Five Days of Fieldwork

BME students test a diagnostic tool in Liberia 

The generator that powers the Redemption Clinic doesn’t click on until 10:30 in the morning, but this delay doesn’t slow the crowd of patients who fill the health care center before 8 a.m. In one corner of the clinic, patients wait on rows of wooden benches as healthcare workers sort through medical files, the walls surrounding them adorned with murals that illustrate basic health instructions: wear a condom to prevent HIV, wash your hands to prevent the spread of germs, sleep with a mosquito net to prevent malaria and yellow fever.

From Duke BME Magazine

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The clinic is adjacent to the larger Redemption Hospital in the densely populated community of New Kru Town, a neighborhood on the outskirts of Monrovia, Liberia’s capital city. Men and women of all ages come in to seek free medical care for a variety of issues, but one corner of the clinic is dedicated to a very specific purpose––screening children for signs of malnutrition.

Staff from the Ministry of Health work their way through the growing queue of mothers and children, measuring the children’s weight, height and arm circumference before recording the measurements onto small squares of paper. The children vary in age, with the youngest in the crowd appearing to be no more than three months old. Some children seem content to calmly watch the process, while others cry when they’re taken out of their mother’s arms, contributing to the orchestra of noise that echoes through the clinic’s hallways. 

Trenton Dailey-Chwalibóg and Issa Kemokai enter the Redemption Clinic.

This was the scene that greeted Daniel Joh and Angus Hucknall when they arrived at Redemption on an early August morning. Joh, an MD-PhD student at Duke University, and Hucknall, a senior research scientist at the university, had traveled from the U.S. two days earlier, making the journey across the Atlantic to complete the next stage of their research––conducting a field test for a diagnostic device that would rapidly test for biomarkers of malnutrition.

“You guys ready to get set up?” asked Trenton Dailey-Chwalibóg, a research project manager from the French non-governmental organization Action Against Hunger/Action Contre la Faim (ACF). For two years, organization has partnered with Liberian health authorities and Duke University Medical Center to investigate the clinical significance of the current diagnostic criteria of malnutrition in hospitals like Redemption. Through his role at ACF, Dailey-Chwalibóg had helped the Duke team coordinate their field test, and was now acting as their unofficial guide to Monrovia.

Joh grabbed his gear. “Let’s get started!”

New Targets and New Tools

Today, more than 25 percent of children under age five are malnourished. According to the United Nations Children’s Fund, more than 16 million of these children suffer from the most extreme form of the condition, severe acute malnutrition (SAM). Children with SAM have very low weights relative to their heights or a very small upper arm circumference. SAM is a major cause of death in children under five, as its victims are more likely to contract diarrhea, pneumonia and other communicable diseases that their immune systems can’t fight off.

But despite the severity and extent of the problem, there is currently no simple test to diagnose malnutrition.

A mother and her child from the out-patient OptiDiag project at the Redemption Clinic in Liberia.

“The World Health Organization recommends that we use two different criteria to diagnose severe acute malnutrition, with the first being the weight-for-height z-score, which is like a measurement for body mass index,” explains Dailey-Chwalibóg. “But we also measure their middle upper arm circumference, called MUAC. If children have a MUAC of less than 115 millimeters, that tells us that they are considered severely acutely malnourished and need rehabilitation, so we conduct more tests to determine if we can take them into our outpatient program. But what’s interesting is that these two measurement criteria don’t always identify the same populations of children as being severely acutely malnourished.”

Based on these measurements, Dailey-Chwalibóg and his colleagues sort children into three categories: children who are considered malnourished based solely on their MUAC score; children who are considered malnourished just based on their z-score; and children who have both a MUAC of less than 11.5 centimeters and a low z-score. 

Focusing on both criteria poses a challenge, as it’s difficult to provide completely consistent measurements in the clinics, especially when a child is protesting and moving around during the measurement process. Even if a child remains still, there is no way to ensure that clinicians around the world have access to the same tools and training that would allow for consistent measurements.

“Being able to use a rapid diagnostic test for malnutrition would relieve the staff of a huge burden,” says Issa Kemokai, a research program manager with ACF based in Liberia. “It would mean that we could help children regardless of the measurement criteria, and it would help us make our rehabilitation efforts more accurate.”

But to make an efficient diagnostic test, researchers would need a single, easy-to-measure biological target.  The problem was that no clear target for malnutrition had been identified.

That changed after Dr. Michael Freemark, the division chief of pediatric endocrinology at the Duke University School of Medicine, conducted a field test in Uganda in 2014 to examine metabolic changes in children who were undergoing treatment for malnutrition.

“We recognized that 35 percent of the deaths of children around the world are either caused by or exacerbated by malnutrition."–Michael Freemark 

“We analyzed comprehensively the hormonal and metabolic changes that occurred before and during recovery--no one had really ever done that before," says Freemark. "The factor that stood out as the most powerful determinant of the risk of death was a low level of a hormone called leptin.”

Leptin, a marker of energy storage, is produced by white adipose tissue. Malnourished children have a low amount of this fatty tissue, making it more difficult for them to maintain the energy needed to keep their bodies properly functioning under severe stress. According to Freemark, this hormone also helps stimulate various components of the immune system, so patients who have lower levels of leptin can also develop an immune deficiency, making them more susceptible to infections.

Freemark presented his findings at the 2014 International Atomic Energy Agency Conference, where he met with several researchers who expressed interest in studying leptin, including Dr. Benjamin Guesdon, a staff scientist for ACF. Together, they discussed forming a collaboration to use leptin as a biomarker for a diagnostic test. 

“Drawing blood from a patient and shipping it back for testing at Duke wasn’t really feasible,” says Freemark. “We needed a test that could work in settings around the world where malnutrition is a significant problem, and a colleague suggested I meet with Ashutosh Chilkoti, the chair of Duke’s biomedical engineering department, to see if he had any ideas about a point-of-care diagnostic test.”

At the time, Chilkoti was working with Hucknall, Joh and other students in his lab to develop a diagnostic test using a unique non-stick coating that acted like Teflon for molecules and cells found in blood. “I told Tosh what I was looking for and asked for his advice, and he suggested using this new tool that he’d helped create,” says Freemark. “We ended up writing a grant with ACF and together we created a portable diagnostic device to measure various proteins including leptin.”

Now, three years after the initial collaboration, they were going to see how the test performed in the field.

Joh, Dailey-Chwalibóg and Jay Gupta perform preliminary verification tests using the D4 assay at the ACF Liberia base.

Trials and Triumphs  

Joh and Hucknall were led down a dark hallway to a room in the back of the clinic, where posters from ACF illustrating symptoms of malnutrition were nailed to the wall. A large wooden desk was in the middle of the room, facing away from a barred window that peered back into the clinic.

“You guys can set up your stuff here. I’ll be back to bring you more samples as our patients come in,” Trenton said, leaving the room to help the staff with measurements.

Joh nodded and began unloading his pack, pulling out two small blue boxes that contained their diagnostic test––the D4 assay

In resource-limited settings, clinicians typically rely on rapid diagnostic tests, called lateral flow tests, which can provide a diagnosis in five minutes and are highly portable. But the trade-off is a diminished sensitivity. Highly sensitive tests, like the ELISA platform, can give researchers a highly accurate analysis of a sample, but they aren't portable or rapid. 

Duke’s D4 assay was designed to combine the sensitivity of an ELISA platform with the portability and speed of a lateral flow assay.

The D4 assay is coated with a novel polymer that prevents non-target proteins from sticking to the surface.

The D4 assay uses a matched pair of antibodies to detect and capture leptin in a biological sample. The array contains two types of antibodies ––immobilized capture antibodies and soluble detection antibodies––which are tagged with a fluorescent marker to allow the researchers to identify how much leptin is present. When a drop of blood is placed on the slide, the detection antibodies dissolve, separate from the array and bind to the leptin in the blood. These fluorescing antibody-leptin pairs then attach to the capture antibodies that are still on the slide. 

Unlike other diagnostic tests, D4 assay’s antibody array is printed on a novel polymer brush coating that works like Teflon to prevent non-target proteins from attaching to the surface of the slide. By preventing unwanted proteins from binding to the assay, the polymer brush makes it easier to detect low levels of target proteins by getting rid of the ‘background noise’ on the chip. Before reading the assay, users wash the chip in a buffer solution and dry it in a centrifuge before viewing the reading with a smartphone camera attachment.

Issa Kemokai examines the assay using a portable microscope.

“This can achieve comparable sensitivity to the ELISA assay within 15 to 30 minutes,” explains Joh. “If we think we need even greater sensitivity then we just let the sample incubate longer.” 

Initially, the team had packed 250 samples of the test, but during verification testing at ACF headquarters, they realized something was wrong. “When we read the slides with the smartphone detector, you should be able to see quantum dots, and those will tell you how much leptin is present in the sample,” says Jay Gupta, a sophomore studying biomedical engineering at Duke and the third member of the Chilkoti lab in Liberia for the field test. “But when we tested the slides we found that most of the tests we packed remained blank, where control dots didn’t even show a reaction.”  

The malfunction may have been caused by the drastic flux in temperatures during the assays’ journey from the USA to the cold cargo hold to Liberia, or by the difference in humidity between Liberia and the laboratory environment at Duke—but it was clear something had interfered with the activity of the biological reagents on the slides.

After testing more samples from the remaining boxes, the team found that two boxes of assays still worked, though the results were faint. To get a more sensitive reading, they decided to try testing the assays with serum rather than whole blood to get a more concentrated sample. They also let the assay sit for an hour instead of 15 minutes to achieve greater sensitivity. By making these changes, the team could still test the D4 assay in the field.

“We anticipated that there would be problems during these field tests,” says Chilkoti. “This iteration of design-build-test-modify and test again is inherent in all tech development, and it’s even more critical when the technology is made for demanding environments where point-of-care tests are used.”

Dailey-Chwalibóg returned with a blood sample from a child who has been recruited into ACF’s OptiDiag project, a study designed to describe and compare the vulnerability of SAM patients based on their diagnostic category (MUAC, z-score or both). 

“Once a child is identified as being a candidate for OptiDiag, ACF workers will help the mother or caretaker fill out a questionnaire about her child’s health history and eating habits, and they’ll collect hair, urine and blood samples that we use in diagnostic tests to identify biomarkers, like leptin,” explains Dailey-Chwalibóg. “These kids then receive a weekly nutritional supplement with the nutrients they need for recovery, and return to the clinic weekly for a medical follow-up until they recover.” 

Joh snapped on the gloves and took the sample. After separating the serum from the blood, he applied it onto one of the D4 slides, setting a timer for an hour. Children peered into the room from the barred window behind Daniel, watching with curiosity as he worked.

After an hour passed, Joh rinsed off the slide in the buffer solution before handing it to Angus, who plugged the slide into the phone attachment and turned on the camera. Although the quantum dots were faint, they were visible.

“I think we’ll get a better reading using the tabletop scanner,” Joh said as he examined the photo. They planned to send the completed assays back to Duke where they could read them with the stronger scanner to get more accurate results.

Once they finished the first slide, Joh and Hucknall cleaned up and prepared to run a second test while they waited for another sample.

Daniel Joh and Angus Hucknall set up their supplies and wait for samples in a closed-off room at the Redemption Clinic.

In addition to testing the D4 assay in Liberia, the team had also decided to collect samples to ship back to Duke so they could test them on freshly printed D4 assays and potentially determine what went wrong with their slides.

Traditionally, biological samples collected in the field are transported by a cold chain process, which requires researchers to keep them frozen or refrigerated to prevent protein degradation. That wouldn’t work in this case, so the team opted to use the Neoteryx Mitra micro-sampler, a unique tool shaped like a Q-Tip that allows researchers to collect and ship biological samples at room temperature. A polymer in the micro-sampler preserves the sample and easily allows it to be reconstituted in the lab.

“We’ll be able to bring these samples back to Duke and process them normally, as well as on the D4 chips as originally intended,” Gupta explained. “In doing so, we’ll be able to generate essential data which we can use to answer questions about how the D4 performs and how sampling methods like the Mitra perform in settings we haven’t tested yet.”

Farewell

Jay Gupta watches as Trenton Dailey-Chwalibóg prepares a sample for the D4 assays.

After a morning of collecting samples from children at the Slipway Clinic across the Mesurado River, Gupta joined Hucknall and Joh at the Redemption Clinic, where he helped run more D4 assay tests. They worked efficiently in a small ACF storeroom, holding vials up to the light to pipette the serum and placing the micro-samplers into their bags for shipment.

Over the next three days, the team continued their work in this manner, with Hucknall and Joh traveling to Redemption to test the assays in the clinic while Gupta gathered samples at Slipway. The novel tests might not have worked as the team intended, but they did have a better understanding of the parameters where these diagnostic tests were needed.

“Even the initial issue with the antibodies on the assay taught us a lot,” Joh said on the last day in Liberia. “The test won’t be useful to anyone if we can’t safely ship it, so it’s good for us to know and address.”

For Chilkoti, the field test provided the team with an important opportunity to test their technology for any weak links or critical issues.

“Now that we’ve had a chance to identify problems, this test gives us the confidence and critical information we need to make crucial modifications to the technology that we are hopeful will lead to success in the next round of field testing,” says Chilkoti.

“No amount of testing at Duke would have brought these issues to the forefront, which is why the trip to Liberia was so useful, despite the setbacks the team faced.”–Ashutosh Chilkoti

The team left nearly 30 micro-samplers with Dailey-Chwalibóg and the ACF team to collect more samples over the coming weeks, with plans to ship them back to Duke when Dailey-Chwalibóg returned to Paris. As everyone said their final goodbyes there was already a discussion about how to improve the assay’s design for the next trip.

“We’re bringing samples back to Duke and we’ll conduct a lot of experiments to see what went wrong with the sensitivity on the slides, but this trip showed us how this tool could be implemented in resource-limited settings,” said Joh.

With their work complete and their bags packed, Gupta, Hucknall and Joh said goodbye to Dailey-Chwalibóg and Kemokai back at ACF Liberia base. “I’m sure we’ll see everyone soon,” Trenton said before everyone piled into the car that would take them back to the airport. “It’s great to see the progress on this.”

“Definitely,” said Joh. “But there is always more to do.”

Gupta shows Kemokai how the micro-samplers are stored so they can be transported without a cold-chain.

Click here to read about other Duke BME researchers working on novel diagnostic tools. 


OptiDiag is supported by the Humanitarian Innovation Fund, a program managed by ELRHA (Enhancing Learning and Research for Humanitarian Assistance), ECHO (European UnionCivil Protection and Humanitarian Aid) and the Fondation ACF pour la Recherche et l’Innovation. The views expressed herein should not be taken, in any way, to reflect the official opinion of these donors and they are not responsible for any use that may be made of the information it contains.