Validação da técnica de imunocromatografia rápida de duplo percurso para o diagnóstico da leishmaniose visceral canina em amostras de sangue total e soro

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Núcleo de Medicina Tropical, 2011.A leishmaniose visceral representa um importante problema em saúde pública para o Brasil, sendo o cão doméstico a principal fonte de infecção para o vetor. O objetivo deste estudo foi validar a técnica de imunocromatografia rápida de duplo percurso para o diagnóstico da leishmaniose visceral canina em amostras de sangue total e soro de cães. A amostra foi composta por 428 cães domiciliados selecionados por conveniência em dois bairros de uma região endêmica na cidade de Fortaleza, Ceará. De cada cão examinado, foram coletados 5µL de sangue periférico da ponta de orelha para realização do teste rápido em sangue total, 5 mL de sangue da veia jugular para obtenção do soro utilizado na realização do teste em condições de laboratório, além de três fragmentos de pele íntegra da região escapular utilizados para exames parasitológicos. Destes, dois foram acondicionados em solução salina estéril com antibiótico e antifúngico para isolamento de Leishmania em cultura e um em formol 10% tamponado para realização do exame histopatológico e de imunohistoquímica. Os animais positivos em pelo menos um desses três exames foram considerados casos e os negativos nos três exames foram considerados controles ou não-casos. Na avaliação do teste rápido no sangue com leitura visual, no soro com leitura eletrônica e no soro com leitura visual, foram estimadas, respectivamente, sensibilidade de 87,5% (IC95% 66,5 a 96,7), 88% (IC95% 67,5 a 96,8) e 88% (IC95% 67,5 a 96,8) e especificidade de 73,3% (IC95% 67,7 a 78,4), 69,2% (IC95% 63,7 a 74,3) e 68,2% (IC95% 62,2 a 74,3). A concordância entre a leitura visual e eletrônica no soro foi classificada como quase perfeita, índice Kappa = 0,88 (IC95% 0,83 a 0,93). O valor preditivo positivo para 7.9% de prevalência observada na amostra do estudo foi 21.9%. A análise de sensibilidade revelou que o valor preditivo positivo permaneceu abaixo de 50% em cenários com prevalência de 20%. O bom desempenho do teste rápido no sangue com leitura visual sugere que o mesmo possa ser indicado como exame de triagem para uso na rotina de campo e que ferramentas para leitura eletrônica não sejam necessárias para confirmação do resultado, porém seu uso associado a outros testes, utilizando estratégias de testagem sequencial ou paralela em algoritmos específicos deve ser avaliado, assim como a sua comparação formal com os testes atualmente disponíveis na rotina, para aproveitar da melhor maneira possível as facilidades da sua aplicação em condições de campo. ______________________________________________________________________________ ABSTRACTVisceral leishmaniasis is a major public health problem in Brazil where the domestic dog is the main source of infection for the vector. The objective of this study was to validate a dual path platform rapid chromatographic immunoassay for diagnosis of canine visceral leishmaniasis in whole blood and serum samples. The study sample consisted of 428 resident dogs selected by convenience from two districts of an endemic region of the city of Fortaleza, Ceará. Five micro liters of peripheral blood were collected from the tip of the ear to perform the rapid test in whole blood; 5 mL of blood from the jugular vein were collected to obtain serum for testing under laboratory conditions, and three fragments of intact skin from the scapular region were obtained for parasitological examination. Two skin fragments were stored in sterile saline with antibiotics and antifungal agents for isolation of Leishmania in culture and one fragment was stored in 10% buffered formalin for performing the histopathology and immunohistochemistry procedures. Positive animals in at least one of those three parasitological tests were classified as cases and negative animals in all the three tests were considered non-cases. Rapid test sensitivities were 87.5% (CI95% 66,5 to 96,7), 88% (CI95% 67,5 to 96,8%) and 88% (CI95% 67,5 to 96,8); and specificities were 73.3% (CI95% 67,7 to 78,4), 69,2% (CI95% 63,7 to 74,3) and 68,2% (CI95% 62,2 to 74,3) in whole blood submitted to visual reading; serum submitted to electronic reading and serum submitted to visual reading; respectively. The agreement between visual and electronic reading of serum samples was classified as almost perfect, Kappa index = 0.88 (CI95% 0.83 to 0.93). Positive predictive value for a 7.9% prevalence observed in the study sample was 21.9%. Sensitivity analysis revealed that positive predicted value remained below 50% in scenarios of prevalence as high as 20%. The performance of the rapid test in whole blood samples submitted to visual reading suggests that it could be indicated as the screening test for routine use in the field and that tools for electronic reading would be unnecessary to confirm the final result. However, its use in conjunction with other serological assays, using sequential or parallel testing strategies in specific algorithms should be evaluated, as well as its forma comparison with the currently available tests for canine visceral leishmaniasis diagnosis

Relato de caso autóctone de leishmaniose visceral canina na zona sul do município do Rio de Janeiro

O Brasil enfrenta uma expansão e urbanização da leishmaniose visceral americana com casos humanos e caninos em várias cidades de grande porte. O presente relato descreve um caso de leishmaniose visceral canina autóctone em uma área não endêmica no município de Rio de Janeiro

A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: evidence for an older and wider sporotrichosis epidemic.

Sporotrichosis is a subcutaneous infection caused by fungi from the genus Sporothrix. It is transmitted by inoculation of infective particles found in plant-contaminated material or diseased animals, characterizing the classic sapronotic and emerging zoonotic transmission, respectively. Since 1998, southeastern Brazil has experienced a zoonotic sporotrichosis epidemic caused by S. brasiliensis, centred in the state of Rio de Janeiro. Our observation of feline sporotrichosis cases in Brasília (Midwestern Brazil), around 900 km away from Rio de Janeiro, led us to question whether the epidemic caused by S. brasiliensis has spread from the epicentre in Rio de Janeiro, emerged independently in the two locations, or if the disease has been present and unrecognized in Midwestern Brazil. A retrospective analysis of 91 human and 4 animal cases from Brasília, ranging from 1993 to 2018, suggests the occurrence of both sapronotic and zoonotic transmission. Molecular typing of the calmodulin locus identified S. schenckii as the agent in two animals and all seven human patients from which we were able to recover clinical isolates. In two other animals, the disease was caused by S. brasiliensis. Whole-genome sequence typing of seven Sporothrix spp. strains from Brasília and Rio de Janeiro suggests that S. brasiliensis isolates from Brasília are genetically distinct from those obtained at the epicentre of the outbreak in Rio de Janeiro, both in phylogenomic and population genomic analyses. The two S. brasiliensis populations seem to have separated between 2.2 and 3.1 million years ago, indicating independent outbreaks or that the zoonotic S. brasiliensis outbreak might have started earlier and be more widespread in South America than previously recognized

A novel Sporothrix brasiliensis genomic variant in Midwestern Brazil: evidence for an older and wider sporotrichosis epidemic.

Sporotrichosis is a subcutaneous infection caused by fungi from the genus Sporothrix. It is transmitted by inoculation of infective particles found in plant-contaminated material or diseased animals, characterizing the classic sapronotic and emerging zoonotic transmission, respectively. Since 1998, southeastern Brazil has experienced a zoonotic sporotrichosis epidemic caused by S. brasiliensis, centred in the state of Rio de Janeiro. Our observation of feline sporotrichosis cases in Brasília (Midwestern Brazil), around 900 km away from Rio de Janeiro, led us to question whether the epidemic caused by S. brasiliensis has spread from the epicentre in Rio de Janeiro, emerged independently in the two locations, or if the disease has been present and unrecognized in Midwestern Brazil. A retrospective analysis of 91 human and 4 animal cases from Brasília, ranging from 1993 to 2018, suggests the occurrence of both sapronotic and zoonotic transmission. Molecular typing of the calmodulin locus identified S. schenckii as the agent in two animals and all seven human patients from which we were able to recover clinical isolates. In two other animals, the disease was caused by S. brasiliensis. Whole-genome sequence typing of seven Sporothrix spp. strains from Brasília and Rio de Janeiro suggests that S. brasiliensis isolates from Brasília are genetically distinct from those obtained at the epicentre of the outbreak in Rio de Janeiro, both in phylogenomic and population genomic analyses. The two S. brasiliensis populations seem to have separated between 2.2 and 3.1 million years ago, indicating independent outbreaks or that the zoonotic S. brasiliensis outbreak might have started earlier and be more widespread in South America than previously recognized