Eliame Mouta-Confort

Visceral leishmaniasis in Rio de Janeiro, Brazil: eco-epidemiological aspects and control

From 1977 (index case) to 2006, 87 cases of visceral leishmaniasis were confirmed in the municipality of Rio de Janeiro, Brazil, in periurban areas on the continental and coastal slopes of the Pedra Branca massif and the continental slopes of the Gericinó massif. The majority (65.5%) of the patients were more than five years old, predominantly males (61.5%), but without any difference between the sexes below the age of 14 years. The overall fatality rate was 10.4%. Two cases of visceral leishmaniasis/human immunodeficiency virus coinfection were detected. Leishmania chagasi was isolated from human and canine cases. The associations between the presence of phlebotomines and human and canine migrations, disorderly occupation involving degradation of environmental preservation areas and poor socioeconomic conditions may have created a favorable setting for the establishment and propagation of the disease. Close epidemiological surveillance associated with traditional control measures and others (active case researches, land clearing and health education), reduced the incidence of human cases from 2.8 per 100,000 inhabitants in 1981 to less than 0.01 per 100,000 since 1997. The canine infection rates decreased from 4.6% in 1984 to 1.6% in 2008. Lutzomyia longipalpis was not detected in some locations where human and canine cases occurred. In the years 2007 and 2008, no new human cases were reported, but there is a persistent and worrisome residual canine seroprevalence.

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.

Accuracy of an ELISA and indirect immunofluorescence for the laboratory diagnosis of American tegumentary leishmaniasis.

We compared the accuracy of ELISA and indirect immunofluorescence (IIF) using Leishmania braziliensis and L. major-like antigens and antigens from the Bio-Manguinhos kit for serological diagnosis of American tegumentary leishmaniasis (ATL). Cut-off values were defined by the area under the receiver-operating characteristic curve. For ELISA, statistical analyses revealed better accuracy [95.7% sensitivity, 100% specificity, 100% positive predictive value (PPV), 97.5% negative predictive value (NPV)] and reliability [intraclass correlation coefficient (ICC): 0.940] for L. braziliensis antigen compared with L. major-like antigen (78.7% sensitivity, 82.8% specificity, 73.3% PPV, 86.6% NPV, ICC: 0.833). ELISA optical density values obtained for both antigens were higher in mucosal forms of ATL. For IIF, sensitivity and specificity were 81.5 and 86.2%, respectively, for the L. braziliensis antigen, compared with 95.4 and 77.7% for the L. major-like antigen and 75.4 and 89.2% for the Bio-Manguinhos kit. No difference in the specificity of the IIF test was observed between antigens, whereas sensitivity differed between the L. braziliensis and L. major-like antigens and the Bio-Manguinhos kit. Parallel ELISA and IIF testing increased sensitivity, irrespective of the antigen employed, and serial testing increased overall specificity. These results support the recommendation that ELISA employing L. braziliensis antigen be used as a diagnostic tool for suspected cases of ATL in L. braziliensis-endemic areas.

Prevalência da infecção canina em áreas endêmicas de leishmaniose tegumentar americana, do município de Paracambi, Estado do Rio de Janeiro, no período entre 1992 e 1993

In the district of Paracambi, State of Rio de Janeiro an epidemiological survey for American tegumentary leishmaniasis in canine population was carried out in endemic localities. A total of 179 dogs was registered and 138 (77.1%) examined for their clinical aspects, development of delayed hypersensitivity (DHS) with Imunoleish® antigen and serological responses by indirect immunofluorescent reaction and enzyme-linked immunosorbent assay. In 9 (6.5%) dogs with active cutaneous lesions or suspect scars, 66.7% were caused by Leishmania sp; 44.4% produced infection in hamsters and showed growth in culture media, which was considered to be compatible with the species of Leishmania braziliensis complex. The molecular characterization (isoenzyme and KDNA restriction profiles) defined two strains with similar profiles for L. (Viannia) braziliensis. The prevalence of canine infection estimated by the cutaneous test, IFR and ELISA was 10.1%, 16.7% and 27.8%, respectively. The presence of clinical / sub-clinical form of ATL in canine population associated with human infections suggested that the dog can act as source of infection as well as for dissemination of the disease.

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.

Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

The Brazilian Ministry of Health recommends the culling and euthanasia of dogs with a positive serological test for canine visceral leishmaniasis (CVL). In the Municipality of Rio de Janeiro, the technique used for the diagnosis of CVL is the indirect fluorescent antibody test (IFAT), using blood samples eluted on filter paper (eluate). A dog survey was conducted over a period of one year in the region of Carapiá, in order to evaluate the diagnosis of CVL in this region. All animals underwent clinical examination, and blood samples (serum and eluate) were collected for analysis by enzyme immunoassay (ELISA) and IFAT. A skin biopsy was obtained for parasitological examination (culture). A total of 305 animals were studied and Leishmania chagasi was isolated from nine animals. Sensitivity and specificity were 100% and 96.6% for ELISA, respectively, 100% and 65.5% for IFAT (cut-off at a 1:40 dilution), 100% and 83.4% for IFAT (cut-off at a 1:80 dilution), and 22.2% and 97.0% for eluate IFAT. In conclusion, ELISA was the best tool for the diagnosis of CVL among the serological techniques tested. The present results suggest the need for a better evaluation of filter paper IFAT as the only diagnostic method for CVL in the Municipality of Rio de Janeiro.

Leishmaniose visceral canina em Maricá, Estado do Rio de Janeiro: relato do primeiro caso autóctone

Visceral leishmaniasis is a zoonosis of public health importance, and dogs represent one of the main problems. This paper describes the first autochthonous case of canine visceral leishmaniasis in the municipality of Maricá. It provides new facts regarding the geographical distribution of Leishmania (Leishmania) chagasi in the State of Rio de Janeiro.

Immunoenzymatic assay for the diagnosis of American tegumentary leishmaniasis using soluble and membrane-enriched fractions from infectious Leishmania (Viannia) braziliensis

The diagnosis of American tegumentary leishmaniasis (ATL) is based on the visualization or isolation of the parasite, which is a time‐consuming and poorly sensitive method. In this study, we evaluated the accuracy and reliability of ELISA for the diagnosis of ATL using soluble (SF) and membrane‐enriched (MF) antigen fractions obtained from an infectious strain of Leishmania (Viannia) braziliensis. A total of 152 serum samples investigated at a referral center in Rio de Janeiro, Brazil, between 2005 and 2007 were studied. Each sample was tested twice with each fraction for the calculation of reliability (intraclass coefficient (ICC)). Cut‐off values of 0.22 (SF) and 0.33 (MF) were defined. The use of the fractions resulted in good discrimination between patients, with a large area under the curve (P<0.0001), but no difference was observed between the two fractions (P=0.45). Sensitivity was 89.5% for each fraction, specificity was 89.5% for SF and 93.4% for MF, and the positive likelihood ratio was 8.5 for SF and 13.6 for MF. The ICCs were excellent (SF: 0.96 and MF: 0.90). The antigens tested provided precision and accuracy for the diagnosis of ATL, with SF being recommended due to its lower cost and greater practicality. J. Clin. Lab. Anal. 24:289–294, 2010.

First encounter of subclinical human Leishmania (Viannia) infection in State of Rio Grande do Sul, Brazil

The objective of the present study was to evaluate the specificity of the Montenegro skin test (MST) in an area in Brazil, state of Grande do Sul State (RS), which was considered to be non-endemic for leishmaniasis. Sixty subjects presented a positive MST and were reevaluated by clinical examination, serology and polymerase chain reaction (PCR) of peripheral blood for the detection of subclinical Leishmania infection. None of the subjects presented clinical signs or symptoms of current leishmaniasis or a history of the disease. Leishmania (Viannia) DNA was detected in blood by PCR and hybridization in one subject. The PCR skin test-positive individual remained asymptomatic throughout the study. Clinical examination showed no scars suggestive of past cutaneous leishmaniasis. Human subclinical infection with Leishmania (Viannia) in RS was confirmed by PCR. This is the first report of subclinical infection with this parasite in the human population of this area.

Differentiation between canine cutaneous and visceral leishmaniasis by the detection of immunoglobulin G specific for Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi antigens using flow cytometry

Flow cytometry employing Leishmania (L.) chagasi (Lc) and L. (Viannia) braziliensis (Lb) antigen was used to establish the differential diagnosis between visceral (VL) and cutaneous leishmaniasis (CL) in dogs. Flow cytometry permitted the detection of Leishmania-specific immunoglobulin G in sera from 19 dogs: nine with CL and 10 with VL. A significant difference in the percentage of positive staining was observed in sera from dogs with CL between the homologous antigen (69% for Lb) and the heterologous antigen (42% for Lc). However, this difference was not significant in sera from dogs with VL (61% for Lb and 73% for Lc). No significant staining was observed in control sera (0.6% for Lb and 0.4% for Lc) consisting of samples from healthy dogs, or in the group with sporotrichosis (1.8% for Lb and 1.5% for Lc), a differential diagnosis of CL. The results suggest that flow cytometry might be useful for the differentiation between CL and VL in dogs, with practical applications in areas where the two infections overlap.