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The study protocol (Protocol # 12–0075) was approved by the Institutional Review Boards (IRB) of the Faculty of Medicine, Pharmacy and Odontostomatology (FMPOS), Bamako, Mali, and of the National Institutes of Allergy and Infectious Diseases (USA). A collective village-wide oral consent was obtained from the villages’ elders, and all adult participants signed individual written informed consent and a parent or guardian of any child participant provided informed consent on their behalf.
The study was carried out in June 2014 in three ecologically distinct districts (Fig 1): 1] The district of Diema in the Region of Kayes, located at 345 km from Bamako in the western part of the country. The study was carried out in 4 villages: Nafadji (9.233399W, 14.557710N), Guemou (9.312490W, 14.546290N), Debo Massassi (9.368320W, 14.579850N) and Tinkare (9.179729W, 14.490520N). In Diema, the undulating topography is dominated by sandy plains and a plateau with a few rocky outcrops, and is an extension of Mont Manding. The climate is typical Sahelian with sandy clayey soil, characterized by the alternation of two seasons with temperatures varying between 15°C and 45°C depending on the season. The rainy season is short lasting from July to October, with rainfall ranging from 400 mm to 800 mm. The dry season lasts from November to June. The site is influenced by the harmattan, a dry wind that blows from the northeast to the southwest, and the monsoon bringing rain. The vegetation is characterized by shrub and tree vegetation. The population is composed mainly of Sarakolé, Bambara, Peulh, Moor, and Kagoro ethnicities.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pntd.0005141.g001 Map of Mali showing the study sites in different districts. 2] The district of Kolokani, in the region of Koulikoro, is located at 105 km northwest of Bamako in central Mali. The study was carried out in the village of Tieneguebougou (8.077450W, 13.57639N). The north of the district is dry, Sahel land, primarily used for livestock. The study site is at the interface between the sahelian and the wetter Sudan to the south. The population is composed mainly of Bambara, Peulh, Mossi, and Dioula ethnicities 3] The district of Kolondieba, region of Sikasso, located at 250 km from Bamako in the southern part of Mali. The study was carried out in the village of Boundioba (6.982890W, 11.040190N). The climate is typically south-savannah type with clear forests and an average of 1250 mm of rain spread over 60 days per year. The average temperatures vary from 20°C to 31°C in the same year. The population is composed of Bambara, Peulh, Senoufos and Sarakolés ethnicities. Based on historical data from the National referral dermatologic hospital of Bamako, CL cases are regularly recorded from Diema, but no population-based prevalence of CL is available. The districts of Kolokani and Kolondieba are known to be endemic for lymphatic filarial (LF) but not for CL.
In each village, 40 to 50 households were randomly selected (with an average of 5–7 persons per household, and 195 to 250 subjects per village) and screened for CL. Households were randomly selected from a list obtained from a census data collected by the study team. Subjects living in the randomly selected households were included in the study: 2–65 years old in the district of Diema (Nafadji, Guemou, Debo Massassi and Tinkare), and 18–65 years old in the districts of Kolokani (Tieneguebougou) and Kolondieba (Boundioba). We targeted adults for recruitment in the districts of Kolokani and Kolondieba due to their low endemicity for CL. After informed consent, all members of the selected households were invited to participate the screening. The screening consisted of clinical examination by a dermatologist, a LST and a finger prick to collect blood samples for immunological studies. The LST (leishmanin, IRC 1228181375, LOT #127; Institute Pasteur of Iran, Tehran) was performed at the beginning of the study as described elsewhere [10]. Briefly, 0.1 ml of leishmanin was injected intradermally in the left forearm. Each ml of preparation contains, 6×106 killed L. major promastigotes (MRHO/IR/75/ER strain) and Thimerosal 0.01% in phosphate buffered saline (PBS) at pH 7.0. Readings were taken 48 to 72 hours after the injection using the ballpoint-pen technique. The induration was measured in two perpendicular directions. A mean of the two measurements of 5 mm or greater was considered as positive [13]. In the case of no reactivity, the test would be observed at 72 h of the injection. All subjects who were not seen by 72 hours post injection of leishmanin were excluded.
Capillary blood samples were collected for anti-sand fly salivary proteins antibody measurement from all study participants screened by LST in each village. The blood collection was made right after LST testing. The finger of each participant was cleaned with isopropyl alcohol and pricked with a sterile single use disposable lancet. A maximum of 5 drops of whole blood (~ 50 μL per drop, 250 μL total) were dropped onto filter papers (Whatman 903 Protein Saver Cards)[14] labeled with the participant’s unique identification number. Thereafter, a subset of fifty individuals out of the 250 collected samples were randomly chosen to test for levels of anti-sand fly saliva antibodies tested.
Filter paper containing the blood sample was obtained using a 6mm disposable punch and eluted in PBS 0.05% Tween and kept at room temperature (RT) overnight. ELISA was performed as described elsewhere [15]. Briefly, ELISA plates were coated with 50 μl of salivary gland homogenate of P. duboscqi diluted to 2μg/ml in Carbonate/Bicarbonate buffer (NaHCO3 0.45 M, Na2CO3 0.02 M, pH 9.6) overnight at 4°C. After three washes with PBS- 0.05% Tween, the plates were blocked with PBS containing 4% bovine serum albumin for 2 hours at room temperature. The plates were washed three times with PBS 0.05% Tween, the sera were added and incubated at RT for one hour. After further washing, the antibody Goat anti-human IgG (H+L) alkaline phosphatase conjugated (Sigma, MO) were added and incubated at 37° for one hour. Following another washing, p-nitrophenyl phosphate substrate (Sigma Sr. Louis, MO) was added and the absorbance was read at 405nm on a Versamax microplate reader after 30 minutes. Values obtained were subtracted from those obtained for the background (i.e. OD values for wells where PBS was added instead of eluted blood).
Active detection of CL in individuals: Interviews and clinical examination were conducted through house-to-house visits by experienced dermatologists to screen for any suspected skin lesions. Interviews included the history of the lesion, while physical examination provided information on the location (head/neck, trunk, upper extremity, lower extremity) and duration (in days up to the date of the visit) of each lesion. Lesions were documented through photography. Non-invasive scrapings of the border of the lesions were performed and tested by both PCR and microscopy to confirm the presence of L. major. All subjects diagnosed with CL, by PCR or microscopy, were treated with Meglumine Antimoniate.
Parasite DNA from suspected lesions was extracted using QIAamp DNA Micro Kit according to the manufacturer’s instructions [16], and stored at -20°C until PCR amplification. Leishmania DNA was detected by PCR using forward and reverse primers for Leishmania sp. (Uni21/Lmj4) as described in [17]. The primer design was based on the published Kinetoplastid DNA minicercle sequence (kDNA) from L. major [18].
The data were recorded on a case form (CRF), entered in iDataFax management (Version 2014.1.0), and analyzed using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA). Descriptive analyses were used to assess the association between LST and demographic variables. Fisher's exact test was used to assess the association between infection and demographic variables. Age means between villages were compared using an independent samples t-test. One-way ANOVA with Bonferroni's multiple comparison test was applied to evaluate the difference in the mean size of the LST reaction between the two study sites.
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