Molecular surveillance of antimalarial drug resistance genes in Plasmodium falciparum isolates in Odisha, India: A decade after CQ withdrawal

doi:10.21203/rs.3.rs-2072289/v1

BACKGROUND: India has targeted to eliminate malaria by 2030. Surveillance of drug-resistant malaria parasites in different endemic settings country is a pressing need to achieve the target in the face of emerging drug resistance. In Odisha, the highest contributor of malaria cases to the national pool has changed the drug policy in 2009 following increasing of failure rate of treatment with chloroquine (CQ). The aim of this study was to determine the prevalence of Plasmodium falciparum molecular markers that are associated with resistance to CQ, S-P, and ART in Odisha 10 years after the institution of the new policy.

METHODS: The study was conducted from July 2018 to November 2020 among the patients attending Government Health facilities, selected randomly in four different physiographical regions of the state. The prevalence of critical point mutations in the genes of Pfcrt (codon 76),

Pfmdr1(codon 86), Pfdhfr (codons, 16, 50, 51, 59, 108, and 164), Pfdhps (codons 436, 437, 540, 581, and 613) and PfK13 gene were examined in parasite isolates.

RESULTS: The prevalence of Pfcrt (K76T) was 2.1% and Pfmdr1 (N86Y) 3.4%. None of the five mutations in the Pfkelch13 gene associated with resistance to artemisininwas detected. The overall prevalence of Pfdhfr mutations was 50.4% with a total number of 12 genotypes. The Pfdhfr C59R mutation was the most common (41.8%), followed by the C50R mutation (40.8%) and S108N mutation (39.2%). The overall prevalence of Pfdhps mutations was 40.1% with a total number of 26 genotypes. The maximum number of mutations was found at codon S436A (26.7%) followed by A613S (17.6%) and K540E(17.2%). No isolate with Pfdhfr triple mutation (N51I/ C59R/S108N) combined with Pfdhps double mutation (A437G/K540E) was found in the studied sample.

CONCLUSION: These results predict the return of susceptibility of P. falciparum to CQ ten years after the change of malaria treatment policy, while confirming the emergence of parasite resistance to S-P in the state of Odisha. Additional surveillance in the same region and other malaria-endemic parts of the country may help to provide evidence for drug policy updates.

Plasmodium falciparum

drug-resistant markers

Chloroquine

Sulphadoxine-Pyrimethamine

Malaria caused by Plasmodium spp. occurs mainly in poor tropical and sub-tropical regions of the world. Of the five species causing human malaria, Plasmodium falciparum is the most lethal and accounts for more than 90% of the world's malaria deaths. Although malaria mortality has been reduced by over a quarter around the world, its transmission still occurs in 99 countries. In 2020, an estimated 241 million malaria cases and 627,000 malaria deaths has occurred worldwide [1]. In Southeast Asia, India alone accounts for 85.2% of malaria cases and 2% of global malaria deaths [2]. While in India, the state of Odisha with 4% of the total population of the country accounted for 32.4% of the total malaria cases, 32.02% of P. falciparum cases, and 9.7% of malaria deaths in 2020 (NVBDCP, Govt. of India). In absence of an effective vaccine, chemotherapy and chemoprophylaxis remain the principal means to minimize the mortality and morbidity burden due to malaria. As in other malaria-endemic countries of the world, chloroquine (CQ) was used in the national programme in India as the first line of treatment of uncomplicated malaria for a prolonged period of time because of its safety profile and cost-effectiveness. After sustained use, the resistance of P. falciparum to CQ emerged during 1973 in Karbi-Anglong district of Assam in India [3] that subsequently spread across the country [4]. Accordingly, Indian drug policy was changed and the Sulphadoxine-Pyrimethamine (S-P) combination was introduced in 1982 as a second line of treatment [5]. But in 2004 the WHO technical advisory group recommended the use of combination antimalarial therapy, particularly with artemisinin derivatives, in member countries for treating P. falciparum to delay the emergence of drug resistance. Consequently, Artemisinin-based combination therapy (Artesunate + Sulphadoxine-Pyrimethamine) was introduced in mid-2009 as the first line of treatment in India (Government of India, 2009). However, for effective implementation of the drug policy continuous monitoring of drug-resistant parasites in the field is required to determine the spread of resistance over wide areas. Since the identification of drug-resistant P. falciparum strains by standard 28 day in-vivo efficacy study is cumbersome, molecular markers have been proposed as alternative tool to monitor resistance. Thus, mutations occurring on the gene Pfcrt (T76), and secondarily Pfmdr1 (Y86) have been reported to have a strong interrelationship and correlation with the resistance of P. falciparum to CQ [6], [7]. Similarly, Pfdhps (A/F436, G437, E540, G581, and S/T613), Pfdhfr (I51, R59, N108, and L164) have been found to be associated with S-P drug resistance [8]. In a major breakthrough mutation in the Pfk13 gene (PF3D7_1343700) has been recently identified to be strongly associated with reduced susceptibility to artemisinin in five countries in South-East Asia: Cambodia, Laos, Thailand, Myanmar, and Vietnam [9, 10].

India and other Asia-Pacific countries have pledged to eliminate malaria by 2030 [11]. In this situation studies on the pattern of mutations in drug-resistant genes of P. falciparum will not only help the early detection of drug resistance to the currently used drugs but also determine the status of chloroquine-resistant P. falciparum after withdrawal of chloroquine for decades, because several studies in different malaria-endemic countries have reported return of CQ susceptibility after cessation of the use of CQ [12, 13]. With this background, the present study was undertaken to investigate the prevalence of Pfcrt K76T and Pfmdr1 N86Y (responsible for CQ resistance), mutations of Pfdhfr and Pfdhps genes responsible for SP drug resistance, and Pfk13 propeller gene polymorphism associated with ART treatment failure. Further evolutionary genetic analysis has been performed in P. falciparum isolates to highlight their immediate relevance to public health.

Study setting and sample collection

This cross-sectional study was conducted from July 2018 to November 2020 among the patients attending government health facilities, selected randomly in four different physiographical regions (northern plateau, central table land, eastern ghat, and coastal belt) of the state (Figure-1). Suspected malaria cases (> 5 years of age) were screened by Pf PAN Ag Rapid Diagnostic Test Kit (SD-Biosensor, India) using finger-prick blood. At least 1ml of venous blood was collected in BD Vacutainer^® EDTA vial from individuals found to be positive for malaria. The blood samples collected in the field were preserved at four 4℃ at the local hospital and transported to the ICMR-RMRC laboratory within 24 h, maintaining a cold chain for further molecular analysis.

Diagnosis and speciation by PCR

Parasite genomic DNA was extracted from 200μl of EDTA blood samples using QIAamp Blood DNA mini kit (QIAGEN, Germany) according to the manufacturer's instructions and eluted in 50μlTE buffer. Nested PCR (nPCR) was performed to confirm the diagnosis and identification of the species using the species-specific primers targeting 18S rRNA and cycling parameters as described by Snounou G. et al. [14] in a Thermal Cycler (Agilent Sure Cycler 8800, USA). Briefly, the primary PCR was performed in a 25μL reaction mixture that contained 0.2U of Taq DNA polymerase (GCC Biotech, India), 0.2mM each dNTP (HIMEDIA Laboratories, India), 0.4mM each primer (GCC Biotech, India) and 2.0mM MgCl₂ (GCC Biotech, India). The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94℃ for 1 min, annealing at 50℃ for 1 min, and extension at 72℃ for 1 min; final extension was at 72℃ for 10 min. The nPCR reaction condition was the same as primary PCR except for the annealing temperature, 55℃. PCR products were visualized under UV light following the electrophoresis on 1.2% (w/v) ethidium bromide-stained agarose gel, and images were captured using a gel documentation system (Alphaimager, USA). Known positive and negative samples from previous malaria-diagnosed or uninfected individuals were used as controls. Of the total 557 RDT-positive malaria samples, 239 samples were confirmed to be positive for P. falciparum mono-infection, 104 mixed-species infections, and 133 non-falciparum malaria (NFM) mono-infections (P. vivax, P.ovale and P.malariae ) as shown in the flow chart Figure-2. All the PCR-confirmed P. falciparum mono-infection samples were included to analyze mutations in P. falciparum genes involved in anti-malarial drug resistance.

Analysis of Pfcrt, Pfmdr1 & PfK13 genes

Genotyping of the resistant markers Pfcrt K76T and Pfmdr1 N86Y was carried out by PCR-RFLP using the genomic DNA isolated from all the enrolled samples (n = 239) followed by sequencing for haplotype analysis [15, 16, 17]. In case of Pfcrt, 212 nucleotide sequence fragments encompassing the K76T mutations responsible for CQ resistance, while 526 nucleotide sequence fragments of Pfmdr1 containing the N86Y mutations responsible for multidrug resistance and 793 nucleotide sequence fragment of PfK13 gene containing N458Y, Y493H R539T, I543T and C580Y known point mutation responsible of ART resistance were sequenced. The sequences of Pfcrt, Pfmdr1, and PfK-13 found in the study have been deposited in Gene Bank via Bankit http://www.ncbi.nlm.nih.gov/Bankit/ (Accession # MZ678763-MZ678766 for Pfcrt, MZ054306, MZ054305, and MZ678767-MZ678769 for Pfmdr1 and MZ151068-MZ151071 for Pfk13 gene).

DNA sequence analysis and summary statistics

The DNA sequences were aligned, and summary statistics were calculated for each gene separately. Manual editing and alignment of DNA sequences were conducted using SeqMan, EditSeq, and MegAlign modules of the Lasergene computer program. [15] All the summary statistics were calculated using the computer program DnaSP version 6 [18].

Analysis of Pfdhfr & Pfdhps genes

Allele-specific Polymerase Chain Reaction (AsPCR) assay was performed to investigate the presence of mutations in dihydrofolate reductase (Pfdhfr: codons 16, 50, 51, 59, 108, and 164) and dihydropteroate synthase (Pfdhps: codons 436, 437, 540, 581, and 613) genes which are associated with antifolate resistance [5].

Ethics and consent

The study has been approved by the Institutional Human Ethics Committee of the Indian Council of Medical Research (ICMR)-Regional Medical Research Centre (RMRC), Bhubaneswar. Before the enrolment, the purpose of the study was explained to the participants in the local language, and verbal consent was obtained for blood sample collection and testing.

Of the total 557 RDT positive samples, 476 samples were found to be positive for malaria infection by nPCR. Amongst the PCR-positive samples, 239 were P. falciparum, 123 were non falciparum malaria (NFM) (P. malariae: 7, P. ovale: 2 and P. vivax: 114) and 114 were P. falciparum mixed with NFM infections. The Figure-2 depicts the flow diagram of the selection of P. falciparum mono infection samples used in the present study for molecular analysis of drug resistance genes.

Analysis of Pfcrt, Pfmdr1 and PfK13 propeller gene

PCR-RFLP analysis performed in 239 P. falciparum isolates have shown Pfcrt K76T point mutation in five isolates (2.1%), Pfmdr1 N86Y point mutation in 8 isolates (3.4%) and no isolate carried Pfcrt K76T + Pfmdr1 N86Y point mutations. Single nucleotide mutations identified through DNA sequencing translated to amino acid substitutions in a subset of samples revealed two different types of haplotypes (CVIET and CVMNT) in isolates having Pfcrt K76T point mutation, the primary determinant of chloroquine (CQ) resistance, while analysis of wild type (Pfcrt K76) samples shown CVMNK haplotype. Of the two different kinds of haplotypes detected in mutant samples during the survey, both the haplotypes (CVIET and CVMNT) were found in the P. falciparum isolates collected from the eastern-ghat area (Raygada and Kandhamal), while the CVIET was found in Sundargarh district of northern plateau region and CVMNT in Bargarh district of central tableland region. Similarly, sequencing of the Pfmdr1 mutant isolates detected by PCR-RFLP showed N86Y and N86Y /Y184F point mutations. Though none of the five validated or established mutations associated with artemisinin resistance detected in P. falciparum isolates subjected for DNA sequencing, six single nucleotide polymorphisms (SNPs) could be identified only in case of changes in nucleotide positions that were synonymous in natures but no changes in protein-coding level indicating the absence of P. falciparum genotype (Pfk13) associated with resistance to artemisinin in Odisha at present.

The summary statistics for all the three genes responsible for antimalarial resistance are displayed in Table 1. While the haplotype diversity was almost similar in all the three genes, the nucleotide diversities, as measured independently by theta (ϴ) and Pi (π), were variable across the three genes. Whereas relatively higher nucleotide diversities were found in the Pfcrt gene for both the parameters theta (ϴ) and Pi (π), the values were found to be lower in the Pfmdr1 and PfK13 genes (Table 1). The test of neutrality as measured by Tajima D, Fu and Li’s D, and Fu and Li’s F tests were not statistically significantly deviated from the model of neutral expectation in either of the three genes. Considering the three genetic fragments utilized in the present study are in the coding regions, it can be predicted that there is no evidence for natural selection in the three-drug resistance genes of P. falciparum of Odisha, India.

Table 1

Details of *P. falciparum* K-13 propeller gene (*Pfk13*), *P. falciparum* chloroquine-resistance transporter (*Pfcrt*) and *P. falciparum* multi drug resistance-1(*Pfmdr-1*) nucleotide fragments and population genetic parameters in *P. falciparum* field isolates of Odisha, India.
Genes	Pfcrt (3107 bp)	Pfmdr-1 (4382 bp)	Pfk13 ( 2417 bp)
	403222–406317	957890–962149	1724817–1726997
Total number of Isolates	17	8	9
Size of the fragment (nucleotide)	212	526	793
Total number of polymorphic (segregating) sites	4	2	6
Total number of singleton mutations	1	1	2
Total number of SNPs	4	2	6
Total number of Haplotypes	5	3	5
Haplotype diversity	0.64	0.679	0.722
Nucleotide diversity π	0.00472	0.00156	0.00266
Nucleotide diversity (ϴ)	0.00558	0.00147	0.00278
Tajima’s D	-0.47378	0.24178	-0.19073
Fu and Li's D	0.23149	-0.14931	0.26719
Fu and Li's F	0.04712	-0.06487	0.17557

Analysis of Pfdhfr and Pfdhps gene

A total of 239 P. falciparum-infected blood samples were analysed for mutations in six codons of the Pfdhfr gene (A16V, C50R, N51I, C59R, S108N/T and I164L) and five codons of the Pfdhps gene (S436F/A, A437G, K540E, A581G and A613S/T) to assess the level of antifolate drug pressure. Out of 239 samples, 232 were PCR positive for Pfdhfr and 221 for Pfdhps, while PCR could detect both Pfdhfr and Pfdhps genes in 119 (49.7%) of the samples. The Pfdhfr C59R mutation was found to be most prevalent (N = 97, 41.8%), followed by the C50R mutation (N = 93, 40.8%) and S108N mutation (N = 91, 39.2%). No isolate had the S108T mutation, while the N51I, I164L, and A16V mutations were found in 17.2% (N = 40) and 3.4% (N = 8) and 9.05% (N = 21) of the isolates respectively. The wild-type Pfdhfr sequence (ACNCSI) at all six codons was prevalent in 49.6% (N = 115) of the isolates. Amongst the total isolates 26 (11.2%) had single mutation, 19 (8.2%) had double, 40 (17.2%) had triple, 23 (9.9%) had quadruple, 8 (3.4%) had pentuplet and 1 (0.43%) had sextuplet mutation. The most frequent triple mutation sequence was ARNRSNI (N = 18, 7.8%)/ ARIR SSI (N = 17, 7.3%) and quadruple mutation sequence was ARIRSNI (N = 12, 5.2%) / VRNRSNI (N = 11, 4.7%). In our sample (N = 232) total 12 different genotypes have been observed in the Pfdhfr gene (Figure-3).

Of the 221 samples PCR positive for Pfdhps, 117 (52.9%) had the wild-type sequences (SAKAA) at all five codons. The maximum number of mutations were found at codon S436A (N = 59, 26.7%), followed by A613S (N = 39, 17.6%), K540E (N = 38, 17.2%), A581G (N = 31, 14.0%), S436F (N = 28, 12.7%), A437G (N = 26, 11.8%) and A613T (N = 23, 10.4%). The double mutations in Pfdhps gene were comparatively more (N = 43, 19.5%) than single (N = 24, 10.9%), triple (N = 21, 9.5%), quadruple (N = 6, 2.7%), pentuplet (N = 6, 2.7%) and sextuple (N = 4, 1.8%) mutations. Amongst the double mutations the sequences with SAAKASA (N = 15, 6.8%) and SAAEAAA (N = 13, 5.9%), and amongst the triple mutations the sequences with SAAKGAA (N = 8, 3.6%), SAAEASA (N = 7, 3.2%), FAGKASA (N = 7, 3.2%), FSGEGAT (N = 5, 2.3%) were common. Genotyping have detected total 26 different genotypes in Pfdhps gene (Figure-4).

Regional distribution of P. falciparum dhfr and dhps mutations

The physiographical region (Northern Plateau, Eastern Ghat, Coastal Belt, and Central Table Land) wise distribution reveals that the prevalence of point mutations in Pfdhfr and Pfdhps gene are more in Eastern Ghat region followed by Northern Plateau, Central Table Land and Coastal Belt (Table- 2). The ARNRSNI and ACNCSNI triple mutation in Pfdhfr gene were present in all four regions, while ARNRSNL, VRNRSNI, ARNRSSI, and ARIRSSI were found in two regions (Northern Plateau and Eastern Ghat). The sextuplet mutations VRIRSNL, pentuplet mutations VRIRSNI and quadruplet mutations ARIRSNI were present only in Eastern Ghat. In case of Pfdhps the single mutation SAAKAAA and the double mutation SAAKASA, SAAEAAA, and SSAKGAT were found in Northern Plateau and in Eastern Ghat, while the multiple mutations like FSGEGST, FAGEGAT, and FAGEGAA were present in Central Table land and Coastal Belt.

P. falciparum dhfr-dhps two-locus mutation analysis

The P. falciparum dhfr-dhps two-locus mutation analysis carried out in 119 (49.79%) isolates have revealed 3 different genotypes in Coastal Belt, 5 in Central Table Land, 29 in Northern Plateau and ≥ 40 in Eastern Ghat regions. However, no isolate with Pfdhfr triple (N51I/ C59R/S108N) mutation in combination with Pfdhps double (A437G/K540E) mutation, a useful predictor of SP treatment failure, was found in the studied sample.

Table 2

Regional distributions of drug-resistance molecular markers in *P. falciparum* isolates in Odisha, India (Period: 2018–2020).
Genes	Pfcrt K76T	Pfmdr1 N86Y	Pfdhfr						Pfdhps
Physiographical regions			A16V	C50R	N51I	C59R	S108N	I164L	S436F	S436A	A437G	K540E	A581G	A613S	A613T
Eastern Ghat	2	1	16	62	35	66	59	6	26	34	17	22	31	20	23
Northern Plateau	1	2	5	26	5	26	20	2	10	17	8	22	8	9	8
Central Table Land	2	1	0	3	0	3	8	0	5	2	3	4	3	2	1
Coastal Belt	0	1	0	2	0	2	4	0	3	2	2	2	2	1	2
Total (239)	5	5	21	93	40	97	91	8	44	55	30	50	44	32	34
%	2.1	2.1	8.9	38.9	16.7	40.6	38.1	3.3	18.4	23.0	12.6	20.9	18.4	13.4	14.2

The National Framework for Malaria Elimination (NFME) in India targets to eliminate malaria from the country by 2030. However, emergence of antimalarial drug resistance especially in P. falciparum has defeated effective antimalarial drug treatment, malaria control, and elimination many times during the past. Therefore, surveillance and regular monitoring of antimalarial drugs is very much essential to check the spread of drug resistance and achieve the goal. With this backdrop the present study was designed to evaluate the prevalence of P. falciparum resistance markers to CQ and S-P, two conventional anti-malarial drugs that have been used for a long time along with artemisinin (ART) the recently introduced anti-malarial drug in the state of Odisha, the highest (~ 40%) contributor of malaria cases to the national pool [19]. This study was intended to watch for a possible return of sensitivity of P. falciparum to CQ on the one hand and on the other hand to describe the current patterns of molecular markers associated with parasite resistance to S-P, used as partner drug in artemisinin-based combination therapy (ACT) for treatment of uncomplicated malaria in the programme.

Artemisinin Combination Therapy (ACT) was introduced as a first-line of treatment for uncomplicated malaria in India in 2008 [20]. Since Western Cambodia, Southern Laos, and North-Eastern Thailand experiencing an increase in artemisinin resistant parasites that are fitter and are able to deprive the partner medication efficacy [21], there is a strong likelihood of translational spreading of these drug-resistant malaria parasites across North-East India to other parts of India including Odisha due to frequent border crossings among mobile populations. Therefore, surveillance of ART drug resistance status is very much essential to provide early warning and to inform national drug strategies to check the spread of artemisinin-resistant P. falciparum in the country. Recently a study conducted among malaria-infected individuals in Eastern India have found that PfK13 G625R and R539T mutations are linked to ART resistance based on the presence of Delayed Parasite Clearance on day 3 (DPC3), which has raised deep concern in the malaria research community in India [22]. However, none of the five established mutations in the PfK13 propeller gene associated with artemisinin resistance could be detected in the present study. Moreover, identification of six synonymous single nucleotide polymorphisms (SNPs) indicates that resistance to artemisinin is not yet developed in P. falciparum of Odisha.

The antifolate combination Sulphadoxine-Pyrimethamine (SP), used as partner drug of ACT for the treatment of uncomplicated malaria in the state over past ten years as part of the national malaria elimination programme, along with other sulpha drugs such as trimethoprim-sulfamethoxazole (co-trimoxazole), used extensively for treatment of bacterial infections including urinary tract infections, middle ear infections (otitis media), bronchitis, and shigellosis (bacillary dysentery), contribute in maintaining drug pressure on malaria parasites [23]. Sequential accumulation of S436F /A, A437G, K540E, A581G, A613S/T mutations in Pfdhps [24] and A16V, C50R, N51I, C59R, and S108T/N mutations in Pfdhfr [25] leads to the development of resistance to sulphadoxine and pyrimethamine respectively in P. falciparum isolates [26]. The primary mutation being A437G/ K540E in dhps and S108N/ C59R in Pfdhfr as per the findings from different malaria endemic regions of the world including India [5, 27, 28, 29]. The high proportion of mutation at codon site C59R (41.4%), C50R (38.4%) and S108N (32.8%) mutations in the Pfdhfr gene than at codon site S436A (26.7%), A613S (17.6%) and K540E (17.3%) mutations in Pfdhps gene indicate that these are the key point mutations and further the overall low prevalence of point mutations in Pfdhps (47.05%) gene sequence compared to Pfdhfr (50.4%) confirms that the mutations associated with parasite resistance to SP appeared earlier on the Pfdhfr than those affecting the Pfdhps (30). The prevalence of single, double, triple, quadruple or quintuple mutation in Pfdhfr and Pfdhps observed in this study reflects the current level of sensitivity of P. falciparum to S-P. Moreover, mutation at codon C59 and S108 along with codons A16, C50 and N51 in Pfdhfr (~ 38%) and codon A437 and K540 along with codons S436, A581 and A613 in Pfdhps (~ 24%) during the present study strongly predicts the decreasing treatment response as reported earlier in Western and Central Africa [31, 32, 33]. Out of the 12 Pfdhfr mutant genotypes found in the P. falciparum isolates in the state, 3 mutant genotypes (ACNCSNI, ACNRSSI, ARNCSNI) have been reported earlier in India including Odisha [5], while 8 mutant genotypes (ARNRSSI ARNRSNI ACNRSSL ARIRSSI ARNRSNL, ARIRSNI, VRIRSNI, VRIRSNL) have been found for the first time in the state. Similar is the situation in case of Pfdhps gene, in which SAAKASA, SAAEASA, FAGKASA, FSGEGAT, FSGEGST, FAGEGAT and FAGEGAA genotypes have been detected for the first time in the state in addition to SAAEAAA and SSAKGAT genotypes reported from India including Odisha [5]. Prevalence of such unique multiple mutations in Pfdhfr as well as Pdhps the state indicates emergence of resistance to S-P drug as observed earlier in Kenya, Thailand and Vietnam [5]. But, absence of mixtures of N51I-C59R-S108N codons in Pfdhfr and A437G-K540E codons in Pfdhps indicates that P. falciparum isolates circulating in this part of the country have not developed RIII (highest) level of resistance [34, 5]. Similarly, absence of A16V-S108T mutation in Pfdhfr responsible for cycloguanil resistance in the present study might be because cycloguanil-proguanil has not yet been introduced for the treatment of malaria in India [5].

The Pfcrt (K76T) has been identified as the primary determinant of CQ resistance, but Pfmdr1 (N86Y and Y184F) can modulate the degree of resistance and also has been found to be linked with differential responses to multiple quinoline-based antimalarial and ART, both in vitro and in vivo [35, 36]. Interestingly high rates of wild alleles of Pfcrt (K76, 98%) and Pfmdr1 (N86 and Y184, 97%) observed in this study compared to our earlier study conducted before CQ withdrawal during 2008–2010 [15] provides evidence of the return of CQ-susceptible P. falciparum strains in Odisha. The results obtained is similar to the observations made in Malawi, Tanzania, Mozambique, Northern Uganda, Saudi Arabia [37, 38, 39, 40, 41] but in contrast to southern Benin [30]. Further DNA sequence polymorphism study that not only informs distribution of different haplotypes, but also the evolutionary potentiality of mutation in the drug-resistant genes that can directly translate to molecular epidemiology of human diseases like malaria. Several studies employing this methodology in the three genes of interest for malaria public health have been conducted worldwide which has immensely helped in determining intervention through therapeutic measures and change in drug policy in different countries [42, 43, 44, 45, 46]. The present study, although limited to a small number of samples, indicates not only the presence of different haplotypes (CVMNK: wild type, CVIET mutant type: believed to be of South-east Asian origin, CVMNT mutant type: believed to be of African Origin) but also inform the high genetic diversity present in field isolates of P. falciparum for these three drug-resistant genes in Odisha, India. Interestingly, the wild type CVMNK haplotype of the Pfcrt gene was found in 76.47% of the isolates, which is in contrast to findings from other studies in Indian P. falciparum. This fact is also documented in earlier studies [47]. It is also argued that Odisha might have served as the epicentre for the distribution of chloroquine-resistant P. falciparum parasites to other parts of India [48]. Combined with the results from other studies of Odisha, it can be therefore hypothesized that chloroquine-sensitive P. falciparum parasites might be getting selective advantages in the absence of chloroquine in the program for the treatment of P. falciparum malaria. However, similar studies with large number of isolates from different malaria endemic pockets needs to be investigated in Odisha as well as other parts of India.

The low prevalence of Pfcrt (K76T) and Pfmdr1 (N86Y and Y184F) indicates return of CQ susceptible P. falciparum parasites after ten years of officially withdrawal of CQ in the treatment of uncomplicated malaria and high prevalence of Pfdhfr / Pfdhps triple and quadruple mutant parasites confirms the emergence of parasite resistance to S-P in the state of Odisha. Additional surveillance may help to assess the dynamics of drug resistant malaria parasites in the same geographic area and other malaria-endemic area of the country. This is required to achieve the goal of malaria elimination by 2030.

Acknowledgments

RKR and MR thank the Director Indian Council of Medical Research (ICMR)-Regional Medical Research Centre Bhubaneswar (RMRC-BBS), Odisha, India, for providing essential laboratory facilities during work. RKR is thankful to the Indian Council of Medical Research (ICMR) for the award of a Senior Research Fellowship (SRF). The authors thank all the laboratory technicians who worked hard during the collection of the P. falciparum field isolate from all over the state, particularly from the tribal inaccessible areas, and also great thanks to all the individuals who participated in this study.

Author Contributions:

RKR, MR, and MSB conceptualized the study, and RKR, MSB, and MR collected the patient samples and obtained relevant malaria epidemiological details. RKR and SS did all laboratory work, and malaria parasite genotyping and PCR-sequencing experiments of the genes. RKR, AD, and NK carried out all the sequencing analysis and bioinformatics work and wrote the interpretations, RKR, AD, and MR wrote the manuscript with contributions from SS, NK, MSB, and SP. All authors agree to the content of the manuscript.

Funding

The Indian Council of Medical Research-Senior Research Fellowship (ICMR-SRF) (Letter Number-Fellowship/25/2018-ECD-II, Dated-29/07/2018) to Ramakanta Rana (RKR) provided financial support for the research and preparation of the article, and study design, the collection, analysis, and interpretation of data.

Ethics and consent

The study has been approved by the Institutional Human Ethics Committee of the Indian Council of Medical Research (ICMR)-Regional Medical Research Centre (RMRC) Bhubaneswar. Before the enrolment, the purpose of the study was explained to the participants in the local language, and verbal consent was obtained for blood sample collection and testing.

Consent for publication

Not applicable

Declaration of competing interest: The authors have no competing interest to declare.

Availability of data and materials

All data generated or analysed during this study are included in the article. The nucleotide sequences, except Pfdhfr and Pfdhps, generated have been deposited in Gene Bank via Bankit http://www.ncbi.nlm.nih.gov/Bankit/ and are available in the database under the accession numbers as indicated in the text.

WHO. World Malaria Report 2021 [Internet]. Organizations WWH, editor. Word Malar. Rep. Geneva World Heal. Organ. (2021). Licence CC. Geneva, Switzerland: WHO Team, Global Malaria Programme; 2021.Availablefrom: https:// www. who.int/ publications/i /item / 9789240040496.
World Health Organization. World Malaria Report: 20 years of global progress and challenges [Internet]. World Heal. Organ. 2020. Available from: https://www.who.int/publications/i/item/9789240015791.
Sehgal PN, Sharma MID, Sharma SL GS. Resistance to chloroquine in falciparum malaria in Assam state, India.e. Journal Commun Dis. 1973; 5:175–180.
Farooq U MR. Drug resistance in malaria. J Vector Borne Dis [Internet]. 2004; 41:45–53. Available from: https://pubmed.ncbi.nlm.nih.gov/15672556/.
Ahmed A, Bararia D, Vinayak S, Yameen M, Biswas S, Dev V, et al. Plasmodium falciparum Isolates in India Exhibit a Progressive Increase in Mutations Associated with Sulfadoxine-Pyrimethamine Resistance. Antimicrob Agents Chemother. 2004; 48:879–89.
Ross LS, Dhingra SK, Mok S, Yeo T, Wicht KJ, Kümpornsin K, et al. Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine. Nat Commun [Internet]. Springer US; 2018; 9:25–8. Available from: http://dx.doi.org/10.1038/s41467-018-05652-0.
Njiro BJ, Mutagonda RF, Chamani AT, Mwakyandile T, Sabas D, Bwire GM. Molecular surveillance of chloroquine-resistant Plasmodium falciparum in sub-Saharan African countries after withdrawal of chloroquine for treatment of uncomplicated malaria: A systematic review. J Infect Public Health [Internet]. Elsevier; 2022; 15:550–7. Available from: https://doi.org/10.1016/j.jiph.2022.03.015.
Svigel SS, Adeothy A, Kpemasse A, Houngbo E, Sianou A, Saliou R, et al. Low prevalence of highly sulfadoxine‐resistant dihydropteroate synthase alleles in Plasmodium falciparum isolates in Benin. Malar J [Internet]. BioMed Central; 2021;20:1–8. Available from: https://doi.org/10.1186/s12936-021-03605-5.
Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505:50–5.
Huang F, Yan H, Xue JB, Cui YW, Zhou S Sen, Xia ZG, et al. Molecular surveillance of pfcrt, pfmdr1 and pfk13-propeller mutations in Plasmodium falciparum isolates imported from Africa to China. Malar J [Internet]. BioMed Central; 2021;20:1–11. Available from: https://doi.org/10.1186/s12936-021-03613-5
Lal AA, Rajvanshi H, Jayswar H, Das A, Bharti P. Malaria elimination: Using past and present experience to make malaria-free India by 2030. J Vector Borne Dis. 2019;56:60–5.
Frosch AEP, Laufer MK, Mathanga DP, Takala-Harrison S, Skarbinski J, Claassen CW, et al. Return of widespread chloroquine-sensitive Plasmodium falciparum to Malawi. J Infect Dis. Oxford University Press; 2014; 210:1110–4.
Lu F, Zhang M, Culleton RL, Xu S, Tang J, Zhou H, et al. Return of chloroquine sensitivity to Africa? Surveillance of African Plasmodium falciparum chloroquine resistance through malaria imported to China. Parasites and Vectors. BioMed Central Ltd.; 2017;10.
Snounou G, Viriyakosol S, Xin Ping Zhu, Jarra W, Pinheiro L, do Rosario VE, et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol. 1993;61:315–20.
Sutar SK Das, Gupta B, Ranjit M, Kar SK, Das A. Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India. Mem Inst Oswaldo Cruz. 2011; 106:78–84.
Chauhan K, Pande V, Das A. DNA sequence polymorphisms of the pfmdr1 gene and association of mutations with the pfcrt gene in Indian Plasmodium falciparum isolates. Infect Genet Evol [Internet]. Elsevier B.V.; 2014; 26:213–22. Available from: http://dx.doi.org/10.1016/j.meegid.2014.05.033.
Rana R, Ranjit M, Bal M, Khuntia HK, Pati S, Krishna S, et al. Sequence Analysis of the K13 -Propeller Gene in Artemisinin Challenging Plasmodium falciparum Isolates from Malaria Endemic Areas of Odisha, India: A Molecular Surveillance Study. Biomed Res Int. 2020; 2020.
Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics. 2003;19:2496–7.
Pradhan M, Meherda P. Malaria elimination drive in Odisha: Hope for halting the transmission. J Vector Borne Dis. 2019; 56:53–5.
Nosten F, White NJ. Artemisinin-based combination treatment of falciparum malaria. Am J Trop Med Hyg. 2007;77:181–92.
Imwong M, Suwannasin K, Kunasol C, Sutawong K, Mayxay M, Rekol H, et al. The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: a molecular epidemiology observational study. Lancet Infect Dis [Internet]. The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY license; 2017;17:491–7. Available from: http://dx.doi.org/10.1016/S1473-3099(17)30048-8.
Das S, Saha B, Hati AK, Roy S. Evidence of Artemisinin-Resistant Plasmodium falciparum Malaria in Eastern India . N Engl J Med. New England Journal of Medicine (NEJM/MMS); 2018;379:1962–4.
National Center for Biotechnology Information. "Pub Chem Compound Summary for CID 358641, Sulfamethoxazole and trimethoprim" PubChem, https://pubchem.ncbi.nlm.nih.gov/ compound/ Sulfamethoxazole-and-trimethoprim. Accessed 4 September, 2022.
Oguike MC, Falade CO, Shu E, Enato IG, Watila I, Baba ES, et al. Molecular determinants of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum in Nigeria and the regional emergence of dhps 431V. Int J Parasitol Drugs Drug Resist [Internet]. Elsevier Ltd; 2016;6:220–9. Available from: http://dx.doi.org/10.1016/j.ijpddr.2016.08.004.
McCollum AM, Poe AC, Hamel M, Huber C, Zhou Z, Shi YP, et al. Antifolate resistance in Plasmodium falciparum: Multiple origins and identification of novel dhfr alleles. J Infect Dis. 2006; 194:189–97.
Hyde JE. Mechanisms of resistance of Plasmodium falciparum to antimalarial drugs. Microbes Infect. 2002;4:165–74.
Ndiaye D, Daily JP SO. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Senegal NIH Public Access. Trop Med Int Heal. 2005;23:1–7.
Roper C, Pearce R, Bredenkamp B, Gumede J, Drakeley C, Mosha F, et al. Antifolate antimalarial resistance in southeast Africa: A population-based analysis. Lancet. 2003;361:1174–81.
Wang P, Lee CS, Bayoumi R, Djimde A, Doumbo O, Swedberg G, et al. Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins. Mol Biochem Parasitol. 1997; 89:161–77.
Ogouyèmi-Hounto A, Ndam NT, Kinde Gazard D, D’Almeida S, Koussihoude L, Ollo E, et al. Prevalence of the molecular marker of Plasmodium falciparum resistance to chloroquine and sulphadoxine/pyrimethamine in Benin seven years after the change of malaria treatment policy. Malar J. 2013; 12:1–8.
Kun JFJ, Lehman LG, Lell B, Schmidt-Ott R, Kremsner PG. Low-dose treatment with sulfadoxine-pyrimethamine combinations selects for drug-resistant Plasmodium falciparum strains. Antimicrob Agents Chemother. 1999; 43:2205–8.
Dunyo S, Ord R, Hallett R, Jawara M, Walraven G, Mesa E, et al. Randomised Trial of Chloroquine/Sulphadoxine-Pyrimethamine in Gambian Children with Malaria: Impact against Multidrug-Resistant P. falciparum. PLoS Clin Trials. 2006; 1:e14.
Picot S, Olliaro P, De Monbrison F, Bienvenu AL, Price RN, Ringwald P. A systematic review and meta-analysis of evidence for correlation between molecular markers of parasite resistance and treatment outcome in falciparum malaria. Malar J. 2009; 8:1–15.
Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, et al. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis. 2002;185:380–8.
Veiga MI, Dhingra SK, Henrich PP, Straimer J, Gnädig N, Uhlemann AC, et al. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun. 2016;7.
Calçada C, Silva M, Baptista V, Thathy V, Silva-Pedrosa R, Granja D, et al. Expansion of a specific plasmodium falciparum pfmdr1 haplotype in southeast asia with increased substrate transport. MBio. 2020;11:1–16.
Laufer MK, Takala-Harrison S, Dzinjalamala FK, Stine OC, Taylor TE, Plowe CV. Return of chloroquine-susceptible falciparum malaria in Malawi was a reexpansion of diverse susceptible parasites. J Infect Dis. 2010;202(5):801-8.
Frosch AEP, Laufer MK, Mathanga DP, Takala-Harrison S, Skarbinski J, Claassen CW, et al. Return of widespread chloroquine-sensitive Plasmodium falciparum to Malawi. J Infect Dis. Oxford University Press; 2014;210:1110–4.
Galatas B, Nhamussua L, Candrinho B, Mabote L, Cisteró P, Gupta H, et al. In-Vivo Efficacy of Chloroquine to Clear Asymptomatic Infections in Mozambican Adults: A Randomized, Placebo-controlled Trial with Implications for Elimination Strategies. Sci Rep. 2017;7:1–10.
Balikagala B, Sakurai-Yatsushiro M, Tachibana SI, Ikeda M, Yamauchi M, Katuro OT, et al. Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda. Malar J [Internet]. BioMed Central; 2020;19:1–12. Available from: https://doi.org/10.1186/s12936-020-03157-0
Madkhali AM, Abdulhaq AA, Atroosh WM, Ghzwani AH, Zain KA, Ghailan KY, et al. The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan region, southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene. Parasitol Res [Internet]. Springer Berlin Heidelberg; 2021;120:3771–81. Available from: https://doi.org/10.1007/s00436-021-07323-4
Hemingway J, Shretta R, Wells TNC, Bell D, Djimdé AA, Achee N, et al. Tools and Strategies for Malaria Control and Elimination: What Do We Need to Achieve a Grand Convergence in Malaria? PLoS Biol. 2016;14:1–14.
Mulenga MC, Sitali L, Ciubotariu II, Hawela MB, Hamainza B, Chipeta J, et al. Decreased prevalence of the Plasmodium falciparum Pfcrt K76T and Pfmdr1 and N86Y mutations post-chloroquine treatment withdrawal in Katete District, Eastern Zambia. Malar J [Internet]. BioMed Central; 2021;20:1–8. Available from: https://doi.org/10.1186/s12936-021-03859-z
Kim J, Tan YZ, Wicht KJ, Erramilli SK, Dhingra SK, Okombo J, et al. Structure and drug resistance of the Plasmodium falciparum transporter PfCRT. Nature [Internet]. Springer US; 2019;576:315–20. Available from: http://dx.doi.org/10.1038/s41586-019-1795-x.
Ecker A, Lehane AM, Clain J, Fidock DA. PfCRT and its role in antimalarial drug resistance. Trends Parasitol. 2012;28:504–14.
Xu C, Wei Q, Yin K, Sun H, Li J, Xiao T, et al. Surveillance of Antimalarial Resistance Pfcrt, Pfmdr1, and Pfkelch13 Polymorphisms in African Plasmodium falciparum imported to Shandong Province, China. Sci Rep. Nature Publishing Group; 2018;8.
Awasthi G, Satya GBK, Das A. Pfcrt haplotypes and the evolutionary history of chloroquine-resistant Plasmodium falciparum. Mem Inst Oswaldo Cruz. 2012; 107:129–34.
Das A. The distinctive features of Indian malaria parasites. Trends Parasitol [Internet]. Elsevier Ltd; 2015; 31:83–6. Available from: http://dx.doi.org/10.1016/j.pt.2015.01.006.

No competing interests reported.

Molecular surveillance of antimalarial drug resistance genes in Plasmodium falciparum isolates in Odisha, India: A decade after CQ withdrawal

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