Genetic Variations of Plasmodium Falciparum Histidine-rich Protein 2 and 3 in Assosa Zone, Ethiopia: Its Impact on the Performance of Malaria Rapid Diagnostic Tests


 Background: Rapid diagnostic tests (RDT) are commonly used for the diagnosis of Plasmodium falciparum malaria. However, false negative results of RDT caused by genetic variations of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. Methods: A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger prick samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. Results: The PfHRP2 repeat type isolates were less conserved compared to the PfHRP3 repeat type. A total of eleven and eight different PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 isolates. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3 % in PfHRP3 sequence isolate at parasitemia levels >250 parasites/µl. Using the Baker model, 100 % of the isolates in group A and 73.7% of the isolates in group B were predicted to be detected by PfHRP2 RDT at parasitemia level> 250 parasite/μl. Conclusion: The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level.


Study area and Period
This study was carried out during the low and high transmission seasons in four selected health facilities: Assosa, Bambasi, Kurmuk and Sherkole Health Centres in Assosa Zone, Benishangul-Gumuz regional state, northwest Ethiopia, April to December 2018. The study area has a high malaria transmission setting. The Assosa Zone is located on the border of Sudan, where transboundary transmission of malaria could occur. The map of the study area is indicated in additional le 1.

Study Design, Sample Size and Sampling Technique
A cross-sectional health facilities based study was conducted to assess genetic variation of pfhrp2/3 in clinical isolates. The study populations were residents of the Assosa Zone and the source of the population was all patients with clinical suspicion of malaria aged ≥ 5 years in the selected health facilities during the study period.
The sample size was calculated based on the single population proportion formula n = z 2 p(1-p)/d 2 [23]; Where, n = the sample size, z = 1.96 at 95% con dence interval (IC), d = margin of error at 5%, p(expected malaria prevalence rate ) is 40% prevalence of symptomatic malaria in a hospital study of the region [24]. As a result, the sample size calculated with 10 % non-response was 406 study participants. A total of 812 study participants were involved in this study, 406 study participants in low and 406 study participants in high transmission seseasons.
The study area, Assosa Health Centre, Bambasi Health Centre, Kurmuk Health Centre and Sherkole Health Centre, was selected using a simple random sampling technique among eight districts in the Assosa zone. Then, allocation of the study participant to each selected health centre was performed based on proportion of con rmed malaria case in each selected district/woreda.

Sample Collection
In a single gure-prick, capillary blood samples were collected from malaria suspected individuals for microscopy, malaria RDTs as well as dried blood spots (DBSs) for molecular assay. Over all work ow of this study indicated in Fig. 1.

Microscopy and Malaria RDT
The CareStart™ malaria RDTs (Pf/PV HRP2/PLDH) were used following manufacturer's instructions. Thick and thin blood smears were stained with a 10% buffer-diluted Giemsa stain working solution for microscopic detection and the measurement of parasite density according to WHO recommendations [25].
Parasite DNA extraction and molecular analysis Genomic DNA was extracted from DBSs using the chelex-saponin method as described previously [26]. Plasmodium falciparum identi cation was con rmed by SYBR Green quantitative PCR (qPCR) assay after ampli cation of 18S ribosomal RNA gene using species-speci c primers [27]. After con rming P. falciparum positive samples, all PCR reaction of pfhrp2 exon2 and pfhrp3 exon3 were performed at 25ul total with 2x Promega Hot Start Master Mix (Promega Corporation, Madison, USA), 0.4µl each of forward and reverse primers, and 3µl of extracted template DNA using PCR conditions previously described [18,22,28]. PCR product of pfhrp2 exon2 and pfhrp3 exon2 were separated by electrophoresis on a 2% agarose gel and visualized in a UV transilluminator and size of the expected amplicons compared to 1kb DNA ladder. Primer sequences, PCR conditions, and expected amplicon size of pfhrp2 exon2 and pfhrp3 exon2 are provided in additional le 2.

Sequencing analyses
A total of 48 pfhrp2 exon2 and 88 pfhrp3 exon2 high quality sequence data were included for molecular analysis of pfhrp2/3 genetic variation. Pfhrp2/3 sequence of this study deposited in the NCBI Gen Bank database (with Accession number: MI050658-MI050792). All amplicons were cleaned using Exosap and sequenced using Sanger technology with ABI BigDyeTM Terminator v 3.1 chemistry (Thermo sher, Santa Clara, CA) and ran on a 3130 Genetic Analyzer (Thermo sher, Santa Clara, CA). Sequences were then cleaned and analyzed using Codon Code Aligner Program V.6.0.2 (Codon Code Corporation, Centerville, MA). Nucleotide sequences were inputted into the ExPASy Translate Tool (Swiss Institute of Bioinformatics Resource Portal) and translated into corresponding amino acids sequence using the correct open reading frame. The amino acid repeat sequences in pfhrp2 and pfhrp3 were given a numeric code system as describe previously [8,11].

Data analysis
The proportions of each amino acid repeat of PfHRP2 and PfHRP3 in the P. falciparum isolates were analyzed using Statistical Package for Social Sciences (SPSS) version 20. The association between PfHRP2 RDT results (sequenced samples could be RDT positive or negative) and certain group of amino acid repeat was tested by Chi square and Fisher's exact test. P-values < 0.05 were interpreted as statistically signi cant.
Bakers' model used for the prediction of PfHRP2 RDT sensitivity, the sequences of PfHRP2 were classi ed into four groups based on combined length of types 2 × type 7 repeats. They include group A (≥ 100, very sensitive), group B (50-99, sensitive), group C (< 43, non-sensitive) and group I (44-49, borderline). The PfHRP2/3 amino acid sequences obtained from Ethiopia were compared with isolates from other countries based on the sequences deposited in the National Center for Biotechnology Information's (NCBI) GenBank using Basic Local Alignment Search Tool for Protein analysis (BLASTP).

Genetic variation of PfHRP2 and PfHRP3 amino acids repeats
The length of pfhrp2 exon2 sequences varied between 453 and 873 base pair (bp) ((ranged from 150 to 290 amino acids (aa)) among the 48 PfHRP2 sequenced samples. A total of 11 different PfHRP2 amino acid repeat types were identi ed (Table 1). Nearly 65% (31/48) of the distinct PfHRP2 amino acid sequence occurred only once, while ve PfHRP2 amino acid sequence patterns (I-V) were found in more than one isolate (Fig. 2). The structural organizations of the amino acid repeats were found at different positions of the PfHRP2 amino acid sequences. PfHRP2 amino acid sequences of all the isolates started with a type 1 repeat, 89.6% of the isolates ended with a type 10 repeat (occurred in ve PfHRP2 patterns and distinct PfHRP2 sequence), and 10.4% of the isolates with a type 12 repeat (occurred only in four distinct PfHRP2 isolates). About 48% (23/48) of the isolates had a PfHRP2 repeat motif composed of types 2, 3, 5, 7, 8, 2, and 7, and this motif was absent in all PfHRP2 patterns. About 15 % (7/48) of the isolates had a PfHRP2 repeat motif composed of types 7, 8, 2, and 7, and this motif was found in all PfHRP2 patterns except pattern III (Fig. 2). Types 2 and 7 were the most frequent repeats among isolates and were broadly distributed in PfHRP2 amino acid sequence.
The type and frequency of each PfHRP2 amino acid repeat varied among parasite isolates at study sites. Each PfHRP2 amino acid sequence contains 15-36 repeats. Repeat types 1, 2 and 7 were found in 100% of the isolates, repeat types 6 and 10 were found in 95.8% of the isolates, and type 5, type 3 and type 8 were found in 75-91.7% of the isolates being sequenced. Lower frequencies of PfHRP2 amino acid repeats were observed in type 4 (found in 20 isolates; 41.7%), as well as types 12 and 13 (found in 5 isolates; 10.4 %). By contrast, types 9 and 11 were not identi ed in any PfHRP2 amino acid sequences in this study (Table 2).  Almost all PfHRP2 repeat types were found in all study sites with a slight difference in the frequency of repeat type within and between the study sites. Repeat types 1, 2 and 7 were found in all isolates of Sherkole, Bambasi, Kurmuk, and Assosa, while repeat types 3, 5, 6, 8 and 10 were found in 66-100% isolates among study sites. On the other hand, repeat types 4 and 12 were occurred in 6.7-62.5% of the isolates among study sites, while type 13 was found only in Sherkole (12.5%; 3/24) and Bambasi ( 13.3 %; 2/15) (Additional File 3).
The length of the pfhrp3 exon2 ranged from 461 to 654 bp (i.e., 153 to 217 aa) among the 88 sequenced samples. Of 88 PfHRP3 sequence isolates, eight different PfHRP2 amino acid repeat types were identi ed (Table 1), while eight distinct PfHRP3 patterns detected in more than one isolate (Fig. 3). The PfHRP3 sequence began with type 1 (present in PfHRP3 pattern I-V), type 16 (present in PfHRP3 pattern VII-VIII), and type 15 (present in PfHRP3 pattern VI) repeats in 93.2%, 4.6%, and 2.3% of the isolates, respectively; and ended with type 4 (present in PfHRP3 pattern I-IV and VII-VIII), type 17 (present in PfHRP3 pattern V), and type 18 (present in PfHRP3 pattern VI) repeats in 87.5%, 11.4%, and 1.1% of the isolates, respectively.
All isolates of the PfHRP3 amino acid sequence in this study contained a non-repeating sequence (ANHGFHFNLHDNNSHTLHHAKANACFDD) between types 7 and 20. Most of the isolates (96.6%; 85/88) had a similar unique PfHRP3 repeat motif with structural organization of repeat types 15, 16, 20, 17, and 18, and this motif was found in all PfHRP3 patterns except type VII (Fig. 3). Comparison of Ethiopian Novel variants of PfHRP2/3 repeat types with global.
A total of 18 novel variants of PfHRP2 repeat types were found in this study (Table 3). Of these novel PfHRP2 amino acid repeat sequences, 15 were not reported previously. Though type 7 (AHHDD) and type 10 (AHHAATHHATD, AHHAAAHHAND) repeats were reported previously (Additional le 6), they occurred at a very low frequency. Similarly, 13 new PfHRP3 repeat types were identi ed among the Ethiopian isolates. Among them, 12 were not previously reported. Only type 20 (SHHDG) was previously reported (Additional le 7) and it occurred at high frequency among the Ethiopian isolates. The asterisks ( †) show deletion position of amino acid code (double letter), Note: bold underline letter with yellow color indicate position of novel repeat with replacement of one or more amino acid compared to known repeat.

Discussion
The present study provide valuable information on genetic variations in the PfHRP2 and PfHRP3 amino acid repeat types, which could affect the performance of PfHRP2 RDT for P. falciparum diagnosis. The structural organization of PfHRP2 amino acid sequences was less conserved than PfHRP3 among the Ethiopia isolates. The sequence length and the number of amino acid repeat types of PfHRP2 and PfHRP3 in this study were comparable to those previously reported in Yemen [29] and Madagascar [16], but lower than those in Kenya [30] and Ghana [31] and higher than in India [32].
Eleven different PfHRP2 repeats and eight different PfHRP3 repeats were found at Sherkole, Bambasi, Kurmuk and Assosa with slight difference in the frequency of the repeat type within and between P. falciparum isolates in Ethiopia, similar to those reported in Africa [30,31] and worldwide [8]. The PfHRP2 repeat types observed in the present study were previously reported in Africa [29][30][31], Asia [8,32], and America [35,36]. BLAST analysis of the Ethiopia PfHRP2 and PfHRP3 amino acid sequences revealed the presence of similarities and shared identity with isolates from Kenya. Interestingly, the amino acid sequences similarities of PfHRP2 and PfHRP3 also extended beyond the border of Ethiopia, India, and Myanmar, which is also in agreement with a recent study from Ghana [31].
On the contrary, PfHRP2 repeat types obtained within this study showed certain difference from African and global reports in a number of ways. First, PfHRP2 type 12 repeat was found in a few isolates (10.5%) this did not align with previous studies from Kenya [30], Ghana [31]and central America [36]. However, studies from Senegal [34] showed the presence of type 12 repeats in a few isolates similar to the present study. Second, we did not nd type 9 and 11 repeats isolates within our study which is consistent with most studies from African countries [29][30][31], but type 11 repeat was reported in a few isolates from Madagascar [16], while type 9 was reported from Senegalese isolates [34]. Third, types 14-27 were completely absent in all isolates in this study. These results disagree with other studies which reported the rare occurrence of type 14 from Nigeria [8] and Madagascar [16], and type 19 from Kenya [30]. The possible explanation for such varied distribution repeat types may be due to random mutation and local selection or directional spread of deleted strains of pfhrp2 /3 throughout the world [37].
Concerning PfHRP3, the majorities of sequences started with type 1 and ended with a type 4 repeats in the Ethiopia isolates, which agrees with previous studies [30,32]. Moreover, all isolates of PfHRP3 amino acid sequence contained a singleton non-repeating sequence [11]. The ndings of PfHRP3 repeat types in the present study are consistent with previous reports from Kenya [30], Yemen [29], Ghana [31], and globally [8]. On the other hand, type 2 repeat was absent in all isolates of PfHRP3 sequence in Ethiopia as well as other parts of Africa [29][30][31], but type 2 repeat has been reported in PfHRP3 in few isolates from Kenya [30] and India [32]. This variation observed in PfHRP2 and PfHRP3 repeat types might be due to differences in geographical and transmission settings [8,38,39], frequency of exposure of drug and level of immunity of study participants [40,41], clinical versus asymptomatic study participants, and methods used for molecular analysis.
Interestingly, of all the novels repeat types identi ed in this study, 15 in PfHRP2 and 12 in PfHRP3 had not been reported elsewhere. Among all novel PfHRP2 repeat types, two unique repeat types (type 7-AHHDD, type 10-AHHAATHHATD) and one unique repeat type (type-AHHAAAHHAND) were found with low frequency in Ethiopia and also in a previous study from Ghana [31] and Kenya [30]. Among all novel PfHRP3 repeat types, a unique repeat type (type 20-SHHDG) was detected with high frequency in Ethiopia as well as in Kenya [30], China-Myanmar border [42], and India [32]. The emergence of novel PfHRP2 and PfHRP3 repeat types at low frequency could be initiated by replacement or deletion of one or more amino acid repeat type [43][44][45].
Density of parasites and genetic variation pfhrp2/3 gene are two most important factors that affect the performance of PfHRP2 RDT [8, 17,22]. In this study, a high proportion of PfHRP2 RDT positive samples was observed with parasitemia > 250 parasites/µl, but substantial numbers of PfHRP2 RDT false negative samples were detected in submicroscopic infections similar to previous reports [8, 46]. Baker's model indicated that the Ethiopian isolates mostly belong to groups A and B were predicated to be detected by PfHRP2 RDT at parasitemia level > 250 parasite/µl and thus aligns with previous studies found in Senegal [34], Madagascar [16], and India [47]. Interestingly, most of P. falciparum isolates in group C were detectable by PfHRP2 RDT at parasitemia level ≥ 200 parasite/µl. This nding partially disagrees with the Baker' model [11] that predicts if the length of repeat types 2 and 7 is below 43 (as in group C), it will alter detection sensitivity of PfHRP2 RDT and lead to false negative results [34].
In this study, the length of amino acid repeat in PfHRP2 (type 1, 2, 7) and shared repeats in PfHRP2 and PfHRP3 (type 1, 4, 7) were not statistically associated with the performance of PfHRP2 RDT, consistent with previous study [30]. Instead, PfHRP2 RDT positivity was signi cantly improved as the length of PfHRP3 repeat type 16 and 17 increased. Types 1, 4 and 7 were common repeat types in both PfHRP2 and PfHRP3 amino acid sequence, whereas types 16 and 17 were identi ed only in PfHRP3 in high frequency among all isolates. In line with this, PfHRP2 and PfHRP3 exhibit structural homology as exon 2 in both pfhrp2 and pfhrp3 encodes similar amino acids and cross reaction may play a role in the diagnosis of falciparum malaria [7,12]. Moreover, novel PfHRP2 and PfHRP3 repeat variants detected in this study might in uence the binding a nity to monoclonal antibody and affect the sensitivity of PfHRP2 RDTs [12]. As results, considerable proportions of false negative results were found in this study by PfHRP2 RDT, 38.5 % of PfHRP2 and 37.5 % of PfHRP3 sequence with novel variants.

Limitations
This study considered two limitations. First, samples represented a limited geographical area from Assoa, Ethiopia. Second, this study did not assess cross reactivity of the possible epitopes based on the amino acid repeat sequence of PfHRP2 and PfHRP2 using speci c monoclonal antibodies.

Conclusion
Page 10/15 The present study indicated, for the rst time, the existence of genetic variability of PfHRP2 and PfHRP3 amino acid repeats including novel repeats in P. falciparum isolates within and between study sites in Ethiopia. There is a need to closely monitor the performance of PfHRP2 RDT and examine the distribution of novel repeat type and shared repeat in PfHRP2/3, and unique repeat found in PfHRP3 broadly in Ethiopia.

Abbreviations
RDT: Rapid diagnostic tests RDT; HRP2: Histidine-Rich Protein 2/3, pfhrp2/3 gene: P. falciparum histidine-rich protein 2 and 3 genes ; DBS: Dried blood spots; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction, SPSS: Statistical Package for Social Sciences; WHO: World Health Organization; NCBI: National Center for Biotechnology Information's; BLASTP: Basic Local Alignment Search Tool for Protein analysis . Figure 1 Study ow chart for molecular analysis of pfhrp2/3 genetic variation. Note: DBS=Dried blood Spot, qPCR=quantitative PCR, Pf=P.falciparum, -Ve = negative, +Ve=Positive, N=number of samples, Figure 2 PfHRP2 structural organization in clinical isolates collected from Assosa Zone, Ethiopia. a. PfHRP2 patterns: Pattern I, IV & V in two isolates; Pattern III in three isolates; Pattern II among eight isolates. b. Thirty-one distinct PfHRP2 amino acid sequence occurred only once with different sample code letter (HShr, Lshr, HBab, LBab, Hkum, Lkum, HAss and LAss ). A total of 11 PfHRP2 amino acid repeat type is differentiated by color block with their respective repeat type number. Unique variant repeat (V) display by yellow color