the post-epidemic group, suggesting a second outbreak due to the same strain, contemporary to a disruption in prevention measures. CONCLUSIONS: These results provide additional data on the role of both PCP and colonized patients as infectious sources. Longitudinal screening of P. jirovecii types in infected patients, including colonized patients, is required in the investigation of the fungus's circulation within hospitals.

[Intrauterine Pneumocystis infection]. / Vnutriutrobnaya pnevmotsistnaya infektsiya.

Pneumocystosis is well known as an opportunistic infection that is presently most frequently registered in patients with HIV infection and in those with other immunodeficiency states. Earlier, after the Second World War, Pneumocystis pneumonia was most commonly detected in debilitated and premature children; nosocomial outbreaks of pneumocystosis were studied in detail in the 1960s and 1970s. The pathogen is transmitted through the air, but a number of references indicate that it can be transmitted through the placenta. Despite the increasing number of publications on pneumocystosis in pediatrics, physicians remain unfamiliar with this disease. The paper provides evidence that Pneumocystis jiroveci can infect the fetus in utero. If unrecognized, the disease can lead to a child's death due to severe respiratory failure. The authors describe their case of generalized pneumocystosis that has developed in a child with evidence of intrauterine infection (detection of the pathogen in the autopsy material and placenta and identification of serological markers in his/her parents). The issues that are associated with intrafamilial infection and a risk for in utero transmission of P. jiroveci are discussed.

Aerially transmitted human fungal pathogens: what can we learn from metagenomics and comparative genomics? / Hongos de transmisión aérea patógenos para el ser humano: conocimientos adquiridos a partir de su metagenómica y genómica comparativa

In the last few decades, aerially transmitted human fungal pathogens have been increasingly recognized to impact the clinical course of chronic pulmonary diseases, such as asthma, cystic fibrosis or chronic obstructive pulmonary disease. Thanks to recent development of culture-free high-throughput sequencing methods, the metagenomic approaches are now appropriate to detect, identify and even quantify prokaryotic or eukaryotic microorganism communities inhabiting human respiratory tract and to access the complexity of even low-burden microbe communities that are likely to play a role in chronic pulmonary diseases. In this review, we explore how metagenomics and comparative genomics studies can alleviate fungal culture bottlenecks, improve our knowledge about fungal biology, lift the veil on cross-talks between host lung and fungal microbiota, and gain insights into the pathogenic impact of these aerially transmitted fungi that affect human beings. We reviewed metagenomic studies and comparative genomic analyses of carefully chosen microorganisms, and confirmed the usefulness of such approaches to better delineate biology and pathogenesis of aerially transmitted human fungal pathogens. Efforts to generate and efficiently analyze the enormous amount of data produced by such novel approaches have to be pursued, and will potentially provide the patients suffering from chronic pulmonary diseases with a better management. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

En las últimas décadas se ha reconocido cada vez más la influencia de los hongos patógenos para el ser humano, y cuya transmisión es aérea, en el curso clínico de afecciones pulmonares crónicas, como el asma, la fibrosis quística o la enfermedad pulmonar obstructiva crónica. Gracias al desarrollo reciente de métodos de secuenciación de alto rendimiento, que no requieren cultivo, en la actualidad los análisis metagenómicos permiten detectar, identificar e incluso cuantificar comunidades de microorganismos procariotas o eucariotas que habitan en las vías respiratorias del ser humano, y acceder a la complejidad de las comunidades microbianas cuya población es de baja densidad, que posiblemente desempeñan un papel en las enfermedades pulmonares crónicas. En la presente revisión examinamos cómo los estudios metagenómicos y genómicos comparativos pueden ayudar a superar los obstáculos de los cultivos de hongos, mejorar nuestros conocimientos sobre la biología fúngica, desvelar el diálogo cruzado (crosstalk) entre el pulmón del huésped y la microbiota fúngica asociada, y adquirir información sobre la influencia patogénica de estos hongos transmitidos por el aire que afectan al ser humano. Revisamos los estudios metagenómicos y los análisis genómicos comparativos de microorganismos cuidadosamente seleccionados, y confirmamos la utilidad de estas estrategias para definir mejor la biología y la patogenia de hongos de transmisión aérea que son patógenos para el ser humano. Los esfuerzos por generar y analizar eficientemente la ingente cantidad de datos obtenidos con estos nuevos métodos deberán continuar, y es posible que ofrezcan un mejor tratamiento de los pacientes portadores de enfermedades pulmonares crónicas.Este manuscrito forma parte de la serie de artículos presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Growth and airborne transmission of cell-sorted life cycle stages of Pneumocystis carinii.

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.

Pneumocystis sp. in bats evaluated by qPCR.

Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.

[Transmission of Pneumocystis infection in humans]. / La transmission des infections à Pneumocystis.

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.

[Current epidemiology of Pneumocystis jirovecii infections]. / Wspólczesna epidemiologia zakazen Pneumocystis jirovecii.

Pneumocystis fungi has been recognized as causative agent of pneumonia in immunocompromised individuals. The epidemiology of Pneumocystis infection has been dynamically changing over the past two decades. In this review, the current understanding of factors contributing to the spreading of Pneumocystis jirovecii in immunocompetent and immunocompromised human populations is highlighted. Primary infections, routs of transmission and colonization issues are discussed. Data concerning P. jirovecii infections in Poland is also presented.

Nosocomial Pneumocystis jirovecii infections.

Airborne transmission of Pneumocystis sp. from host to host has been demonstrated in rodent models and several observations suggest that interindividual transmission occurs in humans. Moreover, it is accepted that the Pneumocystis organisms infecting each mammalian species are host specific and that the hypothesis of an animal reservoir for Pneumocystis jirovecii (P. jirovecii), the human-specific Pneumocystis species, can be excluded. An exosaprophytic form of the fungus cannot be strictly ruled out. However, these data point toward the potential for the specific host to serve as its own reservoir and for Pneumocystis infection in humans as an anthroponosis with humans as a reservoir for P. jirovecii. This review highlights the main data on host-to-host transmission of Pneumocystis in rodent models and in humans by the airborne route and provides a rationale for considering the occurrence of nosocomial infections and measures for their prevention

Pneumocystis species, co-evolution and pathogenic power.

The genus Pneumocystis comprises uncultured, highly diversified microfungal organisms able to attach specifically to type-I alveolar epithelial cells and to proliferate in pulmonary alveoli provoking severe pneumonitis. The pathogenic potential of Pneumocystis species, especially of the human-associated Pneumocystis jirovecii, has stimulated a growing interest in these peculiar microfungi. However, a comprehensive understanding of basic biology and pathogenic power of Pneumocystis organisms calls for their recognition as natural, complex entities, without reducing them to their pathogenic role. For many years, the entity named "Pneumocystis carinii" was considered like an anecdotal pulmonary pathogen able to cause pneumonia in immunosuppressed hosts. Only for the last years, marked genetic divergence was documented among the Pneumocystis strains of different mammals. Cross-infection experiments showed that Pneumocystis species are stenoxenous parasites. Mainly on the basis of the Phylogenetic Concept of Species, Pneumocystis strains were considered as genuine species. Five species were described: P. carinii and Pneumocystis wakefieldiae in rats, P. jirovecii in humans, Pneumocystis murina in mice, and Pneumocystis oryctolagi in rabbits. They also present distinctive phenotypic features. Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals, probably resulting from active airborne horizontal and vertical (transplacental or aerial) transmission mechanisms. Cophylogeny is the evolutionary pattern for Pneumocystis species, which dwelt in the lungs of mammals for more than 100 million years. Consistently, Pneumocystis organisms exhibit successful adaptation to colonize the lungs of both immunocompromised and healthy hosts that can act as infection reservoir. Pneumocystis pneumonia, rarely reported in wild mammals, seems to be a rather unfrequent event. A larger spectrum of Pneumocystis infections related to the heterogeneous level of immune defence found in natural populations, is, however, expected. Pneumocystis infection of immunocompetent hosts emerges therefore as a relevant issue to human as well as animal health.

Epidemiology of Pneumocystis colonization in families.

Whether Pneumocystis colonization is transmitted in families with human immunodeficiency virus (HIV)-infected members is unknown. Using nested polymerase chain reaction of oropharyngeal or nasopharyngeal samples, we detected colonization in 11.4% of HIV-infected adults and in 3.3% of their children, but there was no evidence of clustering.

Transmission of Pneumocystis carinii DNA from a patient with P. carinii pneumonia to immunocompetent contact health care workers.

The transmission of Pneumocystis carinii from person to person was studied by detecting P. carinii-specific DNA in prospectively obtained noninvasive deep-nasal-swab samples from a child with a documented P. carinii pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat P. carinii (P. carinii f. sp. carinii) that amplifies all forms of P. carinii and internal primers specific for human P. carinii (f. sp. hominis). P. carinii f. sp. hominis DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of P. carinii pneumonia, document that person-to-person transmission of P. carinii is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk.

Nosocomial transmission of opportunistic infections.

Immunocompromised patients are at high risk for opportunistic infections. Traditionally, these infections were thought to arise from endogenous reactivation of previously acquired latent infections, and nosocomial transmission therefore was deemed to be so unlikely that no special infection control interventions were needed to prevent transmission in healthcare settings. However, new data have challenged this view and suggest that some opportunistic pathogens are transmissible from one immunosuppressed patient to another. Epidemiological investigations, molecular genotyping, animal studies, and air-sampling experiments lend support to the hypothesis that reinfection with opportunistic pathogens does occur, that airborne transmission is possible, and that nosocomial spread is a plausible explanation for case clusters. Taken together, these observations support the view that some opportunistic infections are exogenous in origin and that additional epidemiological investigations are needed to define the true risk of nosocomial spread and need for isolation.

Detection of Pneumocystis carinii DNA in air samples: likely environmental risk to susceptible persons.

The means by which humans acquire Pneumocystis carinii is not well understood. Whether it can be acquired from specific environmental sources or transmitted from person to person has not been determined. This study was designed to detect nucleic acids of P. carinii in air samples from various locations, including P. carinii-infected patients' homes and hospital rooms, non-P. carinii-infected patients' hospital rooms, empty hospital rooms, offices at Indiana University, and other homes in different locations. DNA was extracted from cellulose-ester filters through which air samples had been filtered, and the P. carinii DNA was amplified by PCR with primers specific for the internal transcribed spacer regions of rRNA. P. carinii DNA was found in 17 of 30 air samples (57%) from the rooms of P. carinii-infected patients. It was also found in 6 of the 21 other hospital rooms sampled (29%) but was not found in any of the offices, storage areas, or control homes. Environmental sampling suggests that the airborne presence of P. carinii genetic material and infectious organisms is plausible. The organism was also detected in locations where P. carinii patients were not immediately proximate, such as the hospital rooms of non-P. carinii-infected patients.

Transmission of Pneumocystis carinii from patients to hospital staff.

BACKGROUND: An extrahuman reservoir of human pathogenic Pneumocystis carinii remains unknown. Host to host transmission has been described in animal studies and in cluster cases among immunodeficient patients. P carinii DNA has recently been detected in air filters from inpatient and outpatient rooms in departments of infectious diseases managing patients with P carinii pneumonia (PCP), suggesting the airborne route of transmission. Exposure of staff to P carinii may occur in hospital departments treating patients with PCP. METHODS: Exposure to P carinii was detected by serological responses to human P carinii by ELISA, Western blotting, and indirect immunofluorescence in 64 hospital staff with and 79 staff without exposure to patients with PCP from Denmark and Sweden. DNA amplification of oropharyngeal washings was performed on 20 Danish staff with and 20 staff without exposure to patients with PCP. RESULTS: There was no significant difference in the frequency or level of antibodies to P carinii between staff exposed and those unexposed to patients with PCP. None of the hospital staff had detectable P carinii DNA in oropharyngeal washings. CONCLUSIONS: There is no difference in antibodies and no detectable P carinii DNA in oropharyngeal washings, which suggests that immunocompetent staff treating patients with PCP are not a potentially infectious source of P carinii for immunocompromised patients.

In utero transmission of Pneumocystis carinii sp. f. oryctolagi.

Although vertical transmission of Pneumocystis in human or animal hosts has often been suspected, no evidence demonstrating this infection route has been furnished until now. This widespread parasite is constantly found in the lungs of rabbits, which spontaneously develop a benign pneumocystosis at weaning. However, the infection source, the method of entry of Pneumocystis organisms into the rabbit and when this mammal is infected, remain to be known. As a few parasites have been microscopically observed and detected by PCR in the lungs of rabbits at birth, in utero Pneumocystis infection has been hypothesized. The presence of Pneumocystis was therefore carefully assessed in 16 pregnant does, their embryos and fetuses by using several detection methods. Pneumocystis was detected by PCR in maternal blood, embryos, amniotic fluid and fetuses. The parasite was also revealed histologically and by immunofluorescence in fetal and maternal lungs and in placentas. The results suggest that vertical transmission of P. carinii sp. f. oryctolagi occurs as early as at the 10th day of pregnancy.