Molecular rapid test for neglected tropical disease

The ICGEB projects COVID-19+ and EXPANDIA are providing support for the development of a molecular rapid test for Zika Virus.

In past years, ICGEB has worked on the development and testing of different diagnostic assays targeting emerging viruses affecting low- and middle-Income countries. Arboviruses, such as Dengue, Zika, West Nile, and Chikungunya virus, are recognised as an emerging threat to many public health systems in Africa, Asia, and South America, where more and more outbreaks are being reported every year. What is new, however, is that arboviruses no longer seem to be confined to tropical areas, as cases of Dengue and Zika have begun to be reported in Europe and in the United States also. The World Health Organization (WHO) declared that almost half of the world’s population, about 4 billion people, live in areas with a risk of Dengue.

Arboviruses are primarily transmitted to humans through mosquito bites and lately, climate change has been increasingly recognized to influence the transmission and prevalence of these viruses worldwide. Rising temperatures and changing precipitation patterns have led to shifts in the geographical distribution of vector species, enabling them to expand into new areas previously unsuitable for their survival. This expansion has increased the risk of arbovirus transmission to previously unaffected regions and populations.

The same concern has also been highlighted by the WHO in their regional situation reports. A multi-country outbreak of dengue virus has been ongoing since 2023, with over 5 million cases detected in more than 80 countries. However, the true number of dengue cases is believed to be much higher due to limited surveillance capacity and the availability of diagnostics.

To address this critical gap, the COVID-19+ and EXPANDIA projects are supporting the development of new cost-effective assays for the detection of arboviruses. By building on the results obtained from the testing of RT-LAMP on SARS-CoV-2, the COVID-19+ consortium, comprising ICGEB and reference laboratories in 10 African nations, highlighted the potential for the test to be adapted for detecting pathogens other than SARS-CoV-2. In this context, the Molecular Virology Lab in ICGEB began working on the development of an RT-LAMP for the detection of Zika virus.

Currently, the gold-standard diagnostic method for detecting Zika is RT-qPCR, which, while being efficient in identifying infected individuals in reference laboratories, is not suitable for routine screening of febrile patients in remote rural areas, where Zika outbreaks often occur. RT-LAMP is a molecular technique based on the amplification of nucleic acids that occurs at a constant temperature without the need for thermal cycling. The colorimetric RT-LAMP ZIKV assay developed by the Molecular Virology Group is intended for broad-spectrum detection of several Zika variants belonging to different phylogenetic lineages. Unlike PCR, RT-LAMP typically uses six primers to amplify the target of interest, making the amplification rather specific. Therefore, primer design is a critical step and the information on circulating genotypes is a fundamental prerequisite for the development of molecular diagnostic tools like LAMP.

Unfortunately, genomic surveillance of Zika virus on the African continent has been heavily neglected and only 22 complete viral genomes from five countries are currently available. Analysis of those sequences revealed that phylogenetically distant Zika strains have appeared in Africa and thus multiple primer versions were designed to ensure high primer homology with a broad range of Zika genomes. Hand in hand with RT-LAMP development, the EXPANDIA project supports whole-genome sequencing of Zika isolates in African partner countries, the results of which will provide valuable information on Zika genotypes and allow us to adapt LAMP primers making them specific for strains currently circulating in Sub-Saharan Africa.

So far, RT-LAMP has shown robustness and reliability, especially considering its intended use in point of care laboratories. In fact, the assay, using a colorimetric formulation provided by New England Biolabs®, is run for 30 min at 65° C and shows no cross-reactivity with other viruses of the same family. Molecular amplification of Zika RNA makes this assay more sensitive than rapid antigenic tests. Moreover, this technology does not require sophisticated assets, as the reaction can be run in a simple thermoblock or water bath. Colour change of the mastermix following target-detection can be read by the naked eye and interpreted by personnel with minimal training, making it an ideal detection method for resource-limited settings. Diagnostic performance of the current assay will soon be evaluated in African partner countries on a set of Zika-positive and negative samples, and we believe that this relatively straightforward protocol with reduced turnaround time has good potential for future implementation in remote areas.

Nevertheless, collaborative efforts between researchers, technology providers, healthcare professionals, and public agencies are crucial for the continued development of these novel diagnostic tools. By improving our ability to rapidly and accurately detect Zika Virus (ZIKV) infections, we can enhance surveillance efforts, guide clinical management decisions, and ultimately mitigate the impact of future outbreaks.