In Cambodia's Ratanakiri Province, main roads project like spokes from the small city of Banlung, population 17,000. Here, next to the local health department, a team of Cambodian and Belgian researchers intent on eliminating malaria parked a mobile PCR lab last fall.
Costing $200,000 and funded in part by the Bill and Melinda Gates Foundation, the mobile lab performed PCR-based malaria assays on just shy of 5,000 dried blood samples over 20 days last fall, detecting 244 cases of asymptomatic Plasmodium infection and delivering treatment within 48 hours.
The results of the proof-of-principle study, published this month in Malaria Journal, push the limits of medical diagnostics in a way especially amenable to low-resource settings. They are also encouraging steps toward Cambodia's goal of malaria elimination by 2025 — a goal becoming increasingly important with the rise of drug-resistant strains of the mosquito-transmitted parasite.
In an interview from Cambodia with PCR Insider, Lydie Canier, lead author on the study and a researcher at the Institut Pasteur du Cambodge in Phnom Penh, said previous work on malaria prevalence in Ratanakiri helped them obtain such high throughput. With the mobile lab stationed in Banlung, five teams of four people each drove out to surrounding villages every day with a target number to recruit to the study. Participants were told the study goals and gave consent to be part of the research. They showed no symptoms for malaria at the time they donated a finger-prick blood sample, so this was active detection, as opposed to passively detecting infection when a person requires treatment for malaria.
"To achieve the goal of elimination we need to detect and treat the asymptomatic people because they constitute a reservoir that is contributing to maintain disease transmission," Canier said.
Malaria-causing Plasmodium parasite is transmitted to humans by mosquito bites, and can lead to fever, anemia, organ failure, and death. Although partial immunity develops after infection, adults can be re-infected, and children without immunity are especially vulnerable. The parasite itself has developed immunity, first to chloroquine in the 1970s and '80s, and, more recently, to another drug family, the artemisinins.
Artemisinin-based combination therapies are still considered the best available treatment, but artemisinin-resistant strains of Plasmodium have now been detected in Cambodia, Myanmar, Thailand, and Vietnam, according to the World Health Organization website. With no new anti-malarials on the horizon, WHO warns that resistant strains reaching India or sub-Saharan Africa could constitute a global public health disaster.
Worldwide, WHO estimates there were 219 million cases of malaria in 2010. The majority of these were in Africa, where malaria killed over 660,000 people, mostly children, that year alone. In Cambodia, malaria is rarer and less fatal and eradication campaigns, including use of insecticide-treated bed nets, have reduced the number of fatalities by 75 percent in the last decade, according to WHO.
However, low rates mean achieving eradication of the reservoir by simply treating the entire population for Plasmodium would be extremely inefficient. As Canier told PCR Insider, "The thing is, the prevalence is not that high … for example, in Ratanakiri, the province where there is the biggest prevalence in Cambodia, it is 4 to 5 persons [per 1,000]."
The mobile lab was an innovative solution to this problem. While there have been emergency and military labs deployed in tents, and easily transported equipment is on the market, to Canier's knowledge theirs was the first completely mobile PCR lab.
Canier said their ability to be compact and yet process such high numbers of samples was due in part to their collection and processing methods. "Our originality is to collect the blood in 96-well plates," she said. "We were doing it already in our lab [at Institut Pasteur]; we just transferred that technology into the mobile lab. I think it's saving a lot of time for the analysis."
Finger prick blood samples were collected in the field onto 4-mm disks of filter paper in 96-well plates, with the remainder dried onto another filter paper and reserved as backup. This so-called blood confetti was then driven over rural dirt roads back to the mobile lab in Banlung. Fast-boiling DNA extraction was done directly in the plates, followed by SYBR green PCR assays. Samples testing positive for Plasmodium were then subjected to nested PCR followed by melt curve analysis to detect four common strains of parasite.
The published study contained a blueprint of the mobile lab, which Canier described to PCR Insider: "We have one centrifuge for the plates, two real time PCR [instruments], a DNA hood with UV light, a fridge, a minus 20 [Celsius] freezer, and a minus 80 freezer. That is for the molecular side."
She added that the mobile lab also has a small culture hood, an incubator, and a water bath to do parasite culture.
Parasite culture was not used much in this particular study, but could be important in the future, said Canier. "The idea is to get the sample in good condition, especially to culture Plasmodium vivex," she said. "This species of Plasmodium is not well known and is becoming more and more important, and … we don't have any good protocol for long-term culture. We think that if we can get the sample as soon as possible, it could help to find a protocol to culture that parasite."
One obstacle to the study was the power required for running PCR in a mobile setting. "We have a generator, but it's just more comfortable if we can [also] have the local electricity to work, because we have a high throughput," said Canier. However, in Banlung, "there are a lot of power cuts … After the first trial we set up the generator to start automatically when there is a power cut off … so, the power is not cut during a PCR run," she said.
The team was able to do their PCR-based Plasmodium detection on an average of 240 samples per day. Added to their extensive quality controls, it meant they ran about 300 samples, such that "we are doing four DNA extractions in PCR plates, plus four PCRs per day. And we can manage to get all the results before four o'clock," said Canier.
The study required a fairly large staff. "For the mobile lab we have a driver that has been trained to drive big vehicles," she said. "Each time there are also two drivers going to the field with the mobile lab, and there is a car going as well to the field with the technicians, and me at the beginning of the mission, to set up and to validate all the equipment before the study starts. And then we have two lab technicians that are permanently in the field doing the PCR." Including staffing, Canier estimated it cost the lab $2.75 per sample for DNA extraction and screening PCR.
Canier said the team intends to train the people locally to use the mobile lab, and then it will be transferred to researchers at the Cambodian National Malaria Center for their use.