Malaria Rapid Diagnostic Test (RDT) being used at the Rabuor Health Center in Kenya / USAID.

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MADRID, Spain — In most parts of the world a double strip in a rapid test does not bring news of a pregnancy, as is customary in developed countries, but something far more ominous: malaria. This is the reality for almost 44% of the population in sub-Saharan Africa, where those who begin to suffer symptoms of fever decide to go to a hospital or pharmacy to undergo a rapid diagnostic test (RDT), according to the latest World Malaria Report the World Health Organization (WHO) published in December 2015.

Although malaria remains the biggest global health scourge, today the “forgotten disease” is gainning visibility. The physician and researcher Pedro Alonso, director of the Global Malaria Programme (GMP), from WHO, reviews the data to EL ESPAÑOL: “There has been remarkable progress in the fight against malaria in the past 15 years: mortality has decreased by 60%, the incidence of clinical cases has been reduced by 37% and six million deaths have been prevented.” But he warns: “huge challenges remain: we still have 440,000 deaths and more than 200 million clinical cases per year.”

“We still have 440,000 deaths and more than 200 million clinical cases per year”

Alonso attributes this “historic” progress to factors such as increased funding, from 960 million dollars in 2005 to 2,500 million in 2014, as well as the mass distribution of insecticide-impregnated mosquito nets and new treatments. But there is more: “We always forget to highlight the crucial role played by rapid diagnostic tests”, he adds.

Fom microscope to paper

In 1880, the French doctor Charles Louis Alphonse Laveran observed under the microscope, for the first time, a parasite in the blood of a malaria patient. Today, optical microscopy of blood smears remains the canon for the diagnosis of the disease. When Laveran was a military doctor in Algeria, cell staining techniques had not yet been developed; but since the invention of Giemsa stain method in 1904 - which facilitates the visual detection of the Plasmodium parasite that causes malaria - the standard procedure has barely changed.

The problem is that the microscopic diagnosis requires expensive laboratory instruments, reagents and specialized personnel. In 1994 Parasight-F, the first rapid diagnostic kit, was brought to market: the first rapid immunochromatographic test based on the same principle as pregnancy tests. Paper chromatography is a method used to separate molecules in a sample by making them run in a solution by capillarity. The sample, in this case a drop of blood obtained by a finger or heel prick, is placed on one end of a filter paper and nitrocellulose strip, where an antibody will bind to a protein (an antigen) of the malaria parasite.

The antibody diffuses along the paper to a strip containing a second antibody also capable of binding simultaneously to the same antigen. The first antibody, which contains a colored element, will only stop in this strip if the antigen is attached. Beyond this strip there is a control, a second strip with a third antibody that grabs the first. Two colored bands mean that the test has worked correctly and that the malaria antigen is present in the blood sample. Normally, the paper strip is enclosed in a plastic cartridge or cassette for ease of use.

Anytime and anywhere

The advantage of RDTs is that they can be used anywhere with no more instrumental than a lancet and a small pipette to collect the drop of blood, and without the intervention of specialized personnel. “What has changed is that now RDTs can reach places where it would take decades to have adequately equipped medical units” says Alonso; “They have enabled a transforming leap in time”. Furthermore, while diagnosis by microscopy can take hours to reach the patient, and even days in places without infrastructure, RDTs offer in situ results in less than one hour, usually in just 20 minutes. But, are they cheaper?

“RDTs can reach places where it would take decades to have adequately equipped medical units”

Not necessarily, if we consider the absolute cost. In 2007, a study estimated the price of a RDT in developing countries between 55 cents and 1, 5 dollars, compared to the 12-40 cents of a smear. RDTs have become cheaper in recent years, reaching today, according to UNICEF, an average price of 44 cents. But the truth is that the cost of microscopic diagnosis is less in places with existing facilities, materials and adequate personnel. For example, in a field study conducted in Ghana and published in February 2016 in Malaria Journal, a private hospital doctor estimated the cost of a microscopic diagnosis in 0,1 cedis (Ghanaian currency), to change about 2 cents euro, while a RDT was 2 cedis, about 45 cents.

Moussa Diagne, Entomologist with the Parasite Control Service in Senegal, performs a RDT for malaria / USAID.

However, experts handle a different parameter for the absolute diagnostic price, and it is cost effective in relation to its results. In this case, the data tip the balance in favor of the RDTs. In 2011, a study undertaken in Uganda placed the actual RDTs cost in $5 for each case, in comparison with the 9, 61 of microscopy diagnosis. More recent studies carried out in Zambia, Afghanistan and Brazil confirm that RDTs are profitable. According to a last year article written by the expert of the NGO Population Services International in Kenya, Cristina Lussiana, “RDTs have been widely recommended by governments and the international community as cost-effective tools for diagnosing malaria, and are currently seen as essential to ensure that the therapy is administered to confirmed malaria patients.”

Lussiana’s words reveal the factor that explains the profitability of RDTs. Up to the end of the twentieth century, the diagnosis of malaria was not considered essential, as classic therapies were cheap and the certification of infection by microscopy was expensive and often inaccessible. However, the indiscriminate use of drugs contributed to the development of resistant strains. Today the tables have turned: the current standard treatment, combination therapy with artemisinin derivatives (ACTs), is more expensive than classical drugs, but very effective; and will remain so as long as its use is restricted to patients really suffering from malaria, which requires a reliable diagnosis. Thus, RDTs facilitate the use of resources and avoid overdosing of the population, which can facilitate the emergence of resistant strains. For this reason, since 2010 the WHO recommends that all people with symptoms of malaria are subject to a diagnosis before starting treatment.

Diagnosis for everyone, treatment for those who need it

Thanks to the WHO campaign, the use of diagnostic methods has spread worldwide. Currently, 78% of patients with symptoms of malaria attending public schools undergo diagnosis, and 71% of these analysis are performed with RDTs. Diagnoses in Africa have grown from 36% suspected cases in 2005 to 65% in 2014. In America, Europe and Southeast Asia 90% of cases are diagnosed. The country making more diagnoses is India, with more than 120 million smears in 2014 and 29% of all tests in the world. RDTs sales amounted to almost 50 million units in 2008, rising to 320 million in 2013 and falling slightly to 314 million in 2014. Of the 1,600 billion dollars spent in 2014 on products for malaria control, 9% went to RDTs, a total of 151 million dollars.

Diagnoses in Africa have grown from 36% suspected cases in 2005 to 65% in 2014

The intensive campaign managed, for the first time, that in 2012 the number of ACT treatments in Africa was slightly less than the number of diagnoses, indicating that the medication begins to focus on patients who actually suffer from malaria. However, WHO warns that there is still room for improvement, for only 44% of diagnoses are positive, so the difference between the number of tests and the number of treatments should be enlarged in the future. The result of these efforts, according to a large study carried out in 2014, is that the introduction of RDTs has reduced by three quarters the prescription of antimalarials, maintaining a similar level of healing that before the existence of these tests.

Thus, RDTs are helping to reduce deaths due to malaria. However, as the GMP expert Jane Cunningham points out to EL ESPAÑOL, “it is difficult to estimate the impact of RDTs in the number of lives saved.” Cunningham cites a 2006 study that pointed an estimate: “It is calculated that high-quality tests at the point of care, such as RDTs, can prevent about half a million malaria-related deaths.” The results translate into numbers in specific places: in Zimbabwe diagnoses have increased fivefold between 2004 and 2014, while from 2003 to 2012 hospital admissions for malaria were reduced by 64% and mortality by 71% in 45 hospitals in that country, according to WHO data. In Papua New Guinea, the introduction of RDTs has raised the official numbers of malaria cases since 2012 due to the increase in the number of diagnoses, but hospital admissions fell by 75% between 2000 and 2014.

The RDTs boom

The ease of use, effectiveness and economy of RDTs have led to a boom in the market, with hundreds of kits available. Due to the proliferation of products, and to keep ongoing surveillance, since 2008 the WHO’s GMP, in collaboration with the Foundation for Innovative New Diagnostics (FIND), the Centers for Disease Control and Prevention (CDC) and other institutions, periodically test the RDTs available on the market that meet international standards of ISO quality.

In the six rounds of tests conducted so far by the WHO-FIND Malaria RDT Evaluation Programme, a total of 247 units of 171 products aimed at detecting one or more species of malaria parasite have been examined. A total of 45 RDTs detect only Plasmodium falciparum, the most dangerous and prevalent form of the parasite in Africa, while 125 also recognize other species of the parasite, in most cases distinguishing between them. A single test is designed only for P. vivax, the most widespread species. The RDTs also vary in the antigen detected: those directed to P. falciparum focus on histidine-rich protein II (HRP II), present only in this species, while the lactate dehydrogenase (pLDH) enzyme and fructose-bisphosphate aldolase (ALDOA or ALDA) appear in all forms of the parasite.

The WHO-FIND evaluation requires adequate detection sensitivity even for low levels of the parasite in blood, with less than 10% false positives and a maximum of 5% of invalid tests. In addition, RDTs must pass a thermal stability test. The results show that the quality of RDTs has progressively increased: in the last round, 92.7% of the tests passed the examination in the detection of P. falciparum, although only 58.6% passed for P. vivax. “In general we believe that certified quality malaria RDTs are suitable for detecting clinically significant infections”, says Cunningham.

The WHO-FIND evaluation serves as a guide to the various UN agencies, governments and organizations such as The Global Fund to Fight AIDS, Tuberculosis and Malaria. In addition, their results are integrated into the WHO Prequalification of In Vitro Diagnostics programme, which already includes 12 malaria rapid tests as prequalified products. FIND website has an interactive tool with evaluation results that allows different organisms and health workers choose the most suitable tests to their needs.

A market dominated by three manufacturers

However, despite the wide range of RDTs available in the market, the vast majority of units sold belong to a small number of manufacturers. According to UNITAID, an initiative sponsored by WHO for access to treatment in developing countries, of the 29 companies with products that have passed the WHO-FIND evaluation, only three of them dominated the 92% of the public sector market in 2013: US Access Bio and Alere, and Indian Premier Medical Corporation. The large multinational Alere entered the RDTs market by acquiring South Korean Standard Diagnostics in order to commercialize its SD Bioline line. Alere also bought the US biotech start-up Binax, manufacturer of the first RDT approved by the Food and Drug Administration (FDA) for laboratory use.

The vast majority of units sold belong to a small number of manufacturers

The volume these three manufacturers handle has enabled them to lower prices, but some experts warn of the risk of relying on such a limited number of suppliers. This market concentration has eliminated much of the competition: from the start of the WHO-FIND evaluations, 51 products have disappeared. In addition, Uniaid warns that falling prices, already approaching production costs, discourage the entry of new competitors and the RDTs innovation and improving, as companies are forced to offer cheaper products that barely pass the evauations. Alonso admits that a healthy market should have more competition, but insists that WHO's concern is that the RDTs are "high-quality, cheap and accessible".

WHO’s worry is that, in many cases, patients who need RDTs recommended by the evaluation program do not have them at their disposal. The private sector, which attends between 30% and 50% of cases of fever in countries where malaria is common, has not adopted the recommendations with the same intensity as the public sector. In a recent article published in the WHO Bulletin, Cunningham and colleagues cited as an example that almost one in five pharmacies in Uganda sell RDTs that have not even been evaluated by the WHO-FIND programme.

According to Lussiana, the problem is a mismatch in the market supply: “The private sector is composed of small clinics, pharmacies and drugstores who can not afford to buy big boxes of 25 or 30 RDTs, as those produced for the public sector”, as the expert exposes to EL ESPAÑOL; “They prefer individual kits which contain a RDT, a cotton swab, a pipette, a lancet and a small vial of solution.” Lussiana explains that these kits are not recommended by WHO due to the rapid obsolescence of the solution. The expert advocates promoting the development of stable and cheap individual kits through a subsidy programme similar to that applied to ACT therapies, which could be financed by The Global Fund and private foundations for the creation of new start-ups. Meanwhile, Alonso suggests that countries regulators could collaborate allowing only the importation of RDTs that meet WHO quality standards.

Future duties

Considering Lussiana’s opinion, Alonso reasons that there could be solutions such as collective buying, and that in any case “now the cost is not a barrier to access”; in fact, he notes, The Global Fund already buys RDTs for nations like Tanzania and Mozambique. Nevertheless, recent studies show that in these countries the presence of diagnostic tests is still poor: 50% of the surveyed centers in Tanzania, 59% in Mozambique and 62% in the Democratic Republic of Congo lacked a RDTs stock. Cunningham acknowledges that “there is still some way to go in some regions to reach the goal of universal access”, a way that, for Alonso, passes through improving distribution networks and coordinating the agents involved.

While the implementation of diagnostic testing progresses, some experts warn that it is important to move towards the development of better tests. WHO promotes the harmonization of products in terms of format, labeling, components and method of use, in order to minimize handling errors, especially when changing from one brand to another. But RDTs have inherent limitations beyond the non-specificity of some tests or possible deterioration caused by storage. Unlike microscopy, which allows a parasite count, rapid tests provide only a qualitative result, all or nothing. Cunningham adds that “they are not able either to monitor response to treatment”, because the HRP II antigen detected by P. falciparum tests can take up to 30 days to disappear from the blood after therapy.

However, currently the main headache for WHO is the detection of strains of P. falciparum lacking HRP II - used as indicator for diagnosis. These variants were first discovered in 2010 in the Peruvian Amazon, but Alonso confirmed to EL ESPAÑOL that recently they have also been found in Africa, the continent most ravaged by malaria. “We do not know yet the magnitude, we are investigating, but we are very concerned”, he stresses.

The GMP director raises the possibility that it could be a selection process: diagnostics recognize HRP II, so these cases are the ones being treated, which could favor the spread of strains without this protein that are invisible to diagnosis by RDTs. Given this possible “very worrying” scenario, Alonso warns that “it would be a total catastrophe to generate distrust towards RDTs”, but suggests the need to promote pLDH-based rapid diagnostic tests as an alternative.

New technologies

Performing a PCR test in the University of Tartu, Estonia / Karl Mumm.

Meanwhile, researchers are exploring new avenues of diagnosis. One of the possible technologies is the detection of nucleic acids, i.e., DNA or RNA of the parasite. This is the basis of the polymerase chain reaction (PCR), a method used only in developed countries, since it is a laboratory technique that requires expensive equipment. A simpler variant is the so called Loop Mediated Isothermal Amplification, or LAMP, which is growing strongly, but that is also inaccessible to field diagnosis.

According to Pranab Goswami, profesor in the Department of Biosciences and Bioengineering at the Indian Institute of Technology Guwahati, a viable path would be the use of biosensors based on short chains of nucleic acids, called aptamers. Goswami assures to EL ESPAÑOL that the use of such molecules “would improve the life of RDTs and cheapen its price” and, at the same time, “would aso avoid results variation due to temperature and humidity, as aptamers are more resistant than antibodies to unfavorable conditions.”

From the front line in the fight against malaria, Alonso welcomes innovation in diagnostic technologies; but also considers that the current RDTs are suitable for clinically significant cases without the need of raising their sensitivity or quantifying infection, as some systems based on band intensity readings or the use of fluorescent labeling try. “We follow with interest the development of nucleic acid based tests, but they are still far”, Alonso values. As a war strategist, the GMP director has clear priorities: “We must define exactly what we want, and my biggest worry is the need to have a diagnosis for every case of malaria”. And he is in the process of doing so. Today, victory is closer than ever.