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Epidemiologia e caracterização de espécies implicadas na microsporidiose humana em Portugal por análise parasitológica e molecular
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Os agentes responsáveis pela microsporidiose e, designados, genericamente, por microsporidia são parasitas intracelulares obrigatórios, ubíquos na natureza. Os microsporidia inicialmente reconhecidos como agentes patogénicos de insectos e peixes,
emergiram nas últimas décadas como um importante grupo de microrganismos responsáveis por infecção no Homem, associada à pandemia da sida. Encontram-se descritas na literatura mais de 1200 espécies de microsporidia, mas apenas um número reduzido é considerado infectante para o Homem. Entre os microsporídios mais
prevalentes, responsáveis por infecção humana, encontram-se as espécies Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem e Encephalitozoon cuniculi. Recentemente foi identificada em Portugal, uma nova espécie patogénica para o Homem - Vittaforma-like, em doentes seropositivos para VIH e em imunocompetentes.
No Homem, os microsporídios causam desde patologia gastrintestinal a
infecções disseminadas variando os aspectos patogénicos da doença consoante a espécie envolvida assim como com a resposta imunitária do hospedeiro. Já foram descritos portadores assintomáticos de microsporídios, quer em imunodeficientes quer em imunocompetentes
Actualmente, são ainda escassos os dados epidemiológicos sobre esta parasitose, e poucos os estudos disponíveis sobre os animais identificados como potenciais hospedeiros dos parasitas infectantes para o Homem. Mais recentemente, o contacto com água infectada passou a ser considerado um factor de risco associado à microsporidiose humana.
A recente aplicação de técnicas moleculares ao diagnóstico e caracterização dos microsporidia, permitindo o reconhecimento de diferenças intraespecíficas, tem contribuído para uma melhor compreensão da epidemiologia destes parasitas.
O presente trabalho tem como objectivos: (i) determinar a frequência de infecção de microsporidia patogénicos para o Homem, numa população de seropositivos e seronegativos para VIH, em animais em estreito contacto com humanos e em água de abastecimento público, a fim de identificar, não só os parasitas infectantes para o Homem, como, também, os seus possíveis reservatórios zoonóticos e ambientais; (ii)caracterizar geneticamente as espécies e genótipos de microsporidia identificados na população humana, animal e em amostras de água de consumo público; (iii) avaliar a importância dos microsporidia na Saúde Pública Humana em Portugal.
No intuito de dar prossecução ao presente estudo, de modo a cumprir os objectivos propostos, foram adoptadas metodologias de diagnóstico e caracterização genética dos microsporidia, seleccionadas após extensa pesquisa bibliográfica. Todas as amostras foram analisadas simultaneamente pela técnica parasitológica de coloração Gram-
Chromotrope e por PCR, com excepção das amostras fecais de animais silváticos de vida livre e da água de consumo público, que atendendo às reduzidas quantidades de amostra disponíveis, só foram estudadas pelas técnicas moleculares.
Para o presente trabalho foi efectuado um estudo retrospectivo e prospectivo, englobando 1989 amostras fecais de 856 doentes (adultos e crianças) seropositivos e seronegativos para VIH. A percentagem de doentes seropositivos para VIH estudados foi de 65,5% (561/856) e seronegativos para VIH foi de 34,5% (295/856). Do total de doentes (586 do sexo masculino e 270 do sexo feminino), 675 eram adultos e 181 crianças. Dos doentes analisados 57,6% apresentavam diarreia e 42,4% não apresentavam quadro diarreico. Nove (3,1%) dos 295 seronegativos para VIH apresentavam outras causas de imunossupressão. Uma frequência de infecção por microsporidia de 12,0% (103/856) foi observada nas fezes da população humana
estudada, sendo que 13,9% e 8,5% das amostras fecais pertenciam a seropositivos e a seronegativos para VIH, respectivamente. A percentagem de crianças e adultos positivos para microsporidia foi de 18,8% e 10,2%, respectivamente. Os resultados obtidos permitiram determinar uma associação significativa entre a presença de infecção
por microsporidia e os seropositivos para VIH, facto este que não constituiu um dado surpreendente em virtude do carácter oportunista largamente atribuído a esta doença, por outros autores. Foram identificados esporos de microsporídios nas fezes de doentes,
que, embora sendo seronegativos para VIH, apresentam outro tipo de deficit imunológico. Através das técnicas de PCR, identificaram-se duas espécies de microsporídios nas amostras fecais dos doentes estudados no presente trabalho. E. bieneusi foi detectada em 6,3% (54/856) e Vittaforma-like em 6,8% (58/856) dos doentes estudados. Na análise dos dados obtidos, não se verificou que a infecção por microsporidia, em geral, nem, especificamente, por nenhuma das duas espécies identificadas nas fezes estivesse, significativamente, associada a doentes com quadro diarreico. A caracterização genética da região ITS rRNA de 48 isolados de E. bieneusi obtidos de humanos, de um total de 54 amplificados por PCR, permitiu identificar seis genótipos diferentes, Tipo IV (37,5%), Peru 6 (29,2%), D (12,5%), A (8,3%), C (6,3%)
e PtEbII (6,3%). Até à data os genótipos A e C só foram identificados no Homem. Em contrapartida os genótipos Tipo IV, Peru 6 e genótipo D já foram descritos numa vasta gama de hospedeiros, incluído o Homem e outros animais (inclusive em cães, gatos e ruminantes em Portugal). A caracterização genética dos 58 isolados de Vittaforma-like revelou a presença de dois genótipos diferentes, previamente descritos em humanos: Isolado 5830 (39 amostras) e Isolado 5843 (19 amostras), com frequências de 67,2% e 32,8%, respectivamente.
Para o estudo de microsporidia em amostras de urina, foram recolhidos 69 amostras, retrospectivamente e durante o decurso do trabalho, pertencentes a 69 doentes (59 doentes seropositivos para VIH [85,5%] e 10 doentes seronegativos para VIH [14,5%] sendo 44 adultos (63,8%) e 25 crianças (36,2%). Do total de amostras de urina 48 (69,6%) eram de doentes do sexo masculino e 21 (30,4%) de doentes do sexo
feminino. Foram identificados microsporídios em 1,5% (1/69) das amostras de urina, correspondente a um seropositivo para VIH (1,7%). A espécie identificada na urina foi E. intestinalis e a caracterização da sequência de um fragmento da região SSU rRNA revelou total homologia com diversas sequências previamente descritas em humanos e outros animais.
Para a identificação e caracterização de microsporídios em secreções
pulmonares, foram analisadas 200 amostras, recolhidas retrospectivamente e durante o decorrer do estudo correspondentes a 124 expectorações induzidas (EI) e 76 lavados broncoalveolares (LBA). As amostras pertenciam a 150 doentes seropositivos para VIH
(75%) e a 50 seronegativos para VIH (25%), sendo 129 (64,5%) do sexo masculino e 71 (35,5%) do sexo feminino. A percentagem de infecção por microsporidia foi de 1,0% (2/200) em secreções pulmonares da população estudada. Dois seropositivos (1,3%)
apresentavam microsporídios, nomeadamente num LBA e numa EI. E. cuniculi e Vittaforma-like foram identificadas nos referidos espécimes. A caracterização da SSU rRNA para E. cuniculi revelou 99% de homologia genética nesta sequência com outras sequências previamente descritas em humanos para E. cuniculi, e para a espécieVittaforma-like registou-se homologia genética com o Isolado 5830, previamente
descrito em humanos em Portugal. Ambos os achados constituem os primeiros registos desta espécie em secreções pulmonares no Homem em Portugal.
A detecção e caracterização dos microsporidia em animais, incluiu estudos retrospectivos e prospectivos em amostras fecais de: 66 animais de companhia (oito cães e 58 gatos) e 50 cães errantes albergados em canis; 100 bovinos, provenientes de explorações leiteiras de várias regiões de Portugal; 103 animais cativos no Jardim
Zoológico de Lisboa (75 espécies de mamíferos, 9 espécies de répteis e 9 espécies de aves); 134 pequenos mamíferos capturados nos concelhos de Moura e Aljustrel, Baixo Alentejo (52 insectívoros de espécie Crocidura russula e 80 roedores Mus spretus e dois Apodemus sylvaticus); 39 aves exóticas de gaiola de diversas espécies pertencentes às Ordens Psittaciformes e Passeriformes (31 de um criador de aves e oito de agregados familiares) e 52 pombos urbanos (Ordem Columbiformes), da região da grande Lisboa.
A maioria dos animais dos diversos grupos estudados não apresentava sintomatologia.
Nos diversos grupos de animais estudados foram identificados microsporidia em amostras fecais de 8,6% de cães, 15,5% de gatos 40,7% de aves de gaiola e pombos urbanos, 6,0% de bovinos e 1,9% de animais silváticos em cativeiro (Jardim Zoológico).
E. bieneusi foi identificada nas amostras fecais de cães e gatos analisados, cuja caracterização das sequências ITS rRNA dos 14 isolados de E. bieneusi, amplificados por PCR, veio revelar a presença de seis genótipos distintos: TipoIV, PtEbIV, Peru6, D, PtEbVIII e PtEbIX. Os genótipos PtEbIV, PtEbVIII e PtEbIX, foram descritos pela primeira vez no decurso deste estudo. Nos cães foram identificados os genótipos D (20%; 1/5), Peru6 (60,0%; 3/5) e PtEbIX (20%;1/5) e nos gatos os genótipos TipoIV (77,8%), PtEbIV (11,1%) e PtEbVIII (11,9%). Verificou-se associação com significância estatística da infecção pelo genótipo Tipo IV ao grupo dos gatos, relativamente aos cães estudados. Também nos bovinos, a espécie E. bieneusi foi identificada nas seis amostras positivas estudadas registando-se três genótipos diferentes(dois dos quais já previamente reportados): TipoIV (50%; 3/6), PtEbXI (33,3%; 2/6) e J (16,7%; 1/6) (AF135837). O genótipo PtEbXI foi caracterizado pela primeira vez no decurso deste estudo. A espécie E. bieneusi foi identificada nas amostras fecais de dois
mamíferos do jardim Zoológico: o saguim de face branca (Callithrix geoffroyi) e ocavalo de Timor (Tragelaphus strepsiceros strepsiceros) e a genotipagem através da região ITS rRNA dos isolados de E. bieneusi permitiu caracterizar, pela primeira vez, durante o decurso deste trabalho, dois genótipos distintos: PtEbV (cavalo de Timor) e PtEbXII (saguim de face branca). A análise da diversidade genética da região ITS rRNA dos 10 genótipos diferentes de E. bieneusi identificados num total de 22 isolados de cães, gatos, bovinos e animais do Jardim Zoológico (um primata não humano e um bovídeo) estudados permitiu verificar a presença na maioria destes mamíferos (cães, gatos e bovídeos) de genótipos patogénicos para o Homem e de outros genótipos específicos de hospedeiro (cão, gato, bovinos e primata não humano), que, possivelmente, não terão impacte na saúde pública humana. Nos 134 pequenos mamíferos (Crocidura russula, Mus spretus e Apodemus sylvaticus não foi observado nenhum animal com infecção por microsporidia.
As espécies E. bieneusi e E. hellem foram identificadas, em 35,2% (32/91) e 13,2% (12/91), respectivamente, das aves de gaiola e pombos de parques públicos de Lisboa estudadas. Registou-se co-infecção por ambas as espécies. Ambos os microsporidia foram identificados nas três Ordens de aves: Psittaciformes (E. bieneusi-
12,5%; E. hellem- 12,5%); Passeriformes (E. bieneusi- 14,3%; E. hellem- 42,9%) e Columbiformes (E. bieneusi- 51,9%; E. hellem- 6,9%). Contudo, nos pombos (Columbiformes) registou-se uma elevada frequência de infecção por E. bieneusi, estatísticamente significativa, comparativamente à observada nas outras duas Ordens de aves. Para a espécie E. hellem a percentagem mais elevada de infecção nos
Passeriformes (42,9%; 3/7), não apresentou significância estatística. A caracterização do locus ITS rRNA, dos 32 isolados, pertencentes à espécie E. bieneusi, revelou a presença do genótipo Peru6 descrito pela primeira vez em humanos, no Peru, e foi caracterizado pela primeira vez o genótipo PtEbII muito semelhante ao Peru6, com uma única alteração nucleotídica (G para A) junto da extremidade 3’ do fragmento amplificado por PCR. Em pombos registou-se a presença simultânea dos dois genótipos. Nos 12 isolados de E. hellem identificados nas amostras fecais e/ou conteúdo intestinal e numa
raspagem da traqueia da população de aves analisada, e caracterizados com recurso à sequenciação nucleotídica do fragmento do gene da SSU rRNA, verificou-se 99% a 100% de homologia genética das sequências obtidas com sequências do gene da SSU rRNA, previamente reportadas no homem e em aves (genótipos 1 e 2). No interior dasregiões alvo, registaram-se três locais polimórficos, que permitem agrupar em três tipos diferentes as 12 sequências dos isolados de E. hellem obtidos neste estudo: PtEhI (genótipo1, previamente descrito); PtEhII (99% homologia genética com genótipo 2) e PtEh III (99%de homologia genética com PtEhII).
No presente trabalho, também com o intuito de determinar a presença de esporos de microsporídios, foram analisadas 184 amostras de água, de locais de amostragem envolvidos no processo conducente ao abastecimento de água da cidade de Lisboa (175 amostras processadas de acordo com o método 1623 da USEPA e purificadas por separação imunomagnética [IMS] para os géneros Cryptosporidim spp. e Giardia e nove sem IMS). Cento e seis eram amostras de água tratada (42 captações subterrâneas, 25 captações superficiais, 39 de água de abastecimento), provenientes de três Estações de Tratamento e reservatórios de água de abastecimento da cidade de Lisboa, e 69 eram amostras de águas não tratadas, obtidas em captações superficiais (30 amostras) e na captação subterrânea (30 amostras com IMS e nove sem IMS). As espécies E. bieneusi e Vittaforma-like foram identificadas em água de abastecimento da cidade de Lisboa,
tanto na água de origem superficial como subterrânea. A genotipagem de Vittaformalike revelou a presença de um genótipo Isolado 5830, descrito em humanos, em Portugal. Também o genótipo Tipo IV de E. bieneusi, potencialmente infectante para o homem e outros animais, foi identificado em água tratada com origem subterrânea.
Em conclusão, o presente trabalho permitiu demonstrar a presença de espécies e/ou genótipos de microsporidia patogénicos para o Homem em seropositivos e
seronegativos para VIH, em animais e em águas de consumo público, em Portugal.
Também na nossa população humana, tal como descrito na literatura, a infecção por estes parasitas, , parece afectar os grupos mais susceptíveis, como os doentes com imunossupressão, crianças e idosos. Por outro lado, a identificação dos mesmos genótipos de microsporidia, com destaque para os genótipos de E. bieneusi, em
humanos e animais, sugere a ocorrência de transmissão zoonótica através de animais em estreito contacto com o Homem. Dos resultados obtidos verificou-se que, quer para os humanos, quer para os animais não foi observada associação directa entre a infecção por microsporidia e a presença de sintomatologia, nomeadamente de diarreia, sugerindo a
existência, em ambos os grupos populacionais estudados, de portadores crónicos assintomáticos e salientando a sua importância no ciclo de transmissão destes parasitas.Em suma a análise dos dados deste estudo sugere um papel relevante a nível da Saúde Pública, para os microsporidia circulantes, entre a população portuguesa, evidenciando a necessidade de implementar a pesquisa de microsporídios no diagnóstico de rotina, com recurso a métodos de referência bem definidos e adequados à sua utilização nos laboratórios de rotina. A metodológica adoptada neste estudo, nomeadamente as técnicas de PCR e sequenciação de DNA, traduziu-se numa abordagem eficaz para atingir os objectivos propostos.
Esperamos que os resultados obtidos neste estudo, contribuam para clarificar a
epidemiologia da microsporidiose em Portugal, mais precisamente, os processos envolvidos na circulação e transmissão destes microrganismos oportunistas à população susceptível.
Microsporidia are small eukaryotic, obligate intracellular parasites with a widespread occurrence in animals. There are over 1200 species of microsporidia, but only a few have been identified as cause of human diseases. Before onset of the AIDS pandemic, microsporidia infections had only been described in few cases, but by the expansion of the pandemic, microsporidia came increasingly under attention. The most frequent human infecting microsporidia species are Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon. cuniculi. Recently it has been identified in Portugal, a new species pathogenic to humans - Vittaforma-like, in HIV-positive patients and in immunocompetent persons. These emerging opportunistic organisms have now become important pathogens that can cause a broad spectrum of intestinal and disseminated pathologies depending on the infectious species involved and also on the immune status of the host. Microsporidia asymptomatic carriage was already described among both imunossupressed and immunocompetent population. The epidemiology of human microsporidia infections is still unclear in many aspects. Routes of transmission and sources of human microsporidia infections have been difficult to ascertain. However, the four most common human microsporidian species have been reported in a wide range of domestic and wild animals. Thus, it has been debated for some time now whether animals could be a source of infection of microsporidiosis to humans. More recently, a risk factor consistently associated with human microsporidiosis in epidemiological studies is the contact with water. The use of molecular methods has provided new tools to detect and characterize microsporidia, and the finding of previously unrecognized intraspecific genetic differences has improved our understanding of the epidemiology and zoonotic transmission of microsporidia. This study intended to: (i) confirm the occurrence and determine the frequency of infection of human patogenic microsporidia in HIV-positive and negative patients, in several animals species with close contact with humans (pet dogs, cats and birds, bovines, captive animals from the zoo and pigeons from public parks) and in environmental samples (water), in order to identify, not only the microsporidian speciesenrolled in Portuguese human infection, but also to determine potential animal reservoirs and environmental sources of infection; (ii) to characterize the parasites found at the genotype level; (iii) to evaluate the public health significance of microsporidia in Portugal. The frequency of microsporidia, genetic diversity, distribution of genotypes and the relationship between observed genotypes and different parameters of infection among humans, animals and environmental samples enrolled in the study were investigated using SSU rRNA and ITS-based PCR, followed by DNA sequencing. In order to achieve the objectives proposed in this study were adopted methodologies for diagnosis and genetic characterization of microsporidia, selected after an extensive literature search. All samples were analyzed by both the parasitological Gram-Chromotrope staining and PCR-techniques, with the exception of fecal samples from free-living animals and water for public consumption. Water samples due to the small amount of samples available, have only been studied by molecular techniques.. For the present work it was carried out a retrospective and prospective study, comprising 1989 fecal samples from 856 patients (adults and children), HIV-positive and negative. The percentage of HIV-positive and negative patients studied was 65.5% (561/856) and 34.5% (295/856), respectively. Of all patients (586 males and 270 females), 675 were adults and 181 children. Of the patients studied 57.6% had diarrhea and 42.4% had no diarrhea. Nine (3.1%) of the 295 HIV-negative had other causes of immunosuppression. A frequency of infection with microsporidia of 12.0% (103/856) was observed in the feces of human population studied; being 13.9% and 8.5% of the fecal samples from HIV-positive and HIV-negative patients, respectively. Percentages of 18.8% and 10.2% of positive children and adults, respectively were observed. The results obtained indicated a significant association between the presence of microsporidia infection and HIV-positive patients. This data was not a surprising fact due to the opportunistic nature attributed to this disease. Microsporidia infection was identified in the feces of patients, which, although HIV-negative, have another type of immunedeficiency. Overall, the infection with microsporidia was significantly associated with patients with diarrhea. E. bieneusi was identified in 6.3% (54/856) and Vittaforma-like in 6.8% (58/856) of the fecal samples from the patients studied. Geneticcharacterization of the ITS rRNA region was performed on 48 isolates of E. bieneusi obtained from humans, from a total of 54 amplified by PCR. Six different genotypes were identified, Type IV (37.5%), Peru 6 (29.2%), D (12.5%), A (8.3 %), C (6.3%) and PtEbII (6.3%). So far, the genotypes A and C were identified only in humans. In contrast, Type IV, Peru 6 and D genotypes have been described in a wide range of hosts, including humans and other animals (including dogs, cats and ruminants in Portugal). A total of 58 isolates of Vittaforma-like were characterized genetically revealing the presence of two different genotypes, previously described in humans: Isolated 5830 (39 samples) and isolate 5843 (19 samples), with frequencies of 67.2% and 32.8 %, respectively. Urine samples both collected retrospectively and during the course of the study were obtained from 69 patients corresponding to 59 (85.5%) HIV-positive patients and 10 (14.5%) HIV-negative patients. Of the total sample, 48 (69.6%) were males and 21 (30.4%) females and 44 (63,8%) were adults and 25 (36,2%) children. Microsporidia were identified in 1.5% (1/69) of the urine samples, corresponding to one HIV-positive patient (1.7%; 1/59). The species identified in the urine was E. intestinalis, and characterization of the sequence of a fragment of the SSU rRNA showed total homology with several sequences previously described in humans and other animals. For the identification and characterization of microsporidia were analyzed 200 pulmonary secretions, collected retrospectively and during the course of the study, corresponding to 124 induced sputa (IS) and 76 bronchoalveolar lavage (BAL) fluids. The samples were collected from 150 (75%) HIV-positive patients and from 50 (25%) HIV-negative patients. The proportion of men and women corresponding to the samples studied was 64.5% and 35.5% respectively. The percentage of microsporidia infection was 1.0% in pulmonary secretions of the population studied. Two HIV-positive patients (1.3%) had microsporidia in a BAL and an EI. E. cuniculi and Vittaforma-like were identified in these specimens. Characterization of SSU rRNA for E. cuniculi revealed 99% homology in this gene sequence with other sequences previously described in humans for E. cuniculi, and for the species Vittaforma-like there was genetic homology with the isolate 5830 that was previously described in humans in Portugal. Both findings are the first records of this species in pulmonary secretions in humans in Portugal.The detection and characterization of microsporidia in animals, included retrospective and prospective studies in fecal samples: 66 companion animals (eight dogs and 58 cats) and 50 stray dogs housed in kennels, 100 cattle from dairy farms in various regions of Portugal, 103 captive animals in Lisbon Zoo (75 species of mammals, nine species of reptiles and nine9 species of birds), 134 small mammals captured in the municipalities of Aljustrel and Moura and Baixo Alentejo (52 insectivores Crocidura Russula and rodents 80 Mus spretus and two Apodemus sylvaticus) and 39 exotic birds cage of several different species belonging to the orders Psittaciformes and Passeriformes (31 from a poultry farmer and eight pets) and 52 urban pigeons (order Columbiformes), from the Lisbon area. Most animals in each group did not display symptoms. In the several groups of animals studied microsporidia were identified in fecal samples from dogs (8.6%), cats (15.5%), caged birds and pigeons (40.7%), bovine (6.0%) and silvatic animals (zoo)(1.9%). E. bieneusi was identified in fecal samples from cats and dogs. The characterization of the ITS rRNA sequences of 14 isolates of E. bieneusi, has revealed the presence of six different genotypes: TypeIV, PtEbIV, Peru6, D, and PtEbVIII PtEbIX. Genotypes PtEbIV, PtEbVIII and PtEbIX were first described during this study. Genotypes D (20%, 1/5), Peru6 (60.0%, 3/5) and PtEbIX (20%, 1/5) were identified in dogs, and TypeIV (77.8%), PtEbIV (11.1%) and PtEbVIII (11.9%) were identified in cats. There was a statistically significant association of infection with genotype Type IV to the group of cats. In cattle, E. bieneusi was identified in the six positive samples. The genetic characterization revealed three different genotypes (including two previously reported): TypeIV (50%, 3/6), PtEbXI (33.3%, 2/6) and J (16.7% 1/6). Genotype PtEbXI was characterized for the first time during this study. The species E. bieneusi was identified in feces of two mammals from the zoo: the white-fronted marmoset (Callithrix geoffroyi) and horse Timor (Tragelaphus strepsiceros strepsiceros). ITS rRNA genotyping of the two E. bieneusi isolates allowed characterizing for the first time during the course of this work, two distinct genotypes: PtEbV (horse Timor) and PtEbXII (white-fronted marmoset). Analysis of genetic diversity of the ITS rRNA of 10 different genotypes of E. bieneusi identified in 22 isolates from dogs, cats, cattle and animals from the zoo (a non-human primate and bovid) demonstrated the presence in most of these mammals (dogs, cats and cattle), of genotypes pathogenic to humans and other host specific genotypes (dog,cat, cattle, and nonhuman primate), which possibly have no impact on human health. In the fecal samples of the 134 small mammals (Crocidura Russula, Mus spretus and Apodemus sylvaticus) studied no microsporidian spores were detected. The species E. bieneusi and E. hellem were identified in 35.2% (32/91) and 13.2% (12/91), respectively, of caged birds and pigeons from public parks in Lisbon studied. Co-infection by both microsporidia species were observed in avian samples. Both microsporidia were identified in three orders of birds studied: Psittaciformes (E. bieneusi-12.5%; E. hellem-12.5%), Passeriformes (E. bieneusi-14.3%; E. hellem-42, 9%) and Columbiformes (E. bieneusi-51.9%; E. hellem-6.9%). However, in pigeons (Columbiformes) there was a high frequency of infection by E. bieneusi, statistically significant compared to that observed in the other two orders of birds. For E. hellem the highest percentage of infection in the Passeriformes (42.9%; 3/7) was not statistically significant. The characterization of the ITS rRNA locus of the 32 isolates belonging to the species E. bieneusi, revealed the presence of genotype Peru6 first described in humans in Peru, and it has been characterized for the first time PtEbII genotype, very similar to Peru6, with a single nucleotide change (G to A) near the 3 'end of the fragment amplified by PCR. The simultaneous presence of the two genotypes was observed in two pigeons. The genetic sequences obtained from the 12 isolates of E. hellem identified in fecal samples and/or intestinal contents and a scraping of the trachea of the bird population, analyzed and characterized using the nucleotide sequencing of the gene fragment of SSU rRNA, showed 99% to 100% homology with sequences of the SSU rRNA gene, previously reported in humans and birds (genotypes 1 and 2). Within the target regions, there were three polymorphic sites, which allow to group the 12 isolates of E. hellem from this study into three different sequences: PtEhI (genotype1 previously described); PtEh II (99% genetic homology with genotype 2) and PtEh III (99% of genetic homology with PtEhII). In the present study 184 water samples were analyzed. These water samples corresponded to sampling sites involved in the process leading to the water supply of Lisbon (175 samples processed according to the USEPA 1623 method and purified by immunomagnetic separation [IMS] and nine without IMS): 106 treated water samples collected at water treatment plants (25 surface and 42 groundwater source and 39 finished water samples from six main entrance points to the city of Lisbon watersupply), as well as 69 raw water samples, obtained in the surface (30 samples) and groundwater source (30 and nine samples with IMS and without IMS treatment, respectively). The species E. bieneusi and Vittaforma-like were identified in water supply of the city, both on the surface and groundwater sources. Genotyping of Vittaforma-like revealed the presence of a genotype (Isolate 5830), described in humans in Portugal. The Type IV E. bieneusi genotype, potentially pathogenic for man and other animals, was detected in treated water from groundwater source. In conclusion, this study confirmed the presence of species or genotypes of microsporidia pathogenic to humans in HIV-positive and negative patients, in animals and in water for public consumption in Portugal. Microsporidia infection, as described in the literature in this study, also appears to affect the most susceptible population, such as patients with immunosuppression, children and the elderly. The identification of human infecting microsporidia genotypes, both in humans and animals, in what concerns the species E. bieneusi suggests zoonotic transmission from animals in close contact with humans. It was not observed a direct association between infection with microsporidia and the presence of symptoms, including diarrhea (for both humans and animals), suggesting the existence in both populations of asymptomatic chronic carriers, and emphasizing their importance in the transmission cycle of these parasites. In summary, the analysis of data from this study suggests an important role at Public Health for the microsporidia circulating among the Portuguese population, highlighting the need to implement the diagnosis of microsporidia in routine laboratories, using standard methods well-defined and suitable for use in these laboratories. The methodology adopted in this study, namely the PCR and DNA sequencing techniques, resulted in an effective approach to achieve the proposed objectives. We hope that the results of this study will contribute to clarify the epidemiology of microsporidiosis in Portugal, more precisely, the processes involved in the circulation and transmission of opportunistic microorganisms to susceptible population.
Microsporidia are small eukaryotic, obligate intracellular parasites with a widespread occurrence in animals. There are over 1200 species of microsporidia, but only a few have been identified as cause of human diseases. Before onset of the AIDS pandemic, microsporidia infections had only been described in few cases, but by the expansion of the pandemic, microsporidia came increasingly under attention. The most frequent human infecting microsporidia species are Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon. cuniculi. Recently it has been identified in Portugal, a new species pathogenic to humans - Vittaforma-like, in HIV-positive patients and in immunocompetent persons. These emerging opportunistic organisms have now become important pathogens that can cause a broad spectrum of intestinal and disseminated pathologies depending on the infectious species involved and also on the immune status of the host. Microsporidia asymptomatic carriage was already described among both imunossupressed and immunocompetent population. The epidemiology of human microsporidia infections is still unclear in many aspects. Routes of transmission and sources of human microsporidia infections have been difficult to ascertain. However, the four most common human microsporidian species have been reported in a wide range of domestic and wild animals. Thus, it has been debated for some time now whether animals could be a source of infection of microsporidiosis to humans. More recently, a risk factor consistently associated with human microsporidiosis in epidemiological studies is the contact with water. The use of molecular methods has provided new tools to detect and characterize microsporidia, and the finding of previously unrecognized intraspecific genetic differences has improved our understanding of the epidemiology and zoonotic transmission of microsporidia. This study intended to: (i) confirm the occurrence and determine the frequency of infection of human patogenic microsporidia in HIV-positive and negative patients, in several animals species with close contact with humans (pet dogs, cats and birds, bovines, captive animals from the zoo and pigeons from public parks) and in environmental samples (water), in order to identify, not only the microsporidian speciesenrolled in Portuguese human infection, but also to determine potential animal reservoirs and environmental sources of infection; (ii) to characterize the parasites found at the genotype level; (iii) to evaluate the public health significance of microsporidia in Portugal. The frequency of microsporidia, genetic diversity, distribution of genotypes and the relationship between observed genotypes and different parameters of infection among humans, animals and environmental samples enrolled in the study were investigated using SSU rRNA and ITS-based PCR, followed by DNA sequencing. In order to achieve the objectives proposed in this study were adopted methodologies for diagnosis and genetic characterization of microsporidia, selected after an extensive literature search. All samples were analyzed by both the parasitological Gram-Chromotrope staining and PCR-techniques, with the exception of fecal samples from free-living animals and water for public consumption. Water samples due to the small amount of samples available, have only been studied by molecular techniques.. For the present work it was carried out a retrospective and prospective study, comprising 1989 fecal samples from 856 patients (adults and children), HIV-positive and negative. The percentage of HIV-positive and negative patients studied was 65.5% (561/856) and 34.5% (295/856), respectively. Of all patients (586 males and 270 females), 675 were adults and 181 children. Of the patients studied 57.6% had diarrhea and 42.4% had no diarrhea. Nine (3.1%) of the 295 HIV-negative had other causes of immunosuppression. A frequency of infection with microsporidia of 12.0% (103/856) was observed in the feces of human population studied; being 13.9% and 8.5% of the fecal samples from HIV-positive and HIV-negative patients, respectively. Percentages of 18.8% and 10.2% of positive children and adults, respectively were observed. The results obtained indicated a significant association between the presence of microsporidia infection and HIV-positive patients. This data was not a surprising fact due to the opportunistic nature attributed to this disease. Microsporidia infection was identified in the feces of patients, which, although HIV-negative, have another type of immunedeficiency. Overall, the infection with microsporidia was significantly associated with patients with diarrhea. E. bieneusi was identified in 6.3% (54/856) and Vittaforma-like in 6.8% (58/856) of the fecal samples from the patients studied. Geneticcharacterization of the ITS rRNA region was performed on 48 isolates of E. bieneusi obtained from humans, from a total of 54 amplified by PCR. Six different genotypes were identified, Type IV (37.5%), Peru 6 (29.2%), D (12.5%), A (8.3 %), C (6.3%) and PtEbII (6.3%). So far, the genotypes A and C were identified only in humans. In contrast, Type IV, Peru 6 and D genotypes have been described in a wide range of hosts, including humans and other animals (including dogs, cats and ruminants in Portugal). A total of 58 isolates of Vittaforma-like were characterized genetically revealing the presence of two different genotypes, previously described in humans: Isolated 5830 (39 samples) and isolate 5843 (19 samples), with frequencies of 67.2% and 32.8 %, respectively. Urine samples both collected retrospectively and during the course of the study were obtained from 69 patients corresponding to 59 (85.5%) HIV-positive patients and 10 (14.5%) HIV-negative patients. Of the total sample, 48 (69.6%) were males and 21 (30.4%) females and 44 (63,8%) were adults and 25 (36,2%) children. Microsporidia were identified in 1.5% (1/69) of the urine samples, corresponding to one HIV-positive patient (1.7%; 1/59). The species identified in the urine was E. intestinalis, and characterization of the sequence of a fragment of the SSU rRNA showed total homology with several sequences previously described in humans and other animals. For the identification and characterization of microsporidia were analyzed 200 pulmonary secretions, collected retrospectively and during the course of the study, corresponding to 124 induced sputa (IS) and 76 bronchoalveolar lavage (BAL) fluids. The samples were collected from 150 (75%) HIV-positive patients and from 50 (25%) HIV-negative patients. The proportion of men and women corresponding to the samples studied was 64.5% and 35.5% respectively. The percentage of microsporidia infection was 1.0% in pulmonary secretions of the population studied. Two HIV-positive patients (1.3%) had microsporidia in a BAL and an EI. E. cuniculi and Vittaforma-like were identified in these specimens. Characterization of SSU rRNA for E. cuniculi revealed 99% homology in this gene sequence with other sequences previously described in humans for E. cuniculi, and for the species Vittaforma-like there was genetic homology with the isolate 5830 that was previously described in humans in Portugal. Both findings are the first records of this species in pulmonary secretions in humans in Portugal.The detection and characterization of microsporidia in animals, included retrospective and prospective studies in fecal samples: 66 companion animals (eight dogs and 58 cats) and 50 stray dogs housed in kennels, 100 cattle from dairy farms in various regions of Portugal, 103 captive animals in Lisbon Zoo (75 species of mammals, nine species of reptiles and nine9 species of birds), 134 small mammals captured in the municipalities of Aljustrel and Moura and Baixo Alentejo (52 insectivores Crocidura Russula and rodents 80 Mus spretus and two Apodemus sylvaticus) and 39 exotic birds cage of several different species belonging to the orders Psittaciformes and Passeriformes (31 from a poultry farmer and eight pets) and 52 urban pigeons (order Columbiformes), from the Lisbon area. Most animals in each group did not display symptoms. In the several groups of animals studied microsporidia were identified in fecal samples from dogs (8.6%), cats (15.5%), caged birds and pigeons (40.7%), bovine (6.0%) and silvatic animals (zoo)(1.9%). E. bieneusi was identified in fecal samples from cats and dogs. The characterization of the ITS rRNA sequences of 14 isolates of E. bieneusi, has revealed the presence of six different genotypes: TypeIV, PtEbIV, Peru6, D, and PtEbVIII PtEbIX. Genotypes PtEbIV, PtEbVIII and PtEbIX were first described during this study. Genotypes D (20%, 1/5), Peru6 (60.0%, 3/5) and PtEbIX (20%, 1/5) were identified in dogs, and TypeIV (77.8%), PtEbIV (11.1%) and PtEbVIII (11.9%) were identified in cats. There was a statistically significant association of infection with genotype Type IV to the group of cats. In cattle, E. bieneusi was identified in the six positive samples. The genetic characterization revealed three different genotypes (including two previously reported): TypeIV (50%, 3/6), PtEbXI (33.3%, 2/6) and J (16.7% 1/6). Genotype PtEbXI was characterized for the first time during this study. The species E. bieneusi was identified in feces of two mammals from the zoo: the white-fronted marmoset (Callithrix geoffroyi) and horse Timor (Tragelaphus strepsiceros strepsiceros). ITS rRNA genotyping of the two E. bieneusi isolates allowed characterizing for the first time during the course of this work, two distinct genotypes: PtEbV (horse Timor) and PtEbXII (white-fronted marmoset). Analysis of genetic diversity of the ITS rRNA of 10 different genotypes of E. bieneusi identified in 22 isolates from dogs, cats, cattle and animals from the zoo (a non-human primate and bovid) demonstrated the presence in most of these mammals (dogs, cats and cattle), of genotypes pathogenic to humans and other host specific genotypes (dog,cat, cattle, and nonhuman primate), which possibly have no impact on human health. In the fecal samples of the 134 small mammals (Crocidura Russula, Mus spretus and Apodemus sylvaticus) studied no microsporidian spores were detected. The species E. bieneusi and E. hellem were identified in 35.2% (32/91) and 13.2% (12/91), respectively, of caged birds and pigeons from public parks in Lisbon studied. Co-infection by both microsporidia species were observed in avian samples. Both microsporidia were identified in three orders of birds studied: Psittaciformes (E. bieneusi-12.5%; E. hellem-12.5%), Passeriformes (E. bieneusi-14.3%; E. hellem-42, 9%) and Columbiformes (E. bieneusi-51.9%; E. hellem-6.9%). However, in pigeons (Columbiformes) there was a high frequency of infection by E. bieneusi, statistically significant compared to that observed in the other two orders of birds. For E. hellem the highest percentage of infection in the Passeriformes (42.9%; 3/7) was not statistically significant. The characterization of the ITS rRNA locus of the 32 isolates belonging to the species E. bieneusi, revealed the presence of genotype Peru6 first described in humans in Peru, and it has been characterized for the first time PtEbII genotype, very similar to Peru6, with a single nucleotide change (G to A) near the 3 'end of the fragment amplified by PCR. The simultaneous presence of the two genotypes was observed in two pigeons. The genetic sequences obtained from the 12 isolates of E. hellem identified in fecal samples and/or intestinal contents and a scraping of the trachea of the bird population, analyzed and characterized using the nucleotide sequencing of the gene fragment of SSU rRNA, showed 99% to 100% homology with sequences of the SSU rRNA gene, previously reported in humans and birds (genotypes 1 and 2). Within the target regions, there were three polymorphic sites, which allow to group the 12 isolates of E. hellem from this study into three different sequences: PtEhI (genotype1 previously described); PtEh II (99% genetic homology with genotype 2) and PtEh III (99% of genetic homology with PtEhII). In the present study 184 water samples were analyzed. These water samples corresponded to sampling sites involved in the process leading to the water supply of Lisbon (175 samples processed according to the USEPA 1623 method and purified by immunomagnetic separation [IMS] and nine without IMS): 106 treated water samples collected at water treatment plants (25 surface and 42 groundwater source and 39 finished water samples from six main entrance points to the city of Lisbon watersupply), as well as 69 raw water samples, obtained in the surface (30 samples) and groundwater source (30 and nine samples with IMS and without IMS treatment, respectively). The species E. bieneusi and Vittaforma-like were identified in water supply of the city, both on the surface and groundwater sources. Genotyping of Vittaforma-like revealed the presence of a genotype (Isolate 5830), described in humans in Portugal. The Type IV E. bieneusi genotype, potentially pathogenic for man and other animals, was detected in treated water from groundwater source. In conclusion, this study confirmed the presence of species or genotypes of microsporidia pathogenic to humans in HIV-positive and negative patients, in animals and in water for public consumption in Portugal. Microsporidia infection, as described in the literature in this study, also appears to affect the most susceptible population, such as patients with immunosuppression, children and the elderly. The identification of human infecting microsporidia genotypes, both in humans and animals, in what concerns the species E. bieneusi suggests zoonotic transmission from animals in close contact with humans. It was not observed a direct association between infection with microsporidia and the presence of symptoms, including diarrhea (for both humans and animals), suggesting the existence in both populations of asymptomatic chronic carriers, and emphasizing their importance in the transmission cycle of these parasites. In summary, the analysis of data from this study suggests an important role at Public Health for the microsporidia circulating among the Portuguese population, highlighting the need to implement the diagnosis of microsporidia in routine laboratories, using standard methods well-defined and suitable for use in these laboratories. The methodology adopted in this study, namely the PCR and DNA sequencing techniques, resulted in an effective approach to achieve the proposed objectives. We hope that the results of this study will contribute to clarify the epidemiology of microsporidiosis in Portugal, more precisely, the processes involved in the circulation and transmission of opportunistic microorganisms to susceptible population.
Descrição
Palavras-chave
Parasitologia médica Microsporidia Parasitas Sida Hospedeiro Epidemia Parasitose Saúde pública Portugal
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical