By Joel Rovnak
We wanted a field test to detect the Zika virus that was so simple, we could run it from the back of a pickup truck while checking mosquito traps. Loop-mediated amplification (LAMP) is an alternative test for the genetic material of any infectious agent, whether it’s a bacteria, virus or parasite. LAMP tests for viruses related to Zika, like dengue, West Nile, and yellow fever, had already been developed for diagnostics. But these assays were overly complicated, as they required sophisticated laboratories with expensive equipment and trained technicians.
We developed a one-step LAMP test that requires a simple heat block with results that are easily visualized. Many disease-specific LAMP primers, the parts of the test that target certain organisms, have already been designed and are ready to go. The main goal for mosquito surveillance is to put test results into the hands of people doing mosquito control in the field. If they find the virus, they can treat with insecticides in a controlled and focused manner. If not, they can move on. This is good for the environment and more economical than broadcast sprays of entire regions.
The goal for human medicine is to put the test into doctor’s office’s, regional clinics, and hospitals. Rapid diagnosis of infectious diseases not only informs the doctor and patient as to the best course of care, but also informs regional health departments as to the presence of the disease in the area. When effective vaccines exist, they can be quickly deployed in the area to block further infection. An economical test can be used more frequently and provide much more information for the initial detection, tracking, and prevention of disease. Many countries with endemic diseases cannot afford to support such efforts. We hope to make it possible to apply these approaches everywhere.
The LAMP test we developed detects the genetic material of the Zika virus, whether in a homogenized, infected mosquito or in fluids from infected patients. Test mosquitoes were raised and infected with Zika in our facility. The development of this rapid test was just a part of the efforts at the Arthropod-borne Infectious Disease Laboratory at Colorado State University in Fort Collins, Colorado. There the personnel and facilities built and maintained the test over many years and made it possible to move rapidly in response to the Zika outbreak.
We are currently trapping mosquitoes in Puerto Rico in collaboration with the US Department of Agriculture Animal and Plant Health Inspection Service, to collect data on naturally infected mosquitoes. The samples will be assayed at Colorado State, but we are seeking collaborators in areas with circulating Zika virus, who will deploy the test in the field.
Human bio-fluids were first spiked with a laboratory-grown virus to try out the test. When that worked well, we obtained samples from real Zika patients in Nicaragua and Brazil, where Zika was circulating. These samples had been previously tested in the standard diagnostic assay when the patients first presented symptoms in hospitals and clinics. Remaining sample materials were de-identified and stored frozen for research purposes with patient consent. We ran the LAMP test without knowing which samples were positive or negative on these stored, coded human samples. We used the rapid LAMP test as well as a more sophisticated LAMP assay and compared these to the standard diagnostic assay called quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
In the fall, we will be running the LAMP test in direct comparison to qRT-PCR on fresh patient samples in Managua, and will include LAMP tests for Zika, dengue, and Chikungunya viruses, that are circulating in the region and can cause similar symptoms. Our collaborators in Managua have ran a cohort study of the incidence of these infectious diseases in children since 2004. Zika was added to their approved research protocols in 2015. This work is supported by a grant from the National Institute of Allergy and Infectious Disease (NIAID) at National Institutes of Health.