By Ed Ricciuti
Scientists at a U.S. Army research center have modified an assay that tests whether or not a sample of mosquitoes harbors the virus responsible for the disease known as chikungunya (CHIKV), long a problem in the Old World tropics but recently established in the Americas.
They did not make the assay — a commercial company called VecTOR Test Systems, Inc. did. Instead, the Army researchers tested the test to prove that it works — no small task when dealing with a virus that comes in multiple guises. Their assay is described in an article in the Journal of Medical Entomology.
Health workers now have what the scientists call a “quick and dirty” way to detect the presence of the CHIKV virus within an hour, rather than waiting for results of laboratory tests that take days, even weeks. It’s done with a chemical dipstick, the same kind of simple tool used in a pregnancy test. If the test is positive, measures to control and contain the disease can be mobilized and started immediately, which is important because no vaccine or specific treatment for chikungunya exists.
Evaluation was carried out under epidemiologist Michael J. Turell, PhD, a principal investigator for 30 years at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) in Fort Detrick, Maryland. Turell’s research has focused on factors impacting the ability of mosquitoes to transmit various arboviruses, a term for viruses transmitted by arthropods such as mosquitoes, sandflies, and ticks. In addition, he has worked on the development of vaccines for several mosquito-borne viruses and on assays that detect arboviruses.
The larvae of mosquitoes that transmit CHIKV find excellent harborage in containers that hold standing water, such as spare tires, empty pails, and saucers under flower pots. Thus, the mosquitoes involved prosper among high concentrations of people. People living in tropical shanty towns, with open windows and no air conditioning, are prime targets.
“Chikungunya” is a term used by people of the Makonde Plateau, between Tanzania and Mozambique, where the disease was discovered in 1952. It means, “that which bends up,” referring to the way arthritis caused by the disease crooks posture of the victim’s body. Symptoms of chikungunya can be as brutal as its name is to pronounce, although it is seldom fatal. Victims experience fever and pain and swelling of muscles and joints. Headache and rash may occur. The disabling impact can last for months.
The chikungunya virus belongs to a group known as alphaviruses, at least 30 of which can infect humans and other vertebrates, causing diseases such as equine encephalitis and a variety of rash-accompanied fevers. CHIKV is transmitted by mosquitoes of the genus Aedes, chiefly Aedes aegypti, the bane of humans in the tropics because it also carries viruses responsible for yellow fever and dengue fever. Scientists have identified three different lineages — genetically-related groups — of CHIKV linked to geographical areas: Asia, West Africa, and East/Central/South Africa. All of the lineages exist outside the geographical areas after which they are named. A member of the Asian lineage, for example, has infected people in the Americas.
Like many tropical diseases, chikungunya has been rampant in the developing world for many years, but only became the focus of intensive research after it threatened western nations. An outbreak on Reunion Island, a French Department in the Indian Ocean, during 2005 and 2006 attracted attention largely because it’s a hot tourist destination for Europeans. Perhaps not by coincidence, the first transmission from mosquitoes in the Americans occurred in December 2013, on the French half of St. Martin, and island in the Caribbean. Reported cases in the Americas have now passed 1.5 million.
It began to show up in the United States during 2014, with nearly 2,500 cases reported from 46 states. Nearly all, however, were in travelers who picked up the infection in the tropics. Eleven people on the U.S. mainland, all in South Florida, contracted the disease directly from mosquitoes in the U.S.
During 2014, more than 4,500 people in Puerto Rico and the U.S. Virgin Islands were infected by mosquitoes, although the number may have been higher because chikungunya was not officially reportable to the federal Centers for Disease Control and Prevention until this year. There have been 77 chikungunya cases reported this year — as of April 7, from 21 states — but all were contracted out of the country.
To date, tests for CHIKV require expensive equipment in a laboratory setting and technicians who have undergone extensive training. Not so the dipstick test. It can be done on site by a neophyte and, importantly, does not require electricity. The dipstick involved is not the kind used to check oil in an automotive motor. It’s a small strip, usually paper-like nitrocellulose, a compound that is used in gunpowder, nail polish, laboratory filter paper, and other products. On the surface of the stick are reagents that will react to CHIKV antigens if the virus is present in the test sample, which is in a liquid solution.
Near its bottom end, the dipstick is coated with antibodies to the virus bonded to gold, a blend known as a “conjugate.” The conjugate is attached only loosely to the dipstick. When the dipstick is placed in the solution test sample, the liquid wicks up the stick. The job of the antibodies in the conjugate is to trap the virus if present in the solution. Just as they do in the human body, if antibodies sense the presence of an antigen — in this case the virus — they stick to it. Thus, if the virus particle is there, the antibodies, the gold, and the virus form what scientists call a “complex.”
The complex in the solution detaches and continues to migrate up through the stick. As it does, the complex encounters another batch of antibodies firmly attached in a band across the strip. The antibodies on the band will attach to the virus in the complex. At this point, the purpose of the gold in the conjugate becomes evident. The gold will reveal the presence of the virus by changing color to reddish purple. If a reddish purple line appears where the band is located, the test is positive for CHIKV. If the virus is not present, the antibody band against CHKV will have nothing to stick to and the reddish-purple line will not form. Further up the strip, a second line, serving as a control, binds antibodies that indicate that the test has functioned properly.
The mechanics may sound complex, but all the field worker has to do is dip the stick and look for a colored line. Obtaining a full and accurate evaluation of the test, in the first place, was much more complex. Viruses mutate, so there are myriad strains of the three CHIKV lineages. Moreover, there are multiple sources of antibodies needed to manufacture the dipstick. Evaluators had to sort out those which worked best for the test.
The company that produced the dipstick sent a variety of samples, with several different combinations of antibodies. The prototypes had been tested on viruses grown in laboratory culture, not in real mosquitoes. So first off, the researchers had to provide the real thing, a solution for testing that amounts to a mosquito soup.
Step one was to inoculate mosquitoes — females, which do the biting — with the virus. The injection site was in the thorax near a spiracle, a tiny opening that admits oxygen into the insect body. Mosquitoes were anesthetized and placed on their side prior to injection, which was made using a glass needle with a point too small for the human eye to see. The inoculated mosquitoes were then placed in an incubator for a week, allowing the virus to grow. The next step was to place the mosquitoes in a small tube with a commercially-made grinding solution and copper-coated BBs, and to mash the insects. The test strip then was inserted into what Dr. Turell calls, the “mosquito squish” for 20 minutes. The researchers then read it while wet, and again after an hour.
Most of the tests were on individual mosquitoes. Healthcare workers in the field, however, often must test several mosquitoes at once, so Dr. Turell’s team also tested groups of 10, 25, and 50 insects, each containing only one infected specimen. The intensity of the colored band on the dipstick version that passed the test with flying colors remained the same, no matter how many mosquitoes went into the soup. The researchers tested the dipsticks with all three lineages and eight different strains of CHIKV. When tested with 22 other arboviruses, some closely related to CHIKV, the antibodies did not react, proving it worked specifically on CHIKV. They also proved its worth at different levels of humidity and temperature.
If an outbreak of chikungunya occurs, the test “could enable public health workers to detect CHIKV in infected mosquitoes rapidly without the need for specialized equipment, expertise, or training, making virus surveillance more expedient,” according to the authors of the Journal of Medical Entomology article. It would mean the difference between nipping an outbreak in the bud and a major public health crisis.
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Ed Ricciuti is a journalist, author, and naturalist who has been writing for more than a half century. His latest book is called Bears in the Backyard: Big Animals, Sprawling Suburbs, and the New Urban Jungle (Countryman Press, June 2014). His assignments have taken him around the world. He specializes in nature, science, conservation issues, and law enforcement. A former curator at the New York Zoological Society, and now at the Wildlife Conservation Society, he may be the only man ever bitten by a coatimundi on Manhattan’s 57th Street.