Long-term follow-up of dogs with leishmaniosis treated with meglumine antimoniate plus allopurinol versus miltefosine plus allopurinol.

BACKGROUND: Visceral leishmaniosis is a potentially life-threatening illness caused by a protozoan parasite of the genus Leishmania. It is found mainly in areas where both the parasite and its vector are endemic and is one of the most challenging infectious diseases in the world to control. HIV infected patients are vulnerable to Leishmania infections, and the main reservoir hosts of Leishmania infantum parasites are domestic dogs. Here, we evaluated the long-term efficacy of treatment with meglumine antimoniate plus allopurinol (G1) compared to miltefosine plus allopurinol (G2) in dogs naturally infected L. infantum. METHODS: Eighteen dogs with leishmaniosis were divided into the following two groups: G1 (n = 9) was treated subcutaneously with meglumine antimoniate (100 mg/kg/day/30 days) plus allopurinol (10 mg/kg/per day/30 days), while G2 (n = 9) was treated orally with miltefosine (2 mg/Kg/day/30 days) plus allopurinol (10 mg/kg/day/30 days). Thereafter, the same dose of allopurinol was administered to both groups for 6 years. Leishmania DNA in lymph node aspirates from the G1 and G2 dogs was quantified by real-time quantitative PCR at baseline and every 3 months for 24 months, and then at 28, 36, 48, 60 and 72 months. At each assessment, the dogs were examined for signs of disease, and their clinical scores were recorded. RESULTS: Both combination therapies produced significant clinical improvements in the dogs, with a significant reduction in the parasitic load in the lymph nodes of the dogs from both groups after 3 months of treatment. Clinical relapses were observed in four dogs from G2 (miltefosine/allopurinol), and just one dog from G1 (meglumine antimoniate/allopurinol). All dogs that relapsed had increased clinical scores, and increased anti-Leishmania antibody titers and parasitic loads in their lymph nodes. CONCLUSIONS: Long-term, the clinical and laboratory findings of the G1 dogs were more stable than those of the G2 dogs, thus indicating that meglumine antimoniate had better clinical efficacy than miltefosine. The results suggest that treatment with allopurinol as a maintenance therapy is crucial for stabilizing the care of canine leishmaniosis.

Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report.

BACKGROUND: Transmission of canine leishmaniasis (CanL), a severe infection caused by L. infantum, usually occurs through the sand fly bite to the vertebrate host. A venereal route of transmission has also been suggested, but this issue is still controversial. FINDINGS: Here, we report a case of a dog affected by orchitis showing a clinical profile of L. infantum infection. By exploiting a real-time PCR assay, we detected a significantly higher DNA load of the parasite in the lymph node and testis than in blood and urine samples collected from the dog. CONCLUSIONS: Our results suggest that: 1) L. infantum infection can be associated with testicular lesions in naturally infected dogs; 2) genital involvement could result in shedding of the parasites in the semen, favoring venereal transmission of the disease.

Evidence for a relationship between Leishmania load and clinical manifestations.

Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmaniainfantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis. The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis. Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis.

Study of efficacy of miltefosine and allopurinol in dogs with leishmaniosis.

Visceral leishmaniosis is a life-threatening disease of medical, social and economic importance in endemic areas. It is an opportunistic infection in immunocompromised patients, including human immunodeficiency virus-positive subjects. Dogs are the main reservoir of Leishmania infantum. The aim of this study was to evaluate the efficacy of miltefosine and allopurinol for the control of human leishmaniosis using the dog as a model. The study included 28 sick dogs treated with miltefosine (2 mg/kg/day PO) administered concurrently with allopurinol (10 mg/kg/day, PO) for 30 days, and then with allopurinol alone, at the same dosage, for 1 year. Eight dogs (four of which relapsed) received a second cycle of miltefosine within 6 months of the first cycle. Efficacy was measured by real-time polymerase chain reaction assay on whole blood samples and lymph node aspirates, collected at baseline and every 3 months for 12 months. Of the total number of animals (28), two showed renal insufficiency and died after the start of therapy with miltefosine. Two other dogs presented some side effects to treatment, such as nausea, vomiting and reduction in white and red blood cell counts, and these animals were excluded from the follow-up. The results showed that the first cycle of therapy with miltefosine and allopurinol induced a drastic and progressive reduction of L. infantum load in lymph node aspirates but the second cycle did not eliminate the parasite.

Interferon-gamma (INF-gamma), IL4 expression levels and Leishmania DNA load as prognostic markers for monitoring response to treatment of leishmaniotic dogs with miltefosine and allopurinol.

In this study, we searched for a connection between Leishmania load and cytokine expression levels in the tissues of Leishmaniainfantum naturally infected dogs and the efficacy of treatment with miltefosine and allopurinol. To this purpose, we exploited a real-time PCR system to detect Leishmania load and the expression levels of IFN-gamma and IL-4 mRNAs at the time of diagnosis and during the follow up post-treatment. In particular, we measured the amount of parasites in blood and lymph node samples, while the expression levels of IFN-gamma and IL-4 cytokines were assessed in the blood of the animals. We employed different targeted real-time PCR assays on 20 naturally infected dogs with clinical signs. Three healthy dogs living in a non-endemic area were selected as negative controls. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels in different kinds of tissue might represent a reliable tool to evaluate the immune response, the efficacy of the therapy and to predict the relapses during the treatment.

Urine sampling for real-time polymerase chain reaction based diagnosis of canine leishmaniasis.

A real-time polymerase chain reaction (PCR) assay was used for quantifying Leishmania infantum DNA in urine samples from naturally infected dogs. Forty-one infected dogs were divided into 3 groups: 22 dogs showing only cutaneous signs (group 1), 12 dogs showing hematuria (group 2), and 7 dogs affected by severe nephropathy (group 3). Groups 2 and 3 dogs showed altered laboratory parameters related to an impairment of renal function. The real-time PCR analysis showed higher levels of Leishmania DNA in the lymph node aspirates from all groups of infected dogs versus those measured in their blood or urine. Interestingly, urine samples from dogs belonging to groups 2 and 3 contained a higher Leishmania DNA load than that detected in their blood. This finding suggests that a real-time PCR analysis of urine from infected dogs could be a useful and noninvasive tool for monitoring the severity of leishmaniasis.

Fluorescence-based detection of Leishmania infantum DNA in phlebotomus vectors.

The geographical diffusion of leishmaniasis can depend on the factors limiting the distribution of sandfly vectors. In Sicily, as in all Mediterranean areas, sandflies are present almost all year round because the climate permits perpetuation of this vector's life cycle. Transmission can occurs in rural and domestic habitats through the bite of infected female phlebotomine sandflies. In Italy, the visceral form of leishmaniasis is commonly found, which is due exclusively to L. infantum. Two species of sandfly are considered the main vectors: Phlebotomus perniciosus and P. perfiliewi. Fluorescence resonance energy transfer technology was used to determine the parasite load in phlebotomine vectors, and the test was targeted on a 117-bp fragment of kinetoplast DNA minicircles. The assay was evaluated, focusing on analytical sensitivity, discriminatory power, and reliability of quantification. During 2005, a total of 1686 sandflies were trapped in various Sicilian provinces and in farms randomly selected using black light traps. We found 20, 30, and 16 sandflies positive for Leishmania for each kind of analyzed phlebotomine sandfly, respectively, corresponding to 6.5% for the gravid, 2.7% for the fed, and 6.3% for other groups. Previously the insects were identified on the basis of morphology and the most prevalent sandfly species were P. Sergentomyia minuta, P. perfiliewi, and P. perniciosus.

Leishmania DNA quantification by real-time PCR in naturally infected dogs treated with miltefosine.

A new drug that has just become available in India for treatment of human visceral leishmaniasis (VL) is miltefosine, an alkyphospholipid that was originally developed as an oral antineoplastic agent. Miltefosine is not only directly toxic for Leishmania parasite, but it also enhances both T cell and macrophage activation and production of microbicidal reactive nitrogen and oxygen intermediates. It is highly effective in the treatment of Leishmania infection in mice and human beings. However, adverse effects in dogs treated with miltefosine have been reported, but there are no data on the efficacy of this drug for the treatment of canine visceral leishmaniasis (CVL). The aim of this study was to use a real-time PCR assay to monitor the Leishmania load in the blood samples and lymph node aspirates of 18 naturally infected dogs before and after treatment with miltefosine (2 mg/kg for 30 days). The results of our study showed that the therapy with miltefosine shows a drastic and progressive reduction of parasite load in lymph node aspirates, but does not suppress the parasite in lymph nodes. In all dogs the real-time PCR assay demonstrated an irregular presence of parasites in blood. Therefore, blood does not seem a suitable substrate for the purpose of quantifying Leishmania DNA.

Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol.

A real-time PCR assay was exploited for monitoring the Leishmania DNA load in different tissues from 18 naturally-infected dogs before and after treatment with a combination of meglumine antimoniate (100mg/kg/day, subcutaneously) and allopurinol (10mg/kg/day, orally) for 30 days. After the combined therapy, allopurinol was continued at the same dose until the end of the observation period. Whole blood samples, lymph node aspirates, and skin biopsies were collected at the time of diagnosis, 1 month after starting therapy, and every 3 months for 2 years. In six dogs parasite load assessments continued every 6 months for a further 3 years. At each assessment, the dogs were examined for signs of disease and a clinical score was recorded. At diagnosis, the highest Leishmania DNA load was detected in lymph node aspirates. From 1-6 months post-therapy a general improvement in clinical conditions was recorded in all dogs, which correlated with a decrease in the parasite DNA load in all tested tissues, even though it was less pronounced in lymph node aspirates. In the period from 9-24 months post-therapy, a re-increase in parasite load was observed in the tissues of some dogs, concomitant with a disease relapse. The results show that the combined therapy with meglumine antimoniate and allopurinol promoted a clinical improvement which was accompanied by a reduction in the parasitic load in the blood, skin and lymph nodes but, even after long period of allopurinol administration alone, Leishmania may persist in dog tissues.

Leishmania DNA load and cytokine expression levels in asymptomatic naturally infected dogs.

The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-gamma, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease.

Comparison of different tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.

In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis.