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Chapter 3.2. The hurdles in making diagnostics accessible to the bottom billions: can lateral flow immunoassays make a difference?

Authors: 

Geik Yong Ang, Choo Yee Yu and Yean Yean Chan

Art work
Title: 
Earthquake
Artist: 
Uli Reinhardt
Children are a particularly vulnerable group
generally excluded from decision-making processes 
and with fewer resources to protect their own health. WHO
Maybe it’s not the right time.
Our era has left us talking to ourselves
Jose Emilio Pacheco 
Critique of poetry. 

Introduction

Designing diagnostics for the poorest billion of the world’s people is feasible, but aside from identifying target disease biomarkers and producing functional test prototypes for evaluation, it is important to understand the targeted population and their predicaments, as well as the diseases that greatly affect their lives. An estimated 2.7 billion impoverished individuals worldwide who live on less than US $2 per day suffer from the greatest risk factor for acquiring and succumbing to diseases: poverty [1,2]. In the developing world, transmission of infection is particularly efficient because 1 billion people lack access to safe drinking water, 2.5 billion have no access to basic sanitation, housing is substandard, and there is inadequate vector control [1,3]. Therefore, it is no coincidence that the world’s poorest billion people are mostly localized in regions plagued by the “big three global diseases” (human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), malaria, and tuberculosis (TB)) as well as the neglected tropical diseases (NTDs). Poverty increases the vulnerability of the poor to communicable diseases. Moreover, the endemicity of certain diseases leads to an increase in coinfection, higher levels of susceptibility to opportunistic diseases, and the mistreatment of disease, which increases morbidity and mortality among the bottom billion population [4].

The parasitic and bacterial diseases that are collectively identified as NTDs are regionally endemic and remain one of the most potent reinforcements of the poverty trap that affects the poorest of the poor [4,5]. Infection by some of the major NTDs is catastrophic for patients and their families, as exemplified by a study in Ghana where the cost of care per patient for people with Buruli ulcer was reported to average of 242% of a household’s annual earnings in the poorest earning quartile [6]. In addition to the heavy direct costs (the cost of diagnosis and treatment), households affected by NTDs become poorer through indirect costs such as reduced agricultural productivity as a result of disfigurement or other sequelae of long-term illness, loss of household income, and sale of assets such as land and livestock [7]. It is not just affected households that suffer from the debilitating consequences of NTDs; the accumulative individual loss of productivity becomes even more significant on a global level, where single NTDs such as lymphatic filariasis cause a loss in productivity worth almost US $1.3 billion [6].

The poverty-causing NTDs exert their hold by incapacitating the younger generation, thereby depriving the family of the prospect of a better economic status. Schistosomiasis and soil-transmitted helminth infections affect the wellbeing of children by reducing child survival and impeding physical development, fitness, and cognitive abilities; this impairs educational attainment and the potential to earn an income [5,8]. More than 1 billion impoverished people suffer from NTDs, and the top thirteen neglected diseases together account for over 0.5 million deaths [9,10]. In contrast to infectious diseases such as acute respiratory infection (ARI), malaria, HIV, and TB, which are the major causes of mortality in the developing world, NTDs are characterized by lengthy periods of suffering and often a lifetime of disability. Such NTDs may cause blindness, partial disability, and total incapacity, and may severely diminish a worker’s productivity [6]. In this sense, the top thirteen NTDs collectively account for a disease burden of 56.6 million disability-adjusted life-years (DALYs), which is even worse than for TB or malaria [9].

In the absence of diagnostics, the underlying causes of the millions of deaths that occur in developing countries such as those in sub-Saharan Africa are generally attributed to the “big three global diseases” [11]. Unfortunately, overestimation and the resulting emphasis on HIV/AIDS, malaria, and TB shift medical attention from other less prevalent diseases. In Ghana, one study found that among 251 children admitted to a tertiary referral center with a World Health Organization (WHO)-defined clinical diagnosis of severe malaria, 51 had bacteremia that was accompanied by a very high mortality rate, especially among infants less than 18 months old [12]. Making diagnostics available and accessible would substantially improve global health by revealing the true prevalence of diseases, and by reducing misdiagnosis and the associated consequences of inappropriate or inadequate treatment. Accurate epidemiological studies and assessment of the disease burden in a particular population would enable policymakers to design appropriate control strategies that address the real needs of the people.

The lateral flow technology to address the need for point of care test

The WHO Sexually Transmitted Diseases Diagnostics Initiative has conceived the term “ASSURED test” (Figure 1) to describe the ideal characteristics of a diagnostic test that is suitable for use in a primary healthcare center and in resource-limited settings [13]. Although an ASSURED test may not have optimal sensitivity, a test that can be carried out on-site ensures that more infected people receive treatment. This is because patients from rural areas may have to walk for hours to reach a health facility, and unless they have high mobility and compliance, most will be lost to follow-up if they are requested to return for their results on another day. The 2004 World Development Report identified this lack of accessibility as one of the major underlying reasons for healthcare failure; more people would have benefited if investment had been channeled into increasing access to diagnostics rather than improving test performance [14]. Therefore, introduction of point-of-care (POC) tests in health facilities will significantly impact the life of patients by allowing health providers to offer evidence-based medical treatment while the patient is still in the facility [15]. Health facility settings in remote areas require simple POC tests with a good level of sensitivity and specificity. The complexity of a test can be gauged by various aspects including the need for technical expertise, equipment, and the number of manual manipulations requiring user intervention and interpretation [4].

Figure 1. ASSURED diagnostic tests criteria.

One such POC test is the direct agglutination test (DAT) for the screening of visceral leishmaniasis (VL). The semi-quantitative test involves preparation of a patient’s serum or diluted blood in a microtiter plate before adding stained (and dead) Leishmania donovani promastigotes. If specific antibodies against the promastigotes are present agglutination will occur, but prolonged incubation of up to 18 hours is required for visualization of the results [16]. The rapid plasma reagin (RPR) test, which is used to screen for syphilis, is also based on antigen–antibody formation, but unlike the DAT for VL, the RPR result can be read after 8 minutes. The antigen in the RPR test is coated on charcoal particles and agglutination as a result of immunocomplex formation is visible as black clumps against the white card [17]. The agglutination-based assay is simpler than many other diagnostic platforms but it still requires equipment such as a mechanical rotator and micropipettes. Well-trained laboratory technicians are needed to perform the assay and interpret the results, and to carry out regular quality control to ensure the accuracy of the test. Moreover, the need to store the antigen at 2–8°C once it has been reconstituted severely limits its usage in peripheral health facilities [18].

Despite recent technological advances and research breakthroughs, the lateral flow immunoassay (LFI) remains the diagnostics platform that is most likely to impact healthcare in low-resource settings because LFIs are cheap to produce, simple to use, produce a rapid visual readout, and often require no equipment [4]. These attributes are well suited to rural primary healthcare facilities that lack the resources to pay for trained personnel, an electricity supply for equipment, and refrigerators for reagent storage. The principle of the lateral flow test is based on the capillary action of an aqueous medium through the interstitial space of a membrane, where the analyte of interest encounters the detector and capture agent, which is dried separately on the strip. The resulting formation of an antigen–antibody complex is captured at the detection zone, whilst unbound reactant continues to migrate to the absorbent sink. The immobilized immunocomplex generates a visible response (usually a colored line), and the intensity of the signal is proportional to the analyte concentration. A typical lateral flow strip comprises several critical components, and is assembled and optimized to function as a single test system, as shown in Figure 2. The various components include a sample pad, a conjugate pad, a membrane, and an absorbent pad. Each component is overlapped and attached to an adhesive plastic backing to allow continuous flow from the proximal to the distal end.

Figure 2. Components of a typical lateral flow immunoassay.

Non-competitive versus Competitive Format

As illustrated in Figure 3, most LFI applications are designed either according to the non-competitive (also known as the sandwich or direct assay) or the competitive (indirect assay) scheme. The choice of format for a particular test is usually dictated by the size (or molecular weight) and antigenicity of the target analyte [19]. Non-competitive assays follow the double antibody sandwich format and are usually used to detect larger analytes with multiple antigenic sites, such as the p24 capsid antigen of HIV. Parpia et al. [20] developed an LFI using two monoclonal anti-p24 antibodies targeting different epitopes of the antigen as capture and detector agents, respectively. The typical layout of the double antibody sandwich scheme is shown in Figure 3 (a). The antibody specific for an epitope of the target antigen is coupled to a signal generator and dried on the conjugate pad of the LFI strip. Another antibody targeting a second epitope of the antigen is immobilized on the reaction pad to serve as the capture reagent. The antigen of interest is introduced to the LFI strip by applying a biological sample such as urine, serum, or whole blood onto the sample pad. If the target antigen is present in the sample, it will bind by its antigenic sites to both detector and capture agent, and the resulting immunocomplex is immobilized on the reaction pad at the test line. The intensity of the signal is directly proportional to the amount of target antigen in the sample. The non-competitive format is also applicable for the detection of antibodies, but a double antigen sandwich is used instead of a double antibody sandwich. The Determine HIV-1/2 kit from Abbott Laboratories is an example of a double antigen sandwich assay; it detects antibodies specific to HIV-1 and HIV-2 viruses in serum, plasma, or whole blood using HIV-1 and HIV-2 recombinant antigens and synthetic peptides [21]. Another strategy for developing a serological assay is to use a pair of antigen- and host-specific antibodies as the capture and detector agents. An example of such a system is the ML Flow test (KIT Biomedical Research), which detects antibodies to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae [22]. The assay uses anti-human IgM antibody as the detector agent and a semisynthetic antigen is used as the capture agent. The non-competitive format is widely used, and most commercialized tests for the detection of infectious diseases are based on this scheme.

The competitive format can be used when testing for small molecules with single antigenic determinants that cannot bind to two antibodies simultaneously. In the competitive assay, the detector agent is often the target analyte that is bound to a signal generator (Figure 3 (b)). As the free analytes and detector agents contained within a sample pass over the test line, they compete with each other for the binding site on the capture agent. As a result, random binding of both free analyte and detector agent occur at the test line. Because a higher concentration of the free analyte in the sample is translated into a higher probability of that analyte binding on the test line compared with the detector agent, the resulting signal intensity is inversely proportional to the analyte concentration. The highest concentration of free analyte in the sample results in no signal generation, whereas absence of the analyte in the sample yields a visible colored test line. The intensity of the signal diminishes as the concentration of the analyte in the test sample increases. The inhibition assay is another example of the competitive format. In this particular assay, the analyte under investigation (or a structurally similar analogue) is immobilized on to the reaction pad (Figure 3 (c)). Antibodies against the analyte are conjugated to a signal generator and used as the detector agent. Thus, in the absence of an analyte, the detector agent binds to the capture agent at the test line, resulting in a colored signal. However, if a sample containing the target analyte interacts with the detector agent, a complex is formed that prevents the detector agent from binding to the capture agent. Because the complexes are not bound at the test line, no signal is generated. Thus, the analyte from the sample inhibits the detector from binding to the capture agent. As with the competitive assay, the intensity of the test signal decreases as the concentration of the analyte in the test sample increases. The competitive and inhibition formats have been used to detect drugs [23,24], toxins [25], heavy metals [26], and pesticides [27,28].

In all LFIs, a second line that functions as the control line is located downstream of the test line. The control line typically consists of species-specific anti-immunoglobulin antibodies that target the antibodies raised by the host, which are conjugated to the signal generator. The control line should be visible irrespective of the presence/absence of the target analyte. The incorporation of a control line in the design of an LFI is of the utmost importance as a functionality and operational control for each individual strip. This built-in control in each LFI ensures that the biochemistry on the strip is not adversely affected by the temperature variation during transport and storage in remote areas, and it ensures that the results are obtained accurately by staff, regardless of their level of training. All LF strips are designed for single use, and should therefore be devoid of any issues involving carry-over or cross-contamination between different patients, assuming the assays are performed according to the manufacturer’s instructions. Used strips should be treated as biohazard waste and disposed of accordingly.

Antibodies as capture and detector agents

A typical LFI employs two different antibodies that bind to distinct antigenic sites or epitopes of a target analyte. An antibody that is immobilized on the surface of a membrane serves as the capture agent, whereas the second antibody is labeled with a signal generator and serves as the detector agent. Theoretically, both polyclonal and monoclonal antibodies may be used, and the choice of antibody pairs to be used as conjugate and capture antibodies can be determined empirically. Nisnevitch and Firer [29] have highlighted several immunological and physical aspects to be considered in the selection of antibodies that are applicable to the development of an LFI. A polyclonal antibody is produced as a response to infiltration by a foreign antigen, and a considerable number of hosts are available including laboratory mice, rabbits, and farm-bred goats and sheep. Compared with monoclonal antibody production, which requires technical expertise and specialized facilities, immunization of animals with the target antigen to obtain a polyclonal antibody is cheaper, quicker, and easier to perform. However, the source of polyclonal antibodies is necessarily finite. This leads to variation in the quantity and quality when different batches of polyclonal antibodies are used. Because hybridoma cells that secrete monoclonal antibodies can be stored in liquid nitrogen and can theoretically provide a standardized source of antibodies for many years, LFI developed using monoclonal antibodies may be more economical in the long term. Commercial diagnostic tools require a reliable source of raw material, so monoclonal antibodies are the greatly preferred choice.

Although monoclonal antibody production is a tedious and relatively costly process, monoclonal antibodies are highly specific, and therefore reduce the occurrence of false-positive results and cross-reactivity with structurally similar substances. Moreover, monoclonal antibody production allows researchers to screen for the desired specificity and binding affinity in accordance with a particular clinical application. A polyclonal antibody preparation containing various epitope-targeting antibodies might contribute to more effective antigen capture, but sensitivity is compromised because other non-specific antibodies and proteins compete for binding sites on the membrane. The homogeneity of a monoclonal antibody composition simplifies the optimization process. In contrast, polyclonal antibodies comprise various isotypes and classes of immunoglobulins, each of which requires slightly different binding conditions; species such as IgA and IgM may even pose potential structural and steric problems during immobilization or conjugation [30]. Therefore, assays involving the use of polyclonal antibodies may require affinity purification to improve the performance of the test.

Figure 3. Schematic diagrams showing the various formats of lateral flow immunoassays.
CL, control line; TL, test line.

The Components in a Lateral Flow Strip

The reaction pad, on which the results are interpreted, is one of the most important components of the LFI. The pore size, porosity, thickness, and unique structural characteristics of the polymer that forms the reaction pad determine the surface area available for immobilization, which in turn reflects the pad’s protein-binding capacity [31]. Although a wide variety of materials such as nylon, polyethersulfone, polyethylene, and glass fiber are available for use as the reaction pad in LFIs, nitrocellulose membrane remains one of the most widely used materials [19]. This can be attributed to its protein-binding properties: nitrocellulose membranes are inherently hydrophobic and neutral, allowing proteins to interact and bind readily through electrostatic interaction between the dipole of the nitrate ester moieties of the membrane and the peptide bonds of the protein [31]. Membranes with various capillary flow rates or capillary flow times are commercially available from various vendors, allowing researchers to optimize the type of membrane to be used. In addition to the flow rate of the membrane, the test performance of an LFI is also affected by the capture reagent buffer, the reagent dispensing equipment, the dispensing rate, the concentration of the capture reagent, the placement of the test line, the ambient humidity during protein application, and the drying process used [30,31]. In many cases, blocking of the reaction pad to prevent non-specific adsorption of the signal generator or analyte is not required because the presence of proteins in the sample or blocking buffer impregnated in the sample pad is sufficient to prevent non-specific adsorption. However, if blocking of the reaction pad is required, researchers may choose from a wide variety of blocking agents such as bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), gelatin, and casein. However, the compatibility and optimal concentration of the blocking agent should be determined empirically [32].

The function of the sample pad is to receive the aqueous sample and regulate its flow to prevent flooding of the reaction pad, which reduces the overall sensitivity of the LFI. The sample pad also serves as a suitable site for the introduction of modifying agents such as proteins, detergents, viscosity enhancers, or buffer salts for various purposes including blocking the reaction pad, increasing the viscosity of the sample, facilitating the resolubilization and release of the signal generator, and altering the chemical nature of the sample by modulating the pH value to promote antigen–antibody binding [31]. Typically, woven meshes or cellulose fiber is used, but the sample pad can be customized according to the type of sample to be analyzed. For instance, if the target analyte is present in a serum sample, a sample pad that can function as a blood separator is appropriate because the red blood cells in whole blood can obscure the signal generated on the reaction pad [30]. An example of such a pad is the FUSION 5 from Whatman (Kent, UK), which has an efficiency approaching 93% in filtering serum from fresh blood.

The signal generator or label onto which the detector agent is conjugated provides a means of visualizing the LFI result. Colloidal gold is used most frequently as a label, followed by colored latex particles, whilst other labels such as carbon, selenium, liposomes, and chemiluminescent or fluorescent nanoparticles are used less often [19]. The many attributes of colloidal gold nanoparticles make them an attractive choice, and a proven track record in various LFI applications has established the suitability of colloidal gold as a signal generator. Colloidal gold is relatively inexpensive, non-toxic, and stable in both liquid and dried forms, and does not succumb to photodecomposition [33]. Colloidal gold nanoparticles can be easily conjugated via physical absorption, or functional groups such as amine or thiol may be used to attach them by covalent bonding [34]. The optimal pH and concentration of antibodies needed to produce stable conjugates can be determined by a flocculation assay [35]. When an electrolyte such as sodium chloride is present at high concentration, the electrostatic repulsion between the negatively charged colloidal particles is significantly reduced to the extent that once the particles come into contact with each other, they aggregate permanently [36]. The instability of a conjugate is indicated by a visual change of color from red to grayish blue accompanied by a change in absorbance, which can be measured using a spectrophotometer [37]. Once the gold conjugate is ready to be immobilized on the conjugate pad, a stabilizer such as sucrose or trehalose is generally required to maintain the structural integrity of the conjugates when proteins are dried, so that the activity is retained once the conjugates are rehydrated [38]. The LFI applications involving the use of colloidal gold conjugates are usually associated with pink to red color signals, but the use of microspheres has provided a plethora of colors for use either alone or in combination in a single LFI.

Porous material such as glass fiber filters, cellulose filters, and surface-treated (hydrophilic) polyester or polypropylene filters are suitable for use as conjugate pads [31]. In addition to providing a matrix to hold the dried signal generator in a stable and functional state, the conjugate pad also ensures even and uniform transfer of the signal generator and test sample onto the membrane [32]. The volume of sample required to release all the signal generators from the conjugate pad determines the amount of analyte that can be analyzed, because subsequent analyte entering the membrane will not contribute to signal generation as only half of the immunocomplex is formed in the absence of the detector agent [31].

Most absorbent pads are made from cellulose fibers and serve as a reservoir to accumulate excess sample and reagents that do not contribute to specific signal generation. The absorbent pad should be able to accommodate the full volume of the sample and the buffer required to release all the conjugates, because capillary flow will be halted once the absorbent pad is full [32].

The membranes used for the construction of LFI are often fragile. Therefore, they are always mounted on an adhesive card, which not only provides tensile strength but also allows the LFI manufacturer and the end-user to handle the strip without breaking it. Proper pressure must be applied when overlapping the various membranes on the adhesive card to ensure that consistent sample flow is achieved [30]. Improper lamination results in an irregular flow front, and in some extreme cases, mechanical over-compression of the porous membrane may completely halt capillary flow [31].
The majority of commercially available LFI strips are placed in a plastic housing or cassette after mounting on the adhesive card. The cassette is designed in such a way that the LFI strip is properly held in position by pressure bars or pins, leaving only the area for sample loading and a viewing window accessible to the end-user [32]. The housing also provides guidance to the user on where to load the sample, and the location of the test line and control line are also indicated on the cassette to facilitate result interpretation. Although the inclusion of a cassette inevitably increases the cost per assay, the plastic housing also protects the LFI strip from physical damage.

The Dilemma of Diagnostics in Low-resource Settings

The laboratory-based diagnostic tests with reasonable sensitivity and specificity that are currently used in industrialized countries may not cater for the needs of the developing world. Technological capabilities and infrastructure requirements, such as access to reliable power and clean water, availability of laboratory equipment and supplies, and highly trained laboratory supervisors and technicians, are often beyond the resources of low-income countries, especially in peripheral health facilities, which serve most of the population [11,15,18,39]. Despite the importance of laboratory testing, the available financial resources have mostly been channeled into disease prevention and provision of care without also developing laboratory capacity [11].

Central hospitals in urban setting, where public utilities, infrastructure, and human resources are usually available, are generally the most advanced in terms of laboratory capability. Although many central hospitals do provide testing services for patients from remote areas, patient access is severely limited and patients tend to be lost to follow-up [39]. Scarcity of trained personnel and extreme staff shortages in combination with an overwhelming number of acutely ill patients in remote settings mean that not all clinical specimens are obtained for laboratory testing. When specimens are obtained, other factors such as the need for sample delivery, long delays between sample collection and testing, and loss of specimens or test results severely impact the benefits of laboratory testing [11]. A study investigating the effectiveness of a bus service and “tuberculosis officers” for transporting sputum specimens found that the specimens from only 40% of 964 patients with recurrent smear-positive pulmonary tuberculosis arrived at the Central Reference Laboratory [40].

Extreme infrastructure and instrumentation constrains, large temperature variations, and staffing limitations can have an adverse effect on test performance. Moreover, the prohibitive cost of sophisticated diagnostic instrumentation and consumables, and the availability of technical support and equipment repair services significantly impact the ability of peripheral laboratories to access many technology platforms [4,41]. In fact, the lack of skilled laboratory technicians and lab managers limits laboratory services at every level. The attrition of human resources in developing countries is the outcome of very limited access to professional training and accreditation programs, and current education and access to hands-on training for specific diagnostic tests are inadequate [42]. In the developing world where 1 billion people are illiterate and the literacy rate can be as low as 50%, retaining qualified healthcare providers is a significant challenge because there is little financial incentive to work in rural district facilities or low-paid government jobs. Professionals may therefore opt to leave for jobs with better working conditions, both locally and internationally [43,44].

In a minimally equipped laboratory, microscopy is the most cost-effective diagnostic platform for detecting live infections, and the ability to provide quantitative results largely capitalizes on the diagnosis of parasitic and mycobacterial infections. However, a lack of good-quality slides, reagents, and well-trained microscopists has resulted in the overall poor performance of microscopy as a tool for the diagnosis of malaria and tuberculosis in low-resource settings [4,18]. This was reflected in a study in Malawi where 40% of the smear-positive pulmonary tuberculosis diagnoses in rural or district hospitals had negative smear results after review at the central reference laboratory [45]. The ripple effects of over a century of poor microscopy performance, and the lack of supervisors with the technical expertise to monitor the accuracy of test results, have contributed to a culture of mistrust and undervaluing of diagnostic test results from healthcare providers in remote settings [4].

Culture is the gold standard for bacteriological diagnoses, and as with microscopy, it facilitates the detection of live infections. Furthermore, the isolation of infectious disease agents enables antimicrobial susceptibilities to be determined, which significantly aids physicians in prescribing antibiotics. However, the culture method is known to be time-consuming, and stringent transport conditions are required to maintain specimen viability. The need for a constant supply of reagents and electricity, well-maintained equipment, and adequately trained and supervised technicians contribute to the high cost of culture. Enzyme immunoassays (EIAs) offer a shorter turnaround time (3–4 hours) for the detection of antigens or antibodies for the diagnosis of infectious diseases, and they are more cost-effective because they allow batch processing of samples [18]. However, EIAs are also heavily dependent on equipment, electricity, and highly trained technicians to perform the multiple manual manipulation steps.

Realizing the need to provide appropriate healthcare to patients presenting at health facilities that do not have access to laboratory testing, WHO has developed guidelines for syndromic management of acute respiratory infections, diarrhea, sexually transmitted infections (STIs), and common childhood diseases. The algorithms are simple to use and implementation of the syndromic approach in areas with high prevalence of a disease is not constrained by laboratory capacity, financial considerations, or the limitations of infrastructure in low-resource settings [46]. Unfortunately, syndromic management is a double-edged sword that captures patients requiring treatment but also unnecessarily treats patients that are misdiagnosed [4]. The practice has been associated with increased mortality when patients do not receive proper treatment as a result of misdiagnosis, and overtreatment has also led to the accelerated emergence of drug resistance [47].

The treatment of malaria provides a striking example of the predicament presented by syndromic management. In the past, the WHO has recommended that chloroquine, which is cheap and widely available, should be given to all febrile children in malaria-endemic areas, because death may occur soon after the onset of symptoms [4]. Subsequently, the efficacy of the drug has diminished with the emergence of chloroquine-resistant strains of Plasmodium falciparum, which have spread rapidly and became prevalent in many parts of the world [48]. Artemisinin-combination therapy (ACT) is currently the recommended first-line treatment for P. falciparum malaria [49], and because it is also more expensive, diagnostic tests for malaria have become cost-effective. In light of these events, the WHO changed its recommendation in 2010 so that malaria treatment should only be administered to children with blood-borne malaria parasites [50]. Such a measure not only minimizes unnecessary drug expenditure but is also vital for extending the useful therapeutic lifetime of current antimalarial drugs [4]. ACT was introduced in 1990 to counter the rise in malaria mortality resulting from P. falciparum strains that had acquired resistance to quinoline and antifolate-based compounds. Such strains originated from the Thai–Cambodian border before spreading across the world. Despite the measures taken to manage the prescription of ACT, artemisinin-resistant malaria has emerged along the border between Thailand and Myanmar since 2003, and has subsequently increased substantially [49]. One of the factors that may have led to the emergence of artemisinin resistance is the unregulated use of artemisinin in Western Cambodia, which may have exerted selective pressure for parasites with greater resistance to oxidative stress [51]. When syndromic management becomes the rule rather than the exception, life-saving treatment for misdiagnosed patients is delayed or denied, resulting in increased morbidity and unnecessary loss of life [11].

The emphasis on providing treatment may even have outweighed the need for diagnostics, as reflected in a report in which it was estimated that 12–23% of patients requiring anti-retroviral therapy were receiving it as of 2005 [52], as opposed to appropriate diagnostic testing, which is only accessible to 3–6% of the developing world [41]. Similarly, rapid-impact packages of drugs containing a combination of four of six drugs: albendazole or mebendazole, praziquantel, ivermectin or diethylcarbamazine, and azithromycin can be readily deployed to populations affected by several different parasites in NTD-endemic regions, with the aims of improving quality of health, rapidly reducing morbidity, and disrupting the chain of transmission [2,53]. Despite the benefits that it can bring, the program is threatened by the rise of drug resistance, and the package is not an all-in-one solution because it does not relieve the disease burden of the three NTDs with the highest rates of death: Chagas disease, human African trypanosomiasis (HAT), and visceral leishmaniasis [2]. An understanding of the clinical interventions available, and of the levels of training of the users and healthcare providers at the healthcare access points, would enable diagnostic tests to be tailored to specific targets. Researchers and product developers should carefully evaluate the point in the chain of healthcare at which a diagnostic test should be introduced. If a diagnostic test is to serve the bottom billion people, all the constraints and limitations of remote peripheral facilities, which serve the majority of the population, must be identified and considered at the onset of product development [4].

Researchers should be clear about the targeted clinical application for which the product is being developed, and should identify and consider the various pathogens or human biomarkers that are associated with the progression and severity of the disease. Whilst specific biomarkers may only be detectable within a limited time frame, it is essential that the selection be made with consideration to the time a patient is most likely to consult a physician and the point of intervention as targeted by the diagnostic tool [4]. Dengue infection is an example where detection of viral antigens is most useful in the early stages of the fever, but high expression levels of dengue-specific IgM after 4–5 days of fever may serve as a more sensitive indication of the disease. Therefore, accurate diagnosis of acute dengue infection may be improved if an LFI has a dual antigen and antibody detection function such as that provided by SD Dengue Duo, which detects both NS1 antigen and IgM [54].

LFI allows the use of non-invasive samples such as oral fluid, nasal swab, sputum, and urine, or a simple procedure such as fingerprick or fingerstick. This greatly simplifies the on-site testing protocol and promotes voluntary testing in rural primary healthcare settings by circumventing the need for a trained phlebotomist to obtain blood by venipuncture [55]. A typical lateral flow test only requires a minimally trained operator to add the test sample followed by a buffer, which is usually provided with the strips in a kit format. The result can be read within minutes. However, accurate timing for result interpretation is not very stringent, and prolonging the time may lead to a false-positive result because the absorbent pad, which holds the excess buffer and unreacted signal generator, acts as a reservoir resulting in a backflow once the sample pad becomes dry. The individual test strip is usually sealed in an aluminum pouch with a desiccant to prevent exposure of the test strip to moisture and humidity. LFI kits are usually stored at 2–30°C. However, the thermal stability of the strips at elevated temperatures such as 37°C or 45°C should be evaluated because temperatures may fluctuate during transportation and storage in rural health facilities, and may rise above 30°C. LFI kits that are stable for more than 18 months are recommended, especially in remote areas where the procurement of supplies is unreliable, and wastage due to expiry is also reduced with such kits [56]. The lateral flow format diagnostic test represents an improvement and is cost-effective compared with current practices in the field. It facilitates more widespread evidence-based diagnostic testing without the need for immediate improvements in infrastructure.

POC diagnostics are essential for patient management, especially in cases where the clinical presentation of a disease lacks distinctive features or specificity. In the case of visceral leishmaniasis (VL), a test is needed to confirm or rule-out the diagnosis because the disease is life threatening and clinical symptoms alone are not sufficiently specific. Because the treatment for VL is expensive and toxic, patients should never be treated presumptively for the disease. The rK39 LFI detects human antibodies against a 39-amino acid repeat that is part of a conserved protein in the L. donovani complex. It is a major breakthrough for VL diagnosis in remote areas because the test is easy to perform, generates a rapid visual result (in 10–20 minutes), is cheap (approximately US$ 1 per test), provides reproducible results [57], and has a performance comparable to the DAT [58,59], which is technically more complex to perform and requires 18 hours for visualization of the results. The rK39 LFI has demonstrated excellent diagnostic performance in India but was less accurate in East Africa, indicating the need for a separate diagnostic test for the African population. rK39 provides a simple alternative tool for VL diagnosis, especially in low-resource settings; it should therefore be promoted and used within an appropriate VL diagnostic algorithm [57].

The other disease that would benefit from the development of a simple LFI kit is African trypanosomiasis. In sub-Saharan Africa, this disease affects more than 66 million people and causes an estimated annual death toll of 250,000–300,000 owing to inadequate diagnosis and treatment. Similar to VL, trypanosomiasis is always fatal in the absence of treatment, and the disadvantages of the therapy (high cost, toxicity, and prolonged courses of parenteral treatment) highlight the necessity for a means of diagnosis to confirm infection [18]. A sufficiently sensitive card agglutination test for trypanosomiasis (CATT) is available as a screening tool in areas where Trypanosoma brucei gambiense is endemic [60]. Although converting the format of the CATT to an LFI would increase the accessibility of testing in remote settings, an even more pressing need is that there is no similar test available for T. b. rhodesiense, which also causes trypanosomiasis. The urgent medical requirement for diagnostics to detect trypanosomiasis has attracted the attention of the Foundation for Innovative New Diagnostics (FIND), and efforts are currently ongoing to screen candidate antigens that can be used to detect both T. b. gambiense and T. b. rhodesiense [61]. The ultimate aim would be to develop a specific and sensitive antibody detection LFI in collaboration with Standard Diagnostics from the Republic of Korea [62]. Moreover, PATH has also collaborated with Laboratórios Lemos to develop an LFI for the detection of human antibodies to Trypanosoma cruzi, which causes Chagas disease (American trypanosomiasis). The outcome of the collaboration is a rapid test (20 minutes) with high sensitivity (99.5%) and specificity (98.5%) that may potentially improve POC screening and diagnosis of Chagas disease, especially in low-resource settings [63].

We conducted an online search of PubMed and Scopus databases for the top 13 parasitic and bacterial NTDs using the web-based search engine GOOGLE. Our search revealed that no commercial LFIs were available for more than half of these diseases (Table 1). Although PATH is currently developing an LFI for human African trypanosomiasis (HAT), it is unknown if the remaining six diseases (onchocerciasis, trachoma, ascariasis, trichuriasis, hookworm, and Buruli ulcer) are receiving similar attention. In the case of onchocerciasis, an antibody card test that detects IgG4 antibodies to recombinant Onchocerca volvulus antigen Ov16, which has a reported sensitivity of 90.6%, has been developed by Weil et al. [64], but it is not yet commercially available. A dipstick assay has also been developed for detecting O. volvulus-specific antigens, but there are substantial differences between dipstick and lateral flow assay tests. The O. volvulus antigen detection dipstick assay described by Ayong et al. [65] involves multiple washing and incubation steps followed by enzymatic color development to produce a visual signal for the interpretation of the result. Nevertheless, development of the dipstick represents a step towards the diagnosis of onchocerciasis using non-invasive specimen collection (tears and urine); it remains to be seen whether the format will be converted to an LFI, which would be simpler, more user-friendly, and accessible in remote settings.

In addition to more effective patient management, diagnosis is also important for improving case finding and disease surveillance. TB is a challenging disease to diagnose and treat, particularly when laboratory support is not accessible or available in peripheral health facilities. Because treatment requires a regimen of several drugs for a minimum of 6 months, the development of POC diagnostic as a screening tool to detect active TB, to improve case finding through contact tracing, and to detect latent infection in asymptomatic patients would be of great value to public health [4,47]. Not all infections result in the presentation of clinical symptoms, so asymptomatic individuals who are at risk should be screened, especially if the infection can lead to serious sequelae such as syphilis in pregnant women, which is a major cause of neonatal mortality and congenital syphilis. POC testing in remote areas would be highly beneficial to pregnant women, many of whom travel great distances for antenatal care, which includes screening for STIs and other infectious diseases [47]. Although LFIs for serological diagnosis of HIV and syphilis have been developed and commercialized, POC tests for other STIs such as gonorrhea and chlamydial infections are lacking [66]. More effective case finding leads to early detection and treatment, which prevents morbidity and the development of long-term complications, and reduces the transmission of disease to other community members.

Table 1. List of commercially available lateral flow immunoassays (LFIs) for the top 13 neglected tropical diseases


Barriers and Limitations of POC Testing

The Limitations of Lateral Flow Immunoassay(s)

LFIs are often designed to give qualitative results in the form of a yes/no answer, and when used in conjunction with clinical diagnosis, such POC testing can substantially assist a physician in indicating or ruling out the presence of certain diseases, as described above. However, simple POC quantitative diagnostic tools are also required such as the disease stage test for HIV-positive patients (CD4 count and HIV viral load), which indicates when antiretroviral treatment should be initiated to sustain life [41]. Quantification of the LFI signal, either optical [67] or electrochemical [68], has been reported, but a strip reader or additional instruments are required to generate an electronic readout for the end user, and this in turn significantly increases the complexity of the test as well as the cost per assay [19]. It is worth noting that a standard nitrocellulose membrane can be no thinner than 100 μm [32], and considering that only immunocomplexes formed less than ~10 μm from the surface of a reaction pad are visible to the naked eyes, almost 90% of the signals generated in an assay are obscured by the opacity of the membrane [31]. Interpretation of the LFI result is also dependent on sufficient lighting, which can be an issue if electric lighting is unavailable in a remote area. Good lighting is imperative for the detection of faint bands that could represent true-positive or false-positive “shadow” lines [48,55]. The detection limit of an LFI may be improved by incorporating an enzyme label [67,69], but in the absence of cold storage facilities the limited shelf lives of the enzyme conjugates and substrates often hamper their usage in peripheral facilities [36]. Moreover, the one-step format is usually lost when it is necessary to add a substrate after the assay has been run for a specified time to amplify the signal [19].

If an LFI were insufficiently sensitive and/or specific for POC use, it could be harmful to a patient rather than beneficial, especially if the treatment involved were toxic and associated with adverse side-effects, such as for VL or HAT. Because POC testing is intended to identify and capture putative infected individuals on-site, Barfield et al. [63] have recommended that a screening test should ideally have a sensitivity of greater than 98% without reducing its specificity to below 95%. In the case of malaria diagnosis using LFI, false-positives have been reported due to persistent HRP2 antigenemia after parasite clearance [70], and have been attributed to the presence of rheumatoid factors in some patients [71]. Conversely, antigen excess in association with high parasite densities [72] and possible HRP2 gene deletion have been linked to false-negative results [48]. As with nucleic acid amplification tests (NAATs), serological LFIs cannot distinguish active infections from past infections because the level of serum antibody may remain detectable for several years after the cure [62]. Furthermore, the seropositivity of a population may also be high in endemic regions as a result of previous or asymptomatic infections [57]. Therefore, the use of a rapid diagnostic tool such as an LFI should always be accompanied by clinical case definition when diagnosing an infection [4].

The Lack of Investments and Funding

For diseases such as NTDs, which affect the poorest of the poor, there is no market incentive for commercial companies to invest in low-cost POC diagnostic product development and therapeutics because without the ability to pay for these technologies and treatments, the needs of these underserved people do not translate into demand [15]. Furthermore, NTDs are only regionally endemic; for example, 90% of new cases of VL occur annually in just five countries (India, Bangladesh, Nepal, Sudan, and Brazil) [10], and small markets may not be attractive to investors [18]. The private sector perceives that there will be a low return on investment in developing, manufacturing, and commercializing products when public health institutions and the population they serve lack resources [47,62]. Even when products are made and marketed, there is a formidable challenge in creating a sustainable environment for the continuing manufacture and deployment of new diagnostics in low-resource countries. The development of diagnostics is often undervalued, as reflected in the inequity in financial support and resources received from both the public and private sectors compared with drug discovery and vaccine development [4,11]. According to the Lewin report, expenditure on diagnostics is less than 5% of hospital costs but impacts 60–70% of healthcare decision-making [73]. Therefore, funding organizations should re-evaluate the allocation of resources and put greater emphasis on the development of laboratory diagnostics and supportive infrastructure [11].

The Lack of Rigour in Diagnostic Evaluation

The inherent differences between academic centers and peripheral health facilities may lead to irreproducible results and performance characteristics under disease-control program conditions [42]. Therefore, it is essential to identify the target population and select a sufficient sample size during field evaluation and clinical trials, so that the newly developed diagnostic test may be assessed under actual conditions representing low-resource settings in most countries [42,56]. The results from local trials assist national decision makers in selecting appropriate diagnostic tools by providing substantial information on test performance, reproducibility, ease of use, heat stability, cost, indications for use, shelf life, and the settings in which tests will be used [56]. Evaluation at local settings can be promoted by offering hands-on training, free products for trials, test protocols, and technical assistance [15]. Moreover, research outcomes that offer improvements over currently available diagnostic methods and techniques can be published so that policymakers can determine whether there is a need to implement changes in the technology and procedures being used [42].

The Lack of Regulatory Policy and Budget Allocation

Even when diagnostic products are marketed and made available to health facilities and physicians in the developing world, there is a lack of regulatory control of the design and conduct of diagnostic evaluations, and of the quality of the diagnostics being sold [56]. Lax regulatory standards have allowed low-quality diagnostic tests with no guarantee of adherence to good manufacturing practice for in vitro diagnostics to be sold and used in many developing countries [62]. There have been calls for the implementation of quality standards in the approval of diagnostic tests because low-quality tests that are unreliable and inaccurate do not only jeopardize patient care but also affect the viability of companies producing and selling high-quality tests. The situation also applies to internet services that promote and sell unproven or counterfeit diagnostic tests, which further emphasizes the need for quality standards and regulation to safeguard the effectiveness and quality of diagnostic tests [47].

In the absence of policy and guidelines on selecting diagnostics and their appropriate use in clinical care, physicians may be left to decide which diagnostic test to purchase based on the product insert or published data, which may contain inflated claims of test performance resulting from inadequate or flawed study designs [47,56]. Furthermore, purchase and use of rapid tests by primary healthcare service providers may not be possible if national health budgets for clinical testing are only allocated to laboratories. Health centers deprived of rapid tests will continue to send samples to the laboratory and forgo the advantages of POC for reasons of economy [15]. Above all, procurement of diagnostic tests that do not deliver as claimed will not reduce the disease burden, leading to the mismanagement of patients and unnecessary waste of scarce public resources.

The Lack of Trust or De-emphasis of Laboratory Testing

Introducing new diagnostic tests to developing countries may not be as straightforward as it seems considering how they consistently underperform in low-resource settings, where quality control and quality assessment are not implemented to ensure the accuracy of rapid tests. Successful integration of diagnostic tests into patient management necessitates the education of healthcare providers so that they can correctly perform the tests and interpret the results. Failure to adequately train the end user leads to poor test accuracy, and when physicians lose confidence the resulting poor clinical sensitivity and specificity of the diagnostic test adds to the challenges of an already constrained healthcare system [74].
An historical mistrust of test results, which is further reinforced by presumptive treatment guidelines, has ingrained the perception that diagnostic tests are unreliable and unhelpful to the extent that laboratory testing remains underutilized and undervalued [4, 11]. There have been occasions in Zambia, Uganda, and Ghana when clinical decisions were made without laboratory confirmation, even when the tests were available, and treatments were prescribed to patients despite laboratory results that apparently contradicted the clinical diagnosis [11]. Rollout of easy POC diagnostic tests will not have an impact on patient care if health providers do not respond to test results, as exemplified in a study where 54% of patients with negative malaria test results as diagnosed by LFI were still treated with antimalarial drugs [75]. Awareness of the necessity of diagnostic tests to differentiate between diseases indistinguishable by clinical syndrome, to indicate the need for antimicrobial therapy and to improve patient care must be instilled in healthcare providers at all levels, and other stumbling blocks, either real or perceived, should be address if the implementation of POC tests is to be a success.

Conclusion

The poverty trap, which is reinforced by infectious diseases, confines the poor to a vicious cycle of suffering and despair. The needs of the world’s poorest billion people cannot be addressed by a single approach but require a synergistic effort of intervention from various perspectives. From improving socio-environmental conditions to providing preventive chemotherapy, the importance of ensuring good-quality diagnostic tests that are accessible in peripheral health facilities should not be disregarded or de-emphasized by political will and funding allocation. Lateral flow technology is a promising diagnostic platform that has enabled the capture and detector reagents to be incorporated in an easy-to-use one-step format that avoids both reagent handling and cold chain transportation and storage. Even though introduction of LFI is unlikely to be affected by any immediate educational barriers or infrastructure limitations in remote settings, there is still much room for improvement in test performance, especially in attaining higher sensitivity and specificity. The future of POC diagnostics may lie with lab-on-a-chip technology, which offers accurate and rapid testing for both high-income and low-resource countries. However, LFI remains a highly viable and sensible alternative for achieving POC testing to impact the healthcare of the bottom billion in the developing world. By investing in the health and wellbeing of the people, a nation receives returns in the form of increased labor productivity and higher educational attainment, which eventually contribute to the development of the country.

Acknowledgements

The first and second authors would like to acknowledge the financial support provided by Universiti Sains Malaysia (USM) through the USM Fellowship Scheme and Vice-Chancellor Award, respectively. We are also grateful for eScienceFund 305/PPSP/6113214, Postgraduate Research Grant Schemes 1001/PPSP/8144014 and 1001/PPSP/8144013, and USM Research University Grant 1001/PPSP/813045.

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