PMC5566360_01
Asexual stages of malaria parasite in PBS (Day 0). (A) Ring forms of P. falciparum and P. vivax. (B) Ring form and gametocytes of P. falciparum. Day 3 - PBS. (C) Ruptured schizonts of P. vivax.
(D) Rings, accole forms, and gametocytes of P. falciparum.. Abbreviation: PBS, peripheral blood smear.
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P. vivax and rings and gametocytes of P. falciparum were seen. Parasite density for P. falciparum was found to be 2% (Figure 1). Polymerase chain reaction (PCR) assay confirmed the presence of both the species (P. falciparum and P. vivax) in the patient. On PBS, ruptured schizonts of P. vivax, rings and gametocytes of P. falciparum were seen, with a rise in parasitemia to 5%. (Figure 1).
(A) 18S PCR results showing mixed infection inpatient samples. Lane 1 contains 100 bp ladder, lane 2 has Day 0 sample, and lane 3 has Day 3 sample. Both samples show mixed infections of P. falciparum at 205 bp and P. vivax at 120 bp, respectively. (B) RE analysis of pfcrt gene. Lane 1 contains Day 0 sample and lane 2 contains Day 3 sample with 50 bp ladder in lane 3. (C) Restriction digestion of pfmdr-1 amplicon. Lanes 1 and 2 are patient samples confirming the SNP (N86Y) in the amplicons (lanes 1 and 2 are Day 0 and Day 3 sample, respectively), lanes 3 and 4 are undigested amplicons, and lane 5 contains 100 bp ladder.. Abbreviations: PCR, polymerase chain reaction; RE, restriction enzyme; SNP, single-nucleotide polymorphism.
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DNA extraction of the patient sample collected on Day 0 and Day 3 was done from filter paper blood spots using a QIAamp DNA Blood Mini Kit (QiagenInc., Hilden, Germany) according to the manufacturer's instructions. Diagnostic confirmation of Plasmodium species was done by 18S rRNA PCR assay, which confirmed the results of microscopy and RMAT, showing mixed infections of P. falciparum and P. vivax in the patient (Figure 2). Restriction site for ApoI enzyme for K76T mutation associated with chloroquine resistance in the pfcrt gene and N86Y SNP for restriction enzyme AflIII in pfmdr-1 gene were seen in both the samples of the patient (Figure 2).