Study design and ethical consideration
The patients and controls were recruited in the municipal hospital of Villa Tunari, a tropical town of Bolivia endemic for intestinal helminthiasis, because did not have a basic sanitation services, in the period 2013 to 2015.
A total of 54 patients with confirmed cutaneous leishmaniasis and 40 control subjects, both permanent residents in the tropical area were invited to participate in the study. But it just started with 44 patients and 30 control subjects. The patients and controls were age-sex matched.
For the patients were considered the following exclusion criteria: pregnancy, chronic non-communicable diseases, concomitant infectious diseases such as Tuberculosis and HIV/AIDS, super infection by bacteria of the cutaneous ulcerative lesions and previous therapeutic failure to antimony compounds treatment. The control subjects were clinically no evidences of any diseases at time of recruitment.
Ethic permission for all procedures involving human volunteers was obtained from the Bolivian Ethic Committee of the Medical Faculty, Universidad Mayor de San Simón.
The patients and controls signed a write consent accepting to participate in the study.
Intestinal helminths and cutaneous Leishmaniasis treatment
The patients were randomly assigned to groups: “A” which received the Albendazole to treat the intestinal helminths and posteriorly the antimony compounds as treatment of cutaneous Leishmaniasis and the group “B” which received only the antimony compounds for the treatment of cutaneous Leishmaniasis.
The Albendazole was administered orally (Dose 400mg) once according to WHO recommendations .
The antimony compounds were administered intramuscularly (Dose/Kg/weight) daily by 20 days according the norms of Bolivian Health ministry .
Venous blood tubes of the Vacutainer® systems (cat no 366430 and 367587) were obtained from Becton Dickinson AB (New Jersey. USA). The Panotic fast staining system (cat no 620529) was obtained from LB (Laborclin, Saõ Paulo, Brazil). The IgE ELISA kit (cat no
2525-300A) was obtained from Monobind Inc. (Lake Forest, California, United States of America). The TNF-α (cat no DTA00C) and IL-17 (cat no D1700) ELISA kits were obtained from R&D systems (Minneapolis, United States of America). The CD4, CD8 kit (cat no 340167) was obtained from Becton Dickinson FACSCount TM (United States of America)
Blood samples collection
The patients were blood sampled twice, once before therapy intervention (T0) and then ten days after ending the treatment with antimony compounds (T1). The control subjects were blood-sampled once, at the beginning of the study.
The blood samples were collected by venepuncture after 12 h of fasting in all of subjects in two tubes. The samples for haematology were processed immediately and one aliquot was sent to Medical Faculty laboratory (LABIMED) for the assessment of CD4/CD8 T cells ratio. The samples collected in tubes without additives were centrifuged, fractioned in aliquots of 500 µL and preserved at -80oC until processing by lab tests.
Laboratory test for diagnostic of cutaneous leishmaniasis
The leishmania parasite (amastigote forms) was detected in stained smears of scrapings
of border of cutaneous ulcerative lesions according with the procedures of Bolivian Health ministry  in the laboratory of Villa Tunari Hospital.
Laboratory test in blood samples
The haematological test was performed in the laboratories of Villa Tunari Hospital. The following parameters were measured: Total number of white blood cells, neutrophils, eosinophil and lymphocytes (as fraction of white blood cells), the Total number of red blood cells, haemoglobin and haematocrit in an autohaematology analyser BC-3000 Plus, MindrayTM (Nanshan, Shenzhen, P. R. China).
The plasma concentration of IgE, IL-17 and TNF-α were determined in the laboratories of CUMETROP by ELISA test kit according with the assay instructions. The detection limit was 0.125 IU/mL for IgE and 3pg/mL for IL-17 and TNF-α (Data supplied by the manufacturers).
The CD4/CD8 T cells ratio was performed in LABIMED laboratory by count of CD4 and CD8 T cells in the BD FACSCountTM and using a kit BD for determination of CD markers according with the assay instructions. Briefly, the procedure consisted in the petrification of the cells with formaldehyde solution, subsequently fluorescein-labelled anti-CD4, CD8 and CD3 monoclonal antibodies were added and the intensity of the fluorescence emitted by the complexes formed was measured in a FACSCountTM. The results were expressed in number and percentage of CD4, CD8 cells and also expressed as CD4 / CD8 ratio.
Lesion healing assessment
The cutaneous lesions of patients were measured twice; once at the beginning of treatment with antimony compounds and ten days after complete the last dose of treatment. The following measurements were made: Area of ulcerative lesion (mm2), presence of raised edge of lesion and presence of humidity. The healing lesions were evaluated as reduction of the area expressed in mm2, absence of raised edge and humidity.
The SPSS software v. 22 was used. The Chi square test was used for comparison of individual characteristics of the lesions of the groups “A” (Albendazole) and “B” (Non Albendazole). Wilcoxon signed rank test was used to compare the same variable of patients in T0 and T1 for the two groups. The Mann-Whitney test was used for testing the significance of differences between two non-normally distributed continuous variables such as area of lesion (groups A and B), plasma concentration of immunological markers and Number of eosinophil and CD4/CD8T cell ratio between patients and controls. It was considered in all of cases as level of statistical significance p<0.05