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].
All PfHRP2 repeat types started with repeat type 1 in the Ethiopian P. falciparum isolates, similar to previous reports in Kenya [30], Yemen [29], Senegal [33], and outside Africa [8]. The majorities of the PfHRP2 repeat ended with type 10 repeat which showed discordance with the previous reports from Africa [29, 30, 33] and Asia [32]. On the other hand, a small proportion of the isolates (10.4%) ended with a type 12 PfHRP2 repeat coincide with reports from Senegal [34]. Two types of PfHRP2 repeat motifs structural organization repeat motifs (2, 3, 5, 7, 8, 2, and 7) and (7, 8, 2, 7), were identified [8, 30].
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 [29–31], 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 find type 9 and 11 repeats isolates within our study which is consistent with most studies from African countries [29–31], 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 findings 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 [29–31], 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 identified 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 [43–45].
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 finding 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 significantly 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 identified 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 influence the binding affinity 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 specific monoclonal antibodies.