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Infection with "Pneumocystis" causes a ≥ 99% depletion of plasma S-adenosylmethionine (AdoMet) levels in both "Pneumocystis" pneumonia (PcP) animal models and patients. AdoMet is a critical cellular metabolic intermediate, with a pivotal role as methyl donor in a myriad of biochemical processes and necessary for the synthesis of the essential polyamines spermidine and spermine. In the target tissue of "Pneumocystis", the lung, levels of AdoMet were previously shown to be depleted experimentally using nicotine. Here we show that chronic administration of nicotine in an animal model of PcP resulted in decreased lung AdoMet content. Since "Pneumocystis" is dependent on this metabolite, PcP burden was also relived. We hypothesized that the underlying mechanism behind nicotine-induced AdoMet depletion was an increased consumption of AdoMet through the polyamine pathway where the increased activity of N-1-spermidine/spermine acetyl transferase raises the catabolic / anabolic cycling of polyamines, a process that utilizes AdoMet. In a critical test of our hypothesis, we found that blockage of polyamine metabolism via inhibition of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) hinders the effect of nicotine on lung AdoMet levels. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of AdoMet toward polyamine synthesis and away from methylation reactions; these shifts are also reversed by inhibition of ODC. Because the nicotine effect on "Pneumocystis" is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection (LCM), we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ODC activity in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on "Pneumocystis" pneumonia. Our studies demonstrate the feasibility of pharmacological manipulation of the polyamine pathway in order to reduce AdoMet levels in the lung and prompted the assessment of compounds alternative to nicotine with the potential to achieve a comparable effect. In vitro evaluation of the polyamine analog DENSPM along with putrescine in type II alveolar cell lines, indicates that although such a combination has the potential to induce polyamine flux, an apparent competition for the same polyamine transport system impairs simultaneous uptake of both compounds at effective concentrations. In conclusion, we showed that chronic nicotine administration causes reduction of AdoMet levels in rat lung following 21 days of treatment, by a mechanism involving the induction of polyamine flux, which is responsible of increased AdoMet utilization for polyamine biosynthesis. According to LCM-based analysis, this effect seems to be confined to the alveolar regions of the lung. / Biochemistry

Le genre Pneumocystis regroupe des microchampignons atypiques qui colonisent par voie respiratoire les alvéoles pulmonaires de nombreux mammifères. C’est un pathogène opportuniste qui s’avère particulièrement dangereux lorsque le système immunitaire de l’hôte est déficient (VIH, greffés) et dans ce cas il provoque une pneumonie, la pneumocytsose, fatale en absence de traitement. Du fait que les Pneumocystis spp restent des microchampignons non cultivables, il est alors impossible de manipuler directement ses gènes et la seule solution pour étudier leurs fonctions et leurs localisations, demeure leur expression en système hétérologue. Situé dans l'espace alvéolaire, Pneumocystis spp. sont exposés au stress oxydatif générés par les macrophages alvéolaires, les granulocytes neutrophiles ainsi que par les espèces réactives de l'oxygène (EROs) produits par le métabolisme respiratoire. Pour se protéger, ces micro-organismes ont développé un système spécifique de détoxification des EROs incluant les SuperOxyde Dismutases (SOD). Dans notre étude, la carence en MnSOD d'une souche de Saccharomyces cerevisiae (EG110) dépourvue du gène Scsod2 a été complétée par l'introduction d'un plasmide portant une version inductible du gène Sod2 de P. carinii (Pcsod2). Une fois exprimée la PcMnSOD était capable de complémenter le défaut de croissance de la souche EG110 qui a été exposés à la ménadione. En bref, notre étude a montré une bonne complémentation du gène Sod2 de P. carinii chez une levure déficiente en MnSOD à savoir (i) la reprise de la culture en conditions de stress oxydant, (ii) la mise en évidence de la protéine traduite (Western Blot) et (iii) l’adressage mitochondriale de la protéine hétérologue. Selon le degré d’altération du système immunitaire, les infections à P. jirovecii peuvent présenter des tableaux cliniques variés allant de la colonisation à la pneumocystose. Ces infections semblent être en grande partie liées à des déficits majeurs de l’immunité cellulaire se traduisant plus précisément par une diminution du nombre des lymphocytes TCD4 (LTCD4). Notre deuxième objectif était de parvenir à une appréciation quantitative du risque de contamination par P. carinii en fonction du degré d’immunodépression (ID) des rats exposés. Nous avons ainsi développé un modèle animal de transmission naturelle de P. carinii où des rats nude développant une pneumocystose (« rats donneurs ») sont mis en contact direct avec des rats Sprague Dawley Pneumocystis-free (« rats receveurs ») présentant différents niveaux d’ID (dexamethasone). Après 2 semaines de contact, le niveau de colonisation des rats graduellement ID est déterminé soit par comptage après coloration au Bleu de Toluidine O, soit par qPCR. Cette étude a permis tout d’abord de valider notre modèle d’ID graduelle chez le rat ; mais surtout, et pour la première fois dans un modèle expérimentale chez le rat, nous avons montré une relation inverse entre le niveau de colonisation par P. carinii et le taux de LTCD4 ou LTCD8 circulants. Enfin, nous avons réalisé la première étude épidémiologique portant sur Pneumocystis au Liban. Ce projet franco-libanais a été mis en place au vue de l’importance majeure de la colonisation par Pneumocystis chez les patients immunocompétents, en particulier chez les patients atteints de pathologies pulmonaires chroniques obstructives tels que la BPCO où la colonisation par Pneumocystis est considérée comme un facteur aggravant de la maladie. Nos résultats montrent une faible prévalence de colonisation (5.2%) et une prédominance du génotype mtLSU2 chez les patients atteints de pathologies respiratoires au Liban. De plus, dans notre cohorte de patient présentant des pathologies respiratoires variées, la BPCO semble être la seule maladie respiratoire associée à un facteur de risque de colonisation par P. jirovecii. / Pneumocystis is an opportunistic pulmonary fungal pathogen that causes Pneumocystis pneumonia (PcP) in immunocompromised individuals such as patients with HIV infection as well as those without HIV infection who are undergoing immunosuppression as a consequence of chemotherapy or organ transplantation. Pneumocystis colonization in immunocompetent individuals has recently been described by the detection of fungal DNA without signs or symptoms of pneumonia, and accumulating evidences underline its clinical importance. Pneumocystis organisms are airborne transmitted and represent a large group of species of atypical fungi that cannot be continuously grown in culture. Consequently, it is impossible to directly manipulate genes in Pneumocystis species. Located in the alveolar space, Pneumocystis organisms are exposed to oxidative burst from phagocytic alveolar macrophages and neutrophils as well as to reactive oxygen species (ROS) produced by the mitochondrial oxygen metabolism. To counteract this, microorganisms have developed a ROS detoxifying system. This includes superoxide dismutases (SOD). In the present study, the MnSOD deficiency of a Saccharomyces cerevisiae mutant strain was complemented by introducing a plasmid carrying an inducible version of the P. carinii Sod2 gene (Pcsod2). Expression of Pcsod2 revealed that the corresponding MnSOD recombinant protein could complement the growth defect in the mutant yeast strain when cells were exposed to menadione. The mitochondrial localization was confirmed by immuno-colocalization of the P. carinii recombinant MnSOD with the yeast mitochondrial Cox4 protein. These results suggest that Pcsod2 encodes an active MnSOD that is targeted to the mitochondrion. This work increases our understanding of the antioxidant defense mechanisms deployed by the Pneumocystis organisms.The adaptive host response to Pneumocystis infection involves humoral and cellular immune responses working in concert to promote the clearance of infection. Depending on the degree of alteration of the immune system, Pneumocystis infections may have various clinical presentations going from colonization to the most severe form (PcP).These infections appear to be largely related to major deficits in cellular immunity and are more closely reflecting a decrease in the number of CD4 T lymphocytes (LTCD4). Our objective was to achieve a quantitative assessment of the risk of contamination by Pneumocystis depending on the degree of immunosuppression (ID) of the exposed host. Thus, we developed an animal model of natural transmission of P. carinii where rats undergoing gradual ID (dexamethasone) named receivers, are cohoused with nude rats developing PcP, named donors. Following contact between receiver and donor rats, the level of colonization by Pneumocystis of receiver rats is determined by toluidine blue O staining or by qPCR. This study allowed us to validate our gradual ID rat model, in the sense that we were able to maintain the level of circulating LTCD4 and LTCD8 stable. Finally, and for the first time in an experimental rat model, we observed an inverse relationship between the level of colonization by P. carinii and the level of circulating LTCD4 and LTCD8.Finally, we aimed to acquire the first data concerning the prevalence of P. jirovecii in the Lebanese population. This Franco-Lebanese project was set up because the colonization by Pneumocystis is probably of major importance in the public health, especially in susceptible patients such as patients with chronic obstructive pulmonary diseases (COPD) where Pneumocystis is considered as a worsening prognosis factor. Our results show a low prevalence of P. jirovecii colonization (5.2%) and the predominance of mtLSU genotype 2 in patients with respiratory diseases in Lebanon. Moreover, in our cohort of patients with various respiratory diseases, COPD was the only respiratory disease associated with a significant increased risk of P. jirovecii colonization.

As doenças respiratórias constituem um sério problema em sistemas intensivos de criação de suínos, causando enormes prejuízos à industria suína no Brasil e no mundo. Estes prejuízos estão freqüentemente relacionados à redução de peso, mortalidade, maior predisposição a doenças entéricas, gastos com vacinas e medicamentos. Os distúrbios respiratórios em suínos são manifestados através de um complexo de doenças, com envolvimento de agentes virais, bacterianos e fúngicos. Dentre estes, a presença do circovírus (PCV2) e o Pneumocystis sp. começa a ser gradativamente caracterizada como uma associação entre um agente causador de imunossupressão e um organismo de ação oportunista. O trabalho objetivou diagnosticar Pneumocystis sp. através das técnicas de imunohistoquímica, Grocott e nested-PCR, em suínos abatidos nos Estados do Rio Grande do Sul (RS) e Mato Grosso (MT), diagnosticar a ocorrência de PCV2 na mesma população de suínos, verificar a associação entre Pneumocystis sp. e PCV2, e determinar as relações filogenéticas entre as amostras de Pneumocystis sp. O estudo avaliou um total de 591 pulmões, 297 com alterações macroscópicas (pneumonia) e 294 normais obtidos em frigoríficos. Foram analisados 292 pulmões procedentes do RS e 299 pulmões do MT Para diagnóstico do Pneumocystis sp. as amostras foram analisadas através das técnicas de Grocott, Imunohistoquímica e nested-PCR (mtLSU e mtSSU rRNA). Do total das amostras, 36,9% foram positivas para Pneumocystis. O índice de positividade para o vírus PCV2 foi de 32,7% na amostra total. Os resultados revelaram uma alta prevalência do vírus (PCV2) em pulmões sem lesões macroscópicas. A co-infecção (PCV2 e Pneumocystis sp.), foi detectada em 28,0% em 564 pulmões examinados. As análises das seqüências dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis sp. nos pulmões analisados, sugerem a presença até o presente momento de 2 espécies diferentes de Pneumocystis no Brasil. Este estudo evidencia a ocorrência da co-infecção de dois agentes (Pneumocystis sp. e PCV2) em animais hígidos, fato que, indica a necessidade de planejamento e implementação de medidas de controle para melhorar a produtividade na suinocultura. / Respiratory diseases are a major problem in intensive systems of swine husbandry. They are a cause for high losses in the swine industry in Brazil and in the world. These losses are often related to weight reduction, mortality, higher vulnerability to enteric diseases, and expenses with vaccines and drugs. Respiratory diseases in swine appear through a complex of diseases, caused by virus, bacteria and fungi; among these, the porcine circovirus 2 (PCV2) and Pneumocystis sp., the former an agent which causes immunosupression and the latter an oportunistic microorganism. Both are being recognized as capable of being associated. The objectives of this study were to: identify Pneumocystis sp. through immunohystochemistry techniques, Grocott and nested-PCR in swine slaughtered in the States of Rio Grande do Sul and Mato Grosso (MT); investigate PCV2 in the same swine population; investigate the association between Pneumocystis sp. and PCV2, and establish a filogenetic relationship between isolated of Pneumocystis sp. The study was carried out with a total of 591 lungs, 297 with macroscopic alterations characteristic of pneumonia, and 294 normal lungs from the industry. 292 lungs came from RS and 299 lungs came from MT. In order, to diagnose Pneumocystis infection, samples were analysed through Grocott technique, immunohystochemistry and nested-PCR (mtLSU and mtSSU rRNA). Among all samples 36,9% were positive for Pneumocystis sp.. PCV2 virus was found in 37,2% of the samples. Results revealed a high prevalence of the PCV2 virus in lungs without macroscopic lesions. Co-infection (PCV2 and Pneumocystis sp.) was found in 28,0% of 564 lungs examined. So far, the analyses of the sequences of nucleotides from the products of PCR from the genes mtlSU rRNA and mtSSU rRNA from Pneumocystis obtained from the examined lungs suggest that, it is possible the existence of two different species of Pneumocystis in Brazil. This study shows co-infection by two agents (Pneumocysts sp. and PCV2) in apparently healthy animals. This fact points out the necessity planning and implementation of control measures in order to improve productiviy in swine husbandry and industry.

Introdução O Pneumocystis jirovecii causa uma grave infecção oportunista nos pacientes imunossuprimidos. Como o P. jirovecii é específico do ser humano, o reservatório deve ser constituído pelos indivíduos que apresentam a pneumonia ou a colonização pelo fungo. A colonização foi estudada somente em grupos específicos (pacientes infectados pelo vírus HIV, pacientes com doenças pulmonares crônicas, crianças com a primoinfecção pelo microorganismo, entre outros) e não foi ainda comprovada a colonização de indivíduos adultos saudáveis na população em geral. A distribuição do reservatório do P. jirovecii na comunidade, portanto, não é conhecida. Objetivos Estudar a colonização pelo P. jirovecii na comunidade da região geográfica de abrangência da Unidade Básica de Saúde (UBS) do Hospital de Clínicas de Porto Alegre (HCPA). Determinar a prevalência da colonização; investigar a associação entre dados clínicos ou demográficos e colonização; verificar se adultos saudáveis são colonizados pelo P. jirovecii. Material e métodos Foram selecionados aleatoriamente 405 indivíduos que residem na área atendida pela UBS/HCPA. O lavado de orofaringe e os dados clínicos e demográficos foram coletados no decorrer do ano de 2010. A presença do P. jirovecii no lavado de orofaringe foi investigada através de uma nested-PCR que amplifica a grande subunidade do RNA ribossômico mitocondrial (mtLSUrRNA), a qual foi realizada no Centro de Pesquisa Experimental do HCPA. Os dados obtidos foram submetidos às análises estatísticas univariada e multivariada. Resultados A colonização pelo P. jirovecii foi observada em 7,7% (31/405) dos indivíduos. A análise estatística univariada demonstrou que a presença do fungo está associada ao tabagismo, à doença respiratória crônica e à idade (as crianças e os idosos apresentam-se mais colonizados que os adultos e adolescentes). Na análise multivariada, o tabagismo e a idade foram variáveis cuja associação com a colonização foi independente. Dentre os indivíduos colonizados, três eram adultos saudáveis. Conclusões Uma prevalência de 7,7% (31/405) de colonização entre os indivíduos da população foi identificada. O tabagismo e a idade (crianças e idosos) estão associados à colonização na análise multivariada, o que confirma evidências prévias. Entre os indivíduos colonizados, cerca de 10% (3/31) são pessoas adultas e saudáveis, um grupo que este estudo revela como parte do reservatório do fungo. A prevalência de colonização identificada no estudo, distribuída em diferentes indivíduos da população, sugere que um grande reservatório do P. jirovecii é dinâmico e é mantido através da transmissão do fungo entre os habitantes da região. / Introduction Pneumocystis jirovecii is the cause of a serious opportunistic infection in immunocompromised patients. As a specifically human microorganism, its reservoir is made up of individuals that have pneumonia or are colonized. Colonization has been studied only in specific groups, such as patients with HIV infection or chronic pulmonary diseases and children with primary infection by this microorganism. Colonization in healthy adults in the general population has not been confirmed. The distribution of the P. jirovecii reservoir in the community is, therefore, unknown. Objectives To study colonization by P. jirovecii in the community of the geographic area of the Primary Health Care Unit of Hospital de Clínicas de Porto Alegre (UBS/HCPA); to determine the prevalence of colonization; to evaluate the association between clinical or demographic data and colonization; to investigate whether healthy adults are colonized by P. jirovecii. Material and methods This study randomly enrolled 405 individuals that lived in the geographic area of UBS/HCPA. Oropharyngeal wash specimens and clinical and demographic data were collected along 2010. The presence of P. jirovecii in oropharyngeal wash specimens was investigated using nested PCR, which amplifies the mitochondrial large subunit ribosomal RNA (mtLSUrRNA). Tests were conducted in the Experimental Research Center of HCPA. Univariate and multivariate statistical analyses were used to evaluate the data collected. Results Colonization by P. jirovecii was found in 7.7% (31/405) of the individuals. Univariate statistical analysis revealed that the presence of the fungus was associated with smoking, chronic respiratory diseases and age; children and older adults were more often colonized than adults and adolescents. In multivariate analysis, only smoking and age were independently associated with colonization. Of the individuals with colonization, three were healthy adults. Conclusions The prevalence of colonization among individuals in the population was 7.7% (31/405). Smoking and age (children and older adults) were associated with colonization in multivariate analysis, which confirms previous findings. About 10% (3/31) of all colonized individuals were healthy adults, a group that seems to be part of the reservoir of the fungus. In this study, colonization was distributed among different individuals in the population, which suggests that a large and dynamic reservoir of P. jirovecii is kept by fungus transmission between inhabitants of the region.

As doenças respiratórias constituem um sério problema em sistemas intensivos de criação de suínos, causando enormes prejuízos à industria suína no Brasil e no mundo. Estes prejuízos estão freqüentemente relacionados à redução de peso, mortalidade, maior predisposição a doenças entéricas, gastos com vacinas e medicamentos. Os distúrbios respiratórios em suínos são manifestados através de um complexo de doenças, com envolvimento de agentes virais, bacterianos e fúngicos. Dentre estes, a presença do circovírus (PCV2) e o Pneumocystis sp. começa a ser gradativamente caracterizada como uma associação entre um agente causador de imunossupressão e um organismo de ação oportunista. O trabalho objetivou diagnosticar Pneumocystis sp. através das técnicas de imunohistoquímica, Grocott e nested-PCR, em suínos abatidos nos Estados do Rio Grande do Sul (RS) e Mato Grosso (MT), diagnosticar a ocorrência de PCV2 na mesma população de suínos, verificar a associação entre Pneumocystis sp. e PCV2, e determinar as relações filogenéticas entre as amostras de Pneumocystis sp. O estudo avaliou um total de 591 pulmões, 297 com alterações macroscópicas (pneumonia) e 294 normais obtidos em frigoríficos. Foram analisados 292 pulmões procedentes do RS e 299 pulmões do MT Para diagnóstico do Pneumocystis sp. as amostras foram analisadas através das técnicas de Grocott, Imunohistoquímica e nested-PCR (mtLSU e mtSSU rRNA). Do total das amostras, 36,9% foram positivas para Pneumocystis. O índice de positividade para o vírus PCV2 foi de 32,7% na amostra total. Os resultados revelaram uma alta prevalência do vírus (PCV2) em pulmões sem lesões macroscópicas. A co-infecção (PCV2 e Pneumocystis sp.), foi detectada em 28,0% em 564 pulmões examinados. As análises das seqüências dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis sp. nos pulmões analisados, sugerem a presença até o presente momento de 2 espécies diferentes de Pneumocystis no Brasil. Este estudo evidencia a ocorrência da co-infecção de dois agentes (Pneumocystis sp. e PCV2) em animais hígidos, fato que, indica a necessidade de planejamento e implementação de medidas de controle para melhorar a produtividade na suinocultura. / Respiratory diseases are a major problem in intensive systems of swine husbandry. They are a cause for high losses in the swine industry in Brazil and in the world. These losses are often related to weight reduction, mortality, higher vulnerability to enteric diseases, and expenses with vaccines and drugs. Respiratory diseases in swine appear through a complex of diseases, caused by virus, bacteria and fungi; among these, the porcine circovirus 2 (PCV2) and Pneumocystis sp., the former an agent which causes immunosupression and the latter an oportunistic microorganism. Both are being recognized as capable of being associated. The objectives of this study were to: identify Pneumocystis sp. through immunohystochemistry techniques, Grocott and nested-PCR in swine slaughtered in the States of Rio Grande do Sul and Mato Grosso (MT); investigate PCV2 in the same swine population; investigate the association between Pneumocystis sp. and PCV2, and establish a filogenetic relationship between isolated of Pneumocystis sp. The study was carried out with a total of 591 lungs, 297 with macroscopic alterations characteristic of pneumonia, and 294 normal lungs from the industry. 292 lungs came from RS and 299 lungs came from MT. In order, to diagnose Pneumocystis infection, samples were analysed through Grocott technique, immunohystochemistry and nested-PCR (mtLSU and mtSSU rRNA). Among all samples 36,9% were positive for Pneumocystis sp.. PCV2 virus was found in 37,2% of the samples. Results revealed a high prevalence of the PCV2 virus in lungs without macroscopic lesions. Co-infection (PCV2 and Pneumocystis sp.) was found in 28,0% of 564 lungs examined. So far, the analyses of the sequences of nucleotides from the products of PCR from the genes mtlSU rRNA and mtSSU rRNA from Pneumocystis obtained from the examined lungs suggest that, it is possible the existence of two different species of Pneumocystis in Brazil. This study shows co-infection by two agents (Pneumocysts sp. and PCV2) in apparently healthy animals. This fact points out the necessity planning and implementation of control measures in order to improve productiviy in swine husbandry and industry.

Introdução O Pneumocystis jirovecii causa uma grave infecção oportunista nos pacientes imunossuprimidos. Como o P. jirovecii é específico do ser humano, o reservatório deve ser constituído pelos indivíduos que apresentam a pneumonia ou a colonização pelo fungo. A colonização foi estudada somente em grupos específicos (pacientes infectados pelo vírus HIV, pacientes com doenças pulmonares crônicas, crianças com a primoinfecção pelo microorganismo, entre outros) e não foi ainda comprovada a colonização de indivíduos adultos saudáveis na população em geral. A distribuição do reservatório do P. jirovecii na comunidade, portanto, não é conhecida. Objetivos Estudar a colonização pelo P. jirovecii na comunidade da região geográfica de abrangência da Unidade Básica de Saúde (UBS) do Hospital de Clínicas de Porto Alegre (HCPA). Determinar a prevalência da colonização; investigar a associação entre dados clínicos ou demográficos e colonização; verificar se adultos saudáveis são colonizados pelo P. jirovecii. Material e métodos Foram selecionados aleatoriamente 405 indivíduos que residem na área atendida pela UBS/HCPA. O lavado de orofaringe e os dados clínicos e demográficos foram coletados no decorrer do ano de 2010. A presença do P. jirovecii no lavado de orofaringe foi investigada através de uma nested-PCR que amplifica a grande subunidade do RNA ribossômico mitocondrial (mtLSUrRNA), a qual foi realizada no Centro de Pesquisa Experimental do HCPA. Os dados obtidos foram submetidos às análises estatísticas univariada e multivariada. Resultados A colonização pelo P. jirovecii foi observada em 7,7% (31/405) dos indivíduos. A análise estatística univariada demonstrou que a presença do fungo está associada ao tabagismo, à doença respiratória crônica e à idade (as crianças e os idosos apresentam-se mais colonizados que os adultos e adolescentes). Na análise multivariada, o tabagismo e a idade foram variáveis cuja associação com a colonização foi independente. Dentre os indivíduos colonizados, três eram adultos saudáveis. Conclusões Uma prevalência de 7,7% (31/405) de colonização entre os indivíduos da população foi identificada. O tabagismo e a idade (crianças e idosos) estão associados à colonização na análise multivariada, o que confirma evidências prévias. Entre os indivíduos colonizados, cerca de 10% (3/31) são pessoas adultas e saudáveis, um grupo que este estudo revela como parte do reservatório do fungo. A prevalência de colonização identificada no estudo, distribuída em diferentes indivíduos da população, sugere que um grande reservatório do P. jirovecii é dinâmico e é mantido através da transmissão do fungo entre os habitantes da região. / Introduction Pneumocystis jirovecii is the cause of a serious opportunistic infection in immunocompromised patients. As a specifically human microorganism, its reservoir is made up of individuals that have pneumonia or are colonized. Colonization has been studied only in specific groups, such as patients with HIV infection or chronic pulmonary diseases and children with primary infection by this microorganism. Colonization in healthy adults in the general population has not been confirmed. The distribution of the P. jirovecii reservoir in the community is, therefore, unknown. Objectives To study colonization by P. jirovecii in the community of the geographic area of the Primary Health Care Unit of Hospital de Clínicas de Porto Alegre (UBS/HCPA); to determine the prevalence of colonization; to evaluate the association between clinical or demographic data and colonization; to investigate whether healthy adults are colonized by P. jirovecii. Material and methods This study randomly enrolled 405 individuals that lived in the geographic area of UBS/HCPA. Oropharyngeal wash specimens and clinical and demographic data were collected along 2010. The presence of P. jirovecii in oropharyngeal wash specimens was investigated using nested PCR, which amplifies the mitochondrial large subunit ribosomal RNA (mtLSUrRNA). Tests were conducted in the Experimental Research Center of HCPA. Univariate and multivariate statistical analyses were used to evaluate the data collected. Results Colonization by P. jirovecii was found in 7.7% (31/405) of the individuals. Univariate statistical analysis revealed that the presence of the fungus was associated with smoking, chronic respiratory diseases and age; children and older adults were more often colonized than adults and adolescents. In multivariate analysis, only smoking and age were independently associated with colonization. Of the individuals with colonization, three were healthy adults. Conclusions The prevalence of colonization among individuals in the population was 7.7% (31/405). Smoking and age (children and older adults) were associated with colonization in multivariate analysis, which confirms previous findings. About 10% (3/31) of all colonized individuals were healthy adults, a group that seems to be part of the reservoir of the fungus. In this study, colonization was distributed among different individuals in the population, which suggests that a large and dynamic reservoir of P. jirovecii is kept by fungus transmission between inhabitants of the region.

As doenças respiratórias constituem um sério problema em sistemas intensivos de criação de suínos, causando enormes prejuízos à industria suína no Brasil e no mundo. Estes prejuízos estão freqüentemente relacionados à redução de peso, mortalidade, maior predisposição a doenças entéricas, gastos com vacinas e medicamentos. Os distúrbios respiratórios em suínos são manifestados através de um complexo de doenças, com envolvimento de agentes virais, bacterianos e fúngicos. Dentre estes, a presença do circovírus (PCV2) e o Pneumocystis sp. começa a ser gradativamente caracterizada como uma associação entre um agente causador de imunossupressão e um organismo de ação oportunista. O trabalho objetivou diagnosticar Pneumocystis sp. através das técnicas de imunohistoquímica, Grocott e nested-PCR, em suínos abatidos nos Estados do Rio Grande do Sul (RS) e Mato Grosso (MT), diagnosticar a ocorrência de PCV2 na mesma população de suínos, verificar a associação entre Pneumocystis sp. e PCV2, e determinar as relações filogenéticas entre as amostras de Pneumocystis sp. O estudo avaliou um total de 591 pulmões, 297 com alterações macroscópicas (pneumonia) e 294 normais obtidos em frigoríficos. Foram analisados 292 pulmões procedentes do RS e 299 pulmões do MT Para diagnóstico do Pneumocystis sp. as amostras foram analisadas através das técnicas de Grocott, Imunohistoquímica e nested-PCR (mtLSU e mtSSU rRNA). Do total das amostras, 36,9% foram positivas para Pneumocystis. O índice de positividade para o vírus PCV2 foi de 32,7% na amostra total. Os resultados revelaram uma alta prevalência do vírus (PCV2) em pulmões sem lesões macroscópicas. A co-infecção (PCV2 e Pneumocystis sp.), foi detectada em 28,0% em 564 pulmões examinados. As análises das seqüências dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis sp. nos pulmões analisados, sugerem a presença até o presente momento de 2 espécies diferentes de Pneumocystis no Brasil. Este estudo evidencia a ocorrência da co-infecção de dois agentes (Pneumocystis sp. e PCV2) em animais hígidos, fato que, indica a necessidade de planejamento e implementação de medidas de controle para melhorar a produtividade na suinocultura. / Respiratory diseases are a major problem in intensive systems of swine husbandry. They are a cause for high losses in the swine industry in Brazil and in the world. These losses are often related to weight reduction, mortality, higher vulnerability to enteric diseases, and expenses with vaccines and drugs. Respiratory diseases in swine appear through a complex of diseases, caused by virus, bacteria and fungi; among these, the porcine circovirus 2 (PCV2) and Pneumocystis sp., the former an agent which causes immunosupression and the latter an oportunistic microorganism. Both are being recognized as capable of being associated. The objectives of this study were to: identify Pneumocystis sp. through immunohystochemistry techniques, Grocott and nested-PCR in swine slaughtered in the States of Rio Grande do Sul and Mato Grosso (MT); investigate PCV2 in the same swine population; investigate the association between Pneumocystis sp. and PCV2, and establish a filogenetic relationship between isolated of Pneumocystis sp. The study was carried out with a total of 591 lungs, 297 with macroscopic alterations characteristic of pneumonia, and 294 normal lungs from the industry. 292 lungs came from RS and 299 lungs came from MT. In order, to diagnose Pneumocystis infection, samples were analysed through Grocott technique, immunohystochemistry and nested-PCR (mtLSU and mtSSU rRNA). Among all samples 36,9% were positive for Pneumocystis sp.. PCV2 virus was found in 37,2% of the samples. Results revealed a high prevalence of the PCV2 virus in lungs without macroscopic lesions. Co-infection (PCV2 and Pneumocystis sp.) was found in 28,0% of 564 lungs examined. So far, the analyses of the sequences of nucleotides from the products of PCR from the genes mtlSU rRNA and mtSSU rRNA from Pneumocystis obtained from the examined lungs suggest that, it is possible the existence of two different species of Pneumocystis in Brazil. This study shows co-infection by two agents (Pneumocysts sp. and PCV2) in apparently healthy animals. This fact points out the necessity planning and implementation of control measures in order to improve productiviy in swine husbandry and industry.

Introdução O Pneumocystis jirovecii causa uma grave infecção oportunista nos pacientes imunossuprimidos. Como o P. jirovecii é específico do ser humano, o reservatório deve ser constituído pelos indivíduos que apresentam a pneumonia ou a colonização pelo fungo. A colonização foi estudada somente em grupos específicos (pacientes infectados pelo vírus HIV, pacientes com doenças pulmonares crônicas, crianças com a primoinfecção pelo microorganismo, entre outros) e não foi ainda comprovada a colonização de indivíduos adultos saudáveis na população em geral. A distribuição do reservatório do P. jirovecii na comunidade, portanto, não é conhecida. Objetivos Estudar a colonização pelo P. jirovecii na comunidade da região geográfica de abrangência da Unidade Básica de Saúde (UBS) do Hospital de Clínicas de Porto Alegre (HCPA). Determinar a prevalência da colonização; investigar a associação entre dados clínicos ou demográficos e colonização; verificar se adultos saudáveis são colonizados pelo P. jirovecii. Material e métodos Foram selecionados aleatoriamente 405 indivíduos que residem na área atendida pela UBS/HCPA. O lavado de orofaringe e os dados clínicos e demográficos foram coletados no decorrer do ano de 2010. A presença do P. jirovecii no lavado de orofaringe foi investigada através de uma nested-PCR que amplifica a grande subunidade do RNA ribossômico mitocondrial (mtLSUrRNA), a qual foi realizada no Centro de Pesquisa Experimental do HCPA. Os dados obtidos foram submetidos às análises estatísticas univariada e multivariada. Resultados A colonização pelo P. jirovecii foi observada em 7,7% (31/405) dos indivíduos. A análise estatística univariada demonstrou que a presença do fungo está associada ao tabagismo, à doença respiratória crônica e à idade (as crianças e os idosos apresentam-se mais colonizados que os adultos e adolescentes). Na análise multivariada, o tabagismo e a idade foram variáveis cuja associação com a colonização foi independente. Dentre os indivíduos colonizados, três eram adultos saudáveis. Conclusões Uma prevalência de 7,7% (31/405) de colonização entre os indivíduos da população foi identificada. O tabagismo e a idade (crianças e idosos) estão associados à colonização na análise multivariada, o que confirma evidências prévias. Entre os indivíduos colonizados, cerca de 10% (3/31) são pessoas adultas e saudáveis, um grupo que este estudo revela como parte do reservatório do fungo. A prevalência de colonização identificada no estudo, distribuída em diferentes indivíduos da população, sugere que um grande reservatório do P. jirovecii é dinâmico e é mantido através da transmissão do fungo entre os habitantes da região. / Introduction Pneumocystis jirovecii is the cause of a serious opportunistic infection in immunocompromised patients. As a specifically human microorganism, its reservoir is made up of individuals that have pneumonia or are colonized. Colonization has been studied only in specific groups, such as patients with HIV infection or chronic pulmonary diseases and children with primary infection by this microorganism. Colonization in healthy adults in the general population has not been confirmed. The distribution of the P. jirovecii reservoir in the community is, therefore, unknown. Objectives To study colonization by P. jirovecii in the community of the geographic area of the Primary Health Care Unit of Hospital de Clínicas de Porto Alegre (UBS/HCPA); to determine the prevalence of colonization; to evaluate the association between clinical or demographic data and colonization; to investigate whether healthy adults are colonized by P. jirovecii. Material and methods This study randomly enrolled 405 individuals that lived in the geographic area of UBS/HCPA. Oropharyngeal wash specimens and clinical and demographic data were collected along 2010. The presence of P. jirovecii in oropharyngeal wash specimens was investigated using nested PCR, which amplifies the mitochondrial large subunit ribosomal RNA (mtLSUrRNA). Tests were conducted in the Experimental Research Center of HCPA. Univariate and multivariate statistical analyses were used to evaluate the data collected. Results Colonization by P. jirovecii was found in 7.7% (31/405) of the individuals. Univariate statistical analysis revealed that the presence of the fungus was associated with smoking, chronic respiratory diseases and age; children and older adults were more often colonized than adults and adolescents. In multivariate analysis, only smoking and age were independently associated with colonization. Of the individuals with colonization, three were healthy adults. Conclusions The prevalence of colonization among individuals in the population was 7.7% (31/405). Smoking and age (children and older adults) were associated with colonization in multivariate analysis, which confirms previous findings. About 10% (3/31) of all colonized individuals were healthy adults, a group that seems to be part of the reservoir of the fungus. In this study, colonization was distributed among different individuals in the population, which suggests that a large and dynamic reservoir of P. jirovecii is kept by fungus transmission between inhabitants of the region.

Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2005. / Pneumocystis jirovecii is a significant contributor to the burden of disease in immunocompromised patients. The polymerase chain reaction (PCR) is more sensitive and specific than microscopy. Cotrimoxazole prophylactic breakthrough and treatment failures have been reported, and associated with mutations at codons 55 and 57 of P. jirovecii dihydropteroate synthase (DHPS). No phylogenetic or population genetic models have been successful in elucidating P. jirovecii intraspecies strain relatedness. Aims: 1) Compare detection rates of nine PCR techniques and immunofluorescence microscopy (IF); 2) Determine the extent of co-infecting pathogens associated with Pneumocystis Pneumonia (PcP); 3) Determine local P. jirovecii ITS1-5.8S-ITS2 rDNA strain types, and model lineage evolution employing a coalescent-theory based statistical parsimony network analysis; 4) Investigate the possible emergence of cotrimoxazole-resistant strains Methods: PCR was evaluated on clinical specimens employing: ITS nested; DHPS single and nested; DHFR nested; major surface glycoprotein (MSG) heminested; mitochondrial large subunit rRNA (mtLSUrRNA) single and nested; 18S rRNA onetube nested, and real-time 5S rRNA PCR. Retrospective analysis of co-infecting pathogens seen in PcP patients was conducted. ITS regions were amplified, cloned and sequenced. Statistical parsimony was applied for coalescence based network genotype analysis. DHPS genome walking was attempted and DHPS and DHFR primer annealing sites explored. Amplified DHPS and DHFR genes were cloned and sequenced. Results: Most sensitive PCR technique was mtLSUrRNA nested followed by 5S realtime PCR. A poor correlation exist between mtLSUrRNA PCR and IF. Review of clinical records suggested a high rate of false-positive IF results. P. jirovecii was detected in 4.3% M. tuberculosis-positive HIV-positive, and 2.5% M. tuberculosispositive HIV-negative patients. P. jirovecii was detected in 45% HIV-negative patients. The most prevalent ITS type was Eg. Four new combinations: Eo, Je, Ge, No; 11 new ITS1 and 13 new ITS2 sequences were identified. A new ITS2 type was detected in three patients and designated u. More than one strain type was detected in 15/19 patients. Analysis of 5.8SrDNA region revealed 13 clones containing 1-2 nucleotide polymorphisms. Of 85 mtLSUrRNA PCR-positive specimens, currently employed primers amplified DHPS and DHFR genes from 53 and 27 specimens, respectively. Newly designed DHPS primers increased detection in 3 / 28 previously DHPS-negative mtLSUrRNA-positive specimens. Of 56 DHPS genes amplified and sequenced, one contained the double mutation (Thr55Aa; Pro57Ser). DHFR Ala67Val was detected in three specimens and a new DHFR genotype (Arg59Gly; C278T) was demonstrated. Conclusions: The study emphasises the need to evaluate PCR primers against local strains. It is recommended that mtLSUrRNA PCR be performed in parallel to IF and discordant results resolved with clinical evaluation. Co-infection with P. jirovecii and M. tuberculosis occurs in South Africa, and treatment for both pathogens is recommended when demonstrated by the laboratory. ITS genotyping employing statistical parsimony network analysis suggests type Eg as major ancestral haplotype, and supports recombination contributing to strain diversity worldwide. DHPS mutations may signal emergence of resistance to cotrimoxazole in South Africa, however, low sensitivity of primers limits surveillance efforts.