High Prevalence of Pfhrp2/3 Gene Deletions and Major Threat to Malaria Control Programs in Ethiopia

1. Background

In the past decade, the world moved forward in the fight against malaria with the implementation of a combination of preventive and control measures including the use of long-lasting insecticide-treated nets (LLIN), community deployment of malaria rapid diagnostic tests (RDTs) and treatment using artemisinin combination-based therapies (ACT), and social behavioral change and communication. Despite global efforts, these numbers have only decreased modestly for the past 5 years [1]. Reasons underlying this include a higher number of hrp2/3 deletions in Plasmodium falciparum that decreases the sensitivity in RDTs [2]. Despite this, according to the 2016 World Health Organization (WHO) report, there were 212 million cases and 429,000 deaths of malaria globally with about 90 percent of the cases and deaths occurring in sub-Saharan Africa [1].

Likewise, malaria is yet among the major public health problem in Ethiopia where Plasmodium falciparum and Plasmodium vivax are the main species accounting for roughly 63.6% and 36.4% of malaria cases. Over the last 10 years morbidity and mortality due to malaria decreases in Ethiopia with the implementation of scale-up universal intervention measures [3]. This effort enabled the country to set malaria elimination strategy with objectives to: reduce malaria case incidence to zero by 2030, reduce malaria mortality rate to zero by 2030 and prevent re-establishment of malaria in all historically malaria free districts [4].

However, one of the main challenges in combating malaria that need to be addressed is inadequate diagnostic capacity towards malaria. In Ethiopia, diagnosis of malaria is mainly relying on microscopic or RDTs with limitations of low sensitivity and problems of detection and identification of the Plasmodium species [5, 6].

In spite of this, RDTs is mainly used in the outskirt areas of the country where there is no access to microscopic diagnosis. Since 2011 the Federal Ministry of Health (FMOH) of Ethiopia has launched a health extension package accompanied with RDTs and a stock of anti-malaria treatments to be deployed in communities for early management and reduction of malaria [7]. In the growing effort of global malaria elimination strategies, WHO recommends countries to use RDTs to support mass screening and treatment of malaria depends on the effect that it brings. Currently available commercial malaria RDTs is mainly targeted to detect histidine-rich protein 2 (HRP2) in the case of P. falciparum and that of plasmodium lactate dehydrogenase (pLDH) for non-falciparum species [8, 9]. HRP2 is only expressed by P. falciparum encoded by Pfhrp2 gene. The gene is located in chromosome 8 with flanking regions of MAL7P1_228 and MAL7P1_230 in the upstream and downstream of Pfhrp2, respectively. P. falciparum also expresses HRP3 protein, which is encoded by Pfhrp3 gene in chromosome 13 [10, 11].

These antigens found in peripheral circulation of P. falciparum infected patients and detected by RDTs, which is tagged with nitrocellulose membrane where monoclonal anti-HPR2 antibody is coated [11]. Although this antibody is supposed to detect mainly HRP2 antigen, structural motif similarity between HRP2 and HRP3 contribute to cross reactivity of the antibody with HRP3 [12]. Recently, different studies across the world reported high prevalence of genetic deletion of Pfhrp2 and Pfhrp3 in P. falciparum species. A study from Colombia revealed that the proportion of Pfhrp2 gene deletion was 38.5% among P. falciparum tested positive samples while that of Pfhrp3 was 45.5%. In these samples, at least 90% of upstream and downstream flanking genes for both Pfhrp2 and Pfhrp3 were also deleted. All these samples were tested negative by malaria RDTs although parasitaemia density was equal or above 500 parasites/μL [13]. A study from Peru had also reported that eight among nine P. falciparum tested positive samples had Pfhrp2 and Pfhrp3 genes deletion and ELISA negative result for HRP2 and HRP3 proteins [14]. Whereas a study from Ghana presented that about 36.2% of confirmed P. falciparum positive samples had Pfhrp2 gene deletion [15]. Polymorphism of HRP2 antigens and alteration of antibody recognition sites is also another challenge in the use of RDTs for the diagnosis of malaria [16–18].

Field performance of malaria RDTs, therefore, is not only about the quality of the kits but also the genotypes of the parasite. WHO puts endeavors to maintain the quality of RDTs and suggests countries to procure only WHO prequalified RDTs, to create a national legal framework for batch testing and quality control activities of RDTs and to investigate suspected false negative RDTs results [2, 9].

Although contradicting results of RDTs as compared to microscopy and molecular techniques have been reported in Ethiopia, information on the current genotype status of Pfhrp2 and Pfhrp3 in P. falciparum is not known in the country. Therefore, it is much worth important to reveal the existing genetic profile of Pfhrp2 and Pfhrp3 genes of P. falciparum from infected patients to provide baseline data about the use of RDTs in Ethiopia to support the country's malaria elimination strategic plan.

2. Methods

3. Results

4. Discussion

Malaria RDTs device is the mainstay of malaria diagnosis, and are sought to support malaria elimination strategies through community mass-screening [9, 22], in resource limited malaria-endemic world such as sub-Saharan African regions, where P. falciparum is the most common cause of illness and death [1]. Malaria HRP2-based RDTs target to detect HRP2 antigen, which is encoded by Pfhrp2 gene of all P. falciparum species and is secreted into peripheral circulation of falciparum-infected patients. Therefore, a defect happen in the Pfhrp2 gene affects the production of normal HRP2 antigen. Recent data from Latin America suggest deletions of Pfhrp2 and both of its flanking genes and associated negative results from RDTs device in the diagnosis of falciparum malaria [13, 14].

Our study revealed deletion of Pfhrp2 gene in 63.6% (21/33) samples and deletion of Pfhrp3 gene in 84.9% (28/33) samples. This supports an increasing widespread of deletion of Pfhrp2 and Pfhrp3 genes of P. falciparum in Africa, where it is being reported most recently. Similar studies in the East African countries such as Eritrea, and Ethiopia revealed a 9.7% and 43%, and 27.3% and 30.5% deletion of Pfhrp2 and Pfhrp3 genes, respectively, in falciparum-positive patient samples [23, 24]. Another study in Djibouti 68.6% [25] 86.5% [26] Pfhrp2/Pfhrp3 gene deletions report.

Among a limited number of samples in our study, we reported a higher proportion of deletion of both Pfhrp2 and Pfhrp3 genes than any other similar studies in East African countries, supporting a previous study in the same region of the country presenting a low sensitivity of RDTs to detect P. falciparum malaria as compared to molecular techniques [5]. Although our study did not provide RDTs results for the entire samples, low sensitivity of RDTs could be contributed from false negative results. Given that false-negative results of RDTs could be due to several factors such as storage condition of the device, RDTs lot type, wrong interpretation of the results, parasite density, and genotype of the malaria parasite and polymorphism of the antigens to be detected [12, 15, 27, 28].

Although RDTs handling and technical errors occur while working with RDTs would be improved through retraining, false-negative results due to alteration of genotype of the malaria parasite may be challenging. The implication is therefore all false–negative results from RDTs device need to be investigated in-depth for the essence of malaria elimination and monitoring programs.

Since the detection of HRP2 protein in the plasma of patients infected with falciparum malaria is confirmed [29, 30], HRP2-directed diagnosis and treatment of falciparum malaria has become common. The fact that HRP2 protein is important in the pathogenesis of P. falciparum [31], and has been suggested to prevent parasite intoxication from hemozoin [32], deletion of Pfhrp2 gene has been reported to occur independently among all P. falciparum species.

Although there is no clear evidence demonstrating the root-cause of deletion of this gene, frequent use of HRP2-based RDTs for the diagnosis and treatment of malaria has been suggested to create selective pressure to the Pfhrp2 gene [11, 33]. Taking the role of HRP2 in the detoxification of heme for the survival of the parasite and the mode of action of coartem on heme during the treatment of falciparum malaria in to consideration [34], presumably continuous use of coartem may induce pressure on Pfhrp2 gene. RDTs device and coartem are introduced in 2004 into Ethiopia and are widely used in the diagnosis and treatment of falciparum malaria, respectively [35].

This may comply with a high prevalence of Pfhrp2 gene deletion in Ethiopia as in our study. Although the first report of deletion of Pfhrp2 gene has come out from Latin America in 2010 [14], the origin of deletion of Pfhrp2 gene has been suggested to be from multiple clonal lineages and supposed to be dated as back as 1998 [11]. In this regard, we are not sure of the origin of deletion of Pfhrp2 gene in Ethiopia in our study. Given that both HRP2 and HRP3 antigens are detected in peripheral circulation of falciparum infected patients, and have structural motif similarity [12], RDTs positivity was reported in the presence of only Pfhrp3 regardless of Pfhrp2 gene [36, 37].

However, it has been suggested that the presence of high parasitaemia level is detrimental for RDTs positivity. Our study revealed only 9 (27.3%) samples which contain at least either Pfhrp2 or Pfhrp3, with high proportion of deletion of Pfhrp3 gene. The fact that the locus of Pfhrp2 and Pfhrp3 genes is in different chromosomes of P. falciparum, their expression seems to be independent to each other. From this point, these genes seem to be evolved independently to attribute both genetic polymorphism and gene deletion.

However, according to recent genetic studies of P. falciparum across the world, deletion of both genes simultaneously at different rate seems to be unclear. A significant number of P. falciparum parasites in Ethiopia, have been found to lack the HRP2 gene [38]. This mutation leads to the absence of HRP2 antigen production, preventing the detection of these parasites in patients' blood using RDTs that are designed specifically for HRP2. HRP2-based RDTs are widely recognized as the most sensitive, stable, and commonly utilized tests for diagnosing P. falciparum malaria.

Our study provides clear evidence of widespread deletions in the Pfhrp2 and Pfhrp3 genes in Ethiopia, thereby confirming anecdotal reports of diagnostic failure with HRP2-based RDTs in the region. The implications of our findings for the existing diagnostic framework, which depends on HRP2-based RDTs to detect P. falciparum in remote regions, may require reevaluation. Initial prospective surveys investigating the deletion of the HRP2 and HRP3 genes in P. falciparum populations have been completed or are nearing completion in Ethiopia.

One of the limitations of our study is that RDTs test was not performed for all the samples analyzed for molecular genotyping of the Pfhrp2 and Pfhrp3 and the respective flanking genes and, therefore, performance of locally available RDTs to these samples is not obtained.

Data Availability Statement

Data are available upon request.

Ethics Statement

This study is an extension from an epidemiological study of malaria among children suspected for malaria that has been ethical approval (Reference number: CMHS 278/04/08) was obtained from Ethical clearance was obtained from the research and ethics review committee of the University of Gondar, Gondar, Ethiopia. Permission was obtained from Zonal Health Administrative and participating health centers. Additionally, after explaining the importance of the study, informed written consent was obtained from the study participant's parent/guardian. Children who were positive for malaria infection were handled by the respective health facility according to the malaria diagnosis and treatment guidelines of the country.

Consent

This study does not contain any individual or personal data.

Disclosure

This abstract was offered at a conference (Abstract 297) [39].

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

S.G. and H.N. conceived the study. S.G., M.B., G.G.M., and A.A. designed the study and involved in the data collection. A.L.M., A.A., G.G.M., and S.G. performed statistical analysis and drafted the manuscript. A.L.M. carried out the molecular analysis of the samples. All authors critically reviewed and approved the manuscript.

Funding

No funding was obtained for this study. The authors would like to thank the University of Gondar for supporting the finance of the study.