Parasitic load and histological aspects in different regions of the spleen of dogs with visceral leishmaniasis.

Leishmania infantum causes from subclinical infection to severe disease in humans and dogs. The spleen is one of the organs most affected by the infection. Although evidence exists that the parasitic load distribution and histological alterations may not be homogeneous in the affected organs of naturally infected individuals, it has not been formally demonstrated using the current techniques used for studying the disease. In six dogs naturally infected with Leishmania, parasitic load and histological changes were compared in samples collected from the lower, middle and upper third of the spleen. Parasitic load in the spleen of the group of dogs was variable, revealing a difference of 61 times between animals with the lowest and the highest parasitism. The set of parasitic load values of each dog showed a cluster trend, when compared to the other animals. Nevertheless, the parasitic load values of each dog showed a variation ranging from 3.2 to 34.7 times between lowest and highest value. Histological changes showed recognizable variation in frequency (granulomas) or intensity (perisplenitis) in the spleen of 2 out of the 6 dogs. The agreement of histological findings between samples collected from the different thirds of the spleen was good (kappa coeficient, 0.61-0.80) very good (0.81-0.99) or perfect (1.00), for most of the parameters analyzed. Variability of parasitic load and, to a lesser extent, histological changes in spleen of dogs with visceral leishmaniasis is observed. Such variability may be taken in account in the design of studies on pathogenesis, vaccine and therapeutic drug development.

Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors.

The sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector.

The Rapid Test Based on Leishmania infantum Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs.

Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that the current protocol performed better than previous protocol primarily by reducing false-positive results.

Evaluation of 17-AAG as a chemotherapeutic potential for the treatment of visceral leishmaniasis in experimentally infected hamsters / Evaluation of 17-AAG as a chemotherapeutic potential for the treatment of visceral leishmaniasis in experimentally infected hamsters

A leishmaniose é causada por protozoários do gênero Leishmania. Considerada endêmica em diversos países, incluindo o Brasil, o tratamento dessa doença consiste na utilização de agentes quimioterápicos. No entanto, esses fármacos apresentam diversos efeitos colaterais, alto custo e aumento progressivo de falha terapêutica com baixas taxas de cura. Tendo em vista a necessidade para desenvolvimento de novos compostos anti-Leishmania, ensaios realizados anteriormente por nosso grupo evidenciaram o potencial do 17 - allylamino 17 - demethoxygeldanamycin (17-AAG) no tratamento in vivo da leishmaniose cutânea (LC). Esse composto inibe a atividade ATPásica da proteína de choque térmico 90 (HSP90), que no parasito é conhecida por exercer funções essenciais. Como consequência, sua inibição pode levar à morte do parasito, resultando no controle da doença. Assim, nosso estudo avaliou o potencial do 17-AAG no tratamento da leishmaniose visceral (LV) utilizando hamster como modelo experimental. Este modelo foi utilizado, pois apresenta características clínico-patológicas semelhantes às observadas em humanos com calazar. Inicialmente foi realizado um ensaio de toxicidade com o 17-AAG utilizando doses intraperitoneais de 20 ou 40 mg/kg diariamente ou em dias alternados, totalizando quinze aplicações. O esquema terapêutico causou toxicidade no fígado dos animais tratados com ambas às doses e no rim dos animais tratados com 40mg/kg. Todos os animais que receberam 40mg/kg e 80% daqueles que receberam 20mg/kg vieram a óbito no final dos dois esquemas terapêutico. Com o intuito de reduzir a toxicidade e avaliar o potencial leishmanicida do 17-AAG, experimentos subsequentes foram realizados utilizando doses menores de 5 ou 10 mg/kg de 17-AAG em intervalos maiores de três dias, totalizando sete aplicações. Os animais do estudo apresentavam alterações bioquímicas e hematológicas, antes mesmo de iniciar o tratamento, permanecendo com essas alterações durante todos os períodos avaliados, que foram igualmente observadas nos animais do grupo controle não tratados com 17-AAG. Com o intuito de avançar na avaliação de toxicidade e eficácia do tratamento, outro ensaio foi realizado utilizando doses de 10 ou 20 mg/kg de 17-AAG em dias alternados, totalizando sete aplicações. A análise histopatológica mostrou que os hamsters tratados não apresentaram alterações em fígado e rim adicionais às observadas no grupo controle. Esses achados mostram que o tratamento com 17-AAG foi tóxico somente para as células hepáticas dos animais, quando aplicado em doses mais elevadas e em intervalos de tempos curtos. Em doses mais baixas e intervalos maiores, as análises histopatológicas do fígado e rim dos animais revelaram alterações similares às causadas pelo diluente. Quando os hamsters foram tratados com as doses toleradas de 10 ou 20 mg/kg de 17-AAG em dias alternados, o 17-AAG não foi capaz de reduzir a carga parasitária no baço, fígado ou linfonodo desses animais. Em conjunto, esses dados mostram que os hamsters não representam um modelo adequado para teste com 17-AAG, pois os mesmos não toleraram doses elevadas deste fármaco que seriam necessárias para o controle da infecção, indicando a necessidade da utilização de outro modelo experimental nos estudos de tratamento da LV

Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis.

Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4-100) in splenic aspirate; 79.2% (CI 68-90.3) in lymph nodes; 77.3% (CI 64.5-90.1) in skin; 75% (CI 63.1-86.9) in blood; 50% (CI 30-70) in bone marrow; 37.5% (CI 24.2-50.8) in left-eye; and 29.2% (CI 16.7-41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7-79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5-96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.