PERSISTENCE OF PLASMODIUM FALCIPARUM HRP2 ANTIGEN AFTER EFFECTIVE ANTIMALARIAL THERAPY

---

O.S. Michael¹ , A.E. Orimadegun² , and C.O. Falade¹

1. Department of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, Nigeria
2. Institute of Child Health, College of Medicine, University of Ibadan, Nigeria

Abstract

Introduction: Histidine Rich Protein 2 based (HRP2-based) malaria rapid diagnostic tests (mRDTs) have been shown to perform as well as routine light microscopy, however, they are limited by some factors including persistence of HRP2 antigenemia. In this paper we report the evaluation of an HRP2-based mRDT in a prospective study that enrolled children and followed them up for 28 days. 

Methods: Children aged below five years, with acute episode of fever/pyrexia, were enrolled. The enrolled participants had expert malaria microscopy and RDT done at enrolment (Day 0), and on days 1, 2, 3, 7, 14, 21, and 28. The malaria RDT test was considered positive when the antigen and control lines were visible in their respective windows, negative when only the control band was visible and invalid when the control band was not visible. Faint test lines were considered positive. The RDT results were compared to those of expert microscopy. 

Results: Two hundred and twenty-six children aged 29.2 ± 15.5 months were enrolled. The proportion of children positive by expert malaria microscopy and RDT was 100% and 95.6% respectively. During the 28 day follow up of the children the proportions positive by microscopy and RDT on days 3, 7, 14, and 28 were 1% and 94.6%, 0% and 93.5%, 0% and 91%, and 16.5% and 80.6% respectively. Gender and age dependent analysis of proportion of positive children were similar. Proportion of children with persistence of HRP2 antigen appeared to be lower in those with parasite density below 200/µL, however, this observation requires further evaluation in larger studies. 

Conclusions: the study revealed a high proportion of persistence of HRP2 antigen in the children 28 days after effective antimalarial therapy. Histidine rich protein 2 based malaria rapid diagnostic tests are not recommended for monitoring of antimalarial therapies.

Keywords: Malaria, RDT, Persistence, HRP2, Nigeria

Introduction

Parasite-based diagnosis is the cornerstone of effective antimalarial therapy.¹ Microscopy and rapid diagnostic tests are currently the most effective methods of malaria diagnosis. Microscopy, which is currently the reference standard for malaria diagnosis, is limited by scarcity of expert microscopists, poorly maintained microscopes, erratic electricity supply, and associated fatigue during the procedure.² However, microscopy retains certain essential advantages. Light microscopy visualizes viable parasitemia, parasites in different stages of malaria life cycle, enables the estimation of parasite density, and visualization of fragmented or isolated components of the parasite (e.g. chromatin dots). In addition, with additional equipment and/or special staining, microscopy can be automated with enhancement of parasite density estimation.^3,4 These advantages have made microscopy retain the status of gold standard for malaria diagnosis. However, despite these advantages, the use of malaria rapid diagnostic tests (mRDTs) as alternatives to microscopy has continued to increase.^5,6

Rapid diagnostic tests are user friendly and results can be read by almost everyone after minimum training. They are instrument-free tests that provide results within minutes and can be used by community health workers. They are useful where: rapid diagnosis of malaria is needed, in communities with irregular electricity supply required for microscopes and where there is scarcity of expert microscopists. Their introduction has yielded many benefits; parasite-based malaria diagnosis is now a possibility at points of care and their use could potentially improve malaria in many settings.⁷ Accurate diagnosis of malaria is an essential aspect of the preservation of our shrinking number of effective antimalarial therapies. Studies evaluating mRDTs have also shown satisfactory accuracy compared to microscopy in diagnosis of malaria.^8,9 All these have led to the growing use of mRDT as a method of diagnosis of malaria worldwide.¹⁰ However, there are some drawbacks with mRDT, one of which poses a significant challenge to their use in endemic countries with high rates of malaria transmission.

Majority of Malaria rapid diagnosis assays are based on three bimolecular pathways for the detection of presence of malaria parasites; parasite specific lactate dehydrogenase (pLDH), aldolase-based, and histidine rich protein2 (HRP2-based mRDTs.¹¹ Histidine rich protein 2-based mRDTs are the most evaluated and deployed for field diagnosis of malaria. ¹² The performance of HRP2-based RDTs has been overwhelmingly shown to have adequate diagnostic accuracies. However, the challenges of using the tests need to be noted by the end user. The limitations of malaria RDTs include poor sensitivity at low parasite densities¹³, susceptibility to the prozone effect¹⁴, false-negative results due to Plasmodium falciparum Histidine Rich Protein 2 (Pf-HRP2) deficiency in the case of PfHRP2 gene deletions^15,16, cross-reactions between Plasmodium antigens and detection antibodies, false-positive results by other infections and susceptibility to heat and humidity.¹⁷

While false positive results have been reported in many studies evaluating HRP2-based mRDTs, persistence of HRP2 antigen has been reported in much fewer studies.18,19 In a prospective study of Pf-HRP2 mRDT conducted at Thailand in 2001, HRP2 antigenemia was found to persist in a large proportion of the participants for over two weeks.18 This poses a serious challenge to countries like Nigeria where malaria prevalence is high and transmission intense. Persistent HRP2 makes it challenging to identify treatment failures and may result in repeated treatment of malaria when such treatment may be unnecessary. In this paper we report the evaluation of an HRP2 mRDT in a prospective study that enrolled children and followed them up for 28 days.