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Siguemoto",slug:"erica-siguemoto"},{id:"89969",title:"M.Sc.",name:"Mariana",middleName:null,surname:"Sefora Sousa",fullName:"Mariana Sefora Sousa",slug:"mariana-sefora-sousa"}]},{id:"29165",title:"Content of Total Polyphenols in Biodynamic Organic Acid Lime (Citrus latifolia Tanaka) Clarified by Microfiltration",slug:"content-of-total-polyphenols-in-biodynamic-organic-acid-lime-citrus-latifolia-tanaka-clarified-by-mi",signatures:"Daniela Soares Viana, Lucia Maria Jaeger de Carvalho, Ediane Maria Gomes Ribeiro and Gisela Maria Dellamora Ortiz",authors:[{id:"97047",title:"Prof.",name:"Lucia Maria Jaeger",middleName:null,surname:"De Carvalho",fullName:"Lucia Maria Jaeger De Carvalho",slug:"lucia-maria-jaeger-de-carvalho"},{id:"97940",title:"MSc.",name:"Daniela Soares",middleName:null,surname:"Viana",fullName:"Daniela Soares Viana",slug:"daniela-soares-viana"},{id:"97942",title:"MSc.",name:"Ediane Maria Gomes",middleName:null,surname:"Ribeiro",fullName:"Ediane Maria Gomes Ribeiro",slug:"ediane-maria-gomes-ribeiro"},{id:"97955",title:"Dr.",name:"Gisela Maria",middleName:null,surname:"Dellamora Ortiz",fullName:"Gisela Maria Dellamora Ortiz",slug:"gisela-maria-dellamora-ortiz"}]},{id:"29166",title:"Advanced Oxidation Processes in Food Industry Wastewater Treatment – A Review",slug:"advanced-oxidation-processes-in-food-industry-wastewater-treatment-a-review",signatures:"Anne Heponiemi and Ulla Lassi",authors:[{id:"94998",title:"MSc.",name:"Anne",middleName:null,surname:"Heponiemi",fullName:"Anne Heponiemi",slug:"anne-heponiemi"},{id:"95000",title:"Prof.",name:"Ulla",middleName:null,surname:"Lassi",fullName:"Ulla Lassi",slug:"ulla-lassi"}]},{id:"29167",title:"X-Ray Microtomography for Food Quality Analysis",slug:"x-ray-microtomography-for-food-quality-analysis",signatures:"Janine Laverse, Pierangelo Frisullo, Amalia Conte and Matteo Alessandro Del Nobile",authors:[{id:"90784",title:"Prof.",name:"Matteo",middleName:null,surname:"Del Nobile",fullName:"Matteo Del Nobile",slug:"matteo-del-nobile"}]},{id:"29168",title:"Corrosion in the Food Industry and Its Control",slug:"corrosion-in-the-food-industry-and-its-control",signatures:"Benjamín Valdez Salas, Michael Schorr Wiener, Margarita Stoytcheva, Roumen Zlatev and Monica Carrillo Beltran",authors:[{id:"65522",title:"Dr.",name:"Benjamin",middleName:null,surname:"Valdez",fullName:"Benjamin Valdez",slug:"benjamin-valdez"}]},{id:"29169",title:"Computer-Based On-Line Assessment of Sterilizing Value and Heat Distribution in Retort for Canning Process",slug:"computer-based-on-line-assessment-of-sterilizing-value-and-heat-distribution-in-retort-for-canning-p",signatures:"Montip Chamchong, Vilasinee Sangsom and Nuttakorn Charoeamkitti",authors:[{id:"100940",title:"Dr.",name:"Montip",middleName:null,surname:"Chamchong",fullName:"Montip Chamchong",slug:"montip-chamchong"}]},{id:"29170",title:"Ice-Temperature Storage Technology of Fruits and Vegetables",slug:"ice-temperature-storage-technology-of-fruits-and-vegetables",signatures:"Liu Bin, Cai Bihao and Shen Jiang",authors:[{id:"103474",title:"Dr.",name:"Liu",middleName:null,surname:"Bin",fullName:"Liu Bin",slug:"liu-bin"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"75673",title:"Point-of-Care Strategies Applied to Malaria Diagnosis",doi:"10.5772/intechopen.96721",slug:"point-of-care-strategies-applied-to-malaria-diagnosis",body:'Malaria is one of the deadliest diseases of poverty. It is estimated that malaria causes 228 million illnesses and 405 thousands deaths each year. Among the sick, children aged under 5 years are the most vulnerable group affected by malaria; in 2018, they accounted for 67% (272 000) of all malaria deaths worldwide [1].
In many countries where malaria is endemic, a lack of access to adequate diagnostic services leads to poor health outcomes for fever patients, as well as poor surveillance of infections and outbreaks, and treatment monitoring [2].
To make matters worse, the appearance of antimalarial resistant parasites including artemisinin derivatives pose a major public health threat [3]. In addition, drugs such as the artemisinin-derivatives are more expensive, leading to an increased demand for patient evaluation by accurate diagnostic tests before treatment [4, 5, 6].
Therefore, it has grown in the last years a general agreement that new diagnostic tests are needed for remote areas in malaria-endemic countries. However, the new tests must show improved performance over existing techniques, so that adequate distribution of anti-malarial drugs can effectively target the disease and its outbreaks, contributing to the reduction of generation of drug-resistant parasite strains [7].
In malaria-endemic countries, the major hurdle for widespread access to malaria diagnostics is the limited health care infrastructure [8, 9]. According to the World Health Organization (WHO) guidelines, the useful diagnostic tool at the point of care (POC) is defined by some characteristics. It should be low cost, deliver sensitive and accurate results in as little time as possible, run on a portable instrument (ideally, should be instrument-free), require minimal external power, require minimal training before use, and not require refrigerated reagent storage and transportation. These guidelines are collectively known by the acronym ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users) [10]. In addition to those requirements, an ideal POC malaria diagnostic device should determine which species is infecting the patient, to establish the level of parasitemia, and be able to detect mixed or low-level infections.
Current POC tests for malaria include the smear microscopy and immunochromatographic rapid tests (RDTs). However, more sensitive and specific techniques based on nucleic acid amplification tests (NAAT) have been praised as the best choice for a successful malaria POC diagnostic test. In this work, we will review the status of the diagnostic technologies that have been used for malaria detection at POC conditions, discussing their main advantages and disadvantages in the POC context.
Microscopy remains the gold standard for malaria diagnosis in most endemic areas. This technique allows the identification of different malaria-causing parasites (
There are two variations of the microscopy technique, the thick drop and the blood smear, both use the Giemsa dye in their preparations and are performed with sample of peripheral blood. Thick droop is made by placing a few drops of blood on a glass slide, allowing the blood to dry, and then lysing the blood (usually with water) before staining. The blood smear is made from a thin layer of cells and are fixed with methanol before reading. The thick drop allows the identification of lower parasitemias, by concentrating the parasites. The blood smear technique is more sensitive in speciating the parasites, however it does not allow the identification of low parasitemias [12].
This technique has the great advantage of being cheap (costs approximately $ 0.20 per sample), fast (approximately 1 hour between collection and the result, if performed by a skilled laboratory technicians), and does not need sophisticated equipment. The number of patients tested by microscopic examination increased an increase of 165 million tests in 2010 [13] to more than 208 million testes in 20171. The global total is dominated by India. The sensitivity of the optical microscopy technique using the thick drop method is50–500 parasites/μL, however, many factors may interfere with the results found in the thick drop technique, such as the quality of the microscope, the quality of the available staining reagents, and the skill of the technician. Several studies have shown that the sensitivity of the microscopy technique may be lower in several areas of transmission depending on the quality of the examination and the expertise of the microscopists, which can increase the number of false negative results [14, 15].
Rapid diagnostic test (RDT) is a quick diagnostic approach to detect malaria among malaria-suspected patients and rule out malaria among individuals without malaria. RDTs detect parasite-specific antigens in a drop of fresh blood through lateral flow immunochromatography using antibodies to detect one or several antigens [16] (Figure 1).
Lateral flow assay architecture. Samples containing the analyte flow through the nitrocellulose membrane by capillary flow (panel A), carrying reporter antibodies (labeled with gold, latex or a fluorophore) until the mixture interacts with the test line (containing antibodies that bind the analyte of interest) and the control line (containing anti-IgG antibodies that bind to human IgG molecules)(panel B). If the control line shows a positive reaction, it is a valid test. If the test line shows a positive reaction, it is a positive sample for the specific analyte (panel C).
The RDTs detect a single species (either
The RDT test is highly sensitive and specific, it is easy to perform, simple to interpret and the results can be read in 15–30 min. These make it suitable for community-level health facilitates in rural areas and other endemic situations where equipment and professional microscopists are not accessible. However, they have a limited shelf life, and need to be kept dry and away from extremes of temperature. They may also fail to detect malaria in cases where there are low levels of parasites in the blood, and false positives are possible due to cross reactions or gametocytemia (infection with the sexual stage of the parasite only).
A brief comparison of WHO pre-qualified RDTs is presented in Table 2. WHO, in collaboration with Special Programme for Research and Training in Tropical Diseases (TDR), FIND, the United States Centers for Disease Control and Prevention (CDC) and other partners, established a protocol to measure the quality of malaria RDTs that are designed to diagnose
Product name | Manufacturer | Panel detection score | False positive rates (%) | Meets WHO performance criteria? | ||||||
---|---|---|---|---|---|---|---|---|---|---|
200 parasites/μL | 2000 parasites/μL | 200 parasites/μL | 2000 parasites/μL | |||||||
Pf | Pv | Pf | Pv | Pf | Pv | Pf | Pv | |||
non-Pf infection | Pf- infection | non-Pf infection | Pf- infection | |||||||
Paracheck Pf - Rapid Test for | Orchid Biomedical Systems – Tulip Diagnostics (P) Ltd | 94.0 | NA | 100.0 | NA | NA | 1.4 | NA | 4.3 | Yes |
One Step test for Malaria Pf/Pan Ag MERISCREEN Malaria Pf/Pan Ag | Meril Diagnostics Pvt. Ltd | 73.0 | NA | 99.0 | NA | NA | 0.7 | NA | 0.0 | No |
Parascreen - Rapid test for Malaria Pan/Pf | Zephyr Biomedicals – Tulip Diagnostics (P) Ltd | 91.0 | 94.3 | 100.0 | 97.1 | 0.0 | 0.7 | 0.0 | 1.4 | Yes |
FalciVax - Rapid test for Malaria Pv/Pf | Zephyr Biomedicals – Tulip Diagnostics (P) Ltd. | 95.0 | 100.0 | 100.0 | 100.0 | 0.8 | 0.0 | 0.0 | 0.0 | Yes |
STANDARD Q Malaria Pf/Pv Ag Tes | SD Biosensor, Inc | 85.0 | 100.0 | 99.0 | 100.0 | 0.5 | 0.0 | 0.5 | 0.0 | Yes |
STANDARD Q Malaria Pf Pan Ag Test | SD Biosensor, Inc. | 88.0 | 100.0 | 99.0 | 100.0 | 0.0 | 0.0 | 0.5 | 0.0 | Yes |
STANDARD Q Malaria Pf Ag Test | SD Biosensor, Inc. | 87.0 | NA | 99.0 | NA | NA | 0.0 | NA | 0.0 | Yes |
First Response Malaria Antigen | Premier Medical Corporation Limited | 95.0 | NA | 100.0 | NA | NA | 0.7 | NA | 0.0 | Yes |
First Response Malaria Ag. pLDH/HRP2 Combo Card Test | Premier Medical Corporation Limited | 85.0 | 73.0 | 100.0 | 100.0 | 0.3 | 0.0 | 0.0 | 0.0 | No |
First Response Malaria Ag. Pf/Pv Card Test | Premier Medical Corporation Limited | 94.0 | 100.0 | 100.0 | 100.0 | 0.8 | 0.7 | 0.5 | 0.0 | Yes |
One Step test for Malaria Pf/Pv Ag MERISCREEN Malaria Pf/Pv Ag | Meril Diagnostics Pvt. Ltd | 78.0 | 85.7 | 100.0 | 100.0 | 0.5 | 0.7 | 0.0 | 1.4 | Yes |
ParaHIT Ver. 1.0 Rapid Test for | ARKRAY Healthcare Pvt. Ltd | 77.0 | NA | 100,0 | NA | NA | 0.0 | NA | 0.0 | Yes |
Malaria RDT phase 2 performances in wild type clinical samples containing
NA = not applied; Pf =
NAAT have the potential to overcome several hurdles for malaria diagnostic at POC, more specifically related to the detection of low limits of infection, as well as the ability to discriminate the species and quantify the parasitemia.
As the most known nucleic acid amplification technique, PCR has long been used for malaria diagnostic in laboratory settings. PCR-based malaria assays for laboratory use are found in many variations: conventional, nested, hydrolysis probe, digital, or high resolution melting [19, 20, 21].
As expected, there is a clear correlation between the level of PCR detection and the parasitemia of a given sample ([22] and Figure 2). The lower the parasitemia, the higher the detection cycle above the baseline/threshold (a value known as Ct). Conversely, the higher the parasitemia, the lower the Ct. Figure 1 shows the detection of different amounts of parasites using two instruments: a lab-based and a portable, hand held thermocycler, the Q3-Plus [23, 24]. The linear regressions show that there is no difference in performance between both instruments using Spearman correlation, with r2 ranging from 0.73 to 0.80, showing the viability of this portable platform for POC malaria diagnosis.
Spearman correlation between PCR detection cycle and parasitemia estimated by optical microscopy. Detection of
PCR is more sensitive than both microscopy and immunochromatographic tests. PCR has been found to be especially useful for identifying low-level infections often missed by other techniques, showing a detection limit in the range of 0.5–5 parasites per μL of sample. PCR can detect multiple targets in the same reaction without losing sensitivity and specificity and can be easily parallelized in high throughput instruments [23, 25, 26, 27, 28]. The higher sensitivity is achieved by targeting multi copy genomic regions, such as 18S ribosomal RNA gene, which might be present in up to 1000 copies per genome and contains enough similarity across the genus
PCR has been coupled with lateral flow strips to create a simple and easy-to-use detection method for the amplified products, called nucleic acid lateral flow immunoassay (NALFIA) [29, 30]. It combines the specificity of the PCR amplification with the simplicity of lateral flow strips, using DNA capture and recognition sequences and antibody-based colorimetric methods to visualize the targets on the nitrocellulose membrane. NALFIA has been evaluated under field condition in Kenya, yielding results as sensitive and specific as laboratory PCR and a limit of detection of less than 3 parasites/μL [29].
However, in general, PCR reagents must be stored and transported at freezing temperatures (−20 °C), which is one of the main factors impairing its widespread use at POC settings all over the world and partially explaining why there are so many few available options for POC use for detection of infectious diseases. Some companies that have developed complete systems that use PCR, either conventional or nested, and melting curve analysis to detect targets of interest in POC conditions. However, neither has a solution targeted to malaria.
Much effort has been done to minimize or eliminate the requirement for freezing temperature for storage and transportation of PCR reagents. Even though some progress was indeed achieved, no commercial product is yet available. Rampazzo and colleagues [23] showed a ready-to-use qPCR that can be stored in the reaction vessel at room temperature for up to 28 days without losing performance. Iglesias and coworkers [31] showed similar results using a nested PCR for malaria detection, providing a comparison with microscopy and rapid diagnostic tests. Kamau and colleagues also reported a ready-to-use multiplex PCR for malaria detection [32]. Taylor and coworkers [33] developed a portable PCR instrument that performed 12 reactions in parallel, using lyophilized reagents and high resolution melting to detect and differentiate
Loop-mediated isothermal amplification (LAMP) is the most common isothermal amplification technique, although other methods have been developed in the last years. Other common isothermal methods, developed after the pioneering technique of the rolling circle amplification [35], are recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), and even transcription mediated amplification (TMA) [36, 37, 38, 39, 40, 41, 42].
LAMP uses a complex set of four primers that bind to the region of interest and its boundaries and, after the initial amplification, create a secondary stem-and-loop structure that serves as a binding site constantly open for a new set of primers to anneal and keep the amplification happening [41, 43]. Amplification by LAMP involves two main repetitive steps of elongation by loop primers: the self-elongation step, where the template is elongated from the stem loop structure that was formed at the 3′ end, and the elongation step, where the formation of the new PCR product actually happens, polymerizing from the primers. That way, the end product is not a single band, but a series of concatamers of different sizes, all containing an amplified sequence of the target. Usually LAMP is performed at 55–65 °C by the enzyme Bst, from
Yamamura and colleagues have developed a rapid diagnostic solution for malaria by using DNA extraction through FTA paper, combined with a LAMP assay and melting curve analysis [44]. Although the solution was analyzed with real samples, the analyses were performed in the lab and not at the POC, so the protocol remains promising until tested in field conditions.
A complete technological solution able to extract DNA, amplify and detect specific DNA sequences from malaria parasites was published [43, 45]. The presented an origami device that vertically processed the sample coupled with a microfluidic lateral flow LAMP amplification and detection platform. The platform was able to detect the presence of the malaria parasite in 98% of infected individuals, with a better performance than standard POC tests, such as optical microscopy and commercial rapid immunochromatographic tests, which detected the parasite in 86 and 83% of the cases, respectively [43, 45].
A high throughput LAMP assay for malaria detection has been shown to work in field conditions, with a clinically relevant sensitivity and, importantly, low cost per analysis [46]. It needs to be evaluated on asymptomatic patients, but it remains one of the most promising candidates for a true POC test.
LAMP has also been coupled with a MiniION sequencer, being able to amplify a genomic target and then differentiate all five
Detection of PCR-amplified products by lateral flow strips has been used for detection of malaria parasites in field samples [29]. In that work, DNA was extracted using a commercial kit. However, considering the difficulties of DNA extraction in the field, we believe that LAMP is the most suitable amplification technique to be conjugated with lateral flow strips as a feasible tool to be implemented in POC settings, and it should be further evaluated. Indeed, a meta-analysis of diagnostic accuracy of LAMP methods revealed that it is robust for diagnosing malaria, both in symptomatic and asymptomatic patients [49].
LAMP has also been used for detection of malaria parasites with a simple 1:1000 sample dilution, thus bypassing the hurdles of nucleic acid extraction in the field [50]. Although the protocol is fast and showed excellent detection limits, it has not been tested with real samples in field conditions, and therefore its positive features remain to be confirmed.
Nucleic acid sequence-based amplification (NASBA) is an isothermal reaction that continuously cycle between the reverse transcriptase activity that copies an RNA sequence into a cDNA, and the activity of a polymerase for subsequent amplification. NASBA generates a high number of copies of the target per cycle, usually reaching detectable concentrations of the product faster than other isothermal methods [42].
NASBA has been used to estimate the prevalence in asymptomatic migrants [51], to estimate gametocytes density [52] and has been shown to have significant correlation with quantitative PCR [53]. Because of its great potential for detecting low-level infections, NASBA has been proposed to be a good alternative to microscopy, especially in low prevalence areas [29, 37]. However, NASBA has not been tested in field conditions, so its performance remains to be evaluated.
In recent year, biological research questions have become the center of convergence of several scientific disciplines, such as physics and engineering, joining the early partner chemistry.
Microfluidics is a general term for engineering techniques that leverage on physics forces at the micro scale to enable the miniaturization, simplification and automation of complicated analytical processes, while consuming less reagents, minimizing waste, and requiring less supporting instrumentation. Working at the microscale, microfluidic devices are portable, easy-to-use, self-contained, and low-cost diagnostic devices that allow the precise manipulation of minute amounts of liquids to be manipulated by miniaturized structures, such as micropumps, micromixers, microtweezers, and microvalves. Most attractive features of microfluidic technology for POC applications are the low volume of reagents required, faster reaction times, and compact/portable platforms [54, 55].
Microfluidics can be divided in conventional and paper-based assays. Conventional microfluidics focuses on miniaturizing regular laboratory protocols, with a special emphasis on integration of all necessary elements for the diagnostic reaction into stand-alone systems [54, 56]. In conventional microfluidic systems, liquid control can be exerted by acoustic, mechanical, magnetic, as well as capillary and centrifugal forces. Paper-based systems, on the other hand, rely mostly on the power of capillary forces, passively controlling liquid flow via hydrophilic and hydrophobic interactions. However, paper-based assays can integrate sample preparation and pre-concentration more easily than conventional microfluidic systems [56].
Mao and colleagues developed a portable multiplex microfluidic array system, which used LAMP to simultaneously amplify and detect malaria-related
Among the microfluidic devices, the centrifugal microfluidic device or lab-on-a-disc (LOAD) has advanced remarkably due to simple operation by rotation, allowing for total integration of protocol steps, and high-throughput capability. LOAD devices have been extensively used for molecular diagnostic assays [58]. Among many targets and applications, one stands out relative to our goals in this text. Choi and colleagues developed a platform consisting of a disposable centrifugal disc and a compact instrument able to perform real time PCR for malaria diagnosis with a relevant limit of detection [59]. Although this and other platforms showed great promise towards POC applications, they have not been tested in field conditions [58].
Aptamers, also known as ‘chemical antibodies’, are short single-stranded oligonucleotides, either DNA- or RNA-based, obtained from synthetic libraries by a process called ‘systematic evolution of ligands by exponential enrichment (SELEX)’, that fold into distinct tertiary structures and are able to bind to the target with high affinity and specificity. Aptamer-coated surfaces, such as beads, sensors, or micro channels have been used to capture malaria parasites, which then can be detected by several techniques.
Fraser and colleagues developed a portable biosensor with a colorimetric approach to detect
Since blood collection is an invasive technique, other samples have been evaluated for their diagnostic capacity, in particular saliva and urine [61]. Although there has been some tests in the field using saliva for serological evaluation of samples with reasonable success, no nucleic acid amplification protocol has been used in conjunction with non-invasive samples in field application to help diagnose malaria [61]. In addition, PCR sensitivity in these samples is lower than other POC methods, impairing their current use as samples in molecular POC tests.
Infected or healthy red blood cells (RBCs) have different paramagnetic behaviors, likely due to the presence of hemozoin crystallites in iRBCs [62], and thus can be separated using magnetic fields, taking advantage of the miniaturization of magnetic resonance relaxometry (MRR) [63].
In fact, iRBCs can be enriched by a variety of methods, such as margination [64], dielectrophoresis [65], or magnetic methods [66] [67]. Margination is a separation method based on the deformability of RBCs, where microfluidic forces within the microchannels allow for the stiffer malaria iRBCs to segregate towards the device’s sidewalls [62, 63, 64, 68]. Dielectrophoresis is a force exerted on any particle when it is subjected to a non-uniform electric field, and particle manipulation is dependent on physical properties of the medium and particle electrical properties, on particle size and shape as well as frequency of electric field [69]. In contrast to dielectrophoresis, microfluidic margination offers the convenience of not needing external electrical and magnetic fields for iRBCs separation. However, both dielectrophoresis as well as magnetic methods have been used for development of diagnostic methods for malaria infection [70]. Magnetic resonance relaxometry has also recently emerged as a very attractive technique to detect ring stage parasites, using a portable permanent magnet, with very encouraging results [62, 68]. However, although promising, dielectrophoresis, magnetic resonance, or margination have not been used in POC devices for malaria diagnosis.
Banoth and colleagues developed a portable device that measures optical absorbance at 405 nm of single RBCs flowing through microfluidic channels. Variation in optical absorbance is then used to discriminate infected from health RBCs from other cellular components present in few microliters of whole blood [71]. Nam and coworkers produced a device with microfluidic channels decorated with a magnetic wire where iRBCs can be separated from hRBCs due to the paramagnetic properties of hemozoin with very high efficiencies [72].
Some malaria diagnostic approaches try to avoid sample collection and processing altogether, such as infrared spectroscopy. Fourier transform infrared (FTIR) spectra are acquired directly from the biological sample without the need for any signal enhancer, such as dyes or reagents highlighting the targets of interest [73, 74]. The acquired FTIR spectra are representative of the molecular composition of the sample, and multivariate data analysis can be used to uncover changes in the FTIR spectra produced by cellular and biochemical changes induced by the presence of a specific pathogen [75, 76]. A work describing the use of this technique for malaria diagnosis employed Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) to detect gametocytes at a level 100 times lower than microscopy-based testing [77]. Heraud and colleagues evaluated this technique using portable infrared spectrometers at four regional clinics in Thailand, comparing against laboratory-based qPCR. The analysis of 318 patients resulted in a high degree of sensitivity and specificity, supporting further testing in POC settings [78]. Recently, Mwanga and colleagues extended previous work [77] and provided the first demonstration that infrared spectroscopy could be coupled with supervised machine learning to accurately diagnose malaria in human dried blood spots [79]. Although more extensive field-testing must be performed, FTIR spectroscopy is indeed a promising method for point of care diagnostic of malaria.
Sample preparation has always been a challenge for molecular assays as well as a bottleneck in translating complex molecular based tests to easy-to-use point of care products. Several prototype devices have been proposed and have shown good results in laboratory settings [80].
For nucleic acid testing, Govindarajan and colleagues reported a low cost μPAD for POC extraction of bacterial DNA from raw viscous samples using microfluidic origami. As demonstrated,
The testing of clinical samples for nucleic acids has also been performed for infectious disease diagnosis in resource-limited environments, for example, to detect Ebola virus from extracted RNA or other infectious diseases [82, 83].
A complete technological solution able to extract DNA, amplify and detect specific DNA sequences from malaria parasites was published [43, 45].
Gan and colleagues developed a plastic microfluidic device that integrated a filter disc for DNA extraction from samples as distinct as whole blood, dried blood on various paper substrates, buccal swabs, saliva, and even cigarette ends. The device produced DNA suitable in quantity and quality for several downstream applications such as sequencing, SNP evaluation, and PCR [84].
Rodriguez and colleagues integrated nucleic acid extraction with isothermal amplification and detection by lateral flow on a foldable paper-based device able to detect RNA from Influenza A (H1N1) in less than one hour [85].
Kastania and coworkers present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction that can be used as a stand-alone device or integrated in a lab-on-chip platform. The microfluidic channels were randomly roughened in the micro-nano scale with oxygen plasma, thereby creating high surface area as well as high density of carboxyl groups (–COOH). The –COOH groups together with an optimized buffer are able to bind DNA on the microchannel surface. DNA was the washed away by changing the solution biochemical properties. DNA extracted by this device was evaluated by conventional PCR, yielding satisfactory results [86].
These are just some examples. For an in-depth analysis of the issues of nucleic acid extraction possibilities and problems, the reader is directed to excellent reviews on the subject [80, 87, 88, 89, 90].
Currently, magnetic beads and solid-phase extraction are the prime choices for nucleic acid extraction in POC devices, even though neither is yet ready for large-scale application [80, 91, 92]. Although both methods rely on the use of chaotropic agents for cell lysis and release the nucleic acids from structural proteins, washing steps are more efficient in beads-based methods in POC prototypes. However, some simplified protocols do not use beads or membranes and rely purely on both chemical and mechanical methods to denature cell membranes as well as scaffold proteins to release nucleic acids in adequate amounts and purity for diagnosis tests [80].
As shown above, despite extensive advances, no extraction method has made its way to the market of POC diagnostic solutions, and this is a major obstacle for nucleic acid-based assays. Although promising, integration of sample preparation, nucleic acid extraction, amplification and detection of genomic targets into microfluidic devices has not yet achieve the maturity to have impacts on the diagnosis of malaria in field settings. This is clear when one considers that FTA cards are primarily used for sample transportation from the field to the lab, instead as part of a complete kit combining their sample preparation and nucleic acid capabilities with the portability, not to mention sensitivity and specificity, of some of the techniques discussed in the prior sections of this work. We are not aware of such commercial kit.
A less-spoken but nonetheless quite important challenge of POC devices is waste disposal. Proper discard of biological waste generated by POC tests is a matter of concern, and open air burning might be the only option in some circumstances. However, the situation is more complex, since some chemical waste require special treatment before disposal, such as guanidine thiocyanate used in nucleic acid extraction protocols [93]. Developers need to take this issue in consideration when developing the assays and user-friendly tests.
An absolute requirement for identification and treatment of all parasite carriers, both symptomatic and asymptomatic, is inherent to all malaria elimination programs. Such identification implies that an active search for asymptomatic patients must be performed in POC conditions: since these patients will not look for medical attention, the medical team must find them. In addition, all techniques described in the present text have intrinsic advantages and disadvantages for application in POC settings, and thus each will fulfill a different aspect of the diagnostic needs, some better than others. Table 2 presents a brief comparison between the different techniques used.
Microscopy | RDT | Nucleic Acid | |
---|---|---|---|
Limit of detection (parasitemia) | 50 parasites/μL | > 100 parasites/μL | 0.5–5 parasites/μL |
Time to perform | 60 min | 15–20 min | 2 hours |
Cost | $0.20 | <$1 to $5 | $1.50 –$20 |
Need for technical training | Yes | No | Yes |
Appropriate for remote field testing | Yes | Yes | Not yet |
Microscopy, for example, requires few and inexpensive equipments, can be operated under virtually any environment conditions and within a few hours of collecting the blood, the microscopy test can provide valuable information. However, microscopy requires a very skilled professional for appropriate reading of the slides, which poses a problem due to the relative scarce availability. Furthermore, a review work found out that microscopy underestimates
RDTs, on the other hand, do not require a skilled professional and also does not require expensive instruments. In fact, RDTs can be used with the naked eye, which makes it the perfect choice for remote and hard-to-reach areas. Although they identify
PCR is very sensitive and specific, as well as a very robust and well understood technique. Although PCR is more sensitive and specific than microscopy or RDTs, it has limited use as a POC diagnostic tool because of its proneness to contamination, relative expensive reagents, delicate instruments, the need for a stable power source and skilled workers [99, 100, 101]. If a portable and robust PCR instrument is developed and is loaded with a malaria detecting reaction as it has been recently done [23, 33], then PCR might have a place in POC settings.
Compared to PCR, LAMP has the advantage of not requiring a complex and sensitive instrument, using less energy and time to achieve a sensitive target detection. However, LAMP is prone to contamination and production of false positive results from non-target amplifications, which decreases the specificity. If these technological hurdles are solved, LAMP is the most promising nucleic acid amplification technique for use in POC settings. NASBA, a sensitive and specific amplification technique, also does not require a complex thermocycler. However, it is high cost, prone to contamination and thus production of false positives, and requires a more extensive sample preparation than LAMP. Although equipment-free, NASBA is still far from POC applications.
Non-invasive methods such as infrared detection of hemozoin are very promising due to the good results in field tests and low cost of the instrument. Together with LAMP, it remains one of the most promising techniques for POC malaria diagnosis.
Finally, yet importantly, microfluidic techniques should collaborate with any nucleic acid amplification technique to make feasible a robust, sensitive and specific malaria POC diagnostic assay. Microfluidics have the capability of overcoming most of the obstacles of sample preparation and adequate amplification and detection of genomic targets. We believe that microfluidics will be in the center of a malaria POC diagnostic assay within a few years.
When one considers the production of the tests and its associated accessories such as required instruments (microscope, thermal cycler, thermal block, to name a few) in the cost analysis, microscopy is the cheapest in the long term, after the cost of the microscope is diluted over the years, and qPCR is the most expensive. The average cost for a microscopy slide test is in the range of $0.20, while RDTs typically have a production cost of $0.50 to $2.00 per test, and nucleic acid amplification techniques harbor a production cost ranging from <$1–$5 for a LAMP assay up to $10–$20 per qPCR or NASBA assay (Table 2). Non-invasive methods have a negligible cost per assay, but its overall cost is expected to be as high as qPCR tests due to instrument costs [30, 61].
Costs associated with the different available tests must be considered not solely in terms of production costs. Cost evaluation models must also include variables such as DALY, patient’s waiting time, cost acceptance (or ‘willingness to pay’) and, perhaps most importantly, adherence to the test result by patients, pharmacists, and health care providers, which is ultimately related to the perceived reliability of the test.
As discussed in the previous sections, microscopy and RDTs are the only current diagnostic tools adapted for use in resource-limited settings. Although samples can be transported to centralized laboratories to be evaluated by nucleic acid amplification techniques, turn-around time are typically too long in rural areas of developing countries, defeating the purpose of point of care testing.
Some works have explored cost effectiveness of available malaria diagnostic options for low-resource areas: presumptive, standard microscopy, and RDTs. Shillcut and colleagues used a decision tree model and probabilistic sensitivity analysis to evaluate costs and effects of RDT-positive patients and RDT-negative non-malaria febrile patients [5]. The authors found that the threshold level at which RDTs are more cost-effective than presumptive treatment is intrinsically related to the disease prevalence in the area. A health care provider can be 95% certain that RDTs are cost-effective relative to presumptive treatment at any prevalence below 62%, while there is a 95% certainty that RDTs are not cost-effective if the prevalence if higher than 90%. Microscopy would be cost-effective with 95% certainty relative to presumptive treatment if prevalence were below 41%, and would not be effective at prevalence rates above 83%. Finally, Shillcut and colleagues determined that RDTs are more than 85% likely to be cost-effective relative to microscopy at any level of disease prevalence [5]. Although the authors clearly show that RDTs are cost-effective, little is known about the impact of the facility of use and adherence to therapy that easy-to-use diagnostic tests might have on the patients [5, 102]. Similar results were observed in a deeper analysis using data from urban and rural areas of Nigeria [103], Kenya [104], and the Brazilian Extra-Amazon Region [105].
However, a recent study showed that patients are not always willing to pay for RDTs [106]. Since subsidies for artemisinin combination therapies (ACTs) already exist, it has been argued that similar subsidies could incentivize patients and health care providers to choose RDTs instead of presumptive treatment [107, 108]. Under that assumption, Bath and colleagues used a more complete decision tree and showed that subsidized RDTs could promote increased use of first-line antimalarials. Therefore, RDTs cost-effectiveness relative to microscopy or presumptive treatment would be true also in high prevalence areas, where a greater proportion of patients would benefit from increased first-line antimalarials use [108].
In summary, there are different methods can be employed for the diagnosis of human malaria. While microscopy remains the gold standard, being the mainstay of parasite-based diagnosis, its characteristics are inadequate for ensuring good sensitivity and specificity of malaria diagnosis in the field, adversely affecting health outcomes and optimal use of resources. An acceptable microscopy service is one that is both cost-effective and provides results that are consistently accurate and sufficiently timely to have a direct impact on treatment. This requires a comprehensive and functioning quality assurance program that is lacking in most malaria-endemic countries. RDT tests, which require little expertise to use and are quality-assured from the factory, are a good alternative for malaria diagnosis in remote areas. However, RDTs have no species-specific capacity to identify all five malaria species, and the parasite can present different mutations that hinder this diagnosis by increasing false-negative results. Nuclei acid-based tests, on the contrary, are specific, sensitive, and flexible to be adapted into a POC assay. Nucleic acid-based POC tests have the potential to reduce inappropriate use of anti malarials in endemic regions, bypassing the time and expertise required for microscopic analysis; however, they are more expensive and have not yet been truly adapted for use in remote areas. However, significant advances have been made to facilitate the use of POC methods, so we firmly believe that, within the coming years, interdisciplinary partnership will certainly blend innovative knowledge in biology, chemistry and physics to overcome the major hurdles impairing widespread use of nucleic acid POC tests.
The authors would like to express gratitude to Mr. Wagner Nagib de Souza Birbeire for the development of the scientific image in Figure 1.
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\\n\\nIntechOpen’s co-founders, both scientists themselves, created the company while undertaking research in robotics at Vienna University. Their goal was to spread research freely “for scientists, by scientists’ to the rest of the world via the Open Access publishing model. The company soon became a signatory of the Budapest Initiative, which currently has more than 1000 supporting organizations worldwide, ranging from universities to funders.
\\n\\nAt IntechOpen today, we are still as committed to working with organizations and people who care about scientific discovery, to putting the academic needs of the scientific community first, and to providing an Open Access environment where scientists can maximize their contribution to scientific advancement. By opening up access to the world’s scientific research articles and book chapters, we aim to facilitate greater opportunity for collaboration, scientific discovery and progress. We subscribe wholeheartedly to the Open Access definition:
\\n\\n“By “open access” to [peer-reviewed research literature], we mean its free availability on the public internet, permitting any users to read, download, copy, distribute, print, search, or link to the full texts of these articles, crawl them for indexing, pass them as data to software, or use them for any other lawful purpose, without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. The only constraint on reproduction and distribution, and the only role for copyright in this domain, should be to give authors control over the integrity of their work and the right to be properly acknowledged and cited” (reference: http://www.budapestopenaccessinitiative.org)
\\n\\nOAI-PMH
\\n\\nAs a firm believer in the wider dissemination of knowledge, IntechOpen supports the Open Access Initiative Protocol for Metadata Harvesting (OAI-PMH Version 2.0). Read more
\\n\\nLicense
\\n\\nBook chapters published in edited volumes are distributed under the Creative Commons Attribution 3.0 Unported License (CC BY 3.0). IntechOpen upholds a very flexible Copyright Policy. There is no copyright transfer to the publisher and Authors retain exclusive copyright to their work. All Monographs/Compacts are distributed under the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Read more
\\n\\nPeer Review Policies
\\n\\nAll scientific works are Peer Reviewed prior to publishing. Read more
\\n\\nOA Publishing Fees
\\n\\nThe Open Access publishing model employed by IntechOpen eliminates subscription charges and pay-per-view fees, enabling readers to access research at no cost. In order to sustain operations and keep our publications freely accessible we levy an Open Access Publishing Fee for manuscripts, which helps us cover the costs of editorial work and the production of books. Read more
\\n\\nDigital Archiving Policy
\\n\\nIntechOpen is committed to ensuring the long-term preservation and the availability of all scholarly research we publish. We employ a variety of means to enable us to deliver on our commitments to the scientific community. Apart from preservation by the Croatian National Library (for publications prior to April 18, 2018) and the British Library (for publications after April 18, 2018), our entire catalogue is preserved in the CLOCKSS archive.
\\n"}]'},components:[{type:"htmlEditorComponent",content:'The Open Access publishing movement started in the early 2000s when academic leaders from around the world participated in the formation of the Budapest Initiative. They developed recommendations for an Open Access publishing process, “which has worked for the past decade to provide the public with unrestricted, free access to scholarly research—much of which is publicly funded. Making the research publicly available to everyone—free of charge and without most copyright and licensing restrictions—will accelerate scientific research efforts and allow authors to reach a larger number of readers” (reference: http://www.budapestopenaccessinitiative.org)
\n\nIntechOpen’s co-founders, both scientists themselves, created the company while undertaking research in robotics at Vienna University. Their goal was to spread research freely “for scientists, by scientists’ to the rest of the world via the Open Access publishing model. The company soon became a signatory of the Budapest Initiative, which currently has more than 1000 supporting organizations worldwide, ranging from universities to funders.
\n\nAt IntechOpen today, we are still as committed to working with organizations and people who care about scientific discovery, to putting the academic needs of the scientific community first, and to providing an Open Access environment where scientists can maximize their contribution to scientific advancement. By opening up access to the world’s scientific research articles and book chapters, we aim to facilitate greater opportunity for collaboration, scientific discovery and progress. We subscribe wholeheartedly to the Open Access definition:
\n\n“By “open access” to [peer-reviewed research literature], we mean its free availability on the public internet, permitting any users to read, download, copy, distribute, print, search, or link to the full texts of these articles, crawl them for indexing, pass them as data to software, or use them for any other lawful purpose, without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. The only constraint on reproduction and distribution, and the only role for copyright in this domain, should be to give authors control over the integrity of their work and the right to be properly acknowledged and cited” (reference: http://www.budapestopenaccessinitiative.org)
\n\nOAI-PMH
\n\nAs a firm believer in the wider dissemination of knowledge, IntechOpen supports the Open Access Initiative Protocol for Metadata Harvesting (OAI-PMH Version 2.0). Read more
\n\nLicense
\n\nBook chapters published in edited volumes are distributed under the Creative Commons Attribution 3.0 Unported License (CC BY 3.0). IntechOpen upholds a very flexible Copyright Policy. There is no copyright transfer to the publisher and Authors retain exclusive copyright to their work. All Monographs/Compacts are distributed under the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Read more
\n\nPeer Review Policies
\n\nAll scientific works are Peer Reviewed prior to publishing. Read more
\n\nOA Publishing Fees
\n\nThe Open Access publishing model employed by IntechOpen eliminates subscription charges and pay-per-view fees, enabling readers to access research at no cost. In order to sustain operations and keep our publications freely accessible we levy an Open Access Publishing Fee for manuscripts, which helps us cover the costs of editorial work and the production of books. Read more
\n\nDigital Archiving Policy
\n\nIntechOpen is committed to ensuring the long-term preservation and the availability of all scholarly research we publish. We employ a variety of means to enable us to deliver on our commitments to the scientific community. Apart from preservation by the Croatian National Library (for publications prior to April 18, 2018) and the British Library (for publications after April 18, 2018), our entire catalogue is preserved in the CLOCKSS archive.
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