Abstract INTRODUCTION The control of reservoirs for Leishmania infantum -induced zoonotic visceral leishmaniasis requires the identification of dogs posing a population risk. Here, we assessed the performance of several assays to identify Lutzomyia longipalpis infectious dogs. METHODS We evaluated 99 dogs that were positive for visceral leishmaniasis based on parasite identification. Serological analyses were performed using an enzyme-linked immunosorbent assay, immunofluorescence antibody tests in 1:40 and 1:80 dilutions, rapid dual path platform tests, immunochromatographic assay with a recombinant rK39 antigen, fast agglutination screening tests, and direct agglutination tests. We also performed PCR to analyze peripheral blood and xenodiagnosis. RESULTS Forty-six dogs infected at least one L. longipalpis specimen. Although the serological test sensitivities were above 85% for detecting L. longipalpis infectious dogs, none showed a satisfactory performance, as both specificity (0.06 to 13%) and the area under the receiver operating characteristic curve (45 to 53%) were low. The PCR results were also weak, with a sensitivity of 30%, specificity of 72%, and an area under the receiver operating characteristic curve of 51%. The infected L. longipalpis proportion was higher among asymptomatic dogs than symptomatic dogs. Among the symptomatic dogs, those with ulceration-free skin diseases were more infectious, with an odds ratio of 9.3 (confidence interval of 1.10 - 428.5). The larger the number of insects fed, the greater the detected infectiousness. CONCLUSIONS Our study supports the imperative to develop novel technologies for identifying the infectious dogs that transmit L. infantum for the benefit of public health.

ABSTRACT Dogs are considered the main reservoir of Leishmania infantum. This protozoan causes visceral leishmaniasis (VL), an uncontrolled urban zoonosis in Brazil. Serological tests and polymerase chain reaction (PCR) on peripheral blood were performed to identify infected dogs in scenarios of higher and lower prevalence of the disease (Teresina and Vitória). One-hundred infected and 57 non-infected animals from Teresina and 100 non-infected animals from Vitória were studied. Animal selection was not dependent on previous serology. The sensitivity (Teresina) and specificity (Teresina and Vitória) were as follows: indirect antibody fluorescence (IFAT) cut-off of 1:40 (IFAT 1:40): 96%, 18%, and 76%; IFAT 1:80: 90%, 33%, and 93%; direct agglutination test (DAT): 96%, 33%, and 98%; fast agglutination screening test (FAST): 93%, 68%, and 100%; immunochromatographic assay with a recombinant rK39 antigen (rK39): 88%, 74%, and 98%; enzyme linked immunosorbent assay (ELISA): 91%, 79%, and 98%; rapid dual-path platform test (TR DPP®): 98%, 60%, and 98%; and blood PCR: 29%, 93%, and 97%, respectively. In the high transmission area, none of the tests adequately discriminated L. infantum-infected from non-infected dogs. However, in the high transmission city, the area under the receiver operating characteristic (ROC) curve of FAST, DAT, ICrK39, ELISA and TR DPP® was high.

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

INTRODUCTION:Human immunodeficiency virus (HIV) coinfection with Leishmania infantum or Leishmania donovani, the agents of visceral leishmaniasis (or kala-azar), has become a fatal public health problem in the tropics where kala-azar is endemic.METHODS:The clinical presentation of patients with HIV and L. infantum coinfection is described using two unique databases that together produce the largest case series of patients with kala-azar infected with HIV in South America. First, a retrospective study paired the list of all patients with kala-azar from 1994 to 2004 with another of all patients with HIV/AIDS from the reference hospital for both diseases in the City of Teresina, State of Piauí, Brazil. Beginning in 2005 through to 2010 this information was prospectively collected at the moment of hospitalization.RESULTS:During the study, 256 admissions related to 224 patients with HIV/L. infantum coinfection were registered and most of them were males between 20-40 years of age. Most of the 224 patients were males between 20-40 years of age. HIV contraction was principally sexual. The most common symptoms and signs were pallor, fever, asthenia and hepatosplenomegaly. 16.8% of the cohort died. The primary risk factors associated to death were kidney or respiratory failure, somnolence, hemorrhagic manifestations and a syndrome of systemic inflammation. The diagnosis of HIV and kala-azar was made simultaneously in 124 patients.CONCLUSIONS:The urban association between HIV and kala-azar coinfection in South America is worrisome due to difficulty in establishing the diagnosis and higher mortality among the coinfected then those with either disease independently. HIV/L. infantum coinfection exhibits some singular characteristics and due to its higher mortality it requires immediate assistance to patients and greater research on appropriate combination therapy.

Leishmania infantum (syn. Leishmania chagasi) is the etiological agent of visceral leishmaniasis (VL) in Brazil. The epidemiology of VL is poorly understood. Therefore, a more detailed molecular characterization at an intraspecific level is certainly needed. Herein, three independent molecular methods, multilocus microsatellite typing (MLMT), random amplification of polymorphic DNA (RAPD) and simple sequence repeats-polymerase chain reaction (SSR-PCR), were used to evaluate the genetic diversity of 53 L. infantum isolates from five different endemic areas in Brazil. Population structures were inferred by distance-based and Bayesian-based approaches. Eighteen very similar genotypes were detected by MLMT, most of them differed in only one locus and no correlation was found between MLMT profiles, geographical origin or the estimated population structure. However, complex profiles composed of 182 bands obtained by both RAPD and SSR-PCR assays gave different results. Unweighted pair group method with arithmetic mean trees built from these data revealed a high degree of homogeneity within isolates of L. infantum. Interestingly, despite this genetic homogeneity, most of the isolates clustered according to their geographical origin.

Embora o tratamento da leishmaniose visceral com antimoniato pentavalente seja normalmente bem sucedido, alguns pacientes requerem uma terapêutica de segunda linha, habitualmente com anfotericina B. Para identificar as características clínicas que possam indicar uma resposta inadequada ao antimônio pentavalente, realizou-se um estudo caso-controle em Teresina, Piauí, Brasil. Em um período de dois anos ocorreram 19 casos de LV para quem os médicos de um hospital prescreveram o tratamento de segunda linha com anfotericina B após determinarem falha com o uso de antimônio pentavalente. O grupo controle foi constituído por 97 pacientes que tiveram o tratamento com antimônio bem sucedido. Foi feita uma análise de prontuários, utilizando-se análise univariada e multivariada. A porporção de cura após o uso de anfotericina B foi 90%. A razão de odds para a prescrição de anfotericina B foi 10,2 para crianças com menos de um ano, quando comparadas com pessoas com mais de 10 anos. Pacientes que tinham tido co-infecção tinham uma RO de 7,1 e aqueles que tinham utilizado antibióticos tinham uma razão de odds de 2,8. Estes dados estão de acordo com o uso mais prolongado de antimônio pentavalente para crianças ou com a recomendação de anfotericina B como droga de primeira escolha para crianças e para pessoas com co-infecções. Sugerem também que se pode indicar a quimioprofilaxia para infecções bacterianas em crianças menores com LV.

Although treatment of visceral leishmaniasis with pentavalent antimony is usually successful, some patients require second-line drug therapy, most commonly with amphotericin B. To identify the clinical characteristics that predict an inadequate response to pentavalent antimony, a case-control study was undertaken in Teresina, Piaui, Brazil. Over a two-year period, there were 19 cases of VL in which the staff physicians of a hospital prescribed second-line therapy with amphotericin B after determining that treatment with pentavalent antimony had failed. The control group consisted of 97 patients that were successfully treated with pentavalent antimony. A chart review using univariate and multivariate analysis was performed. The cure rate was 90% with amphotericin B. The odds ratio for the prescription of amphotericin B was 10.2 for children less than one year old, compared with individuals aged over 10 years. Patients who presented coinfection had an OR of 7.1 while those on antibiotics had an OR of 2.8. These data support either undertaking a longer course of therapy with pentavalent antimony for children or using amphotericin B as a first-line agent for children and individuals with coinfections. It also suggests that chemoprophylaxis directed toward bacterial coinfection in small children with VL may be indicated.