Abstract
Malaria is a major cause of illness and death worldwide. Rapid diagnostic tests are the most widely used tool for detecting malaria infection, however, they only provide binary results and lack the sensitivity needed to detect many asymptomatic infections. Molecular assays for quantifying malaria biomarkers offer higher detection sensitivity, however, they are time-consuming, and require expert training and expensive equipment, making them unsuitable for use in most of Africa. To address the need for simple, accurate and field-deployable malaria diagnostic tests, we have developed a microfluidic point-of-care (mPOC) immunoassay for rapid quantification of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, in whole blood. This device features two diagnostic modes for detecting PfHRP2 at low (100’s pg/mL) and high (1,000’s ng/mL) concentrations, making it useful for multiple diagnostic applications, including the detection of asymptomatic infection, prediction of disease outcomes and diagnosis of cerebral malaria. Measurements of PfHRP2 in blood samples from malaria patients demonstrates that this platform offers similar accuracy as an ultra-sensitive PfHRP2 enzyme-linked immunosorbent assay (ELISA) test, while being 12× faster and simpler to use. This mPOC immunoassay can be deployed in rural health centers to assist clinicians in diagnosing and triaging malaria patients, ultimately improving patient outcomes.
Introduction
Malaria remains an overwhelming health burden worldwide with an estimated 247 million cases and 619,000 deaths in 2021 (WHO, 2021). The majority of deaths are in children under the age of five in sub-Saharan Africa. The most severe form of malaria infection, cerebral malaria (CM), is caused by the Plasmodium falciparum parasite and has a mortality rate of >15% even when appropriately treated (Idro et al., 2010). In addition to mortality, there is a high level of morbidity, with up to 50% of CM survivors suffering some form of sequelae (Langfitt et al., 2019). Most P. falciparum infections are asymptomatic or result in mild disease. However, clinical progression to CM and deterioration occurs rapidly where the time of first symptoms to coma and convulsions often occur within 24 h. This sudden occurrence with disastrous outcome provides a diagnostic challenge. Children often present to a rural health center early in the disease process with fever as the only presenting symptom. Most children treated with a course of oral antimalarial drugs at this stage will recover well. However, a small percentage will progress to CM despite oral medicines. If this subset were able to be identified early in the disease process and referred on to higher acuity care, progression to severe disease with its consequences could be avoided. Unfortunately, many of the primary care health centers are remote and lacking in resources, including laboratory equipment and electricity.
Although disease severity is not tightly linked to peripheral parasitemia, it is highly correlated with total body parasite load. We have shown previously that total body parasite load, as measured by the protein histidine-rich protein 2 (HRP2), can predict which children will progress to severe disease and which will recover on oral medication (Fox et al., 2013). HRP2 is one of the most abundant proteins produced by P. falciparum. It is released from infected erythrocytes and can be measured in the blood of infected individuals. It is the antigen of choice for most of the currently available malaria rapid diagnostic tests (mRDTs). Using PfHRP2-specific antibodies and a lateral flow technique, these tests detect the presence of PfHRP2 in the blood of an individual with a visible line of the antigen-antibody complex. While mRDTs are rapid (∼15 min), require no electricity and are relatively easy to administer, they only provide qualitative results (positive or negative), making them unsuitable for predicting disease outcome. Additionally, mRDTs lack the sensitivity needed to detect low levels (<1 ng/mL) of PfHRP2, which could lead to false negative results (Watson et al., 2019).
In addition to identifying children with uncomplicated malaria who are at risk of developing CM, quantitative measures of PfHRP2 has shown to be useful in diagnosing children with CM with similar accuracy as histology and ocular funduscopy (Fox et al., 2013; Seydel et al., 2012). Prior studies have also shown PfHRP2 quantification as a useful measure of disease severity and progression (Hendriksen et al., 2012; Hendriksen et al., 2013). Currently, PfHRP2 quantification requires a highly sensitive ELISA test, which is expensive, complicated, and time-consuming (∼4 h), making it impractical for use in most of Africa. Here, we report on the development and validation of an off-grid mPOC immunoassay capable of quantifying PfHRP2 in whole blood in 15 min with accuracy comparable to a commercial, ultra-sensitive ELISA kit. The scale up of this test would result in the ability for health care providers in rural settings to measure the total parasite load in children at their first interaction with the health care system. This would serve as objective data to be used by clinicians to make triage decisions that would lead to more rapid treatment of children likely to deteriorate clinically and thus reduce the burden of malaria-associated morbidity and mortality.
Section snippets
Materials and chemicals
Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS, pH 7.4), (ethylenedinitrilo)tetraacetic acid (EDTA), 2-Iminothiolane hydrochloride, horseradish peroxidase (HRP) and TMB/E Ultra-Sensitive substrate were purchased from Sigma-Aldrich (St Louis, MO). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS) were obtained from Thermo Fisher Scientific (Waltham, MA). StabilBlock immunoassay stabilizer, StabilCoat Plus immunoassay stabilizer, and StabilZyme HRP
Design and operation of the mPOC immunoassay
The mPOC immunoassay was designed for use in rural and remote health centers in sub-Saharan Africa which lack many resources, including laboratory equipment and electricity. In particular, this immunoassay is capable of quantifying malaria biomarkers in whole blood, circumventing the need for plasma separation. Additionally, the immunoassay workflow does not require pipetting, complicated sample processing or long incubations, which are typically associated with surface binding assays, such as
Discussion
We have presented the development and validation of a point-of-care diagnostic test for rapid quantification of PfHRP2 in whole blood. While offering many of the same benefits as mRDTs in regards to portability, rapid turnaround times and ease of use, this mPOC immunoassay also offers quantitative biomarker measurements with higher analytical sensitivity and accuracy (comparable to a commercial PfHRP2 ELISA kit), making it a promising diagnostic tool for detecting malaria infection and
Conclusions
We have developed a mPOC immunoassay for rapid quantification of PfHRP2 in whole blood, which overcomes many of the limitations of existing malaria diagnostics. This device features two diagnostic modes for detecting PfHRP2 at low (100’s pg/mL) and high (1,000’s ng/mL) concentrations, making it useful for multiple diagnostic applications, including the detection of asymptomatic infection, prediction of disease outcomes and diagnosis of cerebral malaria. Measurements of PfHRP2 in blood samples…..
