Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017–2019

In 2018, the World Health Organization (WHO) estimated there were 228 million cases and 405,000 deaths globally due to malaria. The WHO African Region continues to contribute a disproportionately high share of the global burden (93% of malaria cases and 94% of malaria deaths) in 2018 alone [1]. Nearly all malaria cases in the region are caused by Plasmodium falciparum. Uganda is ranked among the six highest burden countries [1]. The 2018 and 2014 Uganda national malaria indicator surveys have reported overall malaria parasite prevalence of 9 and 19%, respectively [3, 4]. Plasmodium falciparum is the most predominant parasite in Uganda, accounting for > 95% of malaria infections [3, 5].

The WHO recommends parasitological confirmation of malaria in all suspected cases prior to treatment with artemisinin-based combination therapy (ACT) [6, 7]. The Uganda National Malaria Control Division adopted this policy and shifted from clinical to parasite-based diagnosis with microscopy or rapid diagnostic tests (RDTs) in 2011 [6, 7]. Since the introduction of RDTs in late 2000s, over 800 million RDTs have been used for malaria testing in Uganda which has led to increased access to parasite-based diagnosis [2, 8]. A similar increase has been seen particularly in the African region, where large volumes of histidine rich protein 2 (HRP2)-based RDTs are used due to the predominance of P. falciparum species in this region [1, 2]. However, RDTs must remain effective and accurate in detecting the presence of parasites in order to be useful in supporting diagnostic and surveillance programmes [9‐11]. There are several documented factors that have been known to affect the accuracy and functionality of RDTs that range from product design, transport or storage conditions, parasite factors due to gene deletions, operator-related factors and host parasite densities [12, 13]. Many endemic countries in collaboration with the WHO and the manufacturers have instituted quality assurance systems to address the possible causes of false RDT results [14, 15], however, parasite gene deletions have not been studied at a wider scale in many parts of Africa and evidence remains limited [12, 16]. Studies have suggested the possibility of evolution of gene-deleted parasites by a genetic event due to selective pressure resulting from long-term use of HRP2-based RDTs [17]. Failure of the parasite to express the HRP2 target antigen, alteration in the HRP2 protein sequence or pattern of histidine repeats and variation in the number of repeats have been known to affect the sensitivity of HRP2-based RDTs [18‐20]. Although investigation of other causes of false negative RDTs was outside the scope of this study, several studies have provided possible explanations for their occurrences. They include variation in the composition of pfhrp2 repeat sequence, number of repeat types and the amino acid composition of the HRP2 all of which may have impact on RDT sensitivity [19, 21].

Due to the predominance of P. falciparum in Uganda, the national policy recommends exclusive use of HRP2-based RDTs for malaria diagnosis [7]. The principal target recognized by HRP2-based RDTs are HRP2 antigens although, due to similarity in amino acid sequences, antibodies cross-react with HRP3 [5, 7]. These antigens are not expressed in malaria parasites in some parts of Africa due to the absence of the pfhrp2 and pfhrp3 genes [12, 16, 17, 19, 22‐28]. When P. falciparum parasites express little or no pfhrp2/3 target antigens, they are not detected by HRP2-based RDTs, threatening the usefulness of HRP2-based RDTs as a diagnostic test [11, 12, 25]. This poses a public health threat as a large number of infected patients will go untreated, leading to increased risk of malaria morbidity, mortality and transmission [12, 16, 17, 22].

The WHO recommends routine surveillance of pfhrp2 and pfhrp3 gene deletions in malaria parasites in countries that are neighbouring areas where deletions have been confirmed or where there are reports of false negative RDTs [9‐11, 16]. Surveillance data on parasite gene deletions could potentially inform national malaria diagnostic policies regarding choice of RDTs [11, 12]. A policy switch to more effective, alternative RDTs is recommended when the prevalence of false negative RDT results due to pfhrp2/3- gene deletions exceeds 5% [10, 11]. A threshold of 5% was selected because it is somewhere around this point that the proportion of cases missed by HRP2 RDTs due to non-HRP2 expression may be greater than the proportion of cases that would be missed by less-sensitive pLDH-based RDTs [29].

Parasite pfhrp2/3 gene deletions have been reported in areas neighbouring Uganda, including Kenya, Democratic Republic of Congo (DRC), Rwanda, and Eritrea [17, 28, 30, 31], however data on their occurrence and distribution in Uganda are limited. Only one study in Uganda reported the existence of pfhrp2/3 gene deletions in nine (9/416) PCR-confirmed parasite isolates, however its scope was limited to archived samples in one district [32]. The magnitude, extent of spread and the possible factors associated with the pfhrp2/3 gene deletions in Uganda is poorly understood. To improve understanding of the extent and spectrum of pfhrp2 and pfhrp3 gene deletions in Uganda on a wider scale, surveillance of pfhrp2 and pfhrp3 gene deletions was conducted in P. falciparum parasite populations in 48 districts of Uganda.

This is the first large-scale survey reporting the presence of pfhrp2 and pfhrp3 gene deletions in P. falciparum parasite isolates in Uganda. The methods used in the study are adopted from the WHO-recommended protocol for investigation of pfhrp2 and pfhrp3 gene deletions [11]. Samples were confirmed for the presence of parasite DNA and gene deletion classifications were made following the WHO recommended procedure, i.e., quality assured DNA quality by amplifying single copy genes msp1 and msp2 before performing the pfhrp2 and pfhrp3 gene specific PCRs [11, 12]. These methods have been used and widely published in many studies [12, 16, 17, 25].

The study objectives were to determine the proportion of pfhrp2/3 gene deletions in the parasite isolates, extent of spread and investigate the possible factors associated with these deletions. Overall, it was observed that the gene deletions were present in 9.7% (95% CI 6.6–13.6) of the P. falciparum parasite isolates in the exon1 and exon2 of pfhrp2/3 genes. The gene deletions occurred in both surveyed regions but were disproportionately higher in eastern Uganda 14.7% (9.7–20.9), p = 0.001. The specific gene deletions were pfhrp2(−)/pfhrp3(+) 3.3% (CI 1.6–6.0), pfhrp2(+)/pfhrp3(−) 3.0% (CI 1.4–5.6) and pfhrp2(−)/pfhrp3(−) 3.3% (CI 1.6–6.0). A higher proportion of pfhrp2 and pfhrp3 gene deletions was observed in samples that were RDT negative but microscopy positive (RDT−/microscopy+), 14.5% (9.5–20.9%), p = 0.001. GIS mapping of parasite locations showed clustering of the gene deletions close to the Uganda-Kenya border in eastern Uganda and near the Uganda-DRC border in western Uganda (Fig. 3). Overall, a significant proportion of this P. falciparum parasite population contained the pfhrp2 and pfhrp3 genes 62.0% (55.9–67.2), p = 0.001.

The relatively low proportions of gene deletions observed in this study suggests that most parasite isolates were able to express HRP2 antigen (185/300) 62.0% and therefore HRP2-based RDTs will still be useful for malaria diagnosis in these areas. However, the fact that a proportion (24/300) of the P. falciparum isolates lacked the pfhrp2/3 genes and evaded detection and subsequent treatment is of concern. In view of the fact that the HRP2-based RDTs are widely deployed in Uganda, the occurrence and confirmation of pfhrp2/3 gene deletions in P. falciparum parasites may have implications for malaria case management and surveillance, particularly in areas where they have been mapped and located. It is important to conduct follow-up surveys to monitor their prevalence as recommended by the WHO [9, 11]. However, the proportion of gene deletions observed in Ugandan parasite isolates is lower than what was reported in Eritrea and Rwanda [17, 28]. It is however higher than the levels reported in Kenya, Tanzania, DRC, Ghana, and Mali [22‐24, 26, 27, 30, 31, 39, 40]. The specific gene deletions of pfhrp2(−)/pfhrp3(+), pfhrp2(+)/pfhrp3(−) and pfhrp2(−)/pfhrp3(−) were generally lower compared to what has been reported in neighbouring countries. An important point to note however is that the comparison pfhrp2 and pfhrp3 findings across studies in Africa is challenging due to the wide variations in methods and computations of proportions using different denominators [12, 16]. Harmonization of methods for investigation of gene deletions based on WHO-recommended protocol will allow better comparison between studies [11, 16].

As expected, high proportions of gene deletions were observed in samples that were RDT negative but microscopy positive for malaria (RDT−/micro+) compared to those that were RDT and microscopy positive (RDT+/micro+). This indicates that gene deletions are one of the contributors to false negative RDT results in Uganda. However, the presence of non-P. falciparum species (n = 32) and low parasite densities as indicated by low quality DNA (n = 86) particularly in RDT-/microscopy + samples suggests that the two could have contributed to false negative RDTs. The contribution of gene deletion to false negative RDTs has been observed and reported elsewhere in previous studies [12, 17, 19, 22, 26‐28, 30, 31]. The occurrence of fewer deletions in RDT +/micro + samples supports the assumption that the isolates still harbour the pfhrp2 genes and are therefore able to express the HRP2 antigen. The WHO protocol recommends the RDT-/microscopy+ category as the most suitable samples for analysing gene deletions [10, 11]. However, in this study P. falciparum isolates with pfhrp2/pfhrp3 gene deletions were also detected in the RDT+/microscopy+ category of samples, 3.7% (95% CI 1.2–8.4%). This observation supports the previous findings suggesting cross-reactivity between the HRP2 and HRP3 [11, 12, 16, 17, 25, 41]. The detection of pfhrp2/pfhrp3 gene deletions in the RDT+/microscopy+ category of samples suggests the possibility of underestimation of the true proportions of deletion in studies that limit themselves to the RDT−/microscopy+ samples only.

Despite the occurrence of P. falciparum gene deletions in both surveyed regions, they were significantly higher in eastern Uganda, 14.7% (CI 9.7–20.9), p = 0.001. Using latitude and longitude coordinates, areas where all the P. falciparum gene-deleted isolates occurred were mapped and located. Although the gene-deleted parasites occurred across the two regions, they were more clustered close to the Uganda–Kenya border and in mid-eastern Uganda. The occurrence of gene deletions in the mid-eastern region had been reported previously in one district in 9 isolates (seven pfhrp2 and two pfhrp3 deletions) out of 416 PCR-confirmed samples and this study confirms this finding [32]. Some gene-deletion clustering was also observed near the Uganda-DRC boarder in western Uganda. Geographical clustering of pfhrp2/pfhrp3-deleted parasites could be explained by selection pressure as a result of selective treatment of RDT-positive P. falciparum parasites [30]. In view of these findings, the occurrence of deletions in both western and eastern regions of Uganda may have implications for future consideration of RDT deployment and establishment of surveillance systems. Epidemiologically, some parts of the mid-eastern region of Uganda have persistently reported a higher malaria burden, however clinical diagnosis and non-adherence to RDT results remains one of the highest in the country [4, 8]. Clinical diagnosis has poor specificity and may miss identification of true parasitaemic patients, which potentially allows survival and selective pressure of gene-deleted parasites [12, 17]. Follow-up studies should investigate the role of pfhrp2/3 deletions in causing false negative RDTs, severe disease and sustaining malaria burden around this region. Koita et al. showed the potential of pfhrp2/3 gene-deleted parasites to cause severe disease in Mali [22]. Regional and geographical variations in proportions of gene deletions observed in this study are consistent with what has been reported elsewhere in DRC, India and Eritrea [13, 16, 17, 30]. In Eritrea, pfhrp2/3 deletion varied between hospitals in different locations of the country [17]. In DRC and India, the proportions of gene deletions varied across provinces and states [13, 30]. Geographical clustering of pfhrp2 and pfhrp3 gene-deleted parasites was reported in malaria-endemic regions of eastern DRC and western Kenya suggesting a possibility of cross-border transmission [30, 31]. The gene deletion mapping data obtained in this study could inform better targeting of pfhrp2/3 follow-up surveys to monitor the levels of deletions. WHO recommends the establishment of surveillance systems that particularly focus on catchment areas where deletions have been reported and where false RDTs results are being reported [11, 16].

The study explored the possible factors associated with gene deletion in this study. As reported elsewhere, geographical location was an important factor for gene deletion [30]. The deletions were more likely to be detected in the eastern compared to the western region, aOR 6.25 (95% CI 2.02–23.55), p = 0.003. This observation was seen in similar studies in the DRC, India and Eritrea [13, 17, 30]. The association between geographical location and gene deletion may be explained by the evolutionary mechanism of migration or spontaneous occurrence of genetic events in parasites in a specific locality [17, 19, 25, 42, 43]. Although malaria endemicity and overall gene deletion were not associated, aOR 0.78 (95% CI 0.32–1.91) p = 0.579, the pfhrp2-/pfhrp3+ gene deletions were less likely to be found in moderate- compared to low-transmission areas aOR 0.19 (95% CI 0.03–0.88), p = 0.049. A similar observation was reported by Koita et al. in Mali and Berhari et al. in Eritrea [17, 22]. This observation suggests that pfhrp2/pfhrp3 deleted parasites may be easier to detect in areas of low transmission intensity where polyclonal infections or co-infection with wild-type parasites that could trigger a positive RDT and mask the presence of a pfhrp2 deleted parasite are less likely to occur [12]. The increased risk of gene deletion in parasite isolates in low transmission settings has been explained by reduced multiclonal infections so that parasites with gene deletions are likely single clone infections and can be detected readily by the PCR method [12, 17]. The implication here is that in high transmission settings such as most parts of Uganda where multiclonal infections and co-infections are common will require robust and novel diagnostic tools to investigate gene deletions.

However, this study was not free from limitations. The study was not able to explore and investigate clinical correlates between the pfhrp2/pfhrp3 infected and the naturally occurring wild type P. falciparum parasites due to limited clinical data available. It is important that follow-up studies explore to understand the virulence and pathogenicity of pfhrp2/3 deleted parasites particularly in causing severe disease and if these parasites show different drug susceptibility patterns to the current anti-malarial medicines. Also, other known possible causes of false negative RDTs [12] were not explored in this study that could be considered in follow-up surveys.

Furthermore, the study was limited by the fact that the P. falciparum isolates analysed were obtained from only two regions of Uganda, which leaves the status of pfhrp2/3 gene deletions in other regions unknown. It is recommended that future surveillance programmes should consider a more representative sample covering all regions of Uganda. It is equally important to note that the initial surveys where the P. falciparum isolates were obtained were not specifically designed for the pfhrp2/3 gene surveillance, which could have had impact on the characterization and selection of samples.

It is important to recognize the difficulties associated with detection of deletions in multiclonal and co-infections with wild-type parasites that could trigger a positive RDT and mask the presence of a pfhrp2/3-deleted parasite strain. In this case the HRP2-based RDTs will be positive based on the wild-type parasite that is able to express HRP2 antigen, while the masked pfhrp2/3 gene-deleted parasite remains undetected causing underestimation of deletions. However, this has been a challenge in many published studies due to limitation with the currently available molecular tools [12, 30].

While all the RDT−/microscopy+ samples were included in the analysis, only a random sub-set of the RDT+/microscopy+ was included on assumption that all the other (RDT+/microscopy+) samples contained parasites that expressed the HRP2 antigen. However, it is difficult to ascertain whether the RDT positivity was due to HRP2 or HRP3 expression since the two protein antigens share common epitopes due to high degree of similarity in amino acid sequence. This could have been a potential underestimation of pfhrp3 deletion [12, 13].

Despite PCR confirmation, some samples in the RDT−/microscopy+ sub-set had low-quality DNA and could not amplify the two single copy genes probably due to low parasite densities. As recommended, such samples were not included for pfhrp2/3 gene amplification, which could have led to possible underestimation of deletions [11, 12, 16, 29].

This study provides the first evidence on a large scale of the presence of pfhrp2 and pfhrp3 gene deletions in P. falciparum isolates in Uganda. Deletions occurred in both the eastern and western regions of Uganda but were more marked in the east. Proportions of gene deletions were higher in (RDT−/microscopy−) samples compared to (RDT+/microscopy+). In view of these findings the roll-out of RDTs for malaria diagnosis will need to take into consideration the pfhrp2/3 gene deletions in these regions. Periodic pfhrp2/3 surveys will be important to inform future decisions for deployment of alternative RDTs in Uganda.