A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

Area of Science:

• Parasitology
• Molecular Biology
• Infectious Diseases

Background:

• Human leishmaniasis is a widespread parasitic disease, with Sri Lanka identified as a new focus for Leishmania donovani.
• Early detection and management are crucial for controlling L. donovani and preventing severe outcomes like death or chronic illness sequelae.
• Limitations of light microscopy and in-vitro culture in diagnosing atypical or treated leishmaniasis necessitate improved diagnostic tools.

Purpose of the Study:

• To develop and evaluate a highly sensitive and specific molecular diagnostic method for leishmaniasis.
• To improve the detection rates of leishmaniasis, particularly in cases negative by conventional diagnostic techniques.
• To minimize contamination risks associated with nested PCR procedures.

Main Methods:

• A modified nested PCR (Mo-STNPCR) was developed, utilizing specific outer and inner primers for Leishmania DNA amplification.
• The Mo-STNPCR assay was performed in a single tube to reduce handling and contamination risks.
• The method was validated using clinical samples from patients with cutaneous and visceral leishmaniasis, as well as control groups, and compared against light microscopy and in-vitro culture.

Main Results:

• The Mo-STNPCR method demonstrated 100% sensitivity and 100% specificity for leishmaniasis diagnosis.
• Conventional methods (light microscopy and in-vitro culture) achieved sensitivities of 75.0% and 72.5% respectively, with a combined sensitivity of 87.5%.
• Mo-STNPCR showed no cross-reactivity with control samples and significantly reduced contamination risks, though at a higher per-patient cost (22 USD) compared to microscopy (3 USD) and culture (6 USD).

Conclusions:

• The Mo-STNPCR method is a valuable tool for diagnosing leishmaniasis, especially in cases missed by initial investigations.
• This technique enhances diagnostic accuracy for various forms of leishmaniasis.
• The improved sensitivity and specificity of Mo-STNPCR contribute to better disease control and management strategies.