1. Performance evaluation of nine reference centers and comparison of DNA extraction protocols for effective surveillance of Leishmania-infected Phlebotomine sand flies: Basis for technical recommendations.
- Author
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Prudhomme, Jorian, Delabarre, Aymeric, Alten, Bulent, Berberoglu, Umut, Berriatua, Eduardo, Bongiorno, Gioia, Cristovao, José Manuel, Davidovich-Cohen, Maya, Di Muccio, Trentina, Erisoz Kasap, Ozge, Fiorentino, Eleonora, D. Kirstein, Oscar, Kniha, Edwin, Maia, Carla, Mungan, Mesut, Muñoz-Hernández, Clara, Nalçaci, Muhammed, Oguz Kaskan, Gizem, Ozbel, Yusuf, and Ozensoy Toz, Seray
- Subjects
SAND flies ,NUCLEIC acid isolation methods ,FLIES as carriers of disease ,PUBLIC health surveillance ,LEISHMANIA infantum - Abstract
Background: Leishmaniasis, 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 real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for Leishmania DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing Leishmania DNA detection in sand fly. Methodology/Principal findings: EQA samples consisted of Leishmania infantum and L. major species, at concentrations ranging from 10
1 to 104 parasites/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in Leishmania detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load. Conclusion and significance: This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate Leishmania DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities. Author summary: Leishmaniasis 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 infected 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 Kit and QIAamp 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 and enabling appropriate treatment. [ABSTRACT FROM AUTHOR]- Published
- 2024
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