Assessment of pan-Leishmania detection by recombinase polymerase amplification assay
Chiheb Louizi1, Md Anik Ashfaq Khan2, Khaledul Faisal3
1Institute for Medical Microbiology and Virology, University Medical Center Goettingen, Georg-August University, Göttingen, Germany.
Abstract:
The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.
Insights
This study evaluated recombinase polymerase amplification (RPA) assays for detecting diverse Leishmania species. Combining 18S rRNA-RPA and kinetoplast DNA-RPA offers high sensitivity for diagnosing leishmaniasis.
Area of Science:
- Molecular Biology
- Parasitology
- Infectious Diseases
Background:
- Increasing human mobility and changing vector habitats facilitate the spread of atypical Leishmania species.
- Existing diagnostic methods struggle with rapid detection of Leishmania species outside their narrow target range.
- Accurate and broad-spectrum diagnostics are crucial for timely leishmaniasis treatment.
Purpose of the Study:
- To assess the pan-Leishmania detection capability of isothermal recombinase polymerase amplification (RPA) assays.
- To evaluate RPA assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions.
- To determine the sensitivity and specificity of combined RPA assays for diagnosing leishmaniasis.
Main Methods:
- Isothermal recombinase polymerase amplification (RPA) assays were developed targeting 18S rRNA, Cpb, and kDNA regions of Leishmania.
- The limit of detection for 18S rRNA-RPA and Cpb-RPA assays was determined.
- The performance of 18S rRNA-RPA and kDNA-RPA assays was evaluated on 70 real-time PCR-positive leishmaniasis samples.
Main Results:
- Both 18S rRNA-RPA and Cpb-RPA assays demonstrated broad-spectrum amplification of 7 pathogenic Leishmania species.
- The lowest limit of detection was 12 standard DNA molecules for 18S rRNA-RPA and 17 for Cpb-RPA.
- The kDNA-RPA assay achieved 88.57% sensitivity, while the 18S rRNA-RPA assay showed 35.71% sensitivity; their combined sensitivity reached 98.57%.
Conclusions:
- Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays shows high potential for sensitive and broad-spectrum Leishmania detection.
- These RPA assays can enhance rapid diagnosis of leishmaniasis, especially in cases involving atypical species.
- Further diagnostic assessments are recommended for the combined RPA approach.
