Novel mutations in the antifolate drug resistance marker genes among Plasmodium vivax isolates exhibiting severe manifestations Shilpi Garg a , Vishal Saxena a , Vanshika Lumb b , Deepak Pakalapati a , P.A. Boopathi a , Amit Kumar Subudhi a , Shibasish Chowdhury a , Sanjay K. Kochar c , Dhanpat K. Kochar d , Y.D. Sharma b , Ashis Das a,⇑ a Department of Biological Sciences, Birla Institute of Technology and Science, Pilani 333031, Rajasthan, India b Department of Biotechnology, All India Institute of Medical Sciences, New Delhi 110029, India c Department of Medicine, Sardar Patel Medical College, Bikaner 334003, Rajasthan, India d Department of Neurology, Kothari Medical and Research Institute, Bikaner, Rajasthan, India highlights " Novel mutations were observed in both DHFR and DHPS of Plasmodium vivax. " Presence of novel mutations in PvDHPS gene suggests its polymorphic nature. " Frequency of PvDHFR–PvDHPS two locus mutations were higher among the severe isolates. graphical abstract article info Article history: Received 7 March 2012 Received in revised form 19 August 2012 Accepted 21 September 2012 Available online 5 October 2012 Keywords: Plasmodium vivax Severe manifestations Antifolate drug resistance Novel mutations abstract Plasmodium vivax is the predominant species of the human malaria parasite present in the Indian subcontinent. There have been recent reports on Chloroquine (CQ) resistance and severe manifestations shown by P. vivax from different regions of the world including India. This study focuses on Bikaner, India where during the last few years there have been continuous reports of severe manifestations by both Plas- modium falciparum and P. vivax. This region has a widespread use of Chloroquine and Sulfadoxine–Pyri- methamine for the treatment of malaria, but the resistance profiles of these drugs are not available. We report here the profile of mutations in marker genes associated with Chloroquine and antifolate drug resistance among the P. vivax parasites obtained from patients with severe (n = 30) and non-severe (n = 48) manifestations from this region. Most isolates showed the wild type alleles for both the Chloro- quine and antifolate resistance markers (P < 0.0005). Except for one isolate showing Y976F mutation in the Pvmdr-1 gene, no reported mutation was observed in the Pvmdr-1 or Pvcrt gene. This is in accordance with the fact that till date no Chloroquine resistance has been reported from this region. However, the single isolate with a mutation in Pvmdr-1 may suggest the beginning of the trend towards decreased sus- ceptibility to Chloroquine. The frequency of PvDHFR–PvDHPS two locus mutations was higher among the patients showing severe manifestations than the patient group with non-severe (uncomplicated) malaria (P < 0.003). None of the parasites from patients with uncomplicated P. vivax malaria showed the mutant PvDHPS genotype. Novel mutations in PvDHFR (S117H) and PvDHPS (F365L, D459A and M601I) were observed only in the parasite population obtained from patients exhibiting severe complications. 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.exppara.2012.09.018 ⇑ Corresponding author. Fax: +91 1596244183. E-mail addresses: ashdas28@gmail.com, adas@bits-pilani.ac.in (A. Das). Experimental Parasitology 132 (2012) 410–416 Contents lists available at SciVerse ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr