Performance evaluation of nine reference centers for effective surveillance of Leishmania-infected Phlebotomine sand flies and basis for technical recommendations

BackgroundLeishmaniasis, caused by Leishmania protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of Leishmania-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative PCR (qPCR) conditions and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, there is currently a lack of technical recommendations for Leishmania DNA detection, which needs to be addressed. This study aimed to compare various DNA extraction protocols and conduct a qPCR based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories. Methodology/Principal findingsEQA samples were prepared using Leishmania infantum and L. major strains, at different concentration from 101 to 104 parasites/mL and distributed to participating centers. All centers, except one, detected all Leishmania concentrations, demonstrating diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, although Cq value variations suggested differences in sensitivity due to technical capabilities and/or extraction kit performances. Reported comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus(R) Kit and QIAamp(R) DNA mini-kit as the most efficient, supporting their use for standardized protocols. The study also evaluated the impact of lyophilization and shipment conditions, finding no compromise in Leishmania detection, despite slight Cq value variations. In addition to EQA samples, experimentally infected sand fly have been included to mimic sample field condition. All centers detected positive samples, with variable Cq values, reflecting differences in individual infection load. Conclusion and significanceOverall, the study underscores the importance of standardized protocols and continuous quality assurance to maintain high diagnostic validity, crucial for effective surveillance of leishmaniasis, especially in field settings with low infection densities. Continuous training and calibration are essential to ensure uniform diagnostic performance across laboratories, enhancing epidemiological surveillance and disease control strategies. Author SummaryLeishmaniasis is a disease caused by Leishmania parasites, transmitted by sand flies, and poses a major health risk in the Mediterranean region. Monitoring the spread of nfected sand flies is crucial for controlling the disease. This study focused on improving the methods used to detect Leishmania in sand flies by comparing different DNA extraction techniques and assessing the accuracy of these methods across nine reference laboratories. All centers, except one, efficiently detected all Leishmania concentrations, demonstrating proficiency in diagnostic protocols. Moreover, we found that two specific DNA extraction kits, the EZ1 DSP Virus(R) Kit and QIAamp(R) DNA mini-kit, were the most effective for Leishmania detection. We also tested how sample preparation and shipping conditions affected the results, ensuring that our methods would work in real-world settings. Even under these conditions, the detection methods proved reliable. This work helps to standardize the detection of Leishmania, making surveillance more accurate and consistent. Continuous training and calibration are essential to ensure uniform diagnostic performance across laboratories, enhancing epidemiological surveillance and disease control strategies