Validation and multi-site deployment of a lyophilized qRT-PCR reagent for the molecular diagnosis of avian influenza and rabies in Sub-Saharan African regions
Authors: Drzewnioková Petra , Brian Irene , Mancin Marzia , Fortin Andrea , Gourlaouen Morgane , Angot Angélique , Niang Mamadou , Dah Isaac , Berete Kouramoudou , Diakite Adama , Lo Fatou Tall , Meseko Clement , Go-Maro Emilie , D’Amico Valeria , Valastro Viviana , Soumare Baba , De Benedictis Paola , Monne Isabella , Panzarin Valentina 
Published: January 2025

Molecular methods are widely accepted as gold standard techniques for the laboratory diagnosis of most human and animal pathogens. However, most molecular protocols rely on reagents that need to be transported and stored at a freezing temperature, a requirement that might affect their reliability in areas where the cold chain cannot be guaranteed. Over the years, several lyophilized molecular products have been marketed to circumvent this issue. We therefore evaluated the feasibility of replacing liquid reagents with freeze-dried formulations for the molecular diagnosis of avian influenza (AIV) and rabies (RABV) viruses, two priority zoonotic pathogens widely spread in Sub-Saharan Africa. Among the available kits, we selected the Qscript lyo 1-step kit (Quantabio) due to its easy-to-use features, single-reaction format, and preliminary performance assessment. Through a more in-depth evaluation, we determined its analytical and diagnostic performance and formulation stability, and obtained results comparable to those of standard liquid master mixes. Notably, for the detection of divergent lyssaviruses, the lyophilized reagent’s sensitivity was affected by suboptimal complementarity between the oligonucleotides and the target sequences. Finally, a multi-site evaluation in four veterinary diagnostic laboratories located in Sub-Saharan Africa demonstrated the successful deployment of AIV and RABV assays utilizing freeze-dried reagents, which can interchangeably replace liquid master mixes. Altogether our results indicate that the Qscript lyo 1-step kit (Quantabio) represents a valid alternative to wet reagents for the molecular diagnosis of avian influenza and rabies, and has the potential for broader applications to other relevant infectious diseases upon proper validation.

Author summary

Molecular diagnostic protocols rely on reagents that need to be transported and stored at freezing temperatures. Meeting this requirement can be challenging in areas where the maintenance of the cold chain is not guaranteed, such as in sub-Saharan Africa. Our study aimed to assess the feasibility of using lyophilized reagents as a replacement for liquid reagents in the molecular diagnosis of two widespread zoonotic pathogens in Sub-Saharan Africa, namely avian influenza and rabies. To accomplish this, we selected a commercially available lyophilized reagent based on its format and performance characteristics. We conducted a laboratory validation to assess the use of the lyophilized reagent throughout the entire diagnostic process. We also conducted a reproducibility test involving African laboratories as potential end-users. Our findings confirm that the lyophilized reagent can replace traditional liquid reagents to diagnose rabies and avian influenza and suggest its possible use for a wider range of infectious diseases after undergoing appropriate validation.