Leptospirose: Características da enfermidade em humanos e principais técnicas de diagnóstico laboratorial / Leptospirosis: Characteristics of the human disease and main laboratory diagnostic techniques

A leptospirose é a zoonose de maior distribuição geográfica, com estimativa de cerca de 60.000 mortes por ano. A doença é causada por bactérias do gênero Leptospira, que possui mais de 300 diferentes sorovares e 64 espécies já identificadas, sendo o ambiente a principal fonte de contaminação. A doença em humanos apresenta manifestações clínicas variadas e caráter bifásico, devendo ser confirmada por meio do diagnóstico laboratorial. O objetivo deste trabalho foi reunir conceitos atualizados sobre a leptospirose humana e as principais técnicas de diagnóstico laboratorial empregadas. A MAT é considerada o padrão-ouro para o diagnóstico da leptospirose, mas devido à baixa sensibilidade na fase inicial da doença é necessário o emprego de técnicas mais sensíveis neste período. Baseado em diversos estudos, as metodologias de PCR, ELISA-IgM e teste rápido apresentaram sensibilidade satisfatória nos primeiros dias após o início dos sintomas. Na segunda semana, a MAT apresentou 100% de sensibilidade, mantendo sua alta especificidade em ambas as fases. No geral, os testes sorológicos de ELISA-IgM e teste rápido apresentaram resultados satisfatórios como métodos de diagnóstico precoce, principalmente tratando-se de locais com pouca infraestrutura, diferente dos laboratórios de referência onde é possível empregar as técnicas de PCR e MAT.

Leptospirosis is the most widespread zoonosis, which has a balance of almost 60,000 deaths per year. Bacteria of Leptospira genus, which has more than 300 different serovars and 64 species already identified, cause the disease, being the environment the main source of contamination. The human disease presents a large set of clinical manifestations, showing biphasic presentation, the reason why leptospirosis must be confirmed by laboratory diagnosis. This study aimed to group recent concepts concerning human leptospirosis and the main diagnosis techniques employed at the laboratory. MAT is considered the gold standard for leptospirosis diagnosis, but has low sensitivity on the onset of disease, leading to the use of techniques with higher sensitivity on this period. Based on several studies, PCR, ELISA-IgM and rapid test presented satisfactory sensitivity on the onset of symptoms. In the second week, MAT showed 100% of sensitivity, maintaining its high specificity in both phases. In general, the ELISA-IgM and rapid serological tests showed satisfactory results as methods for early diagnosis, especially in the case of places with poor infrastructure, different from the reference laboratories where it is possible to use the PCR and MAT techniques.

Leptospirosis en pediatría, un diagnóstico a tener en cuenta / Leptospirosis in pediatrics, a diagnosis to be considered

Resumen La leptospirosis humana es una importante causa de enfermedad febril aguda que afecta con mayor frecuencia las zonas tropicales. Su principal reservorio es los roedores por lo que la infección está vinculada con factores de pobreza, déficit sanitario, actividades recreativas y laborales que favorecen el contacto con la orina de animales infectados, agua o suelos contaminados. Las infecciones por Leptospira spp tienen una presentación inespecífica desde cuadros asintomáticos hasta un compromiso multisistémico con alta mortalidad, siendo la sospecha clínica y la presencia de factores de riesgo las variables iniciales para el diagnóstico de la enfermedad. Entre los métodos disponibles para el diagnóstico microbiológico se destaca la observación directa, el cultivo, la reacción de polimerasa en cadena y la serología que permiten la confirmación de éste. El tratamiento de leptospirosis en niños se basa en medidas de soporte y en el inicio de antimicrobianos β-lactámicos o macrólidos. Las medidas de prevención de la enfermedad son fundamentales en la población expuesta al riesgo siendo recursos esenciales intervenciones generales, quimioprofilaxis y vacunación. El conocimiento, la consideración diagnóstica y el tratamiento oportuno en el paciente pediátrico, acelera la recuperación y limita la aparición de complicaciones que pueden impactar en la calidad de vida.

Abstract Human leptospirosis is an important cause of acute febrile disease that most frequently affects tropical areas. Its main reserve is rodents, so the infection is limited by factors of poverty, health deficit, recreational and work activities that favor contact with the urine of infected animals, contaminated water or soil. Leptospira spp infections have a non-specific presentation, ranging from asymptomatic to multisystem compromise with high mortality; being the clinical suspicion and the presence of risk factors the initial variables for the diagnosis of the disease. Among the available methods for microbiological diagnosis, direct observation, culture, polymerase chain reaction and serology that allows confirmation of this are highlighted. The treatment of leptospirosis in children is based on clinical support measures and the antibiotic initiation of β-lactams or macrolides. Disease prevention measures are fundamental in the population exposed to risk being essential resources general interventions, chemoprophylaxis and vaccination. The knowledge, diagnostic consideration, and timely treatment in pediatric patients accelerate recovery and limit the appearance of complications that can impact the quality of life

Leptospira patógena en murciélagos de Campeche y Yucatán, México / Pathogenic Leptospira in bats from Campeche and Yucatán, Mexico

RESUMEN Objetivo. Reportar la infección con Leptospira en ríñones de murciélagos de Campeche y Yucatán, México, a través de la amplificación por PCR de dos fragmentos distintos del gen 16S RNA ribosomal. Materiales y métodos. Se realizaron capturas en un sitio de Campeche y dos de Yucatán. A los murciélagos capturados se les aplicó la eutanasia y se les realizó una necropsia para recolectar tejido renal que se usó en la extracción de ADN total. Se realizaron dos PCR convencionales para la amplificación de los fragmentos de 16S RNA ribosomal. Se obtuvieron las secuencias de algunos productos positivos y se analizaron con herramientas bioinformáticas para identificar la especie infectante de Leptospira. Resultados. Se capturaron 69 murciélagos pertenecientes a cuatro familias y a ocho especies distintas. La familia con mayor diversidad fue Phyllostomidae con cinco especies. La especie con mayor frecuencia de captura fue Artibeusjamaicensis (41, 59.4%). Las PCR arrojaron una frecuencia global de infección de 21.7%. Las especies infectadas fueron A. jamaicensis, Pteronotus parnellii y Chiroderma villosum. El análisis bioinformático arrojó un 99.0% de identidad para Leptospira noguchii, Leptospira borgpetersenii y Leptospira santarosai. Conclusiones. Algunas especies de murciélagos de Yucatán y Campeche son portadores renales de leptospiras patógenas, por lo que podrían participar en el ciclo silvestre de transmisión en la región. La frecuencia de infección encontrada en los riñones de los murciélagos utilizados es mayor en comparación con aquellas obtenidas en otros reservorios de Yucatán y Campeche. Nuevas especies de murciélagos son reportadas como portadores de Leptospira para México.

ABSTRACT Objective. To report the infection with Leptospira in the kidneys of bats from Campeche and Yucatán, Mexico, through the amplification by PCR of two different 16S RNA ribosomal gene fragments. Materials and methods. Bat captures were carried out at one site in Campeche and two sites in Yucatán. Euthanasia was applied to the captured bats and a necropsy was performed to collect a renal tissue sample that was used in the total DNA extraction. Two different conventional PCR were performed for the amplification of the 16S RNA ribosomal gene fragments. Some sequences from positive products were obtained and analyzed with bioinformatics tools to identify the infectious species of Leptospira. Results. Sixty-nine bats belonging to four families and eight different species were captured. The family with the greatest diversity was Phyllostomidae with five species. The most captured species was Artibeus jamaicensis (41, 59.4%). Both PCR showed a global infection frequency of 21.7%. The infected species were A. jamaicensis, Pteronotus parnellii, and Chiroderma villosum. The bioinformatic analysis of the positive products yielded a 99.0% identity for Leptospira noguchii, Leptospira borgpetersenii, and Leptospira santarosai. Conclusions. Some bat species of Yucatán and Campeche, Mexico, are renal carriers of pathogenic Leptospira, therefore participating in the transmission cycle in the region. The frequency of infection found in the renal tissue of the captured bats is higher than the one obtained from other reservoirs captured in Yucatán and Campeche. New species of bats are reported as renal Leptospira carriers in Mexico.

Caracterización clínica de la leptospirosis grave en un hospital de alta complejidad de Cali, Colombia, 2010-2016 / Clinical characterization of patients with severe leptospirosis in a tertiary hospital in Cali, Colombia, 2010-2016

Resumen Introducción. La leptospirosis es una infección bacteriana endémica en Colombia. Su curso clínico puede ser variable y, en ocasiones, fatal. Hay pocos estudios en el país sobre los casos graves de esta enfermedad. Objetivo. Describir las características demográficas y clínicas de los pacientes con diagnóstico de leptospirosis grave hospitalizados en salas generales o atendidos en la unidad de cuidados intensivos de un hospital de cuarto nivel. Materiales y métodos. Se llevó a cabo un estudio observacional descriptivo de los pacientes adultos y niños con diagnóstico serológico de leptospirosis entre el 2010 y el 2016. Resultados. Se analizaron las historias clínicas de 87 pacientes, 74 % de los cuales correspondía a hombres y, el 84 %, a mayores de 18 años. El 35 % tenía alguna comorbilidad y la hipertensión arterial sistémica (16 %) y la diabetes mellitus (9 %) fueron las más comunes. Los síntomas más frecuentes fueron: fiebre, náuseas, astenia, mialgias, artralgias y dolor abdominal. El 34 % requirió atención en la unidad de cuidados intensivos, con una mediana de estancia de 5 días. El 61 % requirió hospitalización en sala general, con una mediana de estancia de 6 días. Todos los casos recibieron tratamiento antibiótico con ceftriaxona o doxiciclina. La tasa de letalidad fue del 1,1 % (n=1). Conclusiones. La infección por Leptospira spp. tiene el riesgo de diagnosticarse de manera tardía por su presentación clínica inespecífica, lo que implica considerar un gran número de diagnósticos diferenciales. La atención temprana de los pacientes con cuadros graves de esta enfermedad en la unidad de cuidados intensivos, puede evitar una mayor incidencia de complicaciones y disminuir la mortalidad.

Abstract Introduction: Leptospirosis is an endemic bacterial infection in Colombia. Its clinical course can be variable and occasionally fatal. There are few studies in the country about severe cases of leptospirosis. Objectives: To describe the demographic and clinical characteristics of patients with a diagnosis of leptospirosis, and their management in a high complexity hospital. Materials and methods: This was a descriptive retrospective study of patients with a serologic diagnosis of leptospirosis between 2010 and 2016. Results: We analyzed 87 patients, 74% of them were men, and 84% were older than 18 years; 35% had a comorbidity, the most common being arterial hypertension (16%) and diabetes mellitus (9%). The most frequent symptoms were fever, nausea, fatigue, myalgia, arthralgia, and abdominal pain. The majority of patients required hospitalization in general wards (61%), with a median stay of six days; 34% required management in the intensive care unit, with a median stay of five days. Mortality was 1.1% (n=1). All patients received treatment with either ceftriaxone or doxycycline. Conclusions: There is a risk of leptospira infections having a late diagnosis given their unspecific clinical presentation, which generates a high number of differential diagnoses. The early management in the intensive care unit could decrease the incidence of complications and the mortality of patients with leptospirosis.

Hibridização in situ fluorescente para diagnóstico de Brachyspira hyodysenteriae e B. pilosicoli ­em suínos / Fluorescence in situ hybridization for diagnosis of Brachyspira hyodysenteriae e B. pilosicoli in swine

Disenteria Suína e Colite Espiroquetal são duas enfermidades importantes em suínos causados pela Brachyspira hyodysenteriae e Brachyspira pilosicoli, respectivamente. O diagnóstico eficaz dessas espécies é extremamente importante para a adoção de estratégias adequadas para o controle. Propõe-se avaliar a técnica de hibridização in situ de fluorescência (FISH) para detecção de B. hyodysenteriae e B. pilosicoli em fragmentos histopatológicos de intestino de suínos e compará-la ao PCR duplex. Foram analisadas amostras de fezes e intestinos de suínos de terminação com histórico de diarreia pelas técnicas de reação em cadeia da polimerase duplex (dPCR), hibridização in situ fluorescente (FISH) para diagnóstico dessas bactérias. Foram utilizadas 34 amostras de intestino de suínos de campo positivos para alguma das duas espécies de Brachyspira sp. nos testes de FISH ou PCR. Das 34 amostras analisadas, foram detectadas 28 (82,35%) positivas na PCR e no FISH. Dentre as 29 amostras positivas para B. hyodysenteriae, 23 (79,3%) foram positivas à PCR e 21 (72,4%) no FISH. Os resultados de FISH e PCR não diferiram estatisticamente entre si. Baseado no fato dessa técnica poder ser realizada em tecidos formolizados, ser prática, rápida e associar a marcação especifica do agente com lesões histológicas, o FISH demonstrou ser mais uma alternativa no diagnóstico de Brachyspira hyodysenteriae e B. pilosicoli.(AU)

Growing and finishing pigs are affected by pathogenic spirochetes of the genus Brachyspira sp., which cause a significant economic impact due to direct and indirect losses. Thus, efficient diagnosis of these species enables better technical intervention to prevent or treat diseases. This study aimed to evaluate the fluorescent in situ hybridization (FISH) for the diagnosis of B. hyodysenteriae and B. pilosicoli in histopathologic fragments of pig's intestine and compare it to the duplex PCR. Thirty-four samples collected from pigs positive for these species in at least one of the tests were used in the study. Out of the 34 analyzed intestine samples, 28 (82.35%) were positive by PCR and FISH. Among the 29 B. hyodysenteriae positive samples, 23 (79.3%) were positive by PCR and 21 (72.4%) by FISH. There was no statistical difference among the detection rate of the used tests. Based on the fact this technique can be performed in formalin fixed tissue samples, it is practical, fast and allows the association of labeling a specific agent with histological lesions, FISH has become an alternative diagnostic method for Brachyspira hyodysenteriae and B. pilosicoli.(AU)

Digital dermatitis of the accessory digits of dairy cows / Dermatite digital nos dígitos acessórios de vacas leiteiras

This report characterizes the digital dermatitis (DD) lesions in the accessory digits of dairy cows and presents data on the applied therapy. Fifteen Holstein cattle with DD affecting the accessory digits of the hindlimbs from four dairy farms with previous history of DD were evaluated. Lesions were excised, the wounds were sutured, and a topical application of oxytetracycline powder covered by bandaging was associated with a single parenteral administration of long acting oxytetracycline IM (20mg/kg). Tissue samples were obtained for histopathology and transmission electronic microscopy (TEM). Lesions from all the animals were recuperated 15 days after surgical procedure. Overal, most DD lesions were papillomatous epidermal projections or wartlike verrucous lesions. Histopathologically, samples revealed hyperplasia of epidermis with hyperkeratosis, several mitoses in the stratum basale and elongated rete ridges in the superficial and middle dermis. TEM revealed long, thin spirochete-like bacteria. Morphologic features of lesions and its response to therapy were comparable to those described for DD.

Esse estudo caracteriza lesões de dermatite digital (DD) nos dígitos acessórios de vacas leiteiras, além de apresentar a terapia aplicada. Foram utilizados 15 bovinos leiteiros da raça Holandês com DD nos dígitos acessórios dos membros pélvicos. Os animais eram provenientes de quatro fazendas leiteiras com histórico prévio de dermatite digital (DD). Todos os quinze animais foram tratados da mesma forma: após excisão das lesões e sutura das feridas cutâneas, aplicou-se oxitetraciclina pó topicamente sob bandagem e oxitetraciclina (20mg/kg) de longa ação, via intramuscular. Obtiveram-se amostras de tecidos para histopatologia, inclusive por microscopia eletrônica de transmissão (MET). Observou-se cicatrização em todos os animais após 15 dias do procedimento cirúrgico. A maioria das lesões macroscópicas foram projeções papilomatosas ou em forma de verrugas. Os achados histopatológicos de todas as amostras revelaram hiperplasia da epiderme com hiperceratose, inúmeras mitoses no estrato basal, com invasões alongadas em forma de rede na derme superficial e intermediária. A MET evidenciou organismos com formas longas, afiladas e espiraladas, presumivelmente espiroquetas. Tanto as características morfológicas, quanto a resposta à terapia das lesões foram comparáveis às descritas para DD.

Borrelia theileri: observação em carrapatos do gênero Boophilus microplus no município de Guaíba, RS, Brasil / Borrelia theileri: observation on Boophilus microplus ticks in Guaiba, RS, Brazil

Espiroquetas da espécie Borrelia theileri identificadas em uma estirpe de carrapatos Boophilus microplus provenientes do município de Guaíba, RS. A observação ocorreu no exame de hemolinfa de fêmeas adultas com 10 dias pós-repleçâo, corada por Giemsa. Não foram observadas espiroquetas em ovos provenientes de teleóginas infectadas. A detecção da estirpe infectada sugere a presença de borreliose em rebanhos bovinos, fato que eventualmente pode interferir em resultados de diagnóstico ou tornar-se motivo de preocupação em produtos derivados de sangue bovino tais como vacinas vivas contra anaplasmose e babesiose bovina.

Spirochetes of species Borrelia theileri were identifica in afield-strain of the caule tick Boophilus microplus, in Guaíba, RS, Brazil. Hemolymph smears from females 10 days post-repletion were collected by gentty section of the tarsal-metatarsaijoint, and dropped onto a microscope slide, and stained by Giemsa. No spirochetes were observed in eggs squashed and stained by Giemsa from the same infected strain. The detection of B. microplus adult females infected with Borrelia theileri suggesfs the likely presence of borreliosis in bovine heras what might eventually interfere with the interpretation of diagnosis results or become cause for concern in blood products such as anaplasmosis and babesiosis live vaccines.