Microbiota oral e retal de calitriquídeos (Callithrix sp) em área antropizada de Mata Atlântica, Rio de Janeiro, Brasil / [Oral and rectal microbiota of callitrichids (Callithrix sp) in an anthropized area of the Atlantic Forest in Rio de Janeiro, Brazil]

A proximidade dos primatas não humanos (PNH) com o ser humano pode ser considerada um fator de risco para transmissão de bactérias entre essas duas populações. Neste estudo, foi investigada a microbiota anfibiôntica aeróbica oral e retal de calitriquídeos em um fragmento de Mata Atlântica localizado no Rio de Janeiro, Brasil, e foram realizados testes fenotípicos para detecção de bactérias multirresistentes nos isolados encontrados. Foram capturados 14 calitriquídeos e coletadas 21 amostras (14 de cavidade oral e sete de cavidade retal) em dois pontos da mata próximos às habitações humanas. As espécies mais frequentes, na cavidade oral, foram Klebsiella oxytoca (50,0%), K. pneumoniae (28,6%), Kluyvera ascorbata (21,4%) e Stenotrophomonas maltophilia (21,4%) e, na cavidade retal, K. pneumoniae (85,7%), Escherichia coli (28,6%) e Enterobacter spp. (42,9%). Todos os 48 isolados da família Enterobacteriaceae foram negativos para ESBL (betalactamase de espectro ampliado), mostrando-se não produtores da enzima nos dois métodos utilizados: disco-aproximação e método de detecção automatizado. Na pesquisa de ERC (enterobactérias resistentes a carbapenêmicos), esses mesmos isolados não apresentaram resistência aos antibióticos imipenem, meropenem e ertapenem. Todas as bactérias isoladas apresentam um potencial zoonótico, o que representa um risco à saúde pública e à conservação das espécies.(AU)

Proximity of nonhuman primates (NHP) to humans can be considered a risk factor for transmission of pathogens between these two populations. This study investigated the oral and rectal aerobic bacterial microbiota of marmosets in an anthropized area of the Atlantic Forest located in Rio de Janeiro, Brazil, and performed phenotypic tests for detection of multidrug-resistant bacteria. Twenty-one samples (14 from the oral cavity and seven from the rectum) were collected from 14 Callithrix sp. captured in two sites of the forest near human dwellings. The most frequent species identified from the oral cavity swabs were Klebsiella oxytoca (50.0%), K. pneumoniae (28.6%), Kluyvera ascorbata (21.4%) and Stenotrophomonas maltophilia (21.4%), whereas the species most commonly identified from the rectum swabs were K. pneumoniae (85.7%), Enterobacter spp. (42.9%) and Escherichia coli (28.6%). All isolates of family Enterobacteriaceae showed no extended spectrum ß-lactamase production by disk-diffusion and automated detection tests. In the search for carbapenem-resistant enterobacteriaceae these isolates presented no resistance to the imipenem, meropenem and ertapenem antibiotics. The isolate of Staphylococcus aureus was susceptible to oxacillin and the isolate of Enterococcus was susceptible to vancomycin. All isolated bacteria showed zoonotic potential, thus posing a risk to species conservation and public health.(AU)

Accuracy of quantitative polymerase chain reaction in samples of frozen and paraffin-embedded healthy skin for the diagnosis of canine visceral leishmaniasis / Acurácia da reação em cadeia da polimerase quantitativa em amostras de pele íntegra congelada e parafinada no diagnóstico da leishmaniose visceral canina

The purpose of the present work was to evaluate the accuracy of quantitative polymerase chain reaction (qPCR) performed on samples of fresh frozen tissue (FT) and formalin-fixed, paraffin-embedded (FFPE) healthy skin. This is a validation study conducted with samples from 46 dogs from an endemic area in Brazil. After sample collection, DNA extractions were conducted using commercial kits and qPCR was oriented to kinetoplast DNA (kDNA) targets of the Leishmania infantum species. The results obtained for the FFPE samples showed 63.6% sensitivity and 77.1% specificity, whereas those obtained for the FT samples showed 100% and 48.6%, respectively. Poor agreement was observed for the results of the qPCR technique with FT and FFPE samples. Our results suggest freezing as the most suitable conservation method for the formation of sample databases considering DNA recovery.(AU)

O objetivo deste trabalho foi avaliar a acurácia da PCR quantitativa (qPCR) realizada em amostras de pele íntegra congelada (FT) e parafinada (FFPE). Trata-se de um estudo de validação, com amostras provenientes de 46 cães de uma área endêmica no Brasil. Após as coletas de amostras, as extrações de DNA foram realizadas utilizando-se kits comerciais, e a qPCR foi orientada para alvos do kDNA da espécie Leishmania infantum. Os resultados obtidos para as amostras FFPE foram 63,6% de sensibilidade e 77,1% de especificidade; para as amostras FT, 100% e 48,6%, respectivamente. A concordância dos resultados da técnica de qPCR com amostras FT e FFPE foi pobre. Os resultados sugerem que o congelamento é o método mais adequado de conservação para banco de amostras para recuperação de DNA.(AU)

Avaliação da confiabilidade entre dois observadores em exames citopatológico e imunocitoquímico de aspirado de medula óssea no diagnóstico da leishmaniose visceral canina / Reliability evaluation between two evaluators in the cytopathologic and immunocytochemical exams from bone marrow aspirate in the canine visceral leishmaniasis diagnosis

Visceral leishmaniasis is a zoonosis in which the dog appears as the main source of infection in urban areas. Its diagnosis is complex and the cytopathological exam is a fast and cheap alternative to parasite direct visualization and its sensitivity can be increased by immunocytochemistry, though with a higher cost. The accuracy of such methods is dependent on the microscopist's experience and therefore, this study evaluated the reliability of such techniques between two observers, from bone marrow aspirates of 50 dogs from an endemic area for the disease. The parasitological culture in Novy-MacNeal-Nicolle medium was used as the reference standard. Among the main findings, the sensitivities obtained by observers I and II were respectively 62.5% and 37.5%, while specificities were 81.1% and 100%. On immunocytochemistry evaluation, the sensitivity was 0% for both evaluators and the specificity 97.3% and 100%. The agreement between evaluators was weak (κ = 0.167) for the cytopathological test and it could not be evaluated for immunocytochemistry, for which there was no detection by the evaluator II. The agreements among the diagnostic methods and the standard reference for the observer I were reasonable (κ = 0.364) for cytopathological examination and bad (κ = -0.041) for immunocytochemistry. For observer II, such agreement could be assessed only for the cytopathological test, being moderate (κ = 0.497). The results point to the possible expertise difference between evaluators, with the evaluator II demonstrating greater experience when interpreting the citopathological test. Although there was the expected sensitivity increase with immunocytochemistry, the technique used in this study was not effective for the diagnosis of infection, regardless of the evaluator.(AU)

Cell-block Immunohistochemistry of Bone Marrow Aspirates: a Novel Tool to Improve the Diagnosis of Leishmania Infection in Dogs.

Parasitological methods are the most specific procedures used for the diagnosis of Leishmania spp. infection, but their limited sensitivity poses a disadvantage and prompts the need for alternatives. The choice of site for sample collection influences diagnostic sensitivity. The combination of an accurate diagnostic method and a technique that allows large-scale field studies is highly desirable to enhance the investigation of Leishmania spp. infection in dogs, especially in endemic regions. The bone marrow is a good target for the detection of Leishmania spp. in dogs. In this context, bone marrow aspiration is rapid and less invasive compared with biopsy procedures, and also enables cell block processing, paraffin wax embedding and the sectioning of samples for further histological and immunohistochemical analyses. The aim of this study was to describe for the first time parasitological methods (immunohistochemistry [IHC] and histopathology) using the cell block technique with bone marrow aspirates for the diagnosis of Leishmania spp. infection in dogs. Bone marrow aspiration was performed in 45 dogs from an area endemic for visceral leishmaniosis for parasitological culture and the cell block technique (histopathology and IHC). Fourteen (31.1%) dogs tested positive for Leishmania spp. by IHC, six (13.3%) by parasitological culture and four (8.9%) by histopathology. Cell block IHC was a useful tool for the diagnosis of canine visceral leishmaniosis. Further studies should be conducted to validate this method for routine epidemiological screening.

Evaluation of 18S rDNA PCR assay using skin fragments as a diagnostic test for Trypanosoma caninum.

Trypanosoma caninum is a new species that has been recently identified in Brazil and infects domestic dogs. To date, no accurate diagnostic assays for this parasite have been established; thus, our aim was to evaluate more than one type of PCR for the diagnosis and molecular screening of T. caninum in 229 dogs living in Rio de Janeiro state. The tests were based on the amplification and sequencing of the 18S ribosomal DNA (rDNA) gene using healthy skin fragments. Additionally, PCR amplification of the kDNA minicircles region specific to the Leishmania genus was performed. The PCR results were compared with those of culture-based analysis performed with the same specimen. Using cultures, T. caninum and Leishmania chagasi were isolated from 11 and 12 dogs, respectively, whereas the 18S rDNA PCR assay detected parasitic infection in 35 dogs. Among these, 25 dogs showed an amplification pattern similar to T. caninum and 10 showed a pattern similar to L. chagasi; these results were confirmed by sequencing analysis. The kDNA PCR analysis showed that 14 dogs were positive for Leishmania infection. Of these, 2 dogs showed negative culture results and 12 were positive for L. chagasi, including 4 with negative 18S rDNA PCR results. Thus far, culture-based testing has been the only tool used successfully for T. caninum diagnosis. Our results demonstrate that 18S rDNA PCR-based test should be a useful diagnostic tool, particularly for distinguishing between T. caninum and L. chagasi infections in areas where these 2 parasites co-exist.

Natural breeding sites of phlebotomine sand flies (Diptera: Psychodidae) on Marambaia Island, Rio de Janeiro State, Brazil.

Immature phlebotomine sand flies develop in soils with essential and ideal characteristics for their life cycle, such as organic matter, humidity, temperature and low levels of light. Information regarding the potential breeding places of these dipterans is fundamental to understand the epidemiology and ecology of leishmaniasis, in addition to its importance to control them. In the present study, we aimed to find natural breeding sites of sand flies on Marambaia Island with the aid of emergence traps and direct search of immature forms using the flotation technique with saturated sugar solution in organic substrates of the region. Both methods were effective, with a total of 42 specimens of six different species - including some species that participate in the transmission cycle of American Tegumentary Leishmaniasis - collected by the emergence traps, and five immature forms obtained by floatation technique. However, further studies are still necessary, mainly with respect to the ecology and biology of immature sandfly stages, so that control measures focused on breeding sites can produce positive sustainable results in natural environments.

Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum.

In order to evaluate if the presence of Trypanosoma caninum can lead to a confuse diagnosis of canine visceral leishmaniasis (CVL), we investigated the serological status of dogs infected by T. caninum and assessed the serological cross-reactivity with CVL. A set of 117 serum samples from dogs infected by T. caninum, Leishmania chagasi and not infected dogs (n=39 in each group) was tested using commercial kits--indirect immunofluorescence (IFI-LVC), ELISA (EIE-LVC) and immunochromatographic test (DPP)--and in house tests with T. caninum (IIF-Tc and ELISA-Tc) and L. chagasi antigens (IIF-Lc and ELISA-Lc). IIF-Tc and ELISA-Tc presented sensitivity of 64.1% and 94.9% and specificity of 23.1% and 35.9%, respectively. The sensitivity of the IFI-LVC, EIE-LVC and DPP tests was 100% and the specificity was 70.5%, 68% and 97.5% respectively. The concordance between the tests was considered as satisfactory. The specificities of IFI-LVC, EIE-LVC and DPP were higher when the group Tc was excluded, with significant values for IFI-LVC (χ2=4.36, P-value=0.036), thus suggesting that the infection by T. caninum can confuse the diagnosis of CVL.

Occurrence of Trypanosoma caninum in areas overlapping with leishmaniasis in Brazil: what is the real impact of canine leishmaniasis control?

Trypanosoma caninum is a parasite of the Trypanosoma genus recently described in the natural infection of dogs in the municipality of Rio de Janeiro, Brazil. Suspecting the existence of a natural cycle and the circulation of this new species, the objective of this study was the taxonomic identification of samples of Trypanosoma spp. isolated from dogs in different Brazilian regions. Parasites were solely obtained from skin fragments culture and characterized by nested-PCR targeting the partial sequence of 18S rRNA gene and PCR products were sequenced. Thirty-three samples, obtained in São Paulo, Minas Gerais, Goiás, Mato Grosso and Rio de Janeiro states were analyzed. PCR and sequencing showed that the isolates were genetically identical or closely similar and confirmed T. caninum identity. This report broadens the geographical distribution of T. caninum in Brazil and discusses the impact of the presence of this parasite in areas of canine leishmaniasis occurrence.

Laboratory tests performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program.

In 2008, in the west zone of Rio de Janeiro municipality-Brazil, the leishmaniasis control program identified 155 dogs with titers ≥ 40 by Indirect ImmunoFluorescence (IIF) on blood collected onto filter paper. The objective of this study was to describe the laboratory test findings performed in dogs euthanized by the leishmaniasis program control of Rio de Janeiro municipality. Dogs were examined, subjected to euthanasia and collection of clinical specimens. Parasite isolation was obtained in 29 animals: Leishmania chagasi was isolated in 14 dogs; Leishmania braziliensis was isolated in five dogs; Trypanosoma caninum was obtained in seven animals and one dog had mixed infection (L. braziliensis and L. chagasi). By Polymerase Chain Reaction, seventeen animals were positive in intact skin fragments. In the serological reassessment of serum samples, 28% and 22% were positive for IIF and enzyme immunoassay, respectively. Ninety-one (59%) dogs were negative for all tests performed in this study. The findings indicate that the visceral leishmaniasis control program needs to be adjusted in order to avoid non-infected dogs from being removed or permit that dogs infected with L. chagasi to remain undetected in endemic areas.

Isolation of Trypanosoma caninum in domestic dogs in Rio de Janeiro, Brazil.

SUMMARY: The domestic dog's involvement with different members of the Trypanosomatidae family has been the focus of several studies due to this animal's close proximity to man. Recently this animal has been infected by a new Trypanosoma species (T. caninum), described in Rio de Janeiro and 19 similar isolates were later obtained. The objective of this study was to identify these isolates. All samples were isolated from intact skin cultures and analysed morphologically, by biochemical isoenzyme electrophoresis assays and by several molecular PCR assays. Additionally, anti-Leishmania sp. antibodies were assessed using the indirect Immunofluorescence Antibody Test (IFAT) in all animals. The methodologies employed to identify the isolates, including partial nucleotide sequences of 18S rRNA gene, indicated patterns identical to T. caninum and patterns different from the other species, including T. cruzi and T. rangeli samples. A phylogenetic tree constructed with the partial 18S ribosomal sequence shows that T. caninum is clustered with T. pestanai. Ten (52.6%) animals presented anti-Leishmania sp. antibodies with titres varying from 1:40 to 1:320. Thus, the hypothesis that this protozoan has disseminated among the dogs in Rio de Janeiro must be considered. The importance of a correct diagnosis in those animals and the possible consequences in the areas where visceral leishmaniasis is found are discussed here.

Response to azolic antifungal agents for treating feline sporotrichosis.

The effectiveness and safety of treatment with ketoconazole and itraconazole were compared in 773 sporotrichosis-infected cats over a four-year period (2002 to 2005). Five hundred and ninety-eight cats received oral ketoconazole and 175 received oral itraconazole. Treatment was successful in 238 (30.8 per cent) cats, of which 171 (28.6 per cent) of 598 received 13.5 to 27.0 mg/kg/day ketoconazole and 67 (38.3 per cent) of 175 received 8.3 to 27.7 mg/kg/day itraconazole. Adverse effects were reported in 306 (39.6 per cent) of the cats, 105 (13.6 per cent) died and 430 (55.6 per cent) dropped out of treatment or were still under treatment at the time of data analysis.

Comparative histopathological study of sporotrichosis and American tegumentary leishmaniasis in dogs from Rio de Janeiro.

Pyogranulomatous lesions from 80 dogs with sporotrichosis and 26 dogs with American tegumentary leishmaniasis (ATL) were compared microscopically in order to identify features that would support the diagnostic suspicion and direct the subsequent search for the aetiological agent of either infection. Odds ratios and their respective 95% confidence intervals were calculated in order to evaluate the impact of the microscopical findings on the diagnosis of either disease. Lesions with well-formed granulomata were 14 times more likely to be due to sporotrichosis than ATL. Marked neutrophil infiltration into granulomata was 12.26 times more likely to be associated with sporotrichosis when compared with lesions having mild neutrophilic infiltration. Absence of lymphocytes and macrophages in the peripheral infiltrate was associated with a 9.71 and 4.93 higher chance, respectively, of being sporotrichosis rather than ATL compared with lesions where these cells were present. Lesions with a perivascular, perifollicular and interstitial peripheral inflammatory infiltrate were 5.48 times more likely to be due to sporotrichosis than ATL when compared with lesions with a diffuse peripheral infiltrate. Histopathological analysis may therefore contribute to the diagnosis of sporotrichosis or ATL skin lesions in dogs since this method permits the identification of features that direct the diagnostic suspicion, thus facilitating the search for the aetiological agent in histological sections, permitting the precise request of subsequent tests and thereby reducing costs and time taken to achieve a definitive diagnosis and the initiation of appropriate therapy.

Efficacy of an indirect immunofluorescence test in the diagnosis of canine leishmaniosis.

Of 146 dogs from a visceral leishmaniosis-endemic area that tested seronegative by indirect immunofluorescence (IIF) on blood samples collected on filter paper (IIFp), 51 (34.9%) and 10 (6.8%) tested positive by IIF on serum samples (IIFs) and enzyme immunoassay, respectively. Three samples (2.0%) tested positive by PCR. Leishmania chagasi was isolated from the skin of five (3.4%) dogs. Amastigote forms were identified in two of these five animals following histopathological and immunohistochemical examination. The findings highlight that detection methods such as IIFp can permit dogs infected with L. chagasi to remain undetected in endemic areas with attendant consequences for the epidemiology of infection both in the canine and human populations.

Histopathology of canine sporotrichosis: a morphological study of 86 cases from Rio de Janeiro (2001-2007).

The present study reports the histopathological findings of 86 skin lesions of dogs with sporotrichosis from Rio de Janeiro. Suppurative granulomatous inflammation was the predominant finding and was observed in 76 (88.37%) cases. Plasma cells surrounding the suppurative granulomas were detected in 68 (89.5%) cases and an inflammatory infiltrate at the periphery of these granulomatous lesions was observed in 63 (82.9%). Fungus-specific staining revealed yeast cells compatible with Sporothrix schenckii in 36 cases. These fungal elements were only detected in lesions characterized by suppurative granulomatous inflammation. Thus, specific staining of serial sections is recommended in the case of dogs with skin lesions whose histopathological presentation is consistent with sporotrichosis. However, due to the generally small number of yeast cells in lesions, the hypothesis of sporotrichosis should not be ruled out even if the result is negative, especially in epidemic areas where correlation with epidemiological data is particularly useful.

Parasitological diagnosis of canine visceral leishmaniasis: is intact skin a good target?

The objective of this study was to evaluate intact skin of seroreactive dogs as a possible target for the parasitological confirmation of canine visceral leishmaniasis (CVL). For this purpose, 394 dogs identified in serological surveys carried out in the metropolitan region of Belo Horizonte were studied. Blood was collected from all animals for serology and a tissue sample was obtained from two sites for parasitological diagnosis. Skin obtained from the ear and scapular region was simultaneously analyzed in 247 animals and lesion samples and ear skin were analyzed in 147 dogs. Leishmania parasites were isolated from 310 (78.7%) animals, and all isolates were identified as Leishmania chagasi. Simultaneous isolation from two sites was possible in 240 of the 310 animals, including ear and scapular skin in 151/247 (61.1%) and ear skin and skin lesions in 89/147 (60.5%). Ours results suggest that intact skin is one of the main target sites for the parasitological confirmation of CVL in seroreactive dogs.

Trypanosoma caninum n. sp. (Protozoa: Kinetoplastida) isolated from intact skin of a domestic dog ( Canis familiaris) captured in Rio de Janeiro, Brazil.

An unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15.2%) and DMEM (9.2%) media containing 5% FCS, with a mean length of these forms of 43.0 and 36.0 mum, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24salpha rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.

First report of natural infection of a bush dog (Speothos venaticus) with Leishmania (Leishmania) chagasi in Brazil.

We report here the first known case of natural infection of a bush dog with Leishmania (Leishmania) chagasi in Brazil. The specimen was captured in the wild in the State of Mato Grosso and is currently being held in captivity at Fundação Jardim Zoológico, Rio de Janeiro, Brazil. The leishmaniasis was diagnosed by culture of promastigote forms in intact skin fragments and their characterization by isoenzyme electrophoresis. This report calls attention to the parasitological and etiological control of certain zoonoses, such as leishmaniasis, in wild animals kept in captivity, especially when animals are exchanged between zoos in Brazil.

Mycological evaluation of bronchoalveolar lavage in cats with respiratory signs from Rio de Janeiro, Brazil.

Twenty-three cats with respiratory signs who had domiciliary contact with cats with sporotrichosis were studied. Sneezing was the predominant extracutaneous sign. Twelve cats had no skin lesions and 11 had ulcerated skin lesions. Mycological culture of material obtained from the nasal cavity, oral cavity, bronchoalveolar lavage (BAL) and skin lesions, when present, was performed for all cats. In the case of autopsy, lung fragments were cultured. Sporothrix schenckii was isolated from four of the 12 cats without skin lesions: BAL (one cat) and oral and/or nasal cavity (three cats). The latter three animals developed nasal and distant skin lesions within the following 2-4 weeks. The cat with S. schenckii isolated from BAL did not develop skin lesions or lower respiratory tract symptoms during the 6 months of follow-up. S. schenckii was isolated from one or more biological samples of all 11 cats with skin lesions: oral cavity (five), nasal cavity (eight), BAL fluid (four), skin lesions (eight), and blood culture (one). No yeast-like structures were observed upon BAL cytology in any of the 23 cats. The results suggest that S. schenckii can cause infection of skin contiguous to the natural facial orifices through colonisation of the mucosal surfaces of the upper airways.

Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil.

We report here the first case of co-infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil. Isoenzyme characterisation identified the parasites isolated in culture from the cutaneous lesion as L. (V.) braziliensis and the isolates from blood and lymph node as L. (L.) chagasi. PCR analysis using specific primers followed by molecular hybridisation for direct Leishmania species identification in tissue fragments confirmed the presence of L. (V.) braziliensis DNA in the cutaneous lesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. This report emphasises the importance of identification of Leishmania species infecting seropositive dogs in endemic areas, and the consequent re-assessment of control and epidemiological surveillance measures for the control of leishmaniasis, as is the case in Brazil.

American cutaneous leishmaniasis in two cats from Rio de Janeiro, Brazil: first report of natural infection with Leishmania (Viannia) braziliensis.

We describe the isolation of Leishmania (Viannia) braziliensis from two female cats with American cutaneous leishmaniasis in Rio de Janeiro, Brazil. The isolates were identified as L. (V.) braziliensis by isoenzyme electrophoresis.