A 17-year-old male presented to the hospital emergency with fever associated with chills for the past 5-6 days. He also had complaints of abdominal pain and vomiting. The patients did not have any complaints of rash, joint pains, cough, difficulty in breathing, and any feature suggestive of urinary tract infection. The history of seizures and bleeding from any site was ruled out. There was no past history of diabetes mellitus, hypertension, tuberculosis, smoking, or alcohol intake. The patient did not give a history of previous treatment or self-medication. The patient resided in a slum area in east Delhi with a drain near his house.
On examination, the patient was sick and his general condition was poor. He was conscious and oriented and had severe pallor. His heart rate was 110 beats/min, respiratory rate was 16 breaths/min, and blood pressure was 110/70 mmHg. On per abdominal examination, abdomen was soft and nontender. Hepatomegaly and massive splenomegaly was detected. On central nervous system examination, patient was lethargic, but no other abnormality was present. On chest and cardiovascular examination, no significant abnormality was detected. On the basis of these findings, a provisional diagnosis of acute undifferentiated fever was made with a differential diagnosis of malaria, dengue fever, and enteric fever. The patient was admitted in the department of internal medicine, and the case was further investigated.
The hemoglobin of the patient was 5 g/dL, total leukocyte count was 3,900/mm3, and platelet count was 78,000/mm3 on Day 0 and 30,000/mm3 on Day 2. Peripheral blood smear (PBS) showed microcytic hypochromic anemia with tear drop cells and thrombocytopenia. Micro-hematuria was detected on urine routine examination. His liver and renal function tests were within normal limits. Tube Widal test titers were <1:40 for TO, TH, and AH. Serum RK-39 was negative. Chinkungunya immunoglobulin M enzyme-linked immunosorbent assay was negative. Dengue immunoglobulin M enzyme-linked immunosorbent assay (NIV, Pune, India) was reactive. Rapid malaria antigen test (RMAT) (Ybio, Goa, India) on Day 0 was positive for P. vivax only. However, on PBS examination, rings of 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. Abdominal ultrasound showed enlarged spleen (30 cm in size), mild hepatomegaly, and ascites. Bilateral pleural effusion was also noted.
A final diagnosis of mixed malarial infection (P. vivax and P. falciparum) with dengue fever coinfection with pancytopenia was made.
The patient was prescribed intravenous (IV) artesunate 2.4 mg/kg on Day 0 then at 12 h and 24 h. He was also administered IV ceftriaxone 600 mg b.d., IV fluids, IV paracetamol, and IV ondansetron and pantoprazole as supportive management on Day 0 of admission. The next day, the patient was switched to oral ACT (1 tablet of artesunate 200 mg and 2 tablets of sulfadoxine/pyrimthamine 750 mg/37.5 mg on Day 1 followed by artesunate 200 mg for next 2 days).
By Day 3 of treatment, the patient's condition had improved, but fever persisted (>37.5 C).
RMAT and PBS were reperformed on Day 3. On PBS, ruptured schizonts of P. vivax, rings and gametocytes of P. falciparum were seen, with a rise in parasitemia to 5%. (Figure 1). RMAT was now positive for both P. vivax and P. falciparum.
Unfortunately, the patient left the hospital against medical advice and was lost to follow-up.
As the patient had rise in parasitemia with fever of (>37.5 C) on Day 3 of treatment, he was diagnosed as a case of early treatment failure (ETF) according to the National Vector Borne Diseases Control Programme guidelines. The patient's blood spots were inoculated on Whatman filter paper no. 1 for further molecular analysis.
Genomic 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). The samples were also genotyped for the drug resistance genes viz: chloroquine resistance (pfcrt), multidrug resistance (pfmd-r1), sulfadoxine (pfdhps), pyrimethamine (pfdhfr), and artemisinin resistance (keltch 13). Single-nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, pfdhps, and keltch 13 genes were assessed by PCR-RFLP and sequencing as described previously. 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). Two mutations in pfdhfr gene at C59R and S108N were observed and there was one mutation in the pdhps gene at A437G. One SNP was seen on aligning with wild-type sequences of pfdhfr (GenBank accession no. 9221804) and pfdhps (GenBank accession no. 2655294) using Clustal W of the BioEdit 7.0 and MEGA (Molecular Evolutionary Genetics Analysis; http://www.megasoftware.net/) 4.0 programs (Table 1). Since this case was diagnosed as a case of ETF and artesunate resistance was suspected, sequence analysis to detect the presence of any SNP in kelch13 was also carried out. However, no SNPs were detected in this region (Table 1).