In the present study, the mean age of the population was 30.5 ± 12.98 years. Though in high endemic areas younger children are more likely to be affected by malaria, there seems to be no such age predilection in low endemic areas where adequate control measures are in place.(Lacerda et al., 2012; “Malaria :: National Center for Vector Borne Diseases Control (NCVBDC),” n.d.) In a study conducted in Gujarat, median age of those with severe P.vivax was 33.5 years (± 23.5) years.(Anvikar et al., 2016) Similarly, in a study by Dayanand et.al in Mangaluru, malaria infections were higher in adults aged 16–45 years than in children or elderly population.(Dayanand et al., 2017) These indicate that young adults are at a higher risk for malaria. A male preponderance for vivax malaria was noted in the study participants with 63% males against 37% females. This male preponderance has been observed in malarial infections across the globe. A study in China reported a male to female ratio of 1.9:1 for P.vivax cases.(Zhang et al., 2014) Higher incidence of malarial infections in males is also reported in studies from India.(Dayanand et al., 2017) Majority of the participants (70.4%) were from the Villupuram district in our study. 40.7% of the total participants were daily wage labourers belonging to a low socioeconomic status followed by 22.2% who were agricultural workers. Villupuram is a district in Tamil Nadu which has rural pockets but is in the phase of urbanisation. The major proportion of people in this region make their living through farming. The entity urban malaria is a rising concern in less developed regions, especially the areas undergoing rapid and unprecedented urbanization.(Donnelly et al., 2005; Knudsen and Slooff, 1992)
Since there is no species specific case definition for severe malaria due to P.vivax, the WHO case definition for severe P. falciparum malaria, excluding parasite counts is used for categorizing patients of P.vivax malaria as uncomplicated or severe malaria. (“WHO Guidelines for malaria,” n.d.) Hence the criteria for severe malaria includes any of the following: impaired consciousness, prostration, multiple convulsions, metabolic acidosis, hypoglycemia, severe anemia, renal impairment, jaundice, pulmonary edema, significant bleeding and shock.(“WHO Guidelines for malaria,” n.d.) The present study had four cases of severe malaria presenting with severe anemia, acute renal failure, severe thrombocytopenia(< 10,000 platelets/mm³) and ARDS. None of the participants had any neurological manifestations like altered sensorium or seizures or coma. A study done in a tertiary care setting in Mumbai reported thrombocytopenia as the most common manifestation (89%) in severe malaria due to P.vivax.(Nadkar et al., 2012) ARDS is considered the most common predictor for mortality in malaria.(Kumari and Ghildiyal, 2014) In a study done in Mumbai by Nadkar et.al, 5 out of the 8 patients with ARDS/ALI succumbed to death.(Nadkar et al., 2012) There was no mortality in any of the severe malaria cases in the present study.
The most widely reported mutations in the pvmdr1 gene are at codons 958, 976, 1076 and 1028. Besides these, there have been reports of mutations involving codon positions 946, 908, 861 and 898 from India.(Joy et al., 2018; Kaur et al., 2020; Matlani et al., 2021) Among all these reported mutations the T958M mutation which results in change of amino acid from threonine to methionine at the codon position 958 is reported invariably in all studies till date. The present study had all the 27 isolates (100%) with the presence of this mutation. The double mutant pvmdr1 gene with the F1076L mutation, denoting replacement of phenylalanine by leucine was observed in 14 isolates(52%). In India, the T958M F1076L double mutant has been reported from Chandigarh, Delhi, Mangalore, Orissa, Jodhpur and Puducherry in the recent past.(Anantabotla et al., 2019; Joy et al., 2018; Kaur et al., 2020; Matlani et al., 2021) In a study from Mangaluru from 2014–2016, 76% isolates were reported to have this double mutant.(Joy et al., 2018) In a multicentric study by Vamsi et.al in four regions- Cuttack, Mangaluru, Jodhpur and Puducherry, this double mutant was reported in 55 out of the 60 isolates (91.6%). Among these regions, all isolates from Cuttack and Jodhpur had the double mutant and isolates from Puducherry had the least. 11 out of the 15 (73%) isolates from Puducherry carried this mutant.(Anantabotla et al., 2019) Reports of highest proportion of pvmdr1 double mutants(99%) in India comes from the study in Chandigarh by Kaur et.al(Kaur et al., 2020).
None of the isolates carried the Y976F mutation which is known to be associated with a 1.7-fold increase in resistance to chloroquine.(Suwanarusk et al., 2007) However, the Y976F mutation alone doesn’t contribute to therapeutic failure with chloroquine.(Faway et al., 2016) The presence of Y976F and F1076L together is reported to affect the treatment outcome.(Faway et al., 2016) Hence the triple mutant T958M Y976F F1076L is the significant mutant associated with therapeutic failure. Reports of this triple mutant comes from Thailand and the region of French Guiana.(Faway et al., 2016; Tantiamornkul et al., 2018) In India, the Y976F mutation was reported from Mangaluru as a triple mutant and as a double mutant along with T958M and/or F1076L.(Joy et al., 2018) Other unique and novel mutations reported in the past were Y1028C, A861E and T898E. (Joy et al., 2018; Kaur et al., 2020; Matlani et al., 2021)
The known mutations in the pvdhfr gene are at codon positions 57(F57L/F57I), 58(S58R), 61(T61M), 99(H deletion), 117(S117N/S117T) and 173. Among these, S58R and S117N are the most commonly reported mutations. In the present study, 18 out of 27 isolates (67%) showed the S58R S117N mutation. The studies from India have shown a relatively higher proportion of this double mutant in southern part of India compared to northern India. In the study by Kaur et.al from Chandigarh, majority of the isolates were of the wild type(78.9%) and few with the double mutant(21.1%) as opposed to the study by Valecha et.al where 86.4% double mutants were reported from Chennai.(Kaur et al., 2020; Valecha et al., 2006) Though there is an increased risk of developing resistance to pyrimethamine, double mutants primarily don’t contribute to therapeutic failure but causes a reduced action of the drug.(Hastings et al., 2004) Two types of quadruple mutant pvdhfr have been reported; I57R58M61T117 and L57R58M61T117. Most reports of these quadruple mutants come from Thailand and Myanmar.(Tantiamornkul et al., 2018) In the study conducted by Tantiamornkul et.al, the quadruple mutant I57R58M61T117 was identified in 47.1% isolates and the L57R58M61T117 quadruple mutant in 2.9% isolates.(Tantiamornkul et al., 2018) In the present study, quadruple mutants were not identified in any of the isolates. This was in agreement with the study from Chandigarh.(Kaur et al., 2020) However, few studies from Goa and Assam have reported quadruple mutants.(Alam et al., 2007)
The known mutations in the pvdhps gene are at codons 382, 383, 512, 553, 580 and 585. Among these the commonly reported mutations are A383G and A553G. In the present study, 16 out of 27 isolates(59%) were carrying the double mutant A383G A553G pvdhps gene. In contrast, the study from Chandigarh had majority of isolates as the wild type(91.4%) with no isolate carrying the A553G mutation.(Kaur et al., 2020) The double mutant drug targets for antifolates thus seems to circulate in higher proportions in the southern part of the country over the northern part. One unique mutation reported in few studies from India is at codon 459 where aspartic acid is replaced by alanine (D459A). This mutation was significantly higher in patients who presented with complications.(Garg et al., 2012; Kaur et al., 2020) The percentages of double mutants in the genes pvmdr1, pvdhfr and pvdhps among the isolates studied is given in Fig. 8.
The results of the analysis of mutation patterns were compared between the two subgroups. Though the number of samples in the two groups was discordant, they were compared to identify any trends in mutation patterns over time. The retrospective subgroup had samples collected in the year 2018 and 2019 and the prospective subgroup had samples from the year 2020–2022.
There were 21 patients (77.8%) enrolled in the retrospective subgroup and six in the prospective subgroup (22.2%). For pvmdr1 and pvdhfr there was a rise in the proportion of double mutants in the prospective subgroup by 40% and 21% respectively. For pvdhps however there was a decrease in the double mutants from 62%(13/21) to 50%(3/6) with a decline by 12%. Though none of these differences had any statistical significance, it is to be noted that there has been a definite rise in the proportion of double mutants of pvmdr1 in the prospective subgroup which might have been of statistical significance if more samples could be included in the prospective subgroup.
Chloroquine-primaquine was given as the therapy in 48% of the participants. Cases with prolonged duration of hospital stay beyond 3 days were taken as cases of poor therapeutic response to chloroquine. Seven out of the 13 individuals who received chloroquine therapy had the double mutant pvmdr1 gene in the P.vivax isolates from their blood. Among these seven, six required a hospital stay beyond 3 days(86%) whereas among the six without the double mutant pvmdr1 gene, only one (16.7%) required a prolonged hospital stay. In the four cases with severe malaria, the double mutant pvmdr1 and pvdhfr was seen in 3(75%), and the double mutant pvdhps in 2(50%) cases indicating that cases of severe malaria is more likely to arise with the double mutant genes especially for pvmdr1 and pvdhfr.
Due to logistical constraints, sequencing was performed on a small number of samples. 27 samples were sequenced as part of the study. A higher number of samples might have given a more credible result. There was uneven distribution in the samples in the retrospective and prospective subgroup. Because of the unanticipated COVID pandemic and the subsequent disruption of hospital services, there was a significant reduction in the number of samples received in the prospective subgroup. Clinical correlation of the laboratory results couldn’t be performed for sulfadoxine and pyrimethamine resistance. However, this was a pilot study from our region where the molecular marker of artemisinin resistance was analysed.