Plasmodium falciparum histidine-rich protein 2 (PfHRP2) diversity in Ghana CURRENT STATUS: UNDER REVIEW

Background : In the absence of microscopy, Plasmodium falciparum histidine-rich proteins 2 (PfHRP2)-based rapid diagnostic tests (RDTs) are recommended for the diagnosis of falciparum malaria, particularly in endemic regions. However, genetic variability of the PfHRP2 gene threatens the usefulness of the test. This study aimed to investigate the diversity of PfHRP2 in malaria cases among children in Ghana. Methods : A cross-sectional study was conducted at the Adidome Government Hospital in the Volta Region of Ghana. A total of 50 children with mean age of 6.6±3.5 years and diagnosed of falciparum malaria were included. Blood samples were collected for complete blood count, malaria parasite identification and counting. DNA samples were amplified and sequenced. Nucleotide sequences were translated in silico to corresponding amino acids and the deduced amino acids sequences were analyzed for diversity. Results : The number of repeats and number of each repeat within PfHRP2 varied between isolates. Twelve rare PfHRP2 repeat types, two of which are previously unreported, were identified in this study. Our HRP2 sequence shared high similarity with isolates from Kenya. Using Baker’s regression model, Group B was the highest occurring type (58.0%). Screening of all sequences for epitopes recognized by PfHRP2-specific monoclonal antibodies (mAbs), we found the predominant motif to be AHHAADAHH, which is recognized by the C1-13 mAbs. Conclusion : This study reports diversity of P. falciparum histidine-rich proteins 2 in samples from Ghanaian children with symptomatic malaria. We highlight the existence of extra amino acid repeat types which adds to the PfHRP2 antigenic variability. The findings of this study will contribute to the understanding of the performance of PfHRP2-based RDTs in the Ghanaian setting.


Introduction
Malaria, which causes substantial morbidity and mortality, is a major public health problem in sub-Saharan Africa, Asia, and Latin America [1]. It claims the life of a child under five years every two minutes in sub-Saharan Africa and has annual infection and mortality rates of 191 million and 395,000 individuals, respectively [2,3]. In Ghana, malaria remains a major cause of loss of days of healthy life,

Study design/setting and participants
A cross-sectional study was conducted between January and June 2019 at the Adidome Government Hospital in the Volta Region of Ghana. The Volta Region has a perennial malaria transmission, with the predominant parasite being P. falciparum. The regional prevalence of P. falciparum malaria is 45.27% among children [24]. In the study, 50 children between the ages of 3-11 diagnosed of falciparum malaria were included. Samples were obtained prior to initiation of antimalarial therapy.

Sample collection and laboratory analysis
Three milliliters (3 ml) of blood was aseptically obtained from each participant and dispensed into K3 EDTA tubes. Complete blood count was performed on the anticoagulated whole blood using Sysmex KX-21N auto analyzer (Sysmex Corporation, Japan). To reassess falciparum malaria, thick and thin blood films were prepared and stained with 10% Giemsa for microscopic identification and counting of parasites. The parasite density was calculated by assuming a standard WBC count of 8000/μl or 4.5 million RBC/μl in accordance with WHO standards [25].
Additionally, 5 drops of the blood were spotted onto Whatman™ Filter Papers (Schleicher and Schuell BioScience, Inc., Keene, New Hampshire), air dried and individually kept in zip-lock plastic bag for subsequent PCR analysis Parasite DNA extraction and molecular analysis DNA isolation from Whatman filter papers was based on the Chelex-based technique as previously described [26]. PCR amplification of the exon 2 of the PfHRP2 gene was performed using the seminested amplification approach as previously described by Baker et al. [16]. PCR reactions were carried out in 25µl volume for the primary and 35µl volume for the semi-nested reactions. The forward and reverse primers targeting the exon 2 of the pfhrp2 gene are shown in Additional file 1: Table S1. For both primary and secondary PCR reactions, DNA was denatured at 96°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 50 seconds, annealing at 55°C for 50 seconds, extension at 68°C for 1 minute and a final extension at 72°C for 5 minutes. Genomic DNA from 3D7 (wild type) P. falciparum and nuclease free water were used as positive and negative controls, respectively. After secondary amplification, amplicons were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide and visualized under UV light. Sequencing of the PfHRP2 gene was performed by Inqaba Biotechnical Industries (Pty) Ltd, South Africa (https://www.inqababiotec.co.za/). Nucleic acid sequences were deposited at the National Center for Biotechnology Information (NCBI) (Genbank accession numbers: MT094447-79)

Data analysis
Mega X version 10.1.6 [27] was used for sequence analysis. Nucleotide sequences were translated in silico to corresponding amino acids using the correct open reading frame. Amino acid repeats were numerically coded based on previous reports [16,18,28]; the frequencies and percentages of each amino acid repeat were estimated. To compare amino acid sequences in this study with previously published data, we performed a protein-protein BLAST (BLASTP) analysis of our sequences with those in the GenBank database at the NCBI. Our PfHRP2 and homologous sequences downloaded from the NCBI were aligned using the ClustalW tool and a cladogram was built using the Maximum Likelihood method and Dayhoff matrix-based model, with bootstrap consensus tree inferred from 1000 replicates. Although rapid diagnostic tests (RDTs) results were unavailable, we sought to determine the diversity of PfHRP2 with respect to RDT sensitivity. To achieve this, the product of repeats type 2 and 7 was calculated; classification and interpretation was based on Baker's regression model [16].
Screening of our sequences for epitopes recognized by PfHRP2-specific monoclonal antibodies (mAbs) [29] were also performed.
Thirty-three different PfHRP2 amino acid sequences were identified among 50 PfHRP2 sequences obtained in this study. The size of PfHRP2 ranged from 225 to 304 amino acids among all isolates and 25 to 38 amino acid repeat types per isolate. The total number of repeats and the number of each repeat within PfHRP2 varied between isolates. Repeat types 2, 6, 7 and 8 were observed in 100% of the isolates. Repeat types 5 and 12 were observed in 98% whereas types 1, 3 and 10 were found in 92-96% of the samples. The repeat types 4 and 13 occurred in 26% and 10% of the isolates, respectively. None of the samples had repeat types 9 and 11 ( Table 2).
Twelve rare PfHRP2 repeat types, two of which are previously unreported, were identified in this study; the two were APDAHHVAD and AHHAAAHDEAALI. Of the rare repeat types, types 2 (AHHAHHAAH) and 7 (AHHAAH) had the highest frequency ( Table 3).
Predominant repeat types in this study were used to model the structural organization of PfHRP2 in Ghana. Although the structural organization of the PfHRP2 repeat types was variable, the repetitive regions found in most of our samples started with type 1 (94.0%) and all PfHRP2 sequences terminated with type 12 (100.0%) (Fig. 1a). Fifty-four percent of our isolates had a semi-conserved PfHRP2 repeat type motif composed of repeat types 2, 3, 5, 7, 8, 2 and 7. Partial amino acid repeat motif comprising types 7, 8, 2 and 7 was found in 34.0% of the isolates.
To explore the similarities between the modelled PfHRP2 sequence obtained from the Ghanaian isolates and those from other regions available at the NCBI, we performed BLASTP analysis of our amino acid sequence. Seven hits (Accession numbers: QBC65525.1, QBC65570.1, QBC65591.1, QBC65640.1, QBC65657.1 and QBC65674.1 from isolates in Kenya and AKO62989.1 from China-Myanmar border area) were obtained ( Fig. 1b; Additional file 1: Table S2). Our modelled HRP2 sequence shared 85-94% similarity with Kenyan isolates and 94% similarity with the isolates from China-Myanmar border area. BLASTP of the sequences from each of the 50 samples revealed that 78.0% (39/50) have high similarities with isolates from Kenya, highlighting possible shared identity between PfHRP2 from Ghana and Kenya (Additional file 1: Table S3).
Although data on rapid diagnostic tests (RDTs) was unavailable, an obvious limitation of the study, we employed the Baker model [16] to determine the distribution on the basis of PfHRP2 diversity with respect to RDT sensitivity. Isolates were classified as Groups A, B, I and C if their Baker repeat (type 2 × type 7) was >100, 50-100, 44-49 and < 43, respectively. Group B was the highest occurring type (58.0%), followed by group C (22.0%) ( Table 4).
Due to the relatively high percentage of group C isolates (22.0%), which have been reported to be associated with RDT non-sensitivity [30] obtained in this study, we explored the distribution of possible epitopes to be targeted by mAb RDTs based on the study by Lee et al. [29]. The predominant motif among the 50 isolates was AHHAADAHH which is recognized by the C1-13 mAb, followed by AHHAHHA, recognized by mAb 3A4. None of our isolates had the AYAHHAHHAAY motif, while the HAHHAHHAADAHH motif, recognized by C2-3, occurred at a lower frequency ( Table 5).

Discussion
In Africa, P. falciparum is the most common malaria-causing parasite. Microscopy is the gold standard for the diagnosis of malaria, however, in the its absence, RDT has been approved by the WHO for use in malaria diagnosis. Majority of the commercially available RDTs target the PfHRP2; however, the future benefit of these RDTs is in jeopardy due to reports of P. falciparum isolates which lack the PfHRP2 gene [15-19, 22, 23]. It is thus of public health significance to assess the diversity of the PfHRP2, especially in different parts of Africa where the disease exerts a high rate of morbidity and mortality, particularly among children.
Here, we present an analysis of the diversity of PfHRP2 among P. falciparum isolates from children in Ghana. To the best of our knowledge, this is the first study to present data on the diversity of PfHRP2 in the Ghanaian context. In contrast with a study by Amoah et al. in Ghana who reported PfHRP2 gene deletion in 33% and 36% of the microscopically-confirmed and PCR-confirmed RDT positive samples, respectively [21], all our isolates achieved successful sequencing of the PfHRP2 gene indicating absence of deletion. This finding is in harmony with studies by Baker et al. [16,20] and Nderu et al. After in silico translation of the PfHRP2 genes into amino acid sequences, our isolates shared some characteristics with previous published data. Repeat types 2, 6, 7 and 8 were found in all isolates whereas types 9 and 11 were absent. Few of our isolates had the type 4 repeat and only 10% had the type 12. All other repeat types were found in over 90% of the isolates. These findings are consistent with previous reports from other countries [18,20,28,[30][31][32].
We found the structural organization of repeat types to be highly diverse. Out of the 33 unqiue PfHRP2 sequences obtained, only a third occurred more than once. However, some repeat organizations were shared between isolates. Most of our sequences started with type 1 and all terminated with type 12 as is consistent with previous studies [18 -20, 28]. Additionally, a semiconserved PfHRP2 repeat type motif (types 2, 3, 5, 7, 8, 2 and 7) and partial repeat motif (types 7, 8, 2 and 7) were found in about half and a quarter of our isolates, respectively. This is similar to the findings of Baker et al. [20] and Nderu et al. [18]. Phylogenetic analysis of the Ghana PfHRP2 revealed striking similarities with isolates from Kenya. In a study along the Chinese-Myanmar border, novel PfHRP2 repeat types arising from replacement of a single amino acid of eight amino acid repeats types were identified [33]. Nderu et al.
[18] also found 39 novel PfHRP2 repeat types. In India, Bharti et al. reported 5 novel repeat types [19]. In this study, we identified two novel PfHRP2 repeat types (APDAHHVAD and AHHAAAHDEAALI) in addition to ten of the recently reported novel repeats which occurred at low frequencies. Together with previous reports, our findings support the presence of yet to be defined repeat types and highlights that these novel types occur at relatively lower frequencies.
PfHRP2 diversity has been repoprted to influence the diagnosis of malaria using PfHRP2-based RDTs.
In 2005, Baker et al. demonstarated, using logistic regression model, that the product of repeat types 2 and 7 affect inter-study sensitivity variation of PfHRP2-based RDTs, especially for samples with parasite densities ≤ 250 parasites/μL [16]. In 2010 however it was found, using isolates from different geographical areas, that type 2 × type 7 was not associated with RDT sensitivity [20]. Nonetheless, subsequent studies by Kumar et al. [30] in 2012 and Wurtz et al. [34] in 2013 observed an association between the Group C category (type 2 × 7 < 43) and RDT false negativity and reduced limit of detection. Most of our isolates were group B (type 2 × 7 = 50-100) as consistent with previous studies in Africa [18,35,36]. To investigate the distribution of possible epitopes to be targeted by monoclonal antibodies in RDTs, we searched for the eleven common epitopes recognized by commercially available mAb [29] among our isolates. The most occurring epitope was AHHAADAHH, recognized by the C1-13 mAb, followed by AHHAHHA, recognized by mAb 3A4. We did not detect the AYAHHAHHAAY motif in our isolates and HAHHAHHAADAHH occurred at a lower frequency. This is consistent with recent reports by Fontecha et al. [28] and Willie et al. [37] and corroborates with an earlier report by Lee et al. that RDTs which employ the C2-3 and Genway mAbs are less sensitive [29].

Conclusion
This study reports diversity of Plasmodium falciparum histidine-rich proteins 2 in samples from Ghanaian children with symptomatic malaria. Additionally, we highlight the existence of extra amino acid repeat types which adds to the PfHRP2 antigenic variability. The findings of this study will contribute to the understanding of the performance of PfHRP2-based RDTs in the Ghanaian setting. Technology. Written consent was obtained from parents and guardians of the children after the objectives of the study had been explained to them.

Consent for publication
Not applicable.

Availability of data and material
The datasets supporting the conclusions of this article are included within the article and its additional file.

Competing interests
The authors declare that they have no competing interests.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.   shows the position where the rare/novel repeat types vary compared to known repeat types.

Supplementary Files
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