Background
Morbidity and mortality due to
Plasmodium falciparum malaria have increased across Indian sub-geographical regions, largely because of the widespread resistance to chloroquine (CQ) and sulfadoxine–pyrimethamine (SP) leading to generation of regional heterogeneity in the target genes [
1,
2]. The rise in the incidences of malaria is largely attributed to the increase in the drug resistance parasites [
3]. The emergence of chloroquine resistance in India led to the change in the drug policy in the year 2005 to artemisinin-based combination therapy (ACT) as first-line of treatment [
4]. The
Pfcrt and
Pfmdr-
1 genes are associated with CQ resistance, whereas the
dhfr and
dhps genes are related with SP resistance in the malaria parasite caused by the mutations present in these drug resistance genes [
5]. The Kelch propeller domain (
k13-propeller) in
P. falciparum have been found to be associated with delayed parasite clearance after ACT therapy [
6].
Plasmodium falciparum is transmitted from the human host to the mosquito vector during the sexual stage via gametocyte. But the transmissibility of
P. falciparum is due to the relationship between the prevalence, duration and density of gametocyte carriage with variable and modulated by the immune response in the human host [
7]. Gametocytes from multi-clone
P. falciparum infections can persist three times longer than single-clone infections indicating that multiplicity of infection (MOI) promotes either longer persistence or continuous production of gametocytes [
8]. The occurrence of sub-microscopic gametocytes in the parasite population play a major role in malaria transmission. It has also been shown that the microscopic diagnosis is more likely to underestimate the prevalence of gametocytes and more so in the sub-patent population of gametocytes [
9]. For the successful elimination of
P. falciparum it is crucial to limit the transmission of malaria parasites which can be achieved by reducing the carriage of viable gametocytes, hence it is important to investigate the drug resistance genes with gametocyte genes [
9].
The aims of the present study were to determine the prevalence of Pfcrt, Pfmdr-1, dhfr and dhps mutations in drug resistance genes with k13 (particularly ACT with SP as partner drug and CQ) and Pfg377 genes with in vitro susceptibility assays in order to understand the genetic complexity of falciparum malaria in natural infections. The Pfcrt, Pfmdr-1, dhfr and dhps are directly implicated in parasite population turning resistant to anti-malarials and Pfg377 gene is considered under neutral/balancing selection. Coupling the analysis of drug resistance genes along with k13 with in vitro studies will give an insight about the circulating parasite population in the region and profiling the chloroquine markers will provide data to know if there is any change at the genetic level for these markers after a change in the treatment policy.
Discussion
The malaria situation continues to prevail even after intensive control efforts and remains persistent in India; as India is a major hot-spot for malaria transmission due to its unique favourable topography with respect to malaria survival [
20]. Drug resistance is a major hurdle in malaria control not only in the country but worldwide too [
21]. Continuous monitoring of the drug resistance provides warning signals to drug policy makers. The emergence of drug-resistant parasites in India is still on the rise even after the introduction of ACT in the country [
4]. Genotyping of
Pfcrt,
Pfmdr-
1,
dhfr, dhps genes and
k13 was carried out in field isolates of
P. falciparum collected from Ranchi, Raipur and Mewat to determine the prevalence of polymorphisms in these genes along with the neutral gametocyte gene viz
Pfg377. Not many such studies have been reported from India.
In the study, non-drug resistant
Pfg377gene showed lot of genetic diversity in the field isolates which agrees with the observed random association between the loci and absence of regional differentiation among the
P. falciparum isolates is in accordance with other reports [
22,
23]. CQ resistance (CQR) was monitored on the point mutation detected in the
Pfcrt and
Pfmdr-
1 genes by PCR–RFLP method where a high prevalence of mutant K76T
Pfcrt and N86Y
Pfmdr-
1 alleles was observed. This holds true with the previous studies stating the role of
Pfcrt gene crucial in detection of CQR in
P. falciparum field isolates [
24‐
26]. No linkage disequilibrium (LD) between the N86Y and K76T mutations in
Pfmdr-
1 and
Pfcrt genes was observed and more number of samples are to be studied to compare the LD association in
P. falciparum isolates. It is extremely important to routinely detect/analyse the pattern of drug resistance in malarial parasites which play not only an important role in the epidemiological surveys but also help in improvising/updating the anti-malarial drug policies of the country. The CQ resistance if
P. falciparum is mainly linked to the mutation in K76T codon of
Pfcrt gene followed by
Pfmdr-
1 gene N86Y [
24,
25].
SP are the partner drugs in the current ACT therapy of the country and we observed a high prevalence of more than one mutations in the
dhfr,
dhps genes of
P. falciparum [
27]. The double mutants 51I 108N in the
dhfr gene were present at a higher frequency in the population as compared to the triple mutant 51I 59R 108N associated with high resistance for SP was only seen in the field isolates from Mewat [
28]. It has been reported from Africa and South East Asia earlier that I51 R59 N108 are highly SP resistant haplotype and the presence of this genotype in the studied population reveals that the parasite population is possibly moving towards SP resistance where evidence of selection is seen earlier in
dhfr gene than
dhps gene [
29,
30]. This indicates the possibility of high resistant genotypes evolving in the parasite population due to constant drug pressure experienced by the parasite [
31]. The π values of nucleotide diversity were higher for
dhfr and
dhps genes in Mewat and similar in Ranchi and Raipur the endemic regions for malaria similarly documented from other places [
32]. Analysing the SNPs in these drug resistance genes to find new mutations will help us to understand genetic changes leading to altered phenotypic traits better and also give an insight into the evolving genetic recombinations with respect to the current drug therapy [
33]. The usage of ACT in the national drug policy since a decade is expected to decrease the CQ resistance and restore the CQ sensitive parasite population as such a change has been reported from several countries and 31.8% wild type of isolates in the parasite population was also seen in the present study [
34].
Similarly, though the majority of isolates were found to be resistant to CQ, the IC
50 observed value for CQ was comparatively low as seen in other reports [
35]. This low value is indicative of the fact that the absence of CQ in malaria treatment has released the drug pressure with a possibility of moving towards being CQ sensitive in future as reported from other parts of the world [
36]. The field isolates were found to be AS sensitive through with high IC
50 values. Due to constant exposure to AS the increase in the drug pressure is likely to be responsible for increase in the IC
50 values and another plausible explanation also could be the indiscriminate use of the drug resulting in slow rise of low level resistance in the parasite population [
37]. In vitro drug sensitivity remains an important tool to assess the efficacy of the anti-malarial drugs. The change of anti-malarial treatment in the country from CQ to ACT is expected to decrease the CQR population and might be leading to restore the sensitivity to CQ as reported from other places [
38]. The levels of parasitaemia varied in the infections and only 44 samples could be culture adapted. The difference in the growth pattern in different field isolates may be due to their difference in commitment to differentiate into asexual or sexual stages [
39]. The expression profile of
Pfg377 gene was correlated with the efficiency of gametocyte production in the above isolates. It was observed that isolates which produce mature gametocytes in vitro also showed an increase in the
Pfg377 gene expression from 1.00 to 4.56-fold when compared to NF54 reference strain. The results suggested that a correlation between
Pfg377 gene expression and the ability to produce gametocyte (mature) by the isolate exists as also reported by others [
40].
The allele typing of
Pfg377-the sexual stage specific gene in the field isolates revealed several allele types present in them. By conventional PCR only the predominant genotype was detected in the PCR results and presence of more than one clone in a single infection were observed on sequence analysis. This shows that this gene can be effectively used for detecting the presence of gametocyte producing multiclones in the field isolates. The presence of different genotypes/clones in the same infection is an important source of transmission [
8]. The large number of repeats found in this gene is responsible for the high variability seen in parasites thus allowing several parasite lines to merge and survive simultaneously leading to transmission dynamics [
41]. It is understood that presence of mixed gametocyte genotypes in the same sample is responsible for harbouring gametocytes for a significantly longer period of time on comparison with a single genotype where it was cleared three times faster than multiple clones [
42]. Natural infections of
P. falciparum seem to harbour multiple genotypes in the same host which could be due to the transmission burden in endemic regions but still, this knowledge does not improve the understanding of existing multiplicity in parasite biology [
43].
By substantiating the results obtained from the genome sequence of P. falciparum and phylogenetic analysis, a basic idea about the population genetic structure was inferred. The phylogenetic trees of all three genes divided the isolates into two clear clades that can potentially be divided into further subgroups. However, no obvious cluster was observed and the distribution of isolates within the subgroups was random. There was no apparent trend for sequences of the same geographic region to be more closely related. The non-availability of wild-type of isolates limits our understanding of wild type parasites vs drug-resistant parasites as most of the infections had at least single/double SNPs in drug resistance genes with extensive diversity present in the Pfg377 gene. More comprehensive studies like this are needed to understand the emerging complexity in the parasite population. The gametocyte data in relation to diversity level is too limited in Indian context. Our main focus to include this data was to generate data for any type of correlation between the mutation in drug resistance genes and gametocyte gene as these gametocyte genes play a crucial role in transmission as it gives baseline data for further work in this area.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.