Classifying the natural history of asymptomatic malaria

Detecting malaria in people who do not have symptoms is critical to public health efforts to better control this tropical disease in places where the mosquito-borne parasite is common. Asymptomatic people harboring the parasite can still transmit the disease or become ill later, after an initially negative test.

The dynamic life cycle of this pathogen means that parasite densities can suddenly drop below the detection level, particularly when older, less sensitive tests are used. Such fluctuations can make it difficult, when testing only at one point in time, to determine whether a seemingly healthy person is in fact infected.

Malaria can produce intense chills alternating with clammy fevers, headaches, nausea, and other distresses. Still, many infected people feel fine.

A recent asymptomatic detection study was conducted in Katawki district, Uganda, where malaria incidence is high.

“We found that parasite dynamics and the parasite species present were highly variable among patients with low-level asymptomatic infections,” said Dr. Sean C. Murphy, a malaria researcher at UW Medicine, l one of the lead scientists on the study. He noted that sampling every other day or every three days was sufficient to detect a similar proportion of infections to daily sampling. However, testing once a week or less frequently, even with sophisticated diagnostics, could misclassify true infection status in up to a third of individuals.

This finding is important, Murphy said, for improving studies of the prevalence of malaria infection and, by extension, for clinical trials of malaria vaccines and treatments. Most of these trials use one-time testing or repeated but infrequent testing to determine the infection status of asymptomatic participants. This approach is likely to miss infections if participants’ parasite densities fall below the limit of the test used.

Murphy is a physician-scientist and professor of laboratory medicine, pathology and microbiology at the University of Washington School of Medicine and chief of pathology and laboratory medicine at Seattle Children’s.

The project was a collaboration between the Murphy laboratory; Dr. Thomas Egwang and his research team, including Tonny Owalla of Med Biotech Laboratories in Kampala, Uganda; and Dr. Jennifer E. Balkus, professor of epidemiology at the UW School of Public Health. Dr. Dianna EB Hergott, who at the time was a graduate student supervised by Murphy and Balkus, and Owalla of Med Biotech led the study.

The Uganda-based team carried out the community parts of the study. Participants were healthy, non-pregnant adults, aged 18 to 59 years, who were not taking antimalarial medications, as well as older children, aged 8 to 17 years.

“We instructed participants on how to collect a drop of dried blood at home every day for up to 29 days,” Owalla explained. Participants would attend the study clinic once a week to turn in that week’s blood draws, obtain new blood draw cards, and undergo traditional blood draws.

Diagnostic tests checked the dried blood spots for the presence, classification and densities of Plasmodium ribosomal RNA, which contributes to the production of parasitic proteins. The testing strategy also used “pooling” of dried blood spots. This approach allowed the team to test more samples cost-effectively, much like strategies employed during the height of the COVID-19 pandemic.

By analyzing the resulting data, the researchers hoped to discern a sampling program comparable to daily testing for reliably identifying asymptomatic cases, but less burdensome. On the other hand, the study team wanted to avoid a schedule that was too infrequent and would end up missing infections.

The scientists classified each participant’s infection trajectory as no infection, newly detected infection, cleared infection, chronic infection, or impossible to determine. By looking at the daily results, they also calculated how many infections would still have been detected if the sampling frequency had been reduced.

About 60% of all participants had a Plasmodium infection discovered at some point during the month-long study. Less than half had an infection detected at the start of the study. The lowest daily ratio during the study period showed a prevalence of 30%.

The results are reported in The Lancet microbe.

Previously, several other studies had questioned the accuracy of a single measurement for identifying infection status. Undetected asymptomatic infections could inadvertently influence research results.

“Serial testing,” the article’s authors suggest, “should be considered when attempting to determine an individual’s true infection status.”

One limitation of their own study, the authors said, was that participants were not asked to collect their dried blood samples at the same time each day. Parasite densities, they noted, could change up to 100 times in six hours.

Owalla is now a graduate student in pathobiology at the UW School of Public Health. He plans to apply his training to develop more advanced solutions against malaria in Africa, the continent most severely affected by the disease.