Mosquitoes Causing Dengue and Yellow Fever are Easily Identified With a New Analyzer

Researchers have developed a new diagnostic tool that can quickly carry out species-identification of a dead mosquito at a simple cost. The test determines if the mosquito belongs to a viral species that carries dangerous diseases like the Zika and dengue. The tool can also identify if the bug has come into contact with a mosquito-control strategy known as Wolbachia.

The research was conducted at The University of Texas at Austin and appears in the journal PLOS Neglected Tropical Diseases.

"Many of these diseases are spreading in areas where they weren't common before," said Sanchita Bhadra, a research associate in the Department of Molecular Biosciences and first author on the paper. "Having surveillance is important in conjunction with any kind of outbreak, and this method allows a rapid test in the field."

Aedes aegypti is a mosquito species that infects about 100 million people worldwide with illnesses like dengue, Zika, chikungunya or yellow fever. These mosquito-transmitted viruses initially affected only people in the tropics, but have grown to become a widespread health menace due to increased global movement and insecticide resistance. The species is the reason mosquito-borne diseases have tripled in the United States since 2004.

In order to curb viral transmission, in countries around the world and in 20 U.S. states where the Aedes aegypti mosquito is found, the researchers working in public health agencies have used a strategy to infect the disease-causing mosquitoes with a biopesticide called Wolbachia, a type of bacteria that keeps mosquitoes from spreading diseases.

However, Aedes aegypti, do not show any visual signs of having Wolbachia infection. Besides, current diagnostic tests are hard to read, expensive and logistically cumbersome.

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Public health groups along with monitoring efforts trap and kill mosquitoes routinely, but the technology that exists to extract nucleic acid from inside mosquitoes is very complicated, requiring them to be dead for days and starting to decay; the method turns out to be expensive and error-prone.

Hence, Sanchita Bhadra of the University of Texas in Austin, and her colleagues developed a probe that along with a smartphone camera can test the nuclear material of Wolbachia, in addition to finding out the mosquito species type. The tool represents a significant step forward to test the effectiveness of Wolbachia.

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LAMP-OSD - Loop-mediated isothermal amplification and oligonucleotide strand displacement

The probes termed "OSD probes" are based on fundamentals of nucleic acid chemistry. The researchers applied the probes to accurately read "LAMP assays" (a relatively simple nucleic acid detection test). The OSD probe will produce a visible signal only if it binds to the target LAMP DNA; in the absence of specific target DNA, no signal is generated.

The new design of the tool yields an accurate Yes/No visual readout on the smartphone and prevents a very common drawback of LAMP - false positive results. Moreover, when using the one-pot assay, the nucleic acid does not need to be removed and purified to analyze the crudely softened individual or pooled mosquitoes and renders the test simple and one that can be done anywhere.

Study

Scientists conducted a blinded test of 90 field-caught mosquitoes

• The cellphone assay specificity was 98 percent and sensitivity was 97 percent in identifying Ae. aegypti mosquitoes, even when the samples were stored at 37 degrees Celsius without a desiccant for 3 weeks.
• The test also readily identified the Wolbachia strain in field-collected mosquitoes without generating any false positive signals

The authors note that there is a possibility of false positives with their assay due to Wolbachia DNA merging with the chromosomes of an uninfected host, such as a fruit fly; however, this problem is not isolated to this assay alone and can happen in all nucleic acid amplification assays, including PCR.

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."

References:

1. Sanchita Bhadra, Timothy E. Riedel, Miguel A. Saldaña, Shivanand Hegde, Nicole Pederson, Grant L. Hughes, Andrew D. Ellington. Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. PLOS Neglected Tropical Diseases, 2018; 12 (8): e0006671 DOI: 10.1371/journal.pntd.0006671
2. Sanchita Bhadra, Timothy E. Riedel, Miguel A. Saldaña, Shivanand Hegde, Nicole Pederson, Grant L. Hughes, Andrew D. Ellington., "Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone" PLOS Neglected Tropical Disease August 30, (2018) https://doi.org/10.1371/journal.pntd.0006671

Source-Medindia