METHODOLOGY

The methodology of this study were executed in accordance with the relevant
regulations and guidelines (Ahmad et al .,2022).

Sample collection:
A total of 29 samples were collected from suspected CL patients in North Waziristan's
central region and DHQ Hospital in Miranshah from June to August 2023. Data of
these patients were received from these regions District Health Office (DHO) in
North Waziristan's central region and DHQ Hospital in Miranshah and then their skin
lesions were collected. A printed questionnaire consisted of the patient’s name,
father/guardian name, age, gender, number, site, duration, and type of lesions,
address, profession, history of the infection (if any), use of insect repellents,
insecticides, impregnated bed nets and, treatment, and follow up and any previous
diagnosis through microscopy or biopsy. Ethanol (70%) will be used to clean the
lesions. A scalpel will used to scrape the skin for oozing out of the blood from the
lesion and usually, a cut was given in the periphery of the inflamed lesion. The
sample will be identified as L. tropica by microscopy, and stored in the laboratory
under appropriate conditions.
Sample processing
Staining

Giemsa stain was used (45-60 min) for staining the slides. The slides were dipped (3-
4 times) in the Giemsa buffer solution after staining and placed upright in a rack to air
dry.

Microscopy

Subsequently staining, each slide was observed with oil immersion objective (10x100
magnifications) under a microscope (CX31; Japan) to detect the intra-macrophagic
amastigotes of L. tropica. On microscopic examination, they appeared as small ovoid
organisms, single or in clusters with faint eosinophilic cytoplasm, dark-colored
nucleus and kinetochore present within the cytoplasm of phagocytic cells and the
remaining samples were kept in sterile Eppendorf tubes for further processing
Material required for data collection:

i. Gloves
ii. Syringes
iii. Glass Slides
iv. Ethanol
v. EDTA Tubes
vi. from the month Scalpel blades