Advanced Techniques and Unusual Samples for Malaria Diagnosis

Abstract

Successful malaria control, treatment, and prevention depends on successful diagnosis using appropriate equipment with high sensitivity and specificity. In most tropical countries where the disease is endemic, malaria diagnosis is still based on the conventional techniques (Microscopy and RDT) which have so many shortcomings, hence the need to switch to the most advanced diagnostic technique for better results. In this review, several serological and molecular malaria diagnostic techniques like Polymerase Chain Reaction (PCR), Flow cytometry, Loop-mediated Isothermal Amplification (LAMP), Indirect Immunofluorescence, Enzyme-Linked Immunosorbent Assay (ELISA), Radioimmunoassay (RIA), Quantitative Buffy Coat (QBC) and Laser Desorption Mass Spectrometry (LDMS) were systematically discussed in simple and direct language for easier understanding of the principle involved in each case scenario. In addition, some unusual samples for malaria diagnosis like Urine and saliva were also discussed.

1. Introduction

1.1 Malaria diagnoses

Successful prevention, treatment, and eradication of malaria infection heavily depend on the successful diagnosis, as a result, the Word Health Organisation (WHO) stresses the need for effective, reliable, and dependable diagnosis and confirmation before any form of intervention [1, 2], thus, discourages any form of intervention before laboratory diagnosis and confirmation [3, 4]. This is because, clinical diagnosis alone is not enough to confirm the presence or absence of malaria infection [5], as the clinical signs and symptoms of the disease mimic many other tropical diseases [6], which could be hardly differentiated from malaria infection [7, 8].

Malaria diagnosis usually involves identifying the parasite, or parasite’s products (antigens) in the blood of the subjects. In most tropical parts of the globe, except for some rare and special cases, malaria diagnosis is based on conventional techniques, like Microscopy [9] which is regarded as the gold standard technique [10] and Rapid Diagnostic Technique (RDT) [11] using either venous blood or hand prick blood as the case may be. To some extent, conventional diagnostic techniques like RDTs are easy to perform as they require less training and expertise, and are cost-effective, with an appreciable level of Sensitivity and Specificity. With all these advantages, conventional diagnostic techniques have some limitations and challenges that in some cases underscore their actual performance. Some of these limitations include detecting sequestered parasites (Plasmodium species) in tissues like the placenta and liver. In addition, they are less reliable when the level of parasitaemia is very low, as the techniques are not able to detect the presence of the parasite below a certain threshold. These and so many other factors led to either false positive or false negative results [12], and the accumulation of such diagnostic results in a population seriously affects the prevention, control, treatment and elimination of malaria infection generally. This is because such diagnostic result, especially false negative always ensures the presence of a reservoir host of malaria infection in a given population, as the infected individuals may go unnoticed and untreated [13].

Advanced malaria diagnostic techniques have the potential to overcome all these shortcomings observed in conventional techniques [14]. Unlike the conventional techniques, they are quite expensive and require special training and expertise, but they produce the much-needed results with a high level of sensitivity and specificity (Accuracy) [15], especially when performed under appropriate and conducive laboratory conditions and also by applying the principles of quality control and quality assurance in handling and treating all samples for malaria diagnosis. To that effect, advanced diagnostic techniques are very much needed especially in countries and places where malaria infection is still endemic (Sub-Sahran Africa), as the techniques always ensure the real confirmation of the deadly parasites (Plasmodium). Therefore, when appropriate treatment is applied to the confirmed positive subjects, this automatically breaks the chain of transmission, which may lead to successful elimination of the disease.

Nevertheless, conventional malaria diagnostic techniques are still very relevant, especially in underdeveloped and developing tropical countries where electricity which is one of the basic requirements for almost all the advanced techniques is still a topical issue. In addition, some of the advanced diagnostic techniques are automatically not suitable for routine laboratory diagnosis, instead, they only serve as confirmatory or screening of blood donors during blood transfusion. For better results, therefore, both conventional and advanced malaria diagnostic techniques should be married together. In this regard, conventional techniques should not be completely ignored at the expense of advanced Techniques. Nevertheless, the selection and adoption of a given diagnostic technique heavily depend on the peculiarities of the area and its inhabitants which include; level of endemicity, geographical accessibility, social and economic characteristics, underlying health infrastructure, available diagnostic tools and prevalence and type of drug resistance [16].

Given the foregoing discourse, this paper aims to systematically review the state of knowledge on different advanced malaria diagnostic techniques like Polymerase Chain Reaction (PCR), Flow cytometry, Loop-mediated Isothermal Amplification (LAMP), Indirect Immunofluorescence, Enzyme-Linked Immunosorbent Assay (ELISA), Radioimmunoassay (RIA), Quantitative Buffy Coat (QBC) and Laser Desorption Mass Spectrometry which will be discussed in simple and direct language for easier understanding and comprehending. In addition, some unusual samples for malaria diagnosis like Urine and saliva will also be looked at in this review.

2. Quantitative buffy coat

The quantitative Buffy Coat Technique is an advanced form of conventional microscopy, unlike conventional microscopy, it is only a qualitative screening technique not quantitative [17]. This diagnostic technique is by far more sensitive (almost 8 times more sensitive) than conventional microscopy [18, 19].

This technique is applied for malaria diagnosis based on: centrifugation, density gradient of infected erythrocytes and fluorescence. The technique is good enough to detect the presence of malaria parasites as low as 5 parasites per μL of blood sample [20, 21, 22]. The QBC technique is performed with the aid of a capillary tube coated with a fluorescence dye, usually, an acridine orange stain is used. This stain when excited to a frequency of 460 nm allows the detection of the parasite at a lower frequency [23].

Briefly, about 3–5 μl of blood is collected into the tube containing anticoagulant and centrifuge at 1200 g for 5 minute, this makes the blood sample separated and differentiated into different components (Plasma, Platelets, Moncyte/Lymphocyte, Red blood cells etc.) inform of layers based on their density gradient and also even the parasites are separated into different layers based on their developmental stages [24]. For example, gametocytes being lighter than schizont, mature trophozoite and ring form are found at the top layer, while ring form being the heaviest of all are found at the extreme lower part of the tube with red blood cells. The stain (acridine orange) contained in the tube stains the genetic material (DNA and RNA) and cytoplasm of the Plasmodium parasite. When observed under blue-violet light through a microscope (fluorescent microscope) the nucleus fluoresces bright green while the cytoplasm fluoresces yellow-green, thus revealing the morphological features of the Plasmodium parasite [19].

Some major drawbacks of this technique are; the expensiveness of the technique, very difficult in determining Plasmodium species at the ring form stage, and very easy in arriving at false positive results/conclusions due to the presence of artefacts such as cell debris [25].

3. Serological technique for malaria diagnosis

In common parlance, serology is the scientific study of serum and other body fluids. Technically, different serological techniques are now available and applied for the diagnosis of several parasitic and infectious diseases. All serological techniques act on the same principle, which is an antigen-antibody reaction [26]. The simple secret behind this technique is that in all foreign bodies (parasites or any infectious agent) whenever they invade a system, the system or body automatically produces antibodies in response to that [27] while the foreign body produces antigens. For example in the case of Plasmodium falciparum malaria, antigens such as Merozoite Surface Protein 2 (MSP-2), Apical Membrane Antigen 1(APM-1), and Erythrocyte Binding Antigen 175 (EBA-175) [28], Histidine Rich Protein 2 (HRP-2) (produced only by Plasmodium falciparum) Plasmodium Lactate Dehyrogenase (pLDH) (produced by the all four species) which are usually found in serum of the infected individuals [29]. Therefore, in serological diagnostic techniques for malaria parasites, such specific antigens produced by the asexual stage of Plasmodium species are the main target in the serum or plasma of the suspected individuals.

Nevertheless, one important point worth mentioning here is that serological techniques for malaria diagnosis, generally are not highly recommended and suitable for routine diagnosis of malaria infection [30]. This is because all serological techniques are based on antibodies generated or produced after initial infection with a given Plasmodium species, and this may take a minimum period of 2 weeks [31], and it may persist in either serum or plasma for about 3–6 months. Therefore, serological techniques are more suitable and recommended in non-endemic areas where the results revealed by other diagnostic technique is doubtful, and for determining prior exposure to the malaria parasite, epidemiological investigations or screening blood donor [32, 33], to prevent transfusion-induced malaria especially when level of parasitaemia is too low to be diagnosed by conventional techniques like microscopy.

3.1 Immunofluorescence antibody testing for malaria diagnosis

3.1.1 Indirect immunofluorescence

In this, a known antigen of Plasmodium species is placed onto a grease-free glass slide, smeared and allowed to air-dry, after which it is then fixed with acetone for 1–2 minute. After which, a serum which is expectedly to contain Plasmodium species antibody is added onto the smeared slide. Therefore, the antigen-antibody complex generated can then be detected by adding anti-human immunoglobulin (IgG) labelled with a fluorescein Isothiocynate (FIT), this binds to the antibody in the serum and when observed under a fluorescence microscope with the aid UV light, a fluorescence is observed, which confirm a positive sample.

Of all the available serological techniques, the immunofluorescence Antibody Technique (IFA) is considered the “Gold Standard Technique” due to its high level of Sensitivity and Specificity compared to other serological techniques, very easy to perform and reproducible technique, to some extent provides good evidence of recent infection [34]. However, it is time-consuming and subjective, especially with a serum that has low antibody titre. The basic principle of this technique, like all other serological techniques is based on antigen-antibody reactions. Normally, two weeks after initial infection with any of the Plasmodium species, antibodies are generated, which persist in the blood for about 2–3 months even after parasite clearance (Treatment) [35]. In this technique, the ratio of IgG to IgM serves as the measure and determinant factor for the positivity of a given sample and also for identifying recent infection, which is vital for epidemiological studies and also one peculiar advantage of the technique [36]. Therefore, in this, when the titre value (IgG: IgM) is greater than 1:20 is regarded as POSITIVE, while a titre value less than 1:20 is regarded as a UNCONFIRMED malaria sample and a sample with a titre value greater than 1:200 is regarded as RECENT INFECTION [35].

3.2 Enzyme-linked immunosorbent assay

Unlike the immunofluorescence Antibody Technique which is time-consuming, ELISA has been considered to be a fast, Sensitive and specific method for the detection of malaria parasites [37]. It works by detecting the presence of different antibodies of Plasmodium species. It is usually used to screen blood donors before transfusion [38] to prevent possible transmission of malaria parasites to the patients. There are different commercially prepared ELISA kits, for example, Trinity Biotech, cellabs, DiaPro, newbio, Novatec [39], etc. The practical application and utilisation of each of the kits strictly depends on the recommendation and description of the protocol/procedure by the manufacturers of the kit. Nevertheless, all ELISA kits work on the same basic principle and contain the same components, which include the following: Positive control, Negative Control, Conjugate, Conjugate dilution, Buffer, Substrate, Wash, Stop and Microplate (comprised of 96 wells). Malaria ELISA kit uses four recombinants antigens in a sandwich test to detect IgG, IgM and IgA of either Plasmodium falcifarum, P. vivix, P. malariae, P.ovale during all stages of malaria infection, but most attention is on IgG and IgM [40].

The basic general principle of ELISA is as follows; Coated microplate with specific antigens binds to the corresponding antibodies of the sample. All unreacted material in the sample, which is Horseradish peroxide are removed, followed by the addition of a conjugate [41]. The conjugate binds to the captured antibodies, this is followed by the second wash to remove all unreacted conjugate. The pure antigen-antibody complex generated is visualised by adding a substrate (Tetramethyl Benzidine), thereby forming a product which is green in colour. The intensity of the colour is always proportional to the amount of specific antibodies present in the test sample. Sulphuric acid (a stop) is added to completely stop the reaction. This gives rise to a yellow colouration of the reaction. Finally, absorbance at 450 nm is read using an ELISA reader.

The following steps summarise the procedural protocol of Enzyme-linked Immunosorbent Assay. Briefly, dispense 50 μL of the sample (serum or plasma) into a coated well of the well of the plate and shake the loaded plate for 30 seconds using a plate shaker. Use the wash buffer to wash five times, between each wash cycle soak for 30 seconds, after which remove the excess liquid. To each well containing the test sample add 50 μL of the already diluted conjugate and incubate for 30 minutes at 37°C, after which make a second (five times) wash and soak again, then also remove the excess liquid. This is followed by the addition of 50 μL of substrate/Chromogene mixture and incubation at room temperature for 30 minutes, after which add 50 μL of stop (Sulphuric acid) to each well. This makes the colour change from blue to yellow, and then finally reads the absorbance of each sample in an ELISA reader at 450 nm.

3.2.1 Validation of the test technique

The validity of the ELISA test heavily depends on meeting the following requirement, and failure to meet the requirement, may consider the test invalid and need to be repeated.

1. absorbance of substrate blank = < 0.1

2. absorbance of Negative control = 0.200 < Cut-off

3. absorbance of Cut-off = 0.15–1.3

4. absorbance of positive control > Cut-off

3.2.2 Cut-off point determination

This can determine by taking the average of the negative control values and adding 0.100 which is

$$\frac{\text{Negative Control}1+\text{Negative Control}2+\text{Negative Control}3}{3}$$E1

3.2.3 Result interpretation

Samples with A₄₅₀ value above the cut-off value are considered as POSITIVE, on the other hand, if the A₄₅₀ is less than the cut-off value, the result is regarded as NEGATIVE.

NB: For all negative samples (A_{450 <} cut-off), if the difference is not that significant, such samples should be interpreted with extra care. Therefore, such samples should be re-tested again.

3.3 Radioimmunoassay

In the 1950s, two New York Scientists Rosalan Yalow and Solomon Berson invented the technique of Radioimmunoassay. It is a serological diagnostic technique that is regarded as highly sensitive with also high level of specificity. The technique has a very good ability to detect the presence of antigen/protein and hormones in a clinical sample as low as one part in a billion (Nanogram of antigen in a serum). Its high level of sensitivity is attributed to the use of Radionuclides while the specificity is solely associated with the immunochemical reaction, hence the name Radioimmunoassay.

The basic principle on which RIA operates, in addition to antigen-antibody reaction which is common to all other serological techniques, also involves a competitive binding to antibodies between labelled and unlabelled antigens to form antigen-antibody complexes. After initial infection with Plasmodium species, antigens are produced, and in return the B-lymphocytes secrete antibodies. In the RIA, the serum containing the antibodies is collected and treated with radioactive antigens followed by non-radioactive antigens. Two main reactions take place in the process of RIA, as shown in the equations below; first, a reaction between the unlabelled antigen (Plasmodium antigen to be determined in serum) and the antibody generated. Second reaction is the reaction between a labelled antigen and the same antibody, thus creating a competition between labelled and unlabelled antigens, but this competition always favours the labelled antigen, as it always over the unlabelled antigen.

$$\text{Unlabelled antigen}\left(\mathrm{Ag}\right)+\mathit{\text{Plasmodium}}\text{antibody}\left(\mathrm{Ab}\right)==\acute{\mathrm{U}}\mathrm{Ag}—\mathrm{Ab}\text{complex}\dots \dots .\left(\mathrm{Rxn}1\right)$$

$$\text{Labelled antigen}\left({\mathrm{Ag}}^{\ast }\right)+\mathit{\text{Plasmodium}}\text{antibody}\left(\mathrm{Ab}\right)==\acute{\mathrm{U}}{\mathrm{Ag}}^{\ast }—\mathrm{Ab}\text{complex}\dots \dots .\left(\mathrm{Rxn}2\right)$$

Or

$$\text{Unlabelled antigen}\left(\mathrm{Ag}\right)+\mathit{\text{Plasmodium}}\text{antibody}\left(\mathrm{Ab}\right)==\acute{\mathrm{U}}\mathrm{Ag}—\mathrm{Ab}\text{complex}\dots \dots .\left(\mathrm{Rxn}1\right)$$

$$\begin{array}{l}+\\ \text{Labelled antigen}\left({\mathrm{Ag}}^{\ast }\right)\\ \downarrow \\ {\mathrm{Ag}}^{\ast }—\mathrm{Ab}\text{complex}\dots \dots .\left(\mathrm{Rxn}2\right)\end{array}$$

Therefore, the amount of the Plasmodium species antigens will be determined by the amount of radioactivity. Therefore, one good aspect of RIA is that it can be applied for both qualitative and quantitative determination of antigens without the use of bioassays.

4. Molecular techniques

4.1 Polymerase chain reaction

Polymerase Chain Reaction is an advanced molecular technique that has been known to be used in the diagnosis of genetic and infectious diseases including malaria, the technique is usually applied for the confirmation of different Plasmodium species in a given sample [42], especially when the outcomes of other conventional technique (e.g Microscopy and or RDTs) indicate negative results [43] and is more sensitive compared other conventional techniques [44], especially when the parasitaemia level is very low [45]. Some studies have shown that PCR has recorded a sensitivity and specificity of 100% each by detecting 1–3 Plasmodium parasite/μL of blood samples and it also has a good ability to detect mixed infection [46]. PCR like other advanced malaria diagnostic techniques is not usually adopted as a routine technique as it uses very expensive reagent/kit (DNA extraction kit) and other equipment (for example thermocycler) [47] and above all, the whole process starts from DNA extraction, DNA amplification and running of the gels all require steady electricity which is still lacking in most tropical countries where the malaria infection is still endemic [48], as it can hardly be effectively adopted in resource-poor endemic countries [49]. PCR-based diagnosis for malaria infection targets the subunit Ribosomal RNA gene (18 s-rRNA) for the detection of different Plasmodium species [50]. This is because the gene is universally present in all Plasmodia species, coupled with the fact that it has a very strong inter and intra-specie conserved region and is always readily available in large quantities [51].

Apart from the conventional PCR-based assay, there are other different forms of PCR which are purposely designed to overcome the shortcomings of other diagnostic techniques, these include Nested PCR, Quantitative PCR (qPCR) and Multiplex PCR [52, 53] and all PCR work on the same basic principle, but one of the main shortcomings of the technique is that it requires much longer time compared to microscopy and RDTs.

Nested PCR involves primary (first) and secondary (second) PCR, in which universal primers are used for the amplification of the conserved sequence of the small subunit rRNA genes from the Plasmodium species, while the secondary PCR is carried out with primers that are specific to each Plasmodium species [54]. Other target sequences used by the nested PCR include the Merozoite surface antigen (MSA) gene and Erythrocyte binding antigen (EBA-175) [55]. In this, the amplicons (PCR products) generated from the primary PCR serve as a DNA template for the secondary PCR. Therefore, by this, two rounds of amplification are generated, which makes it very possible for a single genome of a given Plasmodium species to be detected and reproduce in large quantities. Two important factors that determine the sensitivity and success of whole processes are the quantity and purity of the starting material which is the DNA template.

Quantitative PCR allows the detection and quantification of the target DNA through the use of fluorophore probes. This method has a detection limit of 0.02 p/μl for genus-level identification, and 1.22 parasite/μl for P. falciparum detection, while multiplex PCR, as the name implies, can detect multiple Plasmodium species, with a detection limit of 0.2–5parasite/μl of the suspected sample [56].

4.2 Loop-mediated isothermal amplification

The technique, which was developed 23 years back [57], unlike other molecular techniques like PCR, is simple, less time-consuming, requires less sophisticated equipment and can be adopted even in a field with a high level of accuracy [58]. It has a very good ability to detect positive malaria samples even at a very low level of parasitaemia (1–2 parasite/μL of blood) this serves as an additional advantage over conventional microscopy which cannot detect the infection when the parasite density is very low. Missing such patients in a population always ensures the presence of a reservoir host. LAMP can be used to detect the presence of all four Plasmodia species in a suspected sample by determining the presence of the parasite’s DNA in a single Isothermal step in the presence of an enzyme (Bacillus stearothermophilus Polymerase) [59] that can separate the double-stranded DNA without temperature denaturation and four to six specific primers [60].

For malaria parasite, the first set of primers is designed to target six to eight sites along the 18S ribosomal RNA gene (18SrRNA) of the genus Plasmodium, unlike PCR at a single temperature (60–65°C) [61], this increases the high specificity of the technique, while other primers target mitochondrial DNA, thus, improving the sensitivity of the technique. The auto-cycling strand-displacement DNA generates a large amount of stem-loop DNA copies (roughly 109) of different sizes through an amplification process in less than an hour [62]. This is produced together with insoluble magnesium pyrophosphate (a by-product of DNA synthesis) which is normally generated in proportion to the amplified gene [63].

The insoluble magnesium pyrophosphate produced in the reaction looks turbid and can be seen with the naked eye, this can be used to interpret the LAMP result. Nevertheless, sometimes the visualisation of magnesium pyrophosphate precipitate is a little bit difficult, especially when the DNA concentration is relatively low. A sample is considered to be positive or negative if an obvious increase in the turbidity is observed by the naked eye compared to the negative control, in addition, a LAMP test/result is considered valid if the turbidity is present in the positive control but completely absent in the negative control [64]. The result also can be interpreted by the use of ultraviolet (UV) fluorescent in the presence of an indicator such as Calcein. On the other hand, real-time detection of results is possible using a Loop real-time turbidimeter. In addition, LAMP results can be interpreted using either colorimetic and fluorescent or Lateral flow detection [65]. Detailed procedures and protocol of LAMP technique for malaria diagnosis heavily depend on the specific type of kit used, as there are different commercially available LAMP kits and each with its specifications, for example, Illumigene (Alethia), malaria LAMP, and Loopamp malaria amplification kit [66]. One of the short-comings of this technique it is qualitative, not quantitative, but has a very good sensitivity similar to other sensitive molecular techniques like PCR [67].

4.3 Laser desorption mass spectrometry

Laser Desorption Mass Spectrometry for malaria diagnosis depends on the detection of heme from hemezoin, which is an insoluble microcrystal generated during haemoglobin digestion by the Plasmodium parasite, thus, this serves as a biomarker for malaria infection. The heme produced has a good potential of absorbing ultraviolet laser [68], as a result, it is also called malarial pigment [69]. LDMS is reported to have a very good sensitivity of 52% and specificity of 92% but the technique is not suitable in places where malaria infection is endemic and is not recommended for routine malaria diagnosis as the technique is quite expensive [70]. The detection limit of this technique is 10 parasites/μl of culture and 100 parasites/μl of the blood sample.

Unlike PCR and other molecular techniques, LDMS does not require sophisticated equipment or apparatus, instead only metal slides, sample collection bottles, distilled water and aliquot are required [71]. The LDMS technique is quite simple, straightforward and very fast to produce results (in fact in less than 1 minute), it only involves diluting the collected/suspected blood sample with the appropriate amount of buffer (Phosphate Buffer Saline) and placing it onto a metal slide and allow to air-dry, after which the prepared slide is inserted into a mass spectrometer for sample analysis. Heme-specific ions in RBCs are desorbed from the probe and analysed by their mass/charge ratio, generating a parasite-specific mass spectral signature [72].

4.4 Flow cytometry

For long, flow cytometry using scattering and fluorescent detection methods has been in use for many years as a fundamental instrument for disease diagnosis [73]. For malaria diagnosis, this technique, like Laser Desorption Mass Spectrometry depends on the detection and quantification of heme using a special machine called flow cytometer and also works on the principle of flow cytometry, hence, the name of the technique. This is a haematology analyser that is used for the presumptive diagnosis of malaria and other haematological parameters of the sample [74]. Different flow cytometric methods are used to determine the presence of the infection and also quantify the level of parasitaemia [75]. Flow cytometry is a measurement of characteristic single cells (cyto) suspended in a flowing saline stream, and is the only technique capable of rapidly identifying cells and parasites from large samples. It relies on scattering or fluorescence measurements that are made while the cells or particles pass through a capillary flow cell [76]. Flow cytometry work on three basic coordinated component, which include; fluidics, optical, and electronic systems [77]. Some of the disadvantages of this technique are its low sensitivity compared to other techniques and it also requires specialised equipment [78]. Therefore, this technique is not highly recommended for routine malaria diagnosis, especially in sub-Saharan Africa where the disease is highly endemic area.

5. Unusual samples for malaria diagnosis

Venus or hand prick blood are the well-known samples for malaria diagnosis in most laboratories in the endemic countries, as it is always readily much available except for some special cases/reasons. Most of the time, the blood serves as the preferred sample [79] as the parasite lives intra-cellularly, therefore this allows physical examination of the parasite (Plasmodium), its developmental stages and its other morphological features under a microscope.

Nevertheless, in some cases, the blood sample may not be easily and readily available due to some factors/reasons which include customs and natural beliefs, creed and religious beliefs [80], and fear of pricking or bleeding using a lancet or needle. In addition sometimes the parasite density in the peripheral blood tends to fluctuate, this is normally due to the sequestration of the parasites in tissues and other vital organs like the brain, spleen etc. [81]. Therefore to overcome all these and always make samples readily available for malaria diagnosis, the parasite’s products (antigen for example Histidine Rich Protein-2, Plasmodium Lactate Dehydrogenase (pLDH)) can also be found in other body fluids and products like Urine [82] Saliva, Faeces and hair [83]. Apart from this, several abnormalities in urine like haematuria (possibly due to massive destruction of infected RBC by the parasite, thus leading to excretion of blood and even haemoglobin together with urine) and proteinuria have been attributed to malaria infection [84, 85]. Therefore, any of these (Urine, Saliva, faeces and Hair) samples can serve as a very good alternative sample for malaria diagnosis. The use of any of these non-invasive samples drastically reduces or eliminates the risk of infection to both the screener and the patient [86].

5.1 Urine

Urine Malaria Test (UMT) involves the determination of the presence of the parasite’s antigen (Histidine Rich Protein-2 (HRP-2)) in a given suspected urine sample, this is because the antigen is water-soluble protein, and therefore it can be found in the urine [87]. In addition, urine is an ultra-filtrate of blood, it can be effectively used instead of blood for malaria diagnosis [88]. Like with the blood, there is a Urine-based RDT/Kit for malaria diagnosis, the detailed principle and protocol of the Urine-based RDT was explained in the work of [89]. In addition, DNA of the Plasmodium can also be recovered from the urine sample of the infected individuals, therefore, PCR can be employed to amplify the parasite’s (Plasmodium) DNA from the urine sample [90]. The urine-Malaria Test technique has many additional advantages compared to the conventional blood-based RDT techniques, which include a lack of difficulties (searching for veins, pain during pricking and or bleeding) in obtaining a sample, also the process is non-invasive, thus eliminating the risk of infection and like blood- based RDT techniques is a very simple, easy technique and it requires less time compared to microscopy and other advanced molecular techniques [91].

5.2 Saliva

Saliva is regarded as a less invasive sample for malaria diagnosis, it contains markers such as antigen, DNA, and sequence of 16S RNA that are normally used as the best target for saliva-based RDT kits. Therefore, the presence of these in the saliva provided very good evidence of obtaining products (DNA and or Proteins) in the saliva of malaria-infected individuals [92]. Having saliva as a sample for malaria diagnosis offers many advantages over the conventional blood sample, these include; ease of collection, transportation and storage [93]. Therefore, like in other samples (blood and urine) the parasite’s products can be detected using already known molecular techniques like PCR, nested PCR, qPCR etc.