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

TL;DR

This study shows that freeze-dried reagents can replace traditional liquid reagents for diagnosing avian influenza and rabies in Sub-Saharan Africa, even without a reliable cold chain.

Contribution

The study validates a lyophilized reagent as a reliable alternative to liquid reagents for diagnosing avian influenza and rabies in resource-limited settings.

Findings

Lyophilized reagents showed analytical and diagnostic performance comparable to standard liquid master mixes.

The lyophilized reagent was successfully deployed in four veterinary labs across Sub-Saharan Africa.

Sensitivity was reduced for divergent lyssaviruses due to suboptimal oligonucleotide complementarity.

Abstract

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 six different commercial freeze-dried kits, we selected one reagent 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. However, for the detection of divergent lyssaviruses, the lyophilized reagent’s sensitivity was affected by suboptimal complementarity between the oligonucleotides and 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 the freeze-dried reagent, which can interchangeably replace liquid qRT-PCR/RT-PCR kits. Altogether, our results indicate that the investigated lyophilized master mix represents a valid alternative to liquid 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.

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.

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