PCR-Based Detection of Leishmania DNA in Skin Samples of Post Kala-Azar Dermal Leishmaniasis Patients from an Endemic Area of Bangladesh

Abstract

SUMMARY: Post kala-azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL) and PKDL patients are an important reservoir for anthroponotic transmission of VL. Therefore, diagnosis and treatment of PKDL is important for the kala-azar elimination program in South Asia, including Bangladesh. While definitive diagnosis of PKDL is still based on microscopy, despite the low sensitivity of this method of diagnosis, PCR for identification of kinetoplast DNA (kDNA) from Leishmania parasites is expected to be a rapid and sensitive diagnostic method. We attempted PCR-based diagnosis from skin biopsy specimens and compared PCR to other available detection methods in order to determine the acceptability and feasibility of the PCR diagnostic method in an endemic area of VL in Bangladesh. Both skin biopsy specimens and blood samples were collected from 110 patients suspected to have PKDL from 6 subdistrict health complexes in Mymensingh, Bangladesh. Using microscopy, we identified 32 samples (29.1z) that were positive for Leishmania. Immunochromatography tests indicated that 85 samples (77.3z) were positive for Leishmania. In contrast, a total of 104 (94.5z) samples tested positive using nested PCR, while unaffected portions of skin from PKDL patients tested negative. Sequencing analysis of the PCR products indicated that the amplified portion had more than 98z nucleotide sequence identity to the Leishmania donovani reference strain, D10. These findings indicate that the PCR method using a skin biopsy is highly sensitive and useful for confirmatory diagnosis of PKDL.

Comparative Evaluation of PCR and Imprint Smear Microscopy Analyses of Skin Biopsy Specimens in Diagnosis of Macular, Papular, and Mixed Papulo-Nodular Lesions of Post-Kala-Azar Dermal Leishmaniasis

“…Our study revealed nearly similar results by nested PCR, whereas detection by imprint smear microscopy was much higher than reported in the other studies. We used the rRNA gene of the ITS region, whereas in the Bangladesh, India, and Sudan studies, a minicircle of kinetoplast DNA (kDNA) and the 18S rRNA gene were employed for the PCR (11,12,15).…”

“…In a recent study conducted in Bangladesh, positivity by nested PCR in macular lesions was 93.2%, whereas microscopy had indicated only 2.7% positive samples, with overall positive results of 94.5% and 29.1%, respectively (11). Similar studies from India and Sudan reported positive PCR results in 93% and 82.7% of samples, whereas by microscopy the positive rates were 30.4% and 54%, respectively (12,15).…”

“…Histopathological diagnosis of PKDL is not very sensitive or specific, as visualization of intact parasites in tissue sections is difficult. Immunohistochemical staining is complex and has varied degrees of sensitivity (8,9,10).In recent years, several studies have proved that PCR is a very sensitive and specific technique for detection of leishmania DNA (11,12,13,14,15). Few PCR methods have been developed for PKDL diagnosis, but its efficacy on biopsy specimens from various types of lesions has not been assessed properly (12,15).…”

“…Our study revealed nearly similar results by nested PCR, whereas detection by imprint smear microscopy was much higher than reported in the other studies. We used the rRNA gene of the ITS region, whereas in the Bangladesh, India, and Sudan studies, a minicircle of kinetoplast DNA (kDNA) and the 18S rRNA gene were employed for the PCR (11,12,15).The number of parasites in macular lesions is scanty in comparison to papular and papulo-nodular lesions, and so the chances of detection by microscopy are much lower for macular lesions, even by trained laboratory personnel. Since PCR amplifies multiple copies of a gene of the parasite in the thermal cycles, the possibility of missing a parasite is very rare.…”

“…In recent years, several studies have proved that PCR is a very sensitive and specific technique for detection of leishmania DNA (11,12,13,14,15). Few PCR methods have been developed for PKDL diagnosis, but its efficacy on biopsy specimens from various types of lesions has not been assessed properly (12,15).…”