*Malaria RDT phase 2 performances in wild type clinical samples containing* P. falciparum *and* P. vivax *at low (200) and high (2000) parasite density (parasites/*μ*L) and clean-negative samples. Data modified from [18].*

#### *Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

*Current Topics and Emerging Issues in Malaria Elimination*

**18**

**Product name**

**Manufacturer**

**Panel detection score**

**200**

**parasites/**μ**L**

**Pf**

Paracheck Pf - Rapid Test for

Orchid Biomedical

94.0

NA

100.0

NA

NA

1.4

NA

4.3

Yes

Systems – Tulip

Diagnostics (P) Ltd

Meril Diagnostics Pvt.

73.0

NA

99.0

NA

NA

0.7

NA

0.0

No

Ltd

Zephyr Biomedicals –

91.0

94.3

100.0

97.1

0.0

0.7

0.0

1.4

Yes

Tulip Diagnostics (P)

Ltd

Zephyr Biomedicals –

95.0

100.0

100.0

100.0

0.8

0.0

0.0

0.0

Yes

Tulip Diagnostics (P)

Ltd.

*P. falciparum* Malaria Device (Ver. 3)

One Step test for Malaria Pf/Pan Ag

MERISCREEN Malaria Pf/Pan Ag

Parascreen - Rapid test for Malaria

Pan/Pf

FalciVax - Rapid test for Malaria

Pv/Pf

STANDARD Q Malaria Pf/Pv Ag Tes

STANDARD Q Malaria Pf Pan Ag

STANDARD Q Malaria Pf Ag Test

First Response Malaria Antigen

*P. falciparum* (HRP2) Card Test

First Response Malaria Ag. pLDH/

HRP2 Combo Card Test

First Response Malaria Ag. Pf/Pv

Card Test

SD Biosensor, Inc.

Premier Medical

95.0

NA

100.0

NA

NA

0.7

NA

0.0

Yes

Corporation Limited

Premier Medical

85.0

73.0

100.0

100.0

0.3

0.0

0.0

0.0

No

Corporation Limited

Premier Medical

94.0

100.0

100.0

100.0

0.8

0.7

0.5

0.0

Yes

Corporation Limited

87.0

NA

99.0

NA

NA

0.0

NA

0.0

Yes

Test

SD Biosensor, Inc

SD Biosensor, Inc.

88.0

100.0

99.0

100.0

0.0

0.0

0.5

0.0

Yes

85.0

100.0

99.0

100.0

0.5

0.0

0.5

0.0

Yes

**Pv**

**Pf**

**Pv**

**Pf** **non-Pf**

**Pfinfection**

**non-Pf infection**

**Pf- infection**

**infection**

**Pv**

**Pf**

**Pv**

**2000**

**200**

**parasites/**μ**L**

**parasites/**μ**L**

**False positive rates (%)**

**2000**

**parasites/**μ**L**

**Meets**

**WHO**

**performance**

**criteria?**


#### **Table 2.**

*Comparison of characteristics from currently commercially available malaria diagnostic methods.*

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 *P. falciparum*-only through detection of histidine rich protein 2 (HRP2) and those designed to diagnose and distinguish *P. falciparum* and *P. vivax* or non-falciparum malaria. The protocol assesses the sensitivity and specificity of RDTs against specimen banks consisting of recombinant antigens, culture-derived parasites, wild-type parasites, and parasite-negative blood samples, as well as the stability of RDTs at various temperatures and usage conditions. The protocol also describes aspects of the RDTs that affect ease of use in the field, and methods for recording all information in specific databases. Up-todata information on WHO pre-qualification policies and protocols can be found at WHO's website2 . **Table 1** shows the malaria RDT performances in wild type *P. falciparum* and *P. vivax* clinical samples containing low (200 parasites /μL) and high (2000 parasites /μL) parasite density (parasites/μL) and clean-negative samples, an obligatory step of the WHO standardized tests [18]. The following criteria must be met for a product to be pre-qualified: (i) For the detection of *P. falciparum* in all transmission settings, the panel detection score should be at least 75% at 200 parasites/μL; (ii) For the detection of *P. vivax* in all transmission settings, the panel detection score should be at least 75% at 200 parasites/μL; (iii) The false positive rate should be less than 10%; (iv) The invalid rate should be less than 5%.

#### **3. Nucleic acid amplification-based tests (NAAT)**

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.

**21**

**Figure 2.**

P. falciparum *and* P. vivax *with the Q3-plus.*

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

probe, digital, or high resolution melting [19–21].

this portable platform for POC malaria diagnosis.

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

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

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, 25–28]. 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 *Plasmodium* and yet contains enough specificity to discriminate the

PCR has been coupled with lateral flow strips to create a simple and easy-touse 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

*Spearman correlation between PCR detection cycle and parasitemia estimated by optical microscopy. Detection of* P. falciparum *or* P. vivax *DNA in human blood samples is plotted against the parasitemia estimated by optical microscopy. Samples were evaluated with two PCR instruments, the benchtop ABI7500 (applied Biosystems, Thermo fisher, USA) and the portable Q3-plus (ST microelectronics, Italy). Panels a and C show the detection of* P. falciparum *and* P. vivax *with the ABI7500. Panels B and D show the detection of* 

ranging from 0.73 to 0.80, showing the viability of

**3.1 Polymerase chain reaction (PCR)**

Spearman correlation, with r2

species [23, 25–28].

<sup>2</sup> https://www.who.int/diagnostics\_laboratory/evaluations/en/, accessed on February, 17th, 2021.

#### **3.1 Polymerase chain reaction (PCR)**

*Current Topics and Emerging Issues in Malaria Elimination*

*Some data were compiled from [29, 60].*

**Table 2.**

mia (infection with the sexual stage of the parasite only).

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

*Comparison of characteristics from currently commercially available malaria diagnostic methods.*

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 RDT Nucleic Acid**

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 gametocyte-

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 *P. falciparum*-only through detection of histidine rich protein 2 (HRP2) and those designed to diagnose and distinguish *P. falciparum* and *P. vivax* or non-falciparum malaria. The protocol assesses the sensitivity and specificity of RDTs against specimen banks consisting of recombinant antigens, culture-derived parasites, wild-type parasites, and parasite-negative blood samples, as well as the stability of RDTs at various temperatures and usage conditions. The protocol also describes aspects of the RDTs that affect ease of use in the field, and methods for recording all information in specific databases. Up-todata information on WHO pre-qualification policies and protocols can be found at

. **Table 1** shows the malaria RDT performances in wild type

*P. falciparum* and *P. vivax* clinical samples containing low (200 parasites /μL) and high (2000 parasites /μL) parasite density (parasites/μL) and clean-negative samples, an obligatory step of the WHO standardized tests [18]. The following criteria must be met for a product to be pre-qualified: (i) For the detection of *P. falciparum* in all transmission settings, the panel detection score should be at least 75% at 200 parasites/μL; (ii) For the detection of *P. vivax* in all transmission settings, the panel detection score should be at least 75% at 200 parasites/μL; (iii) The false positive

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

<sup>2</sup> https://www.who.int/diagnostics\_laboratory/evaluations/en/, accessed on February, 17th, 2021.

rate should be less than 10%; (iv) The invalid rate should be less than 5%.

the ability to discriminate the species and quantify the parasitemia.

**3. Nucleic acid amplification-based tests (NAAT)**

**20**

WHO's website2

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 [19–21].

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.

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, 25–28]. 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 *Plasmodium* and yet contains enough specificity to discriminate the species [23, 25–28].

PCR has been coupled with lateral flow strips to create a simple and easy-touse 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

#### **Figure 2.**

*Spearman correlation between PCR detection cycle and parasitemia estimated by optical microscopy. Detection of* P. falciparum *or* P. vivax *DNA in human blood samples is plotted against the parasitemia estimated by optical microscopy. Samples were evaluated with two PCR instruments, the benchtop ABI7500 (applied Biosystems, Thermo fisher, USA) and the portable Q3-plus (ST microelectronics, Italy). Panels a and C show the detection of* P. falciparum *and* P. vivax *with the ABI7500. Panels B and D show the detection of*  P. falciparum *and* P. vivax *with the Q3-plus.*

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 *Plasmodium* parasites, however with low sensitivity. Sun and colleagues [34] were able to pre-store the reagents in a portable device for rapid detection of *Campylobacter* sp. DNA. However, despite the availability of these technologies, we are unaware of a commercial product for malaria diagnosis that make use of them.

#### **3.2 Isothermal amplification methods**

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), helicasedependent amplification (HDA), strand displacement amplification (SDA), and even transcription mediated amplification (TMA) [36–42].

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 *Bacillus stearothermophilus*, and can use either RNA or DNA as templates. The requirement for a single stable temperature is a positive feature for a POC assay [41, 43].

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.

**23**

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

83% of the cases, respectively [43, 45]*.*

settings [47, 48].

remain to be confirmed.

early partner chemistry.

of the most promising candidates for a true POC test.

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

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

LAMP has also been coupled with a MiniION sequencer, being able to amplify a genomic target and then differentiate all five *Plasmodium* species. The procedure is sensitive enough to determine the C580Y mutation of *P. falciparum*, which confers protection against artemisinin and is an emerging threat for malaria eradication efforts. Although the procedure is relatively long because it is necessary to prepare the sequencing library, it is very versatile and shows great potential for POC

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

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

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

In recent year, biological research questions have become the center of convergence of several scientific disciplines, such as physics and engineering, joining the

Microfluidics is a general term for engineering techniques that leverage on physics forces at the micro scale to enable the miniaturization, simplification and

malaria, both in symptomatic and asymptomatic patients [49].

tested in field conditions, so its performance remains to be evaluated.

**4. Microfluidics and other new technologies**

#### *Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

*Current Topics and Emerging Issues in Malaria Elimination*

However, neither has a solution targeted to malaria.

detection of less than 3 parasites/μL [29].

make use of them.

**3.2 Isothermal amplification methods**

even transcription mediated amplification (TMA) [36–42].

in Kenya, yielding results as sensitive and specific as laboratory PCR and a limit of

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.

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 *Plasmodium* parasites, however with low sensitivity. Sun and colleagues [34] were able to pre-store the reagents in a portable device for rapid detection of *Campylobacter* sp. DNA. However, despite the availability of these technologies, we are unaware of a commercial product for malaria diagnosis that

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), helicasedependent amplification (HDA), strand displacement amplification (SDA), and

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 *Bacillus stearothermophilus*, and can use either RNA or DNA as templates. The requirement for a single stable temperature is a positive feature for a

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

**22**

POC assay [41, 43].

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 *Plasmodium* species. The procedure is sensitive enough to determine the C580Y mutation of *P. falciparum*, which confers protection against artemisinin and is an emerging threat for malaria eradication efforts. Although the procedure is relatively long because it is necessary to prepare the sequencing library, it is very versatile and shows great potential for POC settings [47, 48].

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.

#### **4. Microfluidics and other new technologies**

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 *Anopheles* and *Plasmodium* species. The system used a simple color change to discriminate positive from negative results [57].

Among the microfluidic devices, the centrifugal microfluidic device or lab-on-adisc (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].

#### **4.1 DNA-based capture molecules**

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 *P. falciparum* LDH in a device with three separate microfluidic chambers, obtaining high sensitivity and specificity [60]. Aptamers against other proteins, such as pGDH, PfEMP1, of HMGB1, have also been test in laboratory conditions but still largely remain as a promising approach for use in malariadiagnosing POC assays [55].

#### **4.2 Non-invasive samples and detection methods**

Since blood collection is an invasive technique, other samples have been evaluated for their diagnostic capacity, in particular saliva and urine [61]. Although there

**25**

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

magnetic resonance relaxometry (MRR) [63].

of hemozoin with very high efficiencies [72].

for point of care diagnostic of malaria.

malaria diagnosis.

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 meth-

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

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 [62–64, 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

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

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 laboratorybased 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

ods, impairing their current use as samples in molecular POC tests.

#### *Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

*Current Topics and Emerging Issues in Malaria Elimination*

reaction times, and compact/portable platforms [54, 55].

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

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

Mao and colleagues developed a portable multiplex microfluidic array system, which used LAMP to simultaneously amplify and detect malaria-related *Anopheles* and *Plasmodium* species. The system used a simple color change to discriminate

Among the microfluidic devices, the centrifugal microfluidic device or lab-on-adisc (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

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

Fraser and colleagues developed a portable biosensor with a colorimetric approach to detect *P. falciparum* LDH in a device with three separate microfluidic chambers, obtaining high sensitivity and specificity [60]. Aptamers against other proteins, such as pGDH, PfEMP1, of HMGB1, have also been test in laboratory conditions but still largely remain as a promising approach for use in malaria-

Since blood collection is an invasive technique, other samples have been evaluated for their diagnostic capacity, in particular saliva and urine [61]. Although there

POC applications, they have not been tested in field conditions [58].

**24**

systems [56].

positive from negative results [57].

**4.1 DNA-based capture molecules**

several techniques.

diagnosing POC assays [55].

**4.2 Non-invasive samples and detection methods**

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 [62–64, 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 laboratorybased 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.

#### **5. The nucleic acid extraction problem**

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, *Escherichia coli* with a bacterial load as low as 33 CFU/ mL was reliably extracted from pig mucin (simulating sputum) and subsequently detected [81].

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, 87–90].

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 largescale 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

**27**

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

developing the assays and user-friendly tests.

**6. Advantages and disadvantages for POC settings**

commercial kit.

techniques used.

proficiency.

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

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

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

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 *P. falciparum* prevalence by 50% when compared to PCR, the gap being even more significant in low transmission areas [94, 95]. Likewise, Cheng and colleagues described that, on average, 69.5% of infections by *P. vivax* in areas of low transmission are detected only by PCR [94, 96]. Besides, microscopy results are only as reliable as the laboratories performing the tests. In non-endemic areas where the number of cases is low, the laboratorian does not perform this test regularly, and may not be maintaining optimal

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 *P. falciparum*, specifically, RDTs have no species-specific capacity to identify all five malaria species and cannot provide information on developmental stages. The RDTs rely on antibody detection of parasite antigens, which are constantly under selective pressure and evolve over time, resulting in tests with lower affinities and, consequently, lower sensitivity and specificity [97]. It is important to mention that false-positive results are associated with persistence of PfHRP2 in peripheral blood, cross reactivity against human rheumatoid factor, and other infectious diseases and false-negative results are associated with deletions of pfhrp2 and pfhrp3 genes [98]. 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 [99–101]. If a portable and robust PCR instrument is developed

*Current Topics and Emerging Issues in Malaria Elimination*

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

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, *Escherichia coli* with a bacterial load as low as 33 CFU/ mL was reliably extracted from pig mucin (simulating sputum) and subsequently

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

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

Rodriguez and colleagues integrated nucleic acid extraction with isothermal amplification and detection by lateral flow on a foldable paper-based device able to

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

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

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 largescale 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

specific DNA sequences from malaria parasites was published [43, 45]*.*

detect RNA from Influenza A (H1N1) in less than one hour [85].

such as sequencing, SNP evaluation, and PCR [84].

**5. The nucleic acid extraction problem**

results in laboratory settings [80].

detected [81].

satisfactory results [86].

on the subject [80, 87–90].

**26**

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.

#### **6. Advantages and disadvantages for POC settings**

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, 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 *P. falciparum* prevalence by 50% when compared to PCR, the gap being even more significant in low transmission areas [94, 95]. Likewise, Cheng and colleagues described that, on average, 69.5% of infections by *P. vivax* in areas of low transmission are detected only by PCR [94, 96]. Besides, microscopy results are only as reliable as the laboratories performing the tests. In non-endemic areas where the number of cases is low, the laboratorian does not perform this test regularly, and may not be maintaining optimal proficiency.

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 *P. falciparum*, specifically, RDTs have no species-specific capacity to identify all five malaria species and cannot provide information on developmental stages. The RDTs rely on antibody detection of parasite antigens, which are constantly under selective pressure and evolve over time, resulting in tests with lower affinities and, consequently, lower sensitivity and specificity [97]. It is important to mention that false-positive results are associated with persistence of PfHRP2 in peripheral blood, cross reactivity against human rheumatoid factor, and other infectious diseases and false-negative results are associated with deletions of pfhrp2 and pfhrp3 genes [98].

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 [99–101]. 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.

#### **7. Costs and cost effectiveness**

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

**29**

POC tests.

**Acknowledgements**

**8. Conclusion**

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

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

The authors would like to express gratitude to Mr. Wagner Nagib de Souza

Birbeire for the development of the scientific image in **Figure 1**.

#### *Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

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].

#### **8. Conclusion**

*Current Topics and Emerging Issues in Malaria Elimination*

then PCR might have a place in POC settings.

far from POC applications.

diagnostic assay within a few years.

**7. Costs and cost effectiveness**

due to instrument costs [30, 61].

diagnosis.

and is loaded with a malaria detecting reaction as it has been recently done [23, 33],

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

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

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

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

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

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

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

countries, defeating the purpose of point of care testing.

**28**

the test.

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.

#### **Acknowledgements**

The authors would like to express gratitude to Mr. Wagner Nagib de Souza Birbeire for the development of the scientific image in **Figure 1**.

#### **Author details**

Alexandre Dias Tavares Costa1 \*, Anna Caroline Campos Aguiar2 , Angelina Moraes Silva3 and Dhelio Batista Pereira<sup>3</sup>

1 Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz (Fiocruz), Curitiba, PR, Brazil

2 Universidade Federal de São Paulo (UNIFESP), Departamento de Biociências, Santos, SP, Brazil

3 Centro de Pesquisa em Medicina Tropical (CEPEM), Porto Velho, RO, Brasil

\*Address all correspondence to: alexandre.costa@fiocruz.br

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**31**

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

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Switzerland; 2011.

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**Author details**

Santos, SP, Brazil

Brazil

Alexandre Dias Tavares Costa1

Angelina Moraes Silva3

\*, Anna Caroline Campos Aguiar2

and Dhelio Batista Pereira<sup>3</sup>

1 Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz (Fiocruz), Curitiba, PR,

2 Universidade Federal de São Paulo (UNIFESP), Departamento de Biociências,

3 Centro de Pesquisa em Medicina Tropical (CEPEM), Porto Velho, RO, Brasil

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: alexandre.costa@fiocruz.br

provided the original work is properly cited.

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doi:10.1039/c5an01632k

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doi:10.1186/1475-2875-12-277

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**35**

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s150306947

*Point-of-Care Strategies Applied to Malaria Diagnosis DOI: http://dx.doi.org/10.5772/intechopen.96721*

Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens. Anal Chem. 2015;87: 7872-7879. doi:10.1021/acs. analchem.5b01594

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[80] Ali N, Rampazzo RDCP, Costa ADT, Krieger MA. Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics. Biomed Res Int. 2017;2017.

doi:10.1155/2017/9306564

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doi:10.1039/c4lc00686k

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[103] Uzochukwu BS, Obikeze EN, Onwujekwe OE, Onoka CA, Griffiths UK. Cost-effectiveness analysis of rapid diagnostic test, microscopy and syndromic approach in the diagnosis of malaria in Nigeria: implications for scaling-up deployment of ACT. Malar J. 2009;8: 265. doi:10.1186/1475-2875-8-265

[104] Zurovac D, Larson BA, Skarbinski J, Slutsker L, Snow RW, Hamel MJ. Modeling the financial and clinical implications of malaria rapid diagnostic tests in the case-management of older children and adults in Kenya. Am J Trop Med Hyg. 2008;78: 884-91.

[105] de Oliveira MRF, Giozza SP, Peixoto HM, Romero GA. Costeffectiveness of diagnostic for malaria in Extra-Amazon Region, Brazil. Malar J. 2012;11: 390. doi:10.1186/1475-2875-11-390

[106] Ezennia IJ, Nduka SO, Ekwunife OI. Cost benefit analysis of malaria rapid diagnostic test: the perspective of Nigerian community pharmacists. Malar J. 2017;16: 7. doi:10.1186/s12936-016-1648-0

[107] Hansen KS, Lesner TH, Østerdal LP. Optimal price subsidies for appropriate malaria testing and treatment behaviour. Malar J. 2016;15: 534. doi:10.1186/s12936-016-1582-1

[108] Bath D, Goodman C, Yeung S. Modelling the cost-effectiveness of introducing subsidised malaria rapid diagnostic tests in the private retail sector in sub-Saharan Africa. BMJ Glob Heal. 2020;5: e002138. doi:10.1136/ bmjgh-2019-002138

**39**

**1. Introduction**

**Chapter 3**

**Abstract**

Prompt and Accurate Diagnosis,

The concept of malaria elimination is to get rid of local transmission of malaria parasites in a defined geographical area. Among the measures required for malaria elimination is prompt and accurate diagnosis. Malaria diagnostic tools currently in use: clinical diagnosis, Malaria Rapid Diagnostic Tests (mRDT) and molecular diagnosis, have limitations. Clinical diagnosis can be used as first step in making prompt malaria diagnosis, but cannot confirm cases. Malaria RDTs satisfies the need for prompt diagnosis but has low accuracy in confirming cases. Accuracy of microscopy depends on making good blood films, and accurate film interpretation. Molecular diagnosis required for species-specific diagnosis of malaria parasites, and determination of genes that confers drug resistance to *Plasmodium* species is not available for routine use. As part of elimination efforts, there is development of mRDT kits that utilize urine or saliva instead of blood specimen, microscopy digital image recognition and different technologies for molecular diagnosis. So far, none of these diagnostic tools has satisfied the need for prompt and accurate diagnosis. It is therefore recommended that more than one diagnostic tool is needed for malaria elimination to be achieved in a given area. This will ensure early detection and treatment of cases, as well as prevent the re-establishment of transmission.

**Keywords:** malaria, elimination, malaria diagnosis, clinical diagnosis, rapid

Malaria in humans is a parasitic disease caused by four species of *Plasmodium*: *P. falciparum*, *P. vivax*, *P. ovale* and *P. malariae*; with increasing recognition of enzoonotic transmission of a simian species, *P. knowlesi*. In most cases, the malaria parasites are transmitted through the bite of infected female anopheline mosquitoes. The mosquitoes carry infective sporozoites of malaria parasite in their salivary glands, and they inoculate or transfer these sporozoites into the blood stream of humans during blood meals [1]. After inoculation, the individual(s) is /are said to be infected with malaria parasites. The disease, malaria begins once (i) the asexual parasite multiplies within the red blood cells and (ii) the host produces some immunological response against the parasites. On rare occasions, malaria parasite infection can be acquired by transfusion of blood from a malaria patient, also known

diagnostic tests, microscopy, molecular diagnosis

A Veritable Tool in Malaria

Elimination Efforts

*Chukwudi Michael Egbuche*

#### **Chapter 3**

*Current Topics and Emerging Issues in Malaria Elimination*

of malaria rapid diagnostic test: the perspective of Nigerian community pharmacists. Malar J. 2017;16: 7. doi:10.1186/s12936-016-1648-0

Østerdal LP. Optimal price subsidies for appropriate malaria testing and treatment behaviour. Malar J. 2016;15: 534. doi:10.1186/s12936-016-1582-1

[108] Bath D, Goodman C, Yeung S. Modelling the cost-effectiveness of introducing subsidised malaria rapid diagnostic tests in the private retail sector in sub-Saharan Africa. BMJ Glob Heal. 2020;5: e002138. doi:10.1136/

bmjgh-2019-002138

[107] Hansen KS, Lesner TH,

Definitions, parameters, and

doi:10.3389/fmicb.2017.00108

doi:10.1186/1678-9199-20-16

Strategies. J Vis Exp. 2012.

doi:10.3791/3998

[101] Lorenz TC. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization

[102] Loubiere S, Moatti J-P. Economic evaluation of point-of-care diagnostic technologies for infectious diseases. Clin Microbiol Infect. 2010;16: 1070-1076. doi:10.1111/j.1469-0691.2010.03280.x

[103] Uzochukwu BS, Obikeze EN, Onwujekwe OE, Onoka CA, Griffiths UK. Cost-effectiveness analysis of rapid diagnostic test, microscopy and syndromic approach in the diagnosis of malaria in Nigeria: implications for scaling-up deployment

of ACT. Malar J. 2009;8: 265. doi:10.1186/1475-2875-8-265

[104] Zurovac D, Larson BA, Skarbinski J, Slutsker L, Snow RW, Hamel MJ. Modeling the financial and clinical implications of malaria rapid diagnostic tests in the case-management of older children and adults in Kenya. Am J Trop Med Hyg. 2008;78: 884-91.

[105] de Oliveira MRF, Giozza SP, Peixoto HM, Romero GA. Costeffectiveness of diagnostic for malaria in Extra-Amazon Region, Brazil. Malar J. 2012;11: 390. doi:10.1186/1475-2875-11-390

[106] Ezennia IJ, Nduka SO,

Ekwunife OI. Cost benefit analysis

everything. Front Microbiol. 2017;8: 1-9.

[100] Gonçalves-de-Albuquerque SDC, Pessoa E Silva R, de Morais RCS, Trajano-Silva LAM, Régis-da-Silva CG, Brandão-Filho SP, et al. Tracking falsenegative results in molecular diagnosis: proposal of a triplex-PCR based method for leishmaniasis diagnosis. J Venom Anim Toxins Incl Trop Dis. 2014;20: 16.

**38**
