Nationwide spatiotemporal drug resistance genetic profiling from over three decades in Indian Plasmodium falciparum and Plasmodium vivax isolates

Abstract

Background: Drug resistance is a serious impediment to efficient control and elimination of malaria in endemic areas.

Methods: This study aimed at analysing the genetic profile of molecular drug resistance in Plasmodium falciparum and Plasmodium vivax parasites from India over a ~ 30-year period (1993-2019). Blood samples of P. falciparum and/or P. vivax-infected patients were collected from 14 regions across India. Plasmodial genome was extracted and used for PCR amplification and sequencing of drug resistance genes in P. falciparum (crt, dhps, dhfr, mdr1, k13) and P. vivax (crt-o, dhps, dhfr, mdr1, k12) field isolates.

Results: The double mutant pfcrt SVMNT was highly predominant across the country over three decades, with restricted presence of triple mutant CVIET from Maharashtra in 2012. High rates of pfdhfr-pfdhps quadruple mutants were observed with marginal presence of "fully resistant" quintuple mutant ACIRNI-ISGEAA. Also, resistant pfdhfr and pfdhps haplotype has significantly increased in Delhi between 1994 and 2010. For pfmdr1, only 86Y and 184F mutations were present while no pfk13 mutations associated with artemisinin resistance were observed. Regarding P. vivax isolates, the pvcrt-o K10 "AAG" insertion was absent in all samples collected from Delhi in 2017. Pvdhps double mutant SGNAV was found only in Goa samples of year 2008 for the first time. The pvmdr1 908L, 958M and 1076L mutations were highly prevalent in Delhi and Haryana between 2015 and 2019 at complete fixation. One nonsynonymous novel pvk12 polymorphism was identified (K264R) in Goa.

Conclusions: These findings support continuous surveillance and characterization of P. falciparum and P. vivax populations as proxy for effectiveness of anti-malarial drugs in India, especially for independent emergence of artemisinin drug resistance as recently seen in Africa.

Keywords: Drug resistance; India; Malaria; Molecular markers; P. falciparum; P. vivax.

Fig. 1

Timeline of introduction and appearance of resistance to main anti-malarial drugs in the world and India. QN quinine, PPQ piperaquine, PQ primaquine, CQ chloroquine, PG proguanil, MQ mefloquine, ART artemisinin, SP sulfadoxine–pyrimethamine, AQ amodiaquine, ACT artemisinin based combination therapy, AS artesunate, AL artemether–lumefantrine, SLP sulfalene–pyrimethamine, CQR chloroquine-resistant, NE North east states (Sources: [, , –32])

Fig. 2

Map of India showing study areas where Plasmodium isolates were collected. Each pie chart represents the total number of isolates analysed. P. falciparum (blue) and P. vivax (red). The size of pie chart is proportional to sample size. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 3

Electrophoresis gel depicting P. falciparum mono-infection, P. vivax mono-infection and mixed infection (a), and proportion of types of infections with P. falciparum and/or P. vivax (b). In a, the gel image is showing the 18S RNA PCR amplification of Plasmodium species. Lane 1: Plasmodium falciparum (205 bp). Lane 2: Plasmodium vivax (120 bp). Lane 3: mixed infection (Both P. falciparum and P. vivax). Lane L: 100 bp Ladder. In b, the international codes of areas were used. AS Assam, CG Chhattisgarh, DL Delhi, GA Goa, HR Haryana, JH Jharkhand, KA Karnataka, MH Maharashtra, ML Meghalaya, MP Madhya Pradesh, MZ Mizoram, OR Orissa, RJ Rajasthan, UP Uttar Pradesh

Fig. 4

Pfcrt genotypes by year and area. a Proportion of 72S, 73K, 74I, 75E and 76T mutations in pfcrt gene, b proportion of pfcrt haplotypes. Pf: P. falciparum; crt: chloroquine resistant transporter gene. The international codes of areas were used. DL Delhi, GA Goa, HR Haryana, MH Maharashtra, MP Madhya Pradesh, UP Uttar Pradesh. In a, sample size was different for each area. DL (n = 3), GA (n = 4), HR (n = 18), MH (n = 10), MP (n = 4), UP (n = 8). In b the wild type is depicted in green while mutants are depicted in red and pink. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 5

Pfdhfr genotypes by year and area. a Proportion of pfdhfr 16 V, 50R, 51I, 59R, 108N and 164L mutations, b proportion of pfdhfr haplotypes. Pf: P. falciparum; dhfr: dihydrofolate reductase gene; wt: wild type. The international codes of areas were used. CG Chhattisgarh, DL Delhi, GA Goa, HR Haryana, JH Jharkhand, MH Maharashtra, MP Madhya Pradesh, OR Orissa, UP Uttar Pradesh. In a, sample size was different for each area. CG (n = 13 and 26), DL (n = 12, 17, and 37), GA (n = 5), HR (n = 53), JH (n = 22), MH (n = 9), OR (n = 17 and 19), UP (n = 9). In b wild alleles are in lower case and mutant alleles are in upper case. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 6

Pfdhps genotypes by year and area. Pf: P. falciparum; dhps: dihydropteroate synthase gene; wt: wild type. The international codes of areas were used. AS Assam, CG Chhattisgarh, DL Delhi, GA Goa, HR Haryana, JH Jharkhand, KA Karnataka, MH Maharashtra, ML Meghalaya, MP Madhya Pradesh, MZ Mizoram, OR Orissa, RJ Rajasthan, UP Uttar Pradesh. In a, sample size was different for each area. AS (n = 12), CG (n = 13 and 26), DL (n = 27, 21, and 37), GA (n = 8), HR (n = 53), JH (n = 22), KA (n = 5), MH (n = 10), ML (n = 12), MP (n = 6), MZ (n = 3), OR (n = 27 and 19), RJ (n = 10), UP (n = 6). a Proportion of pfdhps 431V, 436A/F, 437G, 540E/N, 581G and 613S mutations, b proportion of pfdhps haplotypes. In b wild alleles are in lower case and mutant alleles are in upper case. Samples from Gautaum Budh Nagar (UP) was excluded from percentage calculation because of low sample size (n = 1). The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 6

Pfdhps genotypes by year and area. Pf: P. falciparum; dhps: dihydropteroate synthase gene; wt: wild type. The international codes of areas were used. AS Assam, CG Chhattisgarh, DL Delhi, GA Goa, HR Haryana, JH Jharkhand, KA Karnataka, MH Maharashtra, ML Meghalaya, MP Madhya Pradesh, MZ Mizoram, OR Orissa, RJ Rajasthan, UP Uttar Pradesh. In a, sample size was different for each area. AS (n = 12), CG (n = 13 and 26), DL (n = 27, 21, and 37), GA (n = 8), HR (n = 53), JH (n = 22), KA (n = 5), MH (n = 10), ML (n = 12), MP (n = 6), MZ (n = 3), OR (n = 27 and 19), RJ (n = 10), UP (n = 6). a Proportion of pfdhps 431V, 436A/F, 437G, 540E/N, 581G and 613S mutations, b proportion of pfdhps haplotypes. In b wild alleles are in lower case and mutant alleles are in upper case. Samples from Gautaum Budh Nagar (UP) was excluded from percentage calculation because of low sample size (n = 1). The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 7

Pfmdr1 genotypes by year and area. a Proportion of pfmdr1 86Y, 184F, 1034C, 1042D and 1246Y mutations, b proportion of pfmdr1 haplotypes. Pf: P. falciparum; mdr1: multidrug resistance protein 1 gene; wt: wild type. The international codes of areas were used. CG Chhattisgarh, DL Delhi, GA Goa, HR Haryana, JH Jharkhand, MH Maharashtra, UP Uttar Pradesh. In a, sample size was different for each area. CG (n = 26), DL (n = 16), GA (n = 4), HR (n = 53), JH (n = 22), MH (n = 9), UP (n = 5). In b wild alleles are in lower case and mutant alleles are in upper case. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 8

Pvdhfr and Pvdhps genotypes by year and area. a Proportion of pvdhfr 57L/I, 58R, 61M, 117N/T and 173F mutations, b proportion of pvdhps 382F/A/C, 383G, 399I, 512N, 585G, 553G, 555R, 585G, and 661V mutations, c proportion of the pvdhfr and pvdhps haplotypes. Pv: P. vivax; dhfr: dihydrofolate reductase; dhps: dihydropteroate synthase; wt: wild type. The international codes of areas were used. DL Delhi, GA Goa, HR Haryana. In a, sample size was different for each area. DL (n = 8, 32, 22, and 20 in years 2008, 2017, 2018 and 2019), GA (n = 22), HR (n = 13). In b, sample size was different for each area. DL (n = 28, 42, 22, and 12 in years 2008, 2017, 2018 and 2019), GA (n = 22). In c wild alleles are in lower case and mutant alleles are in upper case. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 8

Pvdhfr and Pvdhps genotypes by year and area. a Proportion of pvdhfr 57L/I, 58R, 61M, 117N/T and 173F mutations, b proportion of pvdhps 382F/A/C, 383G, 399I, 512N, 585G, 553G, 555R, 585G, and 661V mutations, c proportion of the pvdhfr and pvdhps haplotypes. Pv: P. vivax; dhfr: dihydrofolate reductase; dhps: dihydropteroate synthase; wt: wild type. The international codes of areas were used. DL Delhi, GA Goa, HR Haryana. In a, sample size was different for each area. DL (n = 8, 32, 22, and 20 in years 2008, 2017, 2018 and 2019), GA (n = 22), HR (n = 13). In b, sample size was different for each area. DL (n = 28, 42, 22, and 12 in years 2008, 2017, 2018 and 2019), GA (n = 22). In c wild alleles are in lower case and mutant alleles are in upper case. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)

Fig. 9

Pvmdr1 genotypes by year and area. a Proportion of pvmdr1 845F, 861E, 898E, 908L, 958M, 976F and 1076L mutations, b proportion of pvmdr1 haplotypes. Pv: P. vivax; mdr1: multidrug resistance protein 1, wt: wild type. The international codes of areas were used. DL Delhi, HR Haryana. In a, sample size was different for each area. DL (n = 33, 18 and 24 in years 2017, 2018 and 2019), HR (n = 15). In b wild alleles are in lower case and mutant alleles are in upper case. The map depicted here is taken from official website of Ministry of External Affairs, Government of India (https://mea.gov.in/india-at-glance.htm, accessed 15/11/2021)