ArticleActa parasitologica2026
A Reliable and Accurate In-House kDNA-Based qPCR Assay for Detection of Leishmania DNA in Microscopy-Negative Cutaneous Leishmaniasis.
Article in Acta parasitologica, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
INTRODUCTION AND
objectiveCutaneous leishmaniasis (CL) is an endemic disease in tropical and subtropical regions which caused by Leishmania parasites. Rapid and accurate diagnosis of this disease is crucial for prognosis, control, and prevention. Considering the importance of accurate diagnosis of this disease and preventing possible errors in common methods, we aimed to design an effective method using the quantitative real time PCR (qPCR) for accurate, and feasible diagnosis of this disease. MATERIALS AND
methodsTotal of 109 samples were included: 89 negative smears from patients clinically suspected of CL from endemic areas in Semnan and Golestan provinces, 10 positive samples with varying parasite loads, and 10 samples from patients with other skin diseases mimicking CL to assess test specificity. DNA was extracted using the phenol-chloroform method. The in-house qPCR was performed using designed primers targeting the kinetoplast DNA of Leishmania. In addition, conventional ITS-PCR was used as a complementary method. PCR-positive samples were submitted for Sanger sequencing, and results were compared with patients’ clinical data.
resultsAfter re-examination of the negative sears, 4 cases (4.49%) were found positive using microscopic method. The qPCR identified 9 positive cases (10.58%) among the 85 negative samples. This technique demonstrated the ability to detect very low parasite loads (grade 1+) in clinical conditions. The limit of detection the assay under laboratory conditions was determined 0.001 ng/µl. The conventional ITS-PCR identified 6 positive cases among the qPCR-positive samples. Sequencing analysis confirmed that all isolates belonged to Leishmania major. DISCUSSION AND
conclusionThe findings of this study indicate that in-house qPCR is a highly sensitive and specific tool for diagnosing CL, even in negative smears. It can serve as a valuable complementary and reliable method to microscopic, leading to earlier and more accurate diagnosis and prevention of misdiagnosis or mistreatment. Furthermore, the identification of L. major in all positive cases supports the existing epidemiological data in the studied regions.
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