Background
With the recommendation of the World Health Organization (WHO) that only parasitologically confirmed cases of malaria patients should be treated with appropriate anti-malarial drugs [
1], simple, reliable and species specific diagnostic methods for detecting malaria infections became absolutely necessary. Clinical diagnosis being unreliable and non-specific, it required confirmation through parasitological diagnosis which involved identification of parasites by their direct observation in patient’s blood [such as microscopy and quantitative buffy coat (QBC) test], through identifying parasite nucleic acids by molecular methods [such as PCR, loop-mediated isothermal amplification (LAMP) method, microarray] or parasite specific antigens/antibody by rapid diagnostic tests and other new strategies (such as mass spectrometry and flow cytometry). However, each of these methods had their own limitations and many of these tests are non-amenable for point-of-care diagnosis, particularly in resource-limited endemic regions.
Although microscopy served as the gold standard for malaria diagnosis, given its unavailability in resource-poor settings and requirement of technical expertise [
2], rapid diagnostic tests (RDTs) became the widely adopted alternative choice as onsite test for its ease to perform, comparable sensitivity with microscopy, not requiring electricity and being quick [
2‐
4].
Detection of malaria by RDTs are principally based on identification of one or more of the three antigens, such as histidine-rich protein-2 (HRP2), lactate dehydrogenase (LDH), and aldolase [
5,
6]. Of these, HRP2 is used for specific detection of
Plasmodium falciparum because of its exclusive expression in this species of human
Plasmodium at asexual and sexual phases in the blood stage infection [
7‐
10], while LDH and aldolase are pan-specific as these are produced by all human malaria parasites. Although LDH can also be used for specific detection of
P. falciparum infection, its sensitivity is less compared to HRP2 and, therefore, HRP2 has been used in almost all of the RDTs for
P. falciparum detection [
11]. However, increasing reports on variable test performances of same HRP2-based RDT in different endemic regions and different tests on panels of blood samples targeting PfHRP2 [
12] is of concern, and has been attributed to device-related factors (such as quality of manufacture, storage condition, techniques for carrying out the test and interpretation of the test results) [
13‐
15] and parasite factors (such as parasite density, quantity of parasite antigen produced or its persistence in peripheral blood and variability of target epitopes in antigen structure) [
11,
16,
17]. Of these, the most important parasite factors of the observed variability in sensitivities in recent years have been confined to lack of the
pfhrp2 gene in the parasite species resulting in no expression of the corresponding antigen [
17‐
22] or variability of target epitopes (its presence or absence and copy number variation) within the PfHRP2 antigen due to genetic diversity in the gene [
22‐
27]. Besides, the PfHRP3 antigen which shares structural similarities to some extent with PfHRP2 has been thought to cross-react with PfHRP2 antibody [
12] and may influence the diagnostic performance of PfHRP2-detecting malaria RDTs. While
pfhrp2 gene is located on subtelomeric region of chromosome 7 flanked by a pseudogene (PF3D7_0831900/MAL7P1.230) and a putative heat shock protein 70 gene (PF3D7_0831700/MAL7P1.228),
pfhrp3 is located on subtelomeric region of chromosome 13 and is immediately flanked by a gene of unknown function, (PF3D7_13721000/MAL13P1.485) in the upstream and a gene for acyl-CoA synthetase (PF3D7_1372400/MAL13P1.475) in the downstream. Both PfHRP2 and HRP3 share many structural similarities with signal peptide sequence located in both on exon 1, and exon 2 harbours histidine (H) and alanine (A) rich amino acid repeats [
20].
Since, malaria in Odisha is largely due to
P. falciparum infections (> 85%) followed by
P. vivax and
P. malariae [
28], HRP2-based RDT along with pan-specific LDH or aldolase had been considered the best choice for malaria diagnosis. Further, in Odisha, India, the majority of severe malaria and malaria related deaths are ascribed to
P. falciparum infections with few instances of severe malaria were due to
P. vivax [
29]. In such cases, failure of HRP-2 based RDT could hamper the early diagnosis and case management of severe patients which may largely affect the malaria control. Until now, except one study [
30] documenting the low prevalence of RDT negative
P. falciparum isolates from a small pocket of Odisha, the status of HRP-2 based RDT as a reliable and accurate method of diagnosing malaria in Odisha has not been evaluated systematically. It is unknown whether there exists genetic diversity in the
pfhrp2 or
pfhrp3 genes affecting HRP-2 detection of
P. falciparum infection throughout the state of Odisha or whether significant proportion of parasites are still capable of expressing the HRP2 antigens. In this paper, failure of HRP-2 based RDT in detecting
P. falciparum infections concomitant to gene deletion and genetic diversity in
hrp2/hrp3 genes are reported among clinical isolates across geographically diverse region of Odisha collected during different seasons. The information generated in this study would be useful for guiding malaria control strategies in this endemic region by providing clue for appropriate incorporation of RDTs in the management of malaria.
Discussion
Malaria in Odisha is mostly due to
P. falciparum infections and in majority of the areas microscopy is inaccessible advocating the use of RDT as the choice for diagnosing malaria in case management [
27,
37]. However, substantial evidences of PfHRP2-based RDT failure in diagnosing
P. falciparum infections in recent years from various parts of South America, Africa and Asia including India [
17‐
19,
21,
30,
38‐
40,
49] is worrisome. Although false negativity of HRP2 based RDT or RDT failure has been reported from two district of Odisha, India during rainy season [
30], its prevalence in other parts of the state and during different seasons are not documented yet. Therefore, the present cross-sectional study was conducted in 25/30 districts of the state representing all four geo-physical regions and in different seasons of the year in order to determine the prevalence of HRP2 based RDT failure. Further, the three major factors (parasite density,
pfhrp2/pfhrp3 gene deletion and polymorphisms of the
pfhrp2/pfhrp3 gene) affecting the performance of PfHRP2-RDT were also examined. The results showed RDT failure in approximately 15% of the microscopically confirmed mono-infected
P. falciparum samples with high proportions of this false negativity observed in EG and NP region of the state. Previous study conducted in India has reported up to 11% of RDT failure in certain region with EG region of Odisha has RDT failure documented to be 7.8% [
30], which is much lower than the present study from the same region (i.e. 20.8%). Since, PfHRP2-RDT is often used as the diagnostic tool for malaria treatment in these areas, failure in RDT and subsequent lack of treatment or improper treatment might have led to rapid selection and transmission of these HRP2 negative parasites. Further, incidence of malaria in EG and NP regions of the state being exceptionally high [
28], upon successful treatment of patients carrying RDT sensitive parasites, it is obvious that the proportions of undetected parasites would be comparatively more than other regions of the state as observed in this study. Besides, the involvement of factors affecting RDT performance like age dependent host immunity [
41], parasite density [
42,
43] or other confounding factors including mosquito preferences to RDT sensitive vs. PfHRP2 negative parasite cannot be ruled out for such large difference (7.8 vs. 20.8) in prevalence of HRP negative parasites. In spite of less number of samples collected during summer and winter (Table
1), the proportions of RDT negative for
P. falciparum was higher in these periods compared to samples collected during the peak season of transmission in rain. This observation is consistent with the earlier reports of fall of sensitivity of RDT with decrease in transmission intensity [
39,
44‐
46]. Similar to present findings, high prevalence of PfHRP
2-negative isolates in Mali has been recorded at the end of summer [
39]. Assuming that RDT sensitive and RDT negative parasites are transmitted with equal opportunities;
msp1 genotyping was performed to monitor multiplicity of infections only in RDT negative samples. The results showed low multiplicity of infections in RDT negative samples consistent to previous reports [
39] and that the frequencies of single strain infections were more in off season compared to peak season of malaria transmission (Fig.
3) which is at par with transmission intensity. Although there was no statistical difference in parasitaemia level between RDT sensitive and RDT negative samples, 7/9 RDT negative
P. falciparum infected patients with parasitaemia level well below 200 parasites/µl were recorded in low transmission period and that low parasite density at this level (i.e. < 200 parasites/μl) has been shown to affect PfHRP2 based RDT sensitivity [
36]. Therefore, the seasonal fluctuations in RDT performance in the present study can be attributed to lack or least mixed infection of PfHRP
2-negative isolates with RDT sensitive strain in low transmission period undermining the detection of
P. falciparum infection by RDT, withholding anti-malarial treatment in RDT negative malaria patients leading to rise in transmission of PfHRP2-negative isolates and low parasitaemia level resulting in insufficient production of detectable HRP2 besides other factors.
Genotyping results in RDT negative samples confirmed for the presence of gene deletions and polymorphisms at
hrp2 and
hrp3 genes among natural isolates in symptomatic patients throughout the state at varying proportions. Further, high prevalence of
hrp2 deletion were observed compared to
hrp3 deletion in all four regions. Similar predominant
hrp2 deletion strains have been reported in previous studies from India [
30] and Suriname [
21], which are in contrast to other studies [
17‐
19,
47,
38‐
40,
48,
49], where the frequency of
hrp3 deletion was higher. Interestingly, high proportions of
hrp2 deletion parasites were observed during summer, whereas the proportions of
hrp2 deletion and
hrp3 deletion parasites were nearly the same in peak season of malaria transmission (Table
1). Although the reason for such seasonal fluctuation in frequency distribution of
hrp2 and
hrp3 deletion parasite strains is not known, it is expected that more of the detection failure by RDT in low transmission period of summer would be due to
hrp2 deletion because of its high prevalence in the region compared to
hrp3, reduced co-infection (either with RDT sensitive,
hrp3 negative parasites or both) and successful treatment of patients infected with RDT sensitive parasites or HRP3 negative but HRP2 positive parasites (as HRP2 antigen is the main target for RDT detection) upon detection by RDT. This speculation is further supported by our observation of only SGI in winter, and appearance of MGI (2/9) at low frequency in summer compared to rainy season (17/45) among RDT negative samples despite small sample size. While double negative variants were obtained in 22.4% of RDT negative samples in the present study which is in consistent with the observations from Peru (21.6%) [
18]; other studies reported high prevalence [
49,
50] or lack of double negative isolates [
51]. The overall discrepancy in prevalence of
hrp2 and
hrp3 deletion parasites in different endemic regions could be due to region specific emergence and selection of corresponding deletion variant, transmission intensity and rate of genetic crossing, or geographical spread from neighboring province or countries. Haplotype analysis of singly infected parasites based on presence and absence of
hrp2/hrp3 and their flanking genes revealed 10 different patterns of parasite strain, of which, the four major patterns (Table
2, P1, 7, 8 and 10) were distributed all throughout the state indicating that no specific pattern is confined to any region of the state and the distribution is uniform.
Although low parasitaemia has been shown to influence RDT detection, the observation of gene deletions at
hrp2 and
hrp3 loci in both low and high parasitaemic patient along with no statistical difference at blood parasitaemia level between RDT sensitive and RDT negative isolates suggest that RDT failure in these samples was not due to low parasitaemia rather due to lack of HRP2 protein. This was also evidenced by undetectable PfHRP2 protein in ELISA test. Further, in HRP2 negative isolates (Table
2) confirming the presence of
hrp2/hrp3 and their flanking genes with considerable parasite density, the false negative RDT result could be due to variation in number, composition and types of sequence repeat as observed in this study. Similar variations in PfHRP2 and HRP3 proteins have been shown to influence HRP2 based RDT sensitivity [
12,
36] and have been reported from different malaria endemic regions among RDT negative isolates [
7,
18,
23,
30,
36]. Altogether, the findings of the present study suggest RDT failure in the study period was largely due to failure of parasite to express the antigen and/or due to alteration of PfHRP2/HRP3 protein sequence affecting the RDT performance. However, the role of low parasitaemia contributing to RDT negativity cannot be ruled out as off season samples with RDT failure mostly had low parasitaemia level. The occurrence and transmission of natural parasite isolates lacking both
hrp2 and
hrp3 though has been reported in several studies [
18,
30,
48,
50], the finding of low parasitaemia in these sub-set of patients in the present study suggest for their poor fitness compared to
hrp2 or
hrp3 negative parasites (as failure of producing HRP by one gene could be compensated by another). This assumption needs to be validated experimentally. In absence of definite function, it is difficult to predict the role of HRP in parasite virulence and fitness, however, its presence in all stages of development of parasite [
7‐
9] explain certain survival advantage though not essential for survival. The proposed explanation of detoxification of free haem converting it to haemozoin [
52,
53], modulation of infected RBC [
54] or host immune response [
55] favouring parasite competence to grow with in human host cannot be excluded.
The present study had several limitations. First, the microscopically-confirmed and RDT sensitive
P. falciparum samples have not been examined for quantitative estimation of HRP2 antigens and genotyping for
hrp2/
hrp3 deletions or their polymorphisms. The fact that sensitivity of RDT is greatly affected by HRP2 antigen, than parasitaemia [
56]; however, correlation of the initial level of blood parasitaemia with HRP2 concentration could not be made. Besides, whether absence of
hrp2 or
hrp3 is compensated by the presence of
hrp3 or
hrp2, respectively in RDT performance due to cross reaction in this region could not be determined. In such case, mixed infection with sensitive parasites or parasite with alternate
hrp deletion would underestimate the actual prevalence of
hrp2 and
hrp3 deletions. Moreover, comparison of sequence variation at
hrp2 or
hrp3 between RDT sensitive and RDT negative isolates could have resulted in identifying the region specific potential repeat type, or number of repeats or composition of amino acid sequence as optimal epitopes of RDT performance. Second, only symptomatic patients were screened for malaria diagnosis; however, RDT false negativity has been frequently reported among individuals with low or subpatent level of parasitaemia in asymptomatic patients in some study [
39]. As asymptomatic patients serve as the silent reservoir of malaria transmission, presence of HRP2 negative parasites in these patients could be the source of infection to mosquitoes or in blood transfusion for further transmission and may become an obstruction to elimination efforts of malaria. After all, the strength of the present study was that the natural
P. falciparum isolates from all four geo-physical regions of the state were screened for the prevalence of
hrp negative parasites. Besides, the study was conducted in three different seasons and
hrp negative isolates were detected in all throughout the year which is a major threat to malaria control programmes, if HRP2-based RDT becomes the choice of malaria diagnosis for case management.
Authors’ contributions
MRR conceived the original idea, supervised the project and evaluated the manuscript. PP sample collection, performed the experiments and prepared the first draft of manuscript. GD analyzed and interpreted the data, prepared all figures, manuscript drafting for important intellectual content. MB contributed to sample collection and verified the analytical methods. All authors discussed the results and contributed to the final manuscript. All authors read and approved the final manuscript.