Newly Developed Cellphone-Read Assay Identifies Mosquitoes and Wolbachia

Wolbachia bacteria are widely studied for possible properties to block transmission of viruses like chikungunya, dengue and zika. In a new study with PLOS Neglected Tropical Diseases, researchers have created a new diagnostic tool using cellphone cameras to test mosquitos for their species type and Wolbachia.

Yellow fever mosquitoes, Aedes aegypti, are estimated to infect about 100 million people annually with devastating illnesses such as dengue and zika. Originally thought to affect only tropical regions, mosquito-transmitted viruses are increasingly become a worldwide health challenge due to increased global movement and insecticide resistance. One approach to reduce viral transmission is to simultaneously infect mosquitoes with Wolbachia, a bacterium that can curb pathogen transmission.

Since there are no visual signs of Wolbachia infection in Aedes aegypti, and current diagnostic tests are hard to read and difficult for researchers with limited resources, Sanchita Bhadra of the University of Texas in Austin, and her colleagues, developed a probe that uses a smartphone camera to test mosquitoes' nucleic acid for Wolbachia in addition to the mosquito species type. The researchers used fundamental principles of nucleic acid chemistry to create these probes (termed "OSD probes") and applied them to "LAMP assays" (a nucleic acid detection test that is favored for its relative simplicity). OSD probes help to accurately read LAMP assays. When OSD probes bind to their target LAMP DNA, they produce a visible signal. In the absence of specific target DNA no signal is generated.

The researchers' new design prevents false positive results (a common drawback of LAMP) and yields an accurate yes/no visual read out on the smartphone. Moreover, the one-pot assay is simple to use as it can directly analyze crudely macerated individual or pooled mosquitoes without requiring nucleic acid purification.

In a blinded test of 90 field-caught mosquitoes, the researchers demonstrated 98 percent specificity and 97 percent sensitivity in identifying Ae. aegypti mosquitoes with their new cellphone assay, even after three weeks of storage without desiccant at 37 degrees Celsius. Similarly, their cellphone assay readily identified the Wolbachia strain in field-collected mosquitoes without generating any false positive signals. The authors note that, while there could be potential false positives with their assay due to Wolbachia DNA merging with the chromosomes of an uninfected host, such as a fruit fly, this is an issue of which researchers using all nucleic acid amplification assays, including PCR, should remain vigilant, and is not isolated to their assay alone.

The researchers conclude: "We are currently automating the assays and workflow on low-cost paper and plastic devices that will not only further streamline diagnostic application but will also provide sealed chambers for biohazard and aerosol containment for macerating mosquitoes. Our sample preparation and workflow not only simplify the application of molecular diagnostics for surveillance, but also reduce cost by eliminating the need for nucleic acid extraction and complex instruments for assay incubation and readout."

Reference: Bhadra S, Riedel TE, Saldaña MA, Hegde S, Pederson N, et al. (2018) Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. PLOS Neglected Tropical Diseases 12(8): e0006671. https://doi.org/10.1371/journal.pntd.0006671

This work was supported by a Bill and Melinda Gates Foundation grant (OPP1128792) and a National Science Foundation BEACON grant (DBI-0939454) to Andrew D. Ellington and a National Institutes of Health grant (R43 AI131948) to Ellington and Grant L. Hughes.

Source: PLOS