REVIEW Virological point-of-care testing for the developing world Marycelin M Baba1, Nina Vidergar2 & Alessandro Marcello*,2 ABSTRACT: The goal of point-of-care testing is to provide fast, convenient, and easy-touse diagnostic assays that shorten the turnaround time of intervention. Several diagnostic tests have already migrated from the centralized laboratory to patients’ bedside, physician oices and domestic environments in more developed countries. However, the situation is dramatically diferent in countries of the developing world where lack of facilities and resources still results in diagnosis to be inferred mostly from the symptoms only. Reliable and rapid diagnosis is urgently needed particularly in case of viral diseases with the concrete risk of outbreaks going undetected in the early stages. In this article we will advocate the necessity to implement robust point-of-care testing for viral diseases to overcome the diagnostic gap of less developed countries. .33 RE ING re K Introducing point-of-care testing Point-of-care testing (POCT) is generally defined as medical testing at or near the site of patient care, but encompasses a large variety of technologies for very different environments, which all have in common a quick turnaround time between specimen collection and diagnosis [1,2] . On one side we find very simple dipstick-based immunochromatographic (ICT) assays for self-testing, such as the rapid HIV test kits that provide a result in less than 30 min from an oral swab [3] . On the other side we find relatively sophisticated and expensive instruments that are used in hospitals for molecular diagnosis that also require trained personnel for their use. Remaining on the HIV topic, confirmatory testing of seroconversion, measurement of viral load and CD4 + T cells’ count all require specific complex laboratory instrumentation. The case of HIV infection is of particular interest because, despite a very efficient specific combination antiretroviral therapy, the virus cannot be eradicated [4] . Poor compliance of treatment leads inevitably to a relapse of viremia and the risk of selecting resistant viruses. Hence, regular monitoring of HIV viral loads during combination antiretroviral therapy is highly recommended and would enormously benefit from quick POCT. Between these two extremes there is a wide range of rapid diagnostic tests that can be performed by minimally trained health workers in the community. The global POCT market is expected to grow steadily in the next years [5] . Manufacturers focus on the development of highly sensitive biosensors, less expensive optical systems, and noninvasive methods to be integrated into POCT devices for better performance. Microfluidic systems based on the lab-on-chip (LOC) concept, along with improved biomarkers, may witness a paradigm shift in the clinical diagnostics industry. But POCT has also the potential to overcome the lack of a widely accessible health-care infrastructure in resource-limited settings. As pointed out in a recent report from the American Academy of Microbiology, “in resource-limited KEYWORDS • developing countries • lab-on-chip • LOC • POC • POCT • point-of-care • viral diagnosis 1 Department of Medical Laboratory Science, College of Medical Sciences, University of Maiduguri, P.M.B. 1069, Borno State, Nigeria Laboratory of Molecular Virology, International Centre for Genetic Engineering & Biotechnology (ICGEB), Padriciano, 99 - 34149 Trieste, Italy *Author for correspondence: marcello@icgeb.org 2 10.2217/FVL.14.33 © 2014 Future Medicine Ltd Future Virol. (2014) 9(6), 595–603 part of ISSN 1746-0794 595 REVIEW Baba, Vidergar & Marcello settings, the impact of diagnostic tests that can be provided at the immediate point-of-care (a POC test) is potentially even greater, because the alternative to a POC testing may be no diagnostic support at all” [6] . However, the development of POCT diagnostics for the developing world should not be a mere translation of a product developed for other markets. Instead, POCT devices need to be designed to perform in adverse local conditions of heat, humidity, dust, and possible lack of infrastructures, running water or electricity being operated by poorly experienced users. Finally, even the best POCT device would be useless if not linked to a program of treatment decisionmaking where the output of the assay is properly managed locally and archived centrally. In this review we will focus on the challenges of translating POCT technology for viral disease to resource-limited settings. To this end we will take into consideration the most recent technology advances for virological testing and consider the needs of a tertiary care health institution in central Africa in order to provide a roadmap of the actions to foster this challenging task. Technologies of POCT The ideal POCT has to adhere to a consensus of features that go under the acronym ASSURED: affordable, sensitive, specific, user friendly, rapid and robust, equipment free (or minimal) and deliverable to end users (WHO sexually transmitted diseases diagnostics initiative) [7] . Affordable is a generic concept that depends only in part from the cost of producing and marketing the device, which varies a lot with the embedded technology applied and the scale of production. Also relevant is the overall strategy of health policy makers for funds allocation between centralized laboratories or POCT technology to cover large areas of their countries. Sensitivity and specificity should be compared with their laboratory counterparts (i.e., ELISA for immunoassays and PCR for molecular assays) to establish a suitable tradeoff for the increased numbers of patients being tested. POCT devices should be simple enough to be used by untrained users. Certainly, a striptest is a very simple technology that everyone can apply and interpret. However, nowadays smartphone technology is so widespread that devices operated in the same way are at the ease of most of the world’s population. One note of caution though, the ability to operate the device is only one aspect of the problem since interpretation and subsequent action should be in the hand of 596 Future Virol. (2014) 9(6) qualified healthcare workers that may decide to refer the patient to a doctor for intervention. The device should give a quick response (i.e., in minutes) and should be in a rugged format to resist in a variety of extreme environments. All phases of the procedure should be incorporated in a single device: sample extraction (i.e., oral swab, urine and blood) processing (i.e., extraction of nucleic acids), testing (i.e., ICT or LOC/POCT) and response (i.e., colorimetric and monitor display). Finally, the device should be deliverable to end users. This final concept includes most of the recommendations above and is strictly dependent on the needs of those who will be using it. A bottom–up approach is therefore highly recommended to drive the development of POCT devices for resource-limited settings. Lateral flow ICT devices for detection of antibody or antigen currently dominate POCT [8] . These generally function like the pregnancy test with the sample (urine, blood and saliva) diffusing by capillarity along a strip of porous material. When the analyte (protein, antibody) reaches the immobilized antigen/antibody, a colorimetric reaction develops allowing the visual detection of the signal. A positive control of test efficacy is also included. The response of a strip test is usually purely qualitative (yes/no), although it is possible to measure the intensity of the test line to determine the quantity of analyte in the sample. These kinds of test are also monoparametric, in that they measure only one feature at a time. Finally, recording of results is mostly not an automated process requiring visual inspection and manual annotation. There are several ICT assays for the major viral diseases available on the market with a variety of manufacturers. One great advantage of these tests is that they can be easily produced at a very low cost affordable to many developing countries. However, a major hurdle is the high heterogeneity and poor standardization of strip tests making it very difficult to compare between different studies [9] . In the future, however, there will be an increasing need to develop cost-effective POCT devices that address biomarkers, which may be well established in laboratory settings but are not currently amenable to point-of-care, such as molecular and/or multiparametric quantitative tests. The LOC concept integrates one or several laboratory functions on a single chip of only millimeters to a few square centimeters in size. LOC applied to POCT would deliver a complete package of diagnostic tests in a hand-held miniaturized format future science group Virological point-of-care testing for the developing world for easy handling and interpretation/storage of output. Some of the advantages of a LOC/POCT include: the handling of extremely small fluid volumes (less reagents, lower costs of reagents, less waste and less sample volumes required for diagnostics); faster analysis and response times; and lower fabrication costs from mass production. Furthermore, integration of LOC/POCT with mobile smart phones, which are ubiquitously available and contain most of the components required for a biodetector (i.e., battery, display and processor), would allow availability and accessibility of diagnostic testing also in developing countries exploiting a friendly technology [10] . However, like for most innovative technologies that are not yet fully developed, several hurdles still need to be overcome. This is a fast developing field where a number of solutions are tested but only a natural process of selection will tell which technology will be the more robust and cost effective for a specific LOC/POCT application. Integration of the different fields of immunology, molecular biology, chemistry, electronic engineering, physics and material sciences is required in a multidisciplinary approach for a successful outcome. Perhaps the most widely used laboratory immunoassay technology is the ELISA, which represents the standard for comparison against all newly developed methods. The ELISA takes advantage of the high affinity of the antibody– antigen interaction and involves the immobilization of the antibody–antigen complex onto a solid surface in a 96-wells format with enzymes conjugated to antibodies being used to develop a colorimetric reaction. Critical for the development of ELISA-based LOC/POCT is the detection of the signal since the other steps of the reaction are easily down scalable. However, detection of the antibody–antigen binding cannot be obtained by the classical colorimetric reaction therefore requiring alternative technologies. Electrochemical and optical immunosensors are being developed for this purpose [11] . A very promising cost-effective optical technology for the development of LOC/POCT is based on organic light-emitting diodes (OLED): flat, few millimeters thick surfaces that can be designed to emit light in different wavelengths [12–17] . The molecular architecture and emission spectrum of the OLED can be optimized to match the excitation spectrum of a fluorophore used in the detection reaction. The signal is captured by a detector and processed in real time future science group REVIEW allowing a wide range of applications. Recently, an immunoassay has been developed based on a bottom-emission small molecule-based OLED optimized to obtain the deep-blue wavelength that is required to excite a succimide-based dye conjugated to an antibody [18] . This configuration is currently being tested for the detection of immunoglobulins in sera from patients infected with Flaviviruses such as Dengue virus, tick-borne encephalitis virus and west Nile virus (WNV) [Marcello A et al., Unpublished Data] . Molecular techniques for the detection of viral pathogens aim at the detection of the viral nucleic acid, which can be RNA or DNA, circulating in the patient (viral load). Two levels of output should be considered: the presence of the pathogen/nucleic acid (qualitative) and the amount of pathogen/nucleic acid (quantitative). The latter is of paramount importance, for example, in the HIV field because, together with other critical parameters such as the CD4 + T-cell counts and clinical symptoms, determines the progression towards AIDS. Direct detection by nucleic acid hybridization can be achieved but levels of circulating viruses may be low therefore usually requiring an amplification step to gain in sensitivity. Nucleic acid amplification may be obtained by isothermal amplification or by PCR, which requires several thermal cycles of amplification/denaturation/annealing. While the latter is a challenge for engineers due to the large delta of temperatures required for the reaction, it still remains the most robust technique for viral nucleic acid detection while awaiting further development of the former [19] . Indeed, preliminary studies have shown that in principle it is possible to detect a viral nucleic acid by acquiring in real-time a fluorescent signal from PCR conducted directly on an emitting light surface [Marcello A et al., Unpublished Data] [20] . Paradoxically however, a field-tested portable PCR system that summarizes all the features of a LOC/POCT for developing countries has been originally conceived for military use. The RAZOR EX BioDetection System (RAZOR; BioFire Diagnostics, UT, USA [formerly Idaho Technologies, recently acquired by BioMerieux]) employs real-time PCR technology to identify biological pathogens taking advantage of system-integrated freeze-dried reagents packaged in durable plastic pouches. RAZOR has been designed specifically for field use with compact and lightweight design (size: 25.4 × 11.4 × 19 cm; weight: 4.9 kg including batteries). The www.futuremedicine.com 597 REVIEW Baba, Vidergar & Marcello device can simultaneously test up to ten pathogens with results available in less than 30 min. Originally developed for first responders and front-line military troops, it is easily operated while working in protective equipment under extreme conditions. Further developments in the direction of better integrating DNA/RNA extraction, PCR and real-time detection in a single easy-to-use instrument are underway. POCT for viral diseases The importance of virological POCT for industrialized countries is restricted to conditions that require a rapid turnaround time since most routine testing is performed in well-distributed diagnostic laboratories serving the community. For example, results from conventional HIV serology take between 30 min to a few hours or even days in a busy laboratory while POCT takes as little as 10 min. This turnaround time can be critical since it has been estimated that 30% of HIV-positive individuals and 39% of HIVnegative individuals testing at publicly funded counseling/testing/referral programs do not return for their test results [CDC, Unpublished Data] . Therefore, these individuals cannot receive counseling about medical treatment and behavioral risks. Use of a POCT with immediate results substantially increases the number of people who know their HIV status and receive information about next steps. Aside from HIV infections, respiratory infections can lead to severe complications if viruses such as the respiratory syncytial virus or influenza virus are involved. Rapid immunoassays for these pathogens can be run in an emergency department or clinic in 15 min. Since flu-like symptoms are also typical of many other endemic diseases, quick and accurate diagnosis is important to direct the optimal treatment and to avoid inappropriate antibiotic or antimalaria prescription [21] . The same concept applies to the detection of enterovirus RNA in cerebrospinal fluid for the differential diagnosis of meningitis [22] . The importance of rapid POCT in emerging infectious disease is also reinforced by the recent outbreak of several new pathogens, including West Nile virus in the Americas and the Coronavirus SARS (severe acquired respiratory syndrome outbreak 2002–2003) and MERS (Middle East respiratory syndrome outbreak 2012–2013). The quick spread of SARS from Asia to North America already demonstrated how critical rapid on-thespot diagnosis and treatment decisions could be 598 Future Virol. (2014) 9(6) with regards to disease containment efforts highlighting the importance of being prepared for rapid action to counter the spread of previously unknown agents or known infectious agents that are re-emerging or used as bioweapons. The spectrum of viral infections that could benefit from POCT in the developing world is much wider. Lack of resources and widespread healthcare facilities make immense areas of the world completely uncovered from viral detection. This has severe implications at various levels. First, quick response to viral outbreaks would limit the number of cases of life-threatening diseases such as hemorrhagic fevers from Ebola virus and Lassa virus. Second, a better diagnosis of the etiological agent responsible for generic symptoms like fever or diarrhea would limit the misuse of improper treatments. Third, the more an emerging or re-emerging viral infection remains undetected the less it is likely to be contained avoiding a worldwide pandemic. Hence, the benefit of POCT testing in the developing world goes well beyond the interests of the people directly involved at the outbreak site. POCT for major diseases are available in ICT format for the detection of reactive immunoglobulins in patients’ sera (IgM/IgG). However, a combination of tests is generally required for a complete diagnosis of a single disease, as well as for the coverage of all the possible/most probable causes of a symptomatic state. This is why multiparametric LOC/POCT tests represent the ultimate frontier of delocalized viral diagnostics. But what would be the kind of test that is required? This would depend from the pathogen and the various phases of the disease. A paradigmatic example is Dengue virus (DENV). With more than a third of the world’s population living in areas at risk of infection, DENV is a leading cause of illness and death in the tropics and subtropics [23] . As many as 400 million people are infected yearly with symptoms of increased morbidity: Dengue fever, dengue hemorrhagic fever and Dengue shock syndrome. DENV belongs to the Flavivirus genus of the family Flaviviridae that include also hepatitis C virus (genus: Hepacivirus). Members of the Flavivirus are arthropod-borne viruses (Arboviruses) that include other important human pathogens such as yellow fever virus (YFV), WNV and tick-borne encephalitis virus. There are four distinct serotypes of DENV1–4, which all cause clinical disease. Understanding the dynamics of DENV infection is critical because it has future science group Virological point-of-care testing for the developing world a large impact on the interpretation of diagnostic results. Following the initial infection, DENV replicates to high titers in the blood before patients are unwell enough to present to a physician, with viremia peaking at the time or shortly after the onset of symptoms. During the viremic phase of dengue infection a soluble form of the nonstructural viral protein NS1 circulates in the serum of patients and hence is an excellent diagnostic target for acute Dengue together with the detection of viral RNA and DENV isolation. Dengue IgM antibody is a reliable marker of recent infection but not necessarily acute infection. In primary DENV infections, IgM antibodies develop following the decline of viremia between days 3–5 after the onset of infection and persist for approximately 6 months. However, amplitude and duration of the IgM during secondary infections, that are most common among individuals with previous Flavivirus infections or immunizations against these viruses, are smaller and shorter. The IgG antibody response, which develops a few days after the onset of the IgM antibody response, is serotype specific and may persist for many years following a single infection. During secondary infections the IgG response is before the IgM response or simultaneous. Several ICT tests are available for IgM/IgG detection and a great effort has been put in the improvement of their specificity and sensitivity [24] . However, the most important development in Dengue diagnostics in recent years has been the specific detection of the DENV NS1 antigen. ICT for NS1 and IgM/IgG allows the rapid differential determination of acute versus recent or past DENV infection thus greatly improving diagnosis. ICT tests for DENV suffer from the same problems of their ELISA counterparts for specificity and sensitivity. Flaviviruses are antigenically related and all immuno-based tests show a certain degree of crossreactivity particularly in areas where different members of the genus and/or family are co-existing [25] . Furthermore, differential diagnosis of the four serotypes of DENV would be also important particularly in areas experiencing a shift from one serotype to another. The phenomenon of antibody-dependent enhancement occurs when non-neutralizing antibodies from a previous infection of a particular serotype facilitate entry of the second DENV of a different serotype into the host cells, leading to increased infectivity [26] . In such cases, the clinical course of the disease is more severe future science group REVIEW with higher viremia and increased risk of dengue hemorrhagic fever/Dengue shock syndrome. The golden standard for differentiating viral serotypes is the plaque-reduction neutralization test that measures viral infectivity in the presence of patients’ neutralizing sera [27] . However, its implementation is cumbersome, may last several days (6–10 days depending on the serotype) and requires expert virologists in a containment laboratory. Semiautomated methods are being developed to shorten plaque-reduction neutralization test and making it less operator sensitive but their implementation to POCT appears less likely in the short term [28] . In summary, for Dengue it would require merging in a single test IgM/IgG detection with NS1 or viral RNA. Multiplexing these assays together with their counterparts for a panel of arboviruses that could be responsible for malaria/typhoid-negative febrile symptoms could represent a valid tool in endemic areas as described below. Challenges & solutions for POCT in resources-limited settings Nigeria is the most populous country in Africa with a population exceeding 170 million. The burden of viral diseases in the country is enormous with an estimated 3.3 million living with HIV/AIDS. Few data are available on other viral diseases that could date back several decades with only few more recent systematic reports. Dengue virus was first isolated in the sixties [29,30] and presently seems the leading arbovirus in the country, followed by Yellow fever virus, Chikungunya and West Nile virus [31,32] . Another example is a recent measles outbreak, which caused 36,428 cases and 198 deaths (reported by Medecins Sans Frontiers in 2013) [33] . In the case of rabies, an estimated 10,000 people, mostly children, are exposed to dog bites each year but the burden of the disease is largely underestimated, as it is for most diseases that get underdiagnosed and underreported due to lack of diagnostic facilities and trained personnel. Finally, Nigeria is also one of the few polio-endemic countries left worldwide, with 53 confirmed poliomyelitis cases in 2013, notwithstanding the global polio eradication vaccine initiative promoted by the WHO (Global Polio Eradication Initiative 2014) [34] . The University of Maiduguri Teaching Hospital (UMTH, Maiduguri, Nigeria) is a tertiary health institution that serves as a referral health center for six states in north eastern Nigeria (Borno, Taraba, Adamawa, Yobe, Gombe www.futuremedicine.com 599 REVIEW Baba, Vidergar & Marcello and Bauchi), and neighboring African countries (Chad to the North East, Niger to the North and Cameroon to the East). The institution also hosts one of the two WHO National Polio Laboratories that is part of the polio eradication program [35] . A limited number of viral diseases are being diagnosed at UMTH. Serological procedures such as ELISA or rapid strip tests are used together with virus isolation in cell culture and neutralization assays. Recently, the introduction of real-time PCR allowed molecular detection to take place. However, the high cost of the equipment and reagents, as well as the need for skilled/qualified operators heavily limits its use. UMTH is a paradigmatic example of the challenges of viral diagnosis in Africa. Malaria, for example is usually ascribed to all febrile illnesses unless confirmed through laboratory testing. Febrile patients regularly exhibit symptoms, which are generally believed to be caused by malaria or typhoid, but that are also commonly observed with arbovirus infections. It is a common practice for patients with fever to visit the healthcare facility only if fever persists after self-medication with two or three different antimalaria treatments. At this stage it becomes very difficult to isolate or detect the virus by reverse transcriptase PCR. The use of serology in such cases would be appropriate, but high levels of crossreactivity among these viruses make their differentiation equally difficult. In a pilot study, a total of 310 blood samples were collected from patients exhibiting febrile illness who visited the UMTH. Microscopy screening for the malaria parasite, Widal agglutination reaction for Salmonella Typhi infection and neutralization assays for WNV, YFV, DENV 1–4 and CHKV were used [36] . The results were stunning: of the 310 febrile sera samples tested here, 285 (92%) tested positive for malaria, typhoid and arbovirus infection, or a combination of one or more of these viruses (Figure 1) . Interestingly, 25 (8%) of the 310 patient sera tested were negative for all six pathogens suggesting that other pathogens may also be circulating, possibly including arboviruses such as O’nyong nyong, Bwamba, Tataguine, Thogoto, Wesselsbron, CrimenanCongo hemorrhagic fever, Zika, Lassa or Dugbe [37–40] . Despite the enormous hurdles for viral diagnosis in Africa, there are examples where POCT is being introduced. Recent approaches propose the use of a portable isothermal reverse transcription recombinase polymerase amplification (RT-RPA) 600 Future Virol. (2014) 9(6) assay for the detection of viral RNA [41] . This rapid (15 min) and sensitive technology entails the amplification of cDNA at temperatures of 25–42°C and fluorescence detection of the signal in real time in the ESEQuant tubescanner (Qiagen, Hilden, Germany). This device is portable (weights ∼1 kg) and can be powered by a car adaptor or by batteries rechargeable by solar panels, making it very suitable for resource-limited settings. A mobile RT-RPA unit was set up during a foot-and-mouth disease virus outbreak in Egypt in 2012, allowing for the first successful field test performed directly at the quarantine stations. The control of infected livestock is of paramount importance for the management of FMD outbreaks that cause huge economic losses. However, the technology applies also for human disease as it has been demonstrated for the MERS-CoV and YFV [42,43] . The latter study was conducted in Senegal and included also steps towards the integration of the RT-RPA into a semiautomated LOC/POCT system using a microfluidic cartridge and a small and portable processing device that could be used in the field with minimal manual interaction. Conclusion The number of POCT devices available to detect viral infections is steadily increasing. Although the majority of them worldwide are ICT based, more complex LOC/POCT tests are also being proposed. However, the capillary availability of centralized laboratories in more advanced countries limits the development of POCT for viral infections because of their lower levels of sensitivity and specificity that cannot compete with the high standards of laboratory-based tests. On the other hand, in resource-limited settings, where laboratory testing is lacking or underperforming, the POCT device could represent a convenient trade-off. This opportunity cannot be missed to reduce the burden of disease in those countries where it is more urgent. In order to accelerate the process from the initial design of a POCT to its implementation in developing countries, transversal partnerships should be formed between academic researchers, international organizations and healthcare institutions. Only by involving all the relevant stakeholders we shall move forward in the diagnostic of infectious disease in resourcelimited countries. It is envisaged that availability of appropriate diagnostic tests will not only improve virus surveillance but also reduce costs of inappropriate treatment. Furthermore, they future science group Virological point-of-care testing for the developing world REVIEW DENV1-4 95 WNV 11 11 31 59 0 0 0 1 3 15 CHIKV 25 DENV1-4 + YFV = 0 6 YFV WNV + CHIKV = 15 Figure 1. Seroprevalence of arbovirus infection at the University of Maiduguri Teaching Hospital (Maiduguri, Nigeria). In total, 310 human sera were tested for DENV1–4, WNV, YFV, CHIKV, Plasmodium falciparum and Salmonella Typhi. A total of 272 tested positive for one or more arbovirus while two tested positive for malaria alone, 11 for S. Typhi alone, none for both and 25 were negative for all pathogens tested. CHIKV: Chikungunya; DENV: Dengue virus; WNV: West Nile virus; YFV: Yellow fever virus. Data taken from [36]. will reduce under-reporting and overdiagnosis of these diseases (i.e., malaria in febrile illness) in endemic countries and consequently decrease the potential of uncontrolled spread of enzootic viruses that have recently been rising serious public health concerns. Thinking that this not a problem for developed countries is a dangerous underestimation of the risk for future pandemics. When it comes to infectious diseases no country is an island and everyone would benefit from a better monitoring of the periphery of the world. Future perspective Development of innovative and affordable portable diagnostic assays for developing countries is the most promising way to reduce the turnaround future science group time between disease manifestation and treatment, thus minimizing disease transmission and impact. In the future, we will experience the reinforcement of ICT-based technologies and a technological shift to LOC/POCT multiparametric quantitative interconnected devices. Therefore, strategies should be put in place to implement ICT-based testing for the detection of acute/recent viral infections, standardize ICTbased testing with international guidelines and develop novel LOC/POCT devices that may represent the future of diagnosis in resource-limited settings. Ultimately, LOC/POCT devices should be able to perform a multiparametric analysis, combining both serology and molecular detection of www.futuremedicine.com 601 REVIEW Baba, Vidergar & Marcello viral nucleic acids and/or proteins. These can be directed towards a single pathogen (i.e., Dengue virus) or towards a panel of pathogens with similar symptoms (i.e., respiratory disease and febrile illness). Acknowledgements The authors would like to thank G Corazza for reading the manuscript. Financial & competing interests disclosure M Baba was funded by an ICGEB short-term fellowship programme grant (F/NIG08-5) and a Volkswagen Stiftung ‘Knowledge for tomorrow: Cooperative Research in SubSaharan Africa’ travel grant (24112008). Virological testing at UMTH has been implemented thanks to collaboration with ICGEB, the Bernhard-Nocht-Institute for Tropical Medicine (S Günther) and the ‘Burlo Garofolo’ Hospital in Trieste, Italy (P D’Agaro). N Vidergar is financially supported by OREL.doo. A Marcello is a consultant of OREL.doo for the development of OLED-based devices. His work on LOC/POCT has been funded by: the National R&D project ‘DIA OLED’ financed by Regione Friuli Venezia Giulia of Italy (art. 12, DM 593/2000), in collaboration with Plast-Optica Spa, Eurotech Spa, Euroclone Spa, Alphagenics Srl and University of Trieste; the International R&D Project ‘OLED CHIP’ financed by FP7 ER A NET in collaboration with OREL.doo (Slovenija), Cosylab (Slovenija), Laplace-CNRS (France), LED Engineering (France); the POR-FESR Project ‘FLAVIPOC’ financed by Regione Friuli Venezia Giulia of Italy in collaboration with Euroclone Spa, University of Trieste, Hospital Burlo Garofolo and Cluster of Biomedicine. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. EXECUTIVE SUMMARY Point-of-care testing for viral infections encompass a variety of techniques ● Immunochromatographic-based assays are simple, cheap and dominate the ield but are monoparametric and mostly not quantitative. ● Lab-on-chip (LOC)/point-of-care testing (POCT) are still in the development phase but represent the future of multiparametric quantitative analysis. POCT is the most feasible solution to overcome the diagnostic gap of resource-limited countries ● Lack of a capillary distribution of diagnostic laboratories does not allow early diagnosis and viral outbreak detection in resource-limited countries. ● The implementation of portable diagnostic devices that can be operated with minimal knowledge by healthcare workers represent a valid alternative to the construction of permanent infrastructures. ● There is a window of opportunity to develop POCT speciically for the need of the less developed countries. ● POCT for resource-limited settings should carefully take into account the necessities of the end user in a bottom–up collaborative approach. Future perspective ● ICT-based assays need to be reinforced and standardized. ● LOC/POCT assays for viral infections should be further implemented. ● Immunological and molecular techniques may be combined to provide LOC/POCT formats for the detection of multiple pathogens or multiple parameters of a single infection to guide intervention. References 2 Peeling RW, Mabey D. Point-of-care tests for diagnosing infections in the developing world. Clin. Microbiol. Infect. 16, 1062–1069 (2010). 3 Piwowar-Manning EM, Tustin NB, Sikateyo P et al. Validation of rapid HIV antibody Papers of special note have been highlighted as: •฀of฀interest;฀••฀of฀considerable฀interest. 1 602 Yager P, Domingo GJ, Gerdes J. Point-of-care diagnostics for global health. Annu. Rev. Biomed. Eng. 10, 107–144 (2008). Future Virol. (2014) 9(6) tests in 5 African countries. J. Int. Assoc. Physicians AIDS Care (Chic.) 9, 170–172 (2010). 4 C Van Lint, Bouchat S, Marcello A. HIV-1 transcription and latency: an update. 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