A simple test can quickly detect if a person is infected with a parasite that causes the diarrheal disease cryptosporidiosis.
Lines on paper strips show whether samples taken from the stool of a patient contain genetic DNA from the parasite.
The research is detailed online in a new paper in the journal Analytical Chemistry.
“Diarrheal illness is a leading cause of global mortality and morbidity,” says Rebecca Richards-Kortum, a bioengineer at Rice University and director of the Rice 360˚: Institute for Global Health Technologies. “Parasites such as cryptosporidium are more common causes of prolonged diarrhea. Current laboratory tests are not sensitive, are time-consuming, and require days before results are available.
“A rapid, affordable, accurate point-of-care test could greatly enhance care for the underserved populations who are most affected by parasites that cause diarrheal illness.”
A. Clinton White, director of the Infectious Disease Division at the University of Texas Medical Branch (UTMB) at Galveston, asked Richards-Kortum to help develop a diagnostic test for the parasite.
“I’ve been working with cryptosporidium for more than 20 years, so I wanted to combine her expertise in diagnosis with our clinical interest,” he says. “Recent studies in Africa and South Asia by people using sophisticated techniques show this organism is a very common, under-appreciated cause of diarrheal disease in under-resourced countries.”
The parasite is common in the United States, he says, but less than 5 percent of an estimated 750,000 cases are diagnosed every year. In 1993, an outbreak of cryptosporidium in the water supply sickened 400,000 people in Greater Milwaukee, he says.
Lead author Zachary Crannell, a graduate student based at Rice’s BioScience Research Collaborative, says the disease, usually transmitted through drinking water, accounts for 20 percent of childhood diarrheal deaths in developing countries.
Cryptosporidiosis is also a threat to people with HIV whose immune system is less able to fight it off, he adds.
“In the most recent global burden-of-disease study, diarrheal disease accounts for the loss of more disability-adjusted life years than any other infectious disease, and cryptosporidiosis is the second leading cause of diarrheal illness.” Crannell says. “Anybody, if it’s not treated, can get dehydrated to the point of death.
“There’s a lot of new evidence that even with asymptomatic cases or cases for which the symptoms have been resolved, there are long-term growth deficits,” he says.
Room or body temperature
Current specialized tests that depend on microscopic or fluorescent analysis of stool samples or polymerase chain reactions (PCR) that amplify pathogen DNA are considered impractical for deployment in developing countries because of the need for expensive equipment and/or the electricity to operate it.
The new test depends on recent developments in a recombinase polymerase amplification (RPA) technique that gives similar “gold standard” results to PCR but operates between room and body temperatures.
In Rice’s experiments, samples were prepared with a commercial chemical kit that releases all the DNA and RNA in the small amount of stool tested. The purified nucleic acids are then combined with RPA primers and enzymes tuned to amplify the pathogen of interest, Crannell says.
“If the pathogen DNA is present, these primers will amplify it billions of times to a level that we can easily detect,” he explains. The sample is then flowed over the detection strip, which provides a positive or negative result.
The RPA enzymes are stable in their dried form and can be safely stored at the point of care without refrigeration for up to a year, he says.
Requires little equipment
While current tests might catch the disease in samples with thousands of the pathogens, the new technique detects the presence of very few—even one—parasite in a sample. In their experiments, the researchers reported the presence or absence of the disease was correctly identified in 27 of 28 infected and control-group mice and all 21 humans whose stool was tested.
Crannell says the method requires little equipment, because the enzymes that amplify DNA work best at or near body temperature. “You don’t need a thermal cycler (used for PCR analysis); you don’t need external heating equipment. You can hold the sample under your armpit, or put it in your pocket,” he says.
The research team’s goal is to produce a low-cost diagnostic that may also test for the presence of several other parasites, including giardia, the cause of another intestinal disease. The researchers are working to package the components for use in low-resource settings, Crannell says.
The National Institute of Allergy and Infectious Diseases of the National Institutes of Health and the National Science Foundation Graduate Research Fellowship Program supported the research.
Source: Rice University