Paper Diagnostics in the Tropical World: A Complex Issue

Infectious diseases like Zika and Dengue have been spiking on public health radars for the last few years. They are simply the latest in what is a yearly outbreak of mostly mosquito-borne diseases that regularly strike the tropical and subtropical regions of the world. These are coincidentally the same regions with large populations of the poor. With science moving to create better, faster and cheaper rapid diagnostics, point-of-care testing might alleviate some of the healthcare problems in these areas. However, obstacles abound on either side.

A small, white, rectangular plastic device rests on her palm. It is a rapid diagnostic test for Zika – a tool that can tell you almost immediately if you have the disease or not. This particular test, however, is a failure. Within a small, rectangular window on the front, I see one line that runs perpendicular to the length of the window. There are supposed to be three lines, she tells me. This particular diagnostic device was used to test a woman in her 20s who was positive for Zika. It came back negative; it should have displayed three lines.

As an expert on tropical diseases, Irene Bosch, a scientist at MIT, visited Brazil in early 2016 during the height of the Zika outbreak. This diagnostic test is a souvenir from that trip. In Recife, the heart of the epidemic in Brazil, she remembers being bitten by mosquitoes day and night within the walls of the hospitals. However, good facilities and medical expertise was also available within those same hospitals. The doctors are experienced, understand febrile illnesses and can tell you which one has you all achy and feverish.  Bosch left Venezuela in 1987 at the age of 26 to get her doctoral degree at Harvard. Comfortable in the tropics, her natural environment, she has never truly left them behind as she studies infectious diseases in Colombia, Mexico, India and the Dominican Republic. Hugo Chavez’s presidency eventually led to the shutdown of her research on malaria and dengue in Venezuela in 2009. But she persists in trying to better understand what dengue and other viral fevers are and how they affect people, especially in resource-poor countries around the world.

Zika is not a deadly disease; patients suffer from a mild fever, rash, conjunctivitis and joint pain for two to seven days. Its effects are not that disastrous – that is, until it infects a pregnant woman. Zika has been proven to alter the neurological functioning of infants born to infected mothers. If that Zika positive woman, who received a false negative on her diagnostic test had gone on to conceive a child, unaware of her disease, chances are high she would have had birthed a child with severe developmental problems; an additional burden that may exacerbate already poor conditions.

On a recent trip to Santo Domingo, the capital of the Dominican Republic, Bosch was unprepared for what she saw at a local hospital. Infants who could not see, hear or swallow. Mothers who had travelled three hours so their children would be examined for 15 minutes. As evidenced by the 1000s of cases unattended in the hospitals of Santo Domingo – several hundred of them children suffering from birth defects due to Zika – the bare minimum in healthcare and other services are available.  “We’ve been in towns where there is no safe water or no jobs and you’re going to come and say what, ‘you need this device?’ Yes, you do. But you also need many other things. It is complex,” said Bosch. According to the United Nations’ Universal Declaration of Human Right, medical care is as important as food, shelter, clothing and other services, and just like these others, is out of reach for millions of people in the world.

For many people living in the poorest and most inaccessible areas of the world, basic healthcare is unaffordable. In low and middle-income countries, paper diagnostics that are accurate and easy to use would be invaluable. At last count, the paper diagnostics market was valued at USD 4.7 billion in 2014 – a number that is only expected to rise. Point-of-care or bedside testing is being used in a multitude of ways in developed countries: for example to assess liver and thyroid function and test for diabetes. Paper diagnostics involves testing biological samples (like blood, urine or saliva) on strips of paper treated to recognize a specific molecule. Perhaps the best known point-of-care device is the home pregnancy test that detects the molecule Human Chorionic Gonadotropin present in the urine of pregnant women.

A diagnosis is not a cure, but, it is significant in its own way. For one, diagnostics can help global health professionals identify a region where an outbreak is taking place and tackle the situation – and the mosquitoes – in the area more rapidly. Secondly, even though diagnosis does not guarantee a patient receives treatment, it does mean the patient is more likely to survive if treatment is available in these regions.

Zika and dengue fever are two diseases easily misdiagnosed – each one as the other – in tropical and subtropical areas which are haunted by the two diseases. They can be difficult to differentiate because of the similarity in initial symptoms. Coming from the same genus, these viruses are both mosquito-borne and often transported by the same species, Aedes aegypti. With a disease like dengue, hemorrhagic fever can lead to imminent death in the absence of medical care. Whereas Zika may not be life-threatening, an expectant mother might have to make an important decision.

Today, most Zika or Dengue testing can be serological or molecular. In the first, antibodies to viral proteins are detected in a patient sample. The antibody test is not specific enough – similarities between Dengue and Zika for instance, can trip up the clinician. Alternatively, a Polymerase Chain Reaction (PCR) can look through viral RNA present in the patient’s fluids base by base and recognize a pattern specific to that virus.  PCR, has its own limitations – it requires a machine that can control the temperatures of the various steps that make up one reaction. Thus, it can only be done in places that have the supporting infrastructure.

A major problem with Zika and Dengue rapid testing is the cross-reactivity that causes confusion during diagnosis. A patient who has suffered Dengue might test positive for Zika and vice versa.

While in Brazil, Bosch came across a rapid diagnostic dengue test made by Alere, a manufacturer of rapid diagnostics. Despite being unspecific – it cross-reacts with Zika – the test is still used. Thus, diagnostics available in the market don’t always do what they promised. There is certainly space and necessity for rapid diagnostics that are accurate and do work well.

In poverty-stricken areas of the world, medical care comprises a clinic outpost or a health center with nary a doctor in the vicinity. Healthcare workers, who are minimally trained may work part-time at these centers. Patients often travel hours, losing precious time and often, their daily wages to seek diagnosis and treatment. A difficult situation is made even more so because they are turned away as diagnosis might take weeks or if they are lucky, a day. Patients seeking results on a Zika test would need to wait for four to six weeks for a report from the CDC and few labs qualified to test for the disease. Even within the US, a response from the CDC could take as many weeks because of the sheer number of cases flooding in during an outbreak. During the early days of the Ebola outbreak, patient samples from Africa needed to be send to France for testing; that is the level of expertise in certain countries. The circumstances are not equally poor everywhere.

In the meantime, patients disappear all the time. “Some people have to walk hours to reach a health center. If they don’t understand why it’s important for them to come back they may not return,” said Davidson Hamer, a professor of global health at Boston. When patient needs are not met with a speedy response, those in need will just disappear.

A prototype for a simple paper test for Zika was developed in early 2016. With it Zika can be diagnosed from the blood sample of a patient in three to four hours. This means a patient could know their results on the same day. This distinction is incredibly important for patients in rural areas who travel long distances to get a diagnosis.

Dr. Melissa Takahashi, a postdoctoral associate at MIT, showed me the test that took only six weeks to develop. “We had high school students come in this summer and do it,” she said. The paper disc used for the test was resting inside a big plastic test tube with a cap on.  It was made of Whatman’s filter paper, standard in many biological laboratories. As I peered through the plastic of the tube, I saw the paper had tiny holes punched in concentric circles. These holes were the missing pieces of paper used for the tests. They had been punched out with a simple device called a biopsy punch, a long needle-like device.

The paper strips were ensconced within black, acrylic cartridges – “homemade” devices made by the scientists on a 3D printer. Each test was a circular bit of paper 2mm in diameter and the cartridge consisted of 48 such tests in all, arranged in a 6 by 8 grid. I noticed the faint purple and yellow still visible on the tests – a positive and a negative respectively. These were the results. Every paper test contains a string of RNA – an intermediate in the DNA-to-protein reaction – the genetic material in many viruses.  Detection occurs when the RNA on the test interacts with viral RNA present in the patient’s blood. 

These paper discs are stable at room temperature and are activated only when a patient’s processed sample is added to it, rehydrating it. This system can be easily stored and distributed globally without cause for concern over degradation of the biological elements. Furthermore, the results can be viewed by the naked eye – a simple matter of differentiating between yellow and purple. In addition, the researchers have developed a cheap, battery-powered portable electronic reader that can quantify the result. 

Dr. Keith Pardee, a biologist, who worked on the test tells me that the Zika paper test is going to undergo validation in Ecuador, Colombia and Brazil. The test will be tested in nine labs spread across the Americas to see if it detects Zika to the same level of accuracy that a PCR test would. Commercially producing this test is not yet even on the horizon.  “It is going to take a few years before we are at the stage where it is actually deployed as a tool,” said Pardee.

This $1 solution can be quickly produced, rapidly redesigned to target other viruses and easily transported. It has potential not only because it can be easily modified to suit changing strains and other viruses but also because it provides a cheap and rapid diagnosis. “This kind of test is pretty versatile and you can use it for different applications,” said Dr. Justina Tam who works in neighboring Dr. Lee Gehrke’s lab.

Gehrke’s lab put together a paper diagnostic that can tell a patient if they have Ebola, dengue fever or yellow fever from the same test. This test was developed a year before the Zika paper test. Gehrke’s team has validated the diagnostic with patient samples from Honduras, Cuba, Brazil, Panama and India and nearly two years after they first came up with the test, are looking towards large scale production. Gehrke says there aren’t any defined paths through which researchers in an academic setting can take a prototype diagnostic and sent to those regions that need it the most. In broad strokes, however, these paths can lead to one of two options. The first would mean partnering with a pharma company, an option that doesn’t always end well. Bosch, who works in Gehrke’s lab, had an unsatisfactory interaction with Iquum Inc., a company that she worked with to develop a dengue diagnostic. Apparently, the company used the biological substances provided by Bosch to make a prototype but the project was not completed and the test never deployed.

The other possibility is starting a company – either for profit or non-profit – to produce the device, though this can be cumbersome. “The process with the FDA I find is difficult,” said Gehrke.  More so than it needs to be, he believes. In foreign nations, matters can get even more complex.  Understanding the regulatory procedures of a distant land and finding the right people to discuss approval with can be challenging. Gehrke, who has partners in Brazil, and other Latin American nations as well as India, said that his testing devices have a prayer of being used in these nations only through appropriate partnerships. He uses the word “co-create” time and again. “Don’t think you’re marching in there,” he said.

With hurdles in import and export of biological samples, the best bet for researchers like Gehrke is to establish contacts in afflicted countries who can verify the test. “Helicopter science” or “sample safaris” have led to wariness: foreign scientists have been known to exploit these regions by using samples from patients without acknowledging the researchers who helped in procuring them.

Collaboration can also help institute trust between makers of the diagnostic and the local practitioners who will be using the test. “The clinicians don’t really trust the diagnostic test,” said Gehrke. They need to be reassured that the test is not a replacement for their services but instead an addition to their arsenal of choices.

A recent approximation, reports the World Health Organization by researchers at the University of Oxford, suggests that around 3.9 billion people in 128 countries around the world are at risk of contracting dengue. However, the area of tropical diseases and their epidemics is still an unstable market on which to base a company’s profits. The ebb and flow of the business can be discouraging for investors. “I think that is a disincentive to companies who want to know that we’re going to have a market for these that is going to be long-lasting,” said Gehrke. Bosch’s own experience with investors was that American money would fund only those devices that cater to the Americans – mostly travelers going to and from the tropical nations.

The Foundation for Innovative New Diagnostics or FIND is one not-for-profit organization that aims to help smooth interactions between researchers and companies and ultimately take a device from the bench to a bedside. With funding from the Bill & Melinda Gates Foundation, National Institutes of Health, the EU, governmental and global health funds, FIND has its headquarters in neutral Switzerland.  FIND enables researchers by partnering them with companies like Cepheid, Becton Dickinson and Illumina. “The problem you have is that a company has a hard time seeing a market value for low and middle income countries,” said David Dolinger of FIND. The companies are incentivized by large funding grants procured through FIND. From development to approval, depending on the type of diagnostic, anywhere between $4 million to $40 million dollars is spent.

Given the population sizes at risk for such diseases, even a small profit per test should lead to a huge turnover. However, these rapid diagnostics don’t always succeed in reaching the people who need it most or being used by health care workers. “There’s a lot of implementation challenges,” said Hamer. That is, while the market size is massive, the market penetration is poor. Rapid diagnostic tests for malaria are well established and thus, a good standard for comparison. With increased competition, as there are at least 60 different brands available, production costs have come down. However, problems can still pop up in the distribution leading to shortage of these devices. These same distribution networks can sometimes double the cost of the diagnostic. 

Meanwhile, health care practitioners lose hope waiting for diagnostic tests to arrive, tests that might never get there or even if they do, may not always work well, leading them to fall back on their old ways. Devices manufactured in a vacuum, without the help of the people who will be using it, who are told “this is what you need”, sometime never even get unboxed. It is generally agreed that for a diagnostic to have a shot at working, people from the region must be involved.

Diagnostics is but one step in finding a solution to the rapid spread of infectious diseases in low and middle income countries. They do not guarantee treatment because clinic outposts only have the most basic amenities and drugs on hand. “Why would you want to diagnose someone and tell them, ‘well you’re resistant to the following drugs, so you’re going to have to get on the bus and you’re going to have to travel 6 hours and lose a day’s wages – if not two days wages – to be able to get to the provincial hospital to get those drugs and then come back home’,” questions Dolinger. However, the availability of treatment is a whole other ballgame.

There are clearly several problems that researchers, companies, public health groups and organizations like FIND have to tackle in the course of their undertaking: from companies dumping their poorly made devices to make a quick buck, difficult to understand markets and complications in implementation. “Diagnostics are always viewed as sort of the red-headed step-child. They’re always forgotten about,” said Dolinger. Yet, a good healthcare system must be built on a strong diagnostic foundation. People are now realizing that by “downplaying diagnostics” we might be setting ourselves up poorly. Despite all the obstacles, some people are working to make this human right available to large swathes of the global population. Their work is only just beginning.


Science Writer