Point-of-Care Strategies Applied to Malaria Diagnosis

*Alexandre Dias Tavares Costa, Anna Caroline Campos Aguiar, Angelina Moraes Silva and Dhelio Batista Pereira*

#### **Abstract**

Rapid and specific diagnosis of malaria remains one of the main strategies to fight the disease. The diagnosis is made primarily by the simple and low-cost thick drop technique, considered the gold standard test. However, the requirement for good quality microscopes and well-trained personnel often lead to inaccurate diagnosis, especially in cases of mixed infections or low parasitemia. Although PCR-based tests can help in these situations, this technique requires large and sensitive equipments, being unsuitable for point of care (POC) settings. A myriad of POC diagnostic tests have being developed in the last years, relying on molecular methods but also on novel strategies. New platforms, miniaturization techniques, and multiplexing possibilities promise great potential to improve disease diagnostics through fast and accurate detection of cases, even at remote places. Here, we will address the main POC strategies developed for the diagnosis of malaria, highlighting their strengths and weakness as POC applications.

**Keywords:** point-of-care, diagnosis, malaria

#### **1. Introduction**

Malaria is one of the deadliest diseases of poverty. It is estimated that malaria causes 228 million illnesses and 405 thousands deaths each year. Among the sick, children aged under 5 years are the most vulnerable group affected by malaria; in 2018, they accounted for 67% (272 000) of all malaria deaths worldwide [1].

In many countries where malaria is endemic, a lack of access to adequate diagnostic services leads to poor health outcomes for fever patients, as well as poor surveillance of infections and outbreaks, and treatment monitoring [2].

To make matters worse, the appearance of antimalarial resistant parasites including artemisinin derivatives pose a major public health threat [3]. In addition, drugs such as the artemisinin-derivatives are more expensive, leading to an increased demand for patient evaluation by accurate diagnostic tests before treatment [4–6].

Therefore, it has grown in the last years a general agreement that new diagnostic tests are needed for remote areas in malaria-endemic countries. However, the new tests must show improved performance over existing techniques, so that adequate distribution of anti-malarial drugs can effectively target the disease and its outbreaks, contributing to the reduction of generation of drug-resistant parasite strains [7].

In malaria-endemic countries, the major hurdle for widespread access to malaria diagnostics is the limited health care infrastructure [8, 9]. According to the World Health Organization (WHO) guidelines, the useful diagnostic tool at the point of care (POC) is defined by some characteristics. It should be low cost, deliver sensitive and accurate results in as little time as possible, run on a portable instrument (ideally, should be instrument-free), require minimal external power, require minimal training before use, and not require refrigerated reagent storage and transportation. These guidelines are collectively known by the acronym ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users) [10]. In addition to those requirements, an ideal POC malaria diagnostic device should determine which species is infecting the patient, to establish the level of parasitemia, and be able to detect mixed or low-level infections.

Current POC tests for malaria include the smear microscopy and immunochromatographic rapid tests (RDTs). However, more sensitive and specific techniques based on nucleic acid amplification tests (NAAT) have been praised as the best choice for a successful malaria POC diagnostic test. In this work, we will review the status of the diagnostic technologies that have been used for malaria detection at POC conditions, discussing their main advantages and disadvantages in the POC context.

#### **2. Currently available POC tests**

#### **2.1 Smear microscopy**

Microscopy remains the gold standard for malaria diagnosis in most endemic areas. This technique allows the identification of different malaria-causing parasites (*P. falciparum, P. vivax*, *P. malariae* and *P. ovale*), their various parasite stages, including gametocytes, and the quantification of parasite density to monitor response to treatment [11].

There are two variations of the microscopy technique, the thick drop and the blood smear, both use the Giemsa dye in their preparations and are performed with sample of peripheral blood. Thick droop is made by placing a few drops of blood on a glass slide, allowing the blood to dry, and then lysing the blood (usually with water) before staining. The blood smear is made from a thin layer of cells and are fixed with methanol before reading. The thick drop allows the identification of lower parasitemias, by concentrating the parasites. The blood smear technique is more sensitive in speciating the parasites, however it does not allow the identification of low parasitemias [12].

This technique has the great advantage of being cheap (costs approximately \$ 0.20 per sample), fast (approximately 1 hour between collection and the result, if performed by a skilled laboratory technicians), and does not need sophisticated equipment. The number of patients tested by microscopic examination increased an increase of 165 million tests in 2010 [13] to more than 208 million testes in 2017<sup>1</sup> . The global total is dominated by India. The sensitivity of the optical microscopy technique using the thick drop method is50–500 parasites/μL, however, many factors may interfere with the results found in the thick drop technique, such as the quality of the microscope, the quality of the available staining reagents, and the skill of the technician. Several studies have shown that the sensitivity of the

**17**

**Figure 1.**

*analyte (panel C).*

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

**2.2 Rapid diagnostic tests (RDTs)**

but not differentiating between them [17].

antigens [16] (**Figure 1**).

increase the number of false negative results [14, 15].

microscopy technique may be lower in several areas of transmission depending on the quality of the examination and the expertise of the microscopists, which can

Rapid diagnostic test (RDT) is a quick diagnostic approach to detect malaria among malaria-suspected patients and rule out malaria among individuals without malaria. RDTs detect parasite-specific antigens in a drop of fresh blood through lateral flow immunochromatography using antibodies to detect one or several

The RDTs detect a single species (either *P. falciparum* or *P. vivax*), some detect multiple species (*P. falciparum*, *P. vivax*, *P. malariae* and *P. ovale*) and some further distinguish between *P. falciparum* and non-*P. falciparum* infection, or between specific species. The most commonly used antibodies react to histidine-rich protein-2 (HRP2), aldolase and *Plasmodium* lactate dehydrogenase (pLDH). HRP-2 is a marker for *P. falciparum*, while pLDH antibodies can be specific for *P. falciparum*, or *P. vivax*. Aldolase antibodies are pan-specific, detecting all types of malaria parasite

*Lateral flow assay architecture. Samples containing the analyte flow through the nitrocellulose membrane by capillary flow (panel A), carrying reporter antibodies (labeled with gold, latex or a fluorophore) until the mixture interacts with the test line (containing antibodies that bind the analyte of interest) and the control line (containing anti-IgG antibodies that bind to human IgG molecules)(panel B). If the control line shows a positive reaction, it is a valid test. If the test line shows a positive reaction, it is a positive sample for the specific* 

<sup>1</sup> https://www.who.int/malaria/areas/diagnosis/microscopy/en/, accessed on February, 17th, 2021.

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

microscopy technique may be lower in several areas of transmission depending on the quality of the examination and the expertise of the microscopists, which can increase the number of false negative results [14, 15].

#### **2.2 Rapid diagnostic tests (RDTs)**

*Current Topics and Emerging Issues in Malaria Elimination*

In malaria-endemic countries, the major hurdle for widespread access to malaria diagnostics is the limited health care infrastructure [8, 9]. According to the World Health Organization (WHO) guidelines, the useful diagnostic tool at the point of care (POC) is defined by some characteristics. It should be low cost, deliver sensitive and accurate results in as little time as possible, run on a portable instrument (ideally, should be instrument-free), require minimal external power, require minimal training before use, and not require refrigerated reagent storage and transportation. These guidelines are collectively known by the acronym ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users) [10]. In addition to those requirements, an ideal POC malaria diagnostic device should determine which species is infecting the patient, to establish the level of parasitemia, and be able to detect mixed or low-level

Current POC tests for malaria include the smear microscopy and immunochromatographic rapid tests (RDTs). However, more sensitive and specific techniques based on nucleic acid amplification tests (NAAT) have been praised as the best choice for a successful malaria POC diagnostic test. In this work, we will review the status of the diagnostic technologies that have been used for malaria detection at POC conditions, discussing their main advantages and disadvantages in the POC

Microscopy remains the gold standard for malaria diagnosis in most endemic areas. This technique allows the identification of different malaria-causing parasites (*P. falciparum, P. vivax*, *P. malariae* and *P. ovale*), their various parasite stages, including gametocytes, and the quantification of parasite density to monitor

There are two variations of the microscopy technique, the thick drop and the blood smear, both use the Giemsa dye in their preparations and are performed with sample of peripheral blood. Thick droop is made by placing a few drops of blood on a glass slide, allowing the blood to dry, and then lysing the blood (usually with water) before staining. The blood smear is made from a thin layer of cells and are fixed with methanol before reading. The thick drop allows the identification of lower parasitemias, by concentrating the parasites. The blood smear technique is more sensitive in speciating the parasites, however it does not allow the identifica-

This technique has the great advantage of being cheap (costs approximately \$ 0.20 per sample), fast (approximately 1 hour between collection and the result, if performed by a skilled laboratory technicians), and does not need sophisticated equipment. The number of patients tested by microscopic examination increased an increase of 165 million tests in 2010 [13] to more than 208 million testes in 2017<sup>1</sup>

The global total is dominated by India. The sensitivity of the optical microscopy technique using the thick drop method is50–500 parasites/μL, however, many factors may interfere with the results found in the thick drop technique, such as the quality of the microscope, the quality of the available staining reagents, and the skill of the technician. Several studies have shown that the sensitivity of the

<sup>1</sup> https://www.who.int/malaria/areas/diagnosis/microscopy/en/, accessed on February, 17th, 2021.

**16**

infections.

context.

**2. Currently available POC tests**

**2.1 Smear microscopy**

response to treatment [11].

tion of low parasitemias [12].

Rapid diagnostic test (RDT) is a quick diagnostic approach to detect malaria among malaria-suspected patients and rule out malaria among individuals without malaria. RDTs detect parasite-specific antigens in a drop of fresh blood through lateral flow immunochromatography using antibodies to detect one or several antigens [16] (**Figure 1**).

The RDTs detect a single species (either *P. falciparum* or *P. vivax*), some detect multiple species (*P. falciparum*, *P. vivax*, *P. malariae* and *P. ovale*) and some further distinguish between *P. falciparum* and non-*P. falciparum* infection, or between specific species. The most commonly used antibodies react to histidine-rich protein-2 (HRP2), aldolase and *Plasmodium* lactate dehydrogenase (pLDH). HRP-2 is a marker for *P. falciparum*, while pLDH antibodies can be specific for *P. falciparum*, or *P. vivax*. Aldolase antibodies are pan-specific, detecting all types of malaria parasite but not differentiating between them [17].

#### **Figure 1.**

.

*Lateral flow assay architecture. Samples containing the analyte flow through the nitrocellulose membrane by capillary flow (panel A), carrying reporter antibodies (labeled with gold, latex or a fluorophore) until the mixture interacts with the test line (containing antibodies that bind the analyte of interest) and the control line (containing anti-IgG antibodies that bind to human IgG molecules)(panel B). If the control line shows a positive reaction, it is a valid test. If the test line shows a positive reaction, it is a positive sample for the specific analyte (panel C).*


**19**

**Product name**

**Manufacturer**

**Panel detection score**

**200 parasites/**μ**L**

**Pf**

One Step test for Malaria Pf/Pv Ag MERISCREEN Malaria Pf/Pv Ag

ParaHIT Ver. 1.0 Rapid Test for *P. falciparum* Malaria Device

*NA = not applied; Pf = Plasmodium falciparum; Pv = Plasmodium vivax.*

**Table 1.**

*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].*

Meril Diagnostics Pvt. Ltd

ARKRAY Healthcare Pvt. Ltd

77.0

NA

100,0

NA

NA

0.0

NA

0.0

Yes

78.0

85.7

100.0

100.0

0.5

0.7

0.0

1.4

Yes

**Pv**

**Pf**

**Pv**

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

**Pf- infection**

**non-Pf infection**

**Pf- infection**

**Pv**

**Pf**

**Pv**

**2000 parasites/**μ**L**

**200 parasites/**μ**L**

**False positive rates (%)**

**2000 parasites/**μ**L**

**Meets WHO performance criteria?**

*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*

