PARTICIPATION OF TICKS IN THE INFECTIOUS CYCLE OF CANINE VISCERAL LEISHMANIASIS, IN TERESINA, PIAUÍ, BRAZIL

In this study, we detected Leishmania spp. infection in R. sanguineus collected from dogs that were naturally infected with L. (L.) infantum. We examined 35 dogs of both sexes and unknown ages. The infected dogs were serologically positive by the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Quick Test-DPP (Dual Path Platform), as well as parasitological examination of a positive skin biopsy or sternal bone marrow aspiration. Ten negative dogs were included as controls. The ticks that infested these dogs were collected in pools of 10 adult females per animal. The PCR was performed with specific primers for Leishmania spp., which amplified a 720-bp fragment. Of the 35 analyzed samples, a product was observed in eight samples (8/35; 22.9%). We conclude that the presence of parasite DNA suggests that ticks participate in the zoonotic cycle of canine visceral leishmaniasis, in the city of Teresina, Piauí.

GENOTYPE CHARACTERIZATION OF Leishmania (Viannia) braziliensis ISOLATED FROM HUMAN AND CANINE BIOPSIES WITH AMERICAN CUTANEOUS LEISHMANIASIS

Introduction:American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo.Methods:Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus.Results:Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample.Conclusion:These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci.

CANINE VISCERAL LEISHMANIASIS CASE INVESTIGATION IN THE JACARE REGION OF NITEROI, RIO DE JANEIRO, BRAZIL

SUMMARY American visceral leishmaniasis is a vector-borne zoonosis in expansion in Brazil. Dogs are the main urban reservoir. Departing from a case of canine visceral leishmaniasis (CVL) in Jacaré, Niterói, Rio de Janeiro State, an epidemiological canine and entomological study was performed to assess the extension of the disease at the location. Sample was collected around the case and the dogs identified by serological tests (rapid double platform immunochromatographic exams, immunoenzymatic assay/ELISA, indirect immunofluorescence/IFAT). The parasitological diagnosis was performed in animals positive in at least one of these tests. The entomological study was carried out by using light traps and manual collection. The associations between canine variables and outcome (ELISA and IFAT reagents) were assessed by the chi-square test and adjusted by multivariate logistic regression for those associations with p < 0.1 in the bivariate analysis. Seventeen cases of CVL were detected among 110 evaluated dogs (prevalence of 15.5%). Presence of ectoparasites (OR 6.5; 95% CI 1.1-37.4), animals with clinical signs (OR 9.5; 95% CI 1.2-76.6), and previous cases of CVL in the same house (OR 17.9; 95% CI 2.2-147.1) were associated with the outcome. Lutzomyia longipalpiswas not detected. Our results are indicative of an ongoing transmission in the area.

GEOGRAPHICAL EXPANSION OF CANINE VISCERAL LEISHMANIASIS IN RIO DE JANEIRO STATE, BRAZIL

SUMMARY Visceral Leishmaniasis (VL) is a vector-borne disease that affects humans, and domestic and wild animals. It is caused by the protozoan Leishmania (Leishmania) infantum (syn = Leishmania chagasi). The domestic dog (Canis familiaris) is considered the main reservoir of the etiologic agent of VL in domestic and peridomestic environments. In the past three years, although control actions involving domestic dogs are routinely performed in endemic areas of the Rio de Janeiro State, new cases of canine visceral leishmaniasis (CVL) have been reported in several municipalities. The objective of this short communication was to describe the geographical expansion of CVL in the Rio de Janeiro State, Brazil, through its reports in the scientific literature and studies performed by our group. From 2010 to 2013, autochthonous and allochthonous cases of CVL were reported in the municipalities of Mangaratiba, Marica, Niteroi, Barra Mansa, Cachoeiras de Macacu, Volta Redonda, Resende and Rio de Janeiro. These reports demonstrate that CVL is in intense geographical expansion around the state; therefore, a joint effort by public agencies, veterinarians and researchers is needed in order to minimize and/or even prevent the dispersion of this disease.

EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.

EPIDEMIOLOGY OF PARACOCCIDIOIDOMYCOSIS

SUMMARYThe epidemiological characteristics of paracoccidioidomycosis were reviewed and updated. The new endemic areas in Brazil were discussed in the section regarding the geographic distribution of the mycosis. Subclinical infection with Paracoccidioides brasiliensis was discussed on the basis of skin test surveys with antigens of the fungus, seroepidemiological studies, and disease cases outside Latin America. Large case series permitted a comparison of the prevalence of the mycosis in different regions, its estimated incidence and risk factors for the development of the disease. Aspects modulating the expression of the clinical forms of paracoccidioidomycosis are also presented. This review also deals with diseases associated with the mycosis, opportunistic paracoccidioidomycosis, lethality, mortality and infection and disease in animals.

PARACOCCIDIOIDOMYCOSIS: CHALLENGES IN THE DEVELOPMENT OF A VACCINE AGAINST AN ENDEMIC MYCOSIS IN THE AMERICAS

SUMMARYParacoccidioidomycosis (PCM), caused by Paracoccidioides spp, is an important endemic mycosis in Latin America. There are two recognized Paracoccidioides species, P. brasiliensis and P. lutzii, based on phylogenetic differences; however, the pathogenesis and disease manifestations of both are indistinguishable at present. Approximately 1,853 (~51,2%) of 3,583 confirmed deaths in Brazil due to systemic mycoses from 1996-2006 were caused by PCM. Antifungal treatment is required for patients with PCM. The initial treatment lasts from two to six months and sulfa derivatives, amphotericin B, azoles and terbinafine are used in clinical practice; however, despite prolonged therapy, relapses are still a problem. An effective Th1-biased cellular immune response is essential to control the disease, which can be induced by exogenous antigens or modulated by prophylactic or therapeutic vaccines. Stimulation of B cells or passive transference of monoclonal antibodies are also important means that may be used to improve the efficacy of paracoccidioidomycosis treatment in the future. This review critically details major challenges facing the development of a vaccine to combat PCM.

PARACOCCIDIOIDOMYCOSIS TREATMENT

SUMMARYConsidered to be an emerging endemic mycosis in Latin America, paracoccidioidomycosis is characterized by a chronic course and involvement of multiple organs in immunocompromised hosts. Infection sequelae are mainly related to pulmonary and adrenal insufficiency. The host-parasite interaction results in different expressions of the immune response depending on parasite pathogenicity, fungal load and genetic characteristics of the host. A few controlled and case series reports have shown that azoles and fast-acting sulfa derivatives are useful treatment alternatives in milder forms of the disease. For moderate/severe cases, more prolonged treatments or even parenteral routes are required especially when there is involvement of the digestive tract mucosa, resulting in poor drug absorption. Although comparative studies have reported that shorter treatment regimens with itraconazole are able to induce cure in chronically-infected patients, there are still treatment challenges such as the need for more controlled studies involving acute cases, the search for new drugs and combinations, and the search for compounds capable of modulating the immune response in severe cases as well as the paradoxical reactions.

IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

PARACOCCIDIOIDOMYCOSIS IN A RENAL TRANSPLANT RECIPIENT

Paracoccidioidomycosis (PCM) is the most common endemic mycosis in Latin America. The etiological agents, which comprise two species, Paracoccidioides brasiliensis and P. lutzii, are thermodimorphic fungi that usually affect previously healthy adults. They primarily involve the lungs and then disseminate to other organs. Such mycosis is rare in organ transplant recipients; there have been only three cases reported in literature, until now. We report a case of PCM in a renal transplant recipient with an unusual dermatological presentation.

A modification of the indirect fluorescence test (IFT) for Paracoccidioidomycosis

A modification of the indirect fluorescence test (IFT) for serological diagnosis of Paracoccidioidomycosis is described in which formolized budding forms (yeast cells) of the fungus Paracoccidioides brasiliensis are used as antigen. A further modification introduced is a less eiaborate technical procedure of the test without lowering the sensitivity of the reaction. The test may be considered as adequateiy accurate and easy to perform in any laboratory with immunofluorescence facilities.

Experimental paracoccidioidomycosis in the mouse. III. Histopathological and immunological findings after intravenous infection in the presence or absence of previous immunization

Fifty male white Swiss mice aged 4 weeks were inoculated with 5 x 10(5) viable yeast forms of Paracoccidioides brasiliensis (strain 18). Ten of these animals had been previously immunized with particulate P. brasiliensis antigenfor 4 weeks by intradermal injection. The controls consisted of 10 animals that were only immunized and 10 animals submitted to no treatment. The animals were sacrificed 2, 4, 7,11 and 16 weeks later. We studied: 1) the anti-P. brasiliensis delayed hypersensitivity response measured by the footpad test 24 hours prior to sacrifice; 2) the specific antibody production measured by double immunodiffusion in agar gel; 3) the histopathology of lungs, liver, spleen, adrenals and kidneys. We observed that: a) the immunized animals developed more intense cell-immune responses than the infected ones; b) infection reduced the cell- immune response of the immunized animals; c) intravenous infection of mice with P. brasiliensis was characterized by a systemic and progressive granulomatous inflammation. The animals infected after previous immunization showed less extensive lung inflammation, with smaller granulomas and fewer fungi. The results indicate that the present murine model mimics some findings of the human subacute form of paracoccidioidomycosis (systemic disease with depressed cellular immunity) and that the extrapulmonary immunization scheme was able to induce a certain degree of protection of the lung from infection with P. brasiliensis

Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females) aged 17 to 58 years. Twenty one (53.84%) of the patients presented a slow acetylatingphenotype and 18(46.16%) a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD) acti vity was decreased in 5(23.80%) slow acetylators and in 4(22.22%) fast acetylators. Glutathione reductase activity was decreased in 14 (66.66%) slow acetylators and in 12 (66.66%) fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p < 0.02). Analysis of the resultspermitted us to conclude that serum sulfadoxin levels are related to the acetylatorphenotype. Furthermore, sulfadoxin levels were always above 50 µg/ml, a value considered therapeutic. Glutathione reductase deficiency observed in 66% of patients may be related to the intestinal malabsorption of nutrients, among them riboflavin, a FAD precursor vitamin, inpatients with paracoceidioidomycosis.

Influence of canine brain decomposition on laboratory diagnosis of rabies

Canine brains infected with rabies virus were submitted to decomposition by being left at room temperature of 25 to 29oC for up to 168h. At 24h intervals, brain fragments were analyzed by immunofluorescence (IF) and by the mouse intracerebral inoculation (MI) test to confirm the diagnosis of rabies and to measure the putrefaction effect on the accuracy of the diagnosis. Forty eight h after the beginning of the experiment, the MI test showed signs of impairment with four negative results, while after 72h, 100% of the results were negative to the MI test and only one result was negative to the IF test, indicating that the threshold period for accurate diagnosis is 24 to 48h before putrefaction. The authors recommend the shipment of suspected cases of rabies to the laboratory for confirmation, but the use of putrid materials for diagnosis is meaningless because of false-negative results.