A 53-year-old previously healthy Danish woman was admitted to a hospital in Zealand, Denmark with a seven-day history of fever, night sweats, nausea and severe headache. She had been experiencing fever up to 39 ºC every 24 hours, each attack lasting for two hours. Prior to falling ill, the patient had spent 14 days in the Bornean state of Sabah, Malaysia. Most of her vacation involved trekking in highlands and rainforest areas in wildlife nature resorts of Sepilok and Tabin. A detailed itinerary revealed that the patient had spent several days in two “hotspot” divisions in the state of Sabah, where P. knowlesi transmission is predicted to intensify (4). The last two days of her vacation were spent near coastal areas. She only slept indoors but not under a bed net. Despite using a topical mosquito repellent, the patient noticed several mosquito bites and did not take any anti-malarial chemoprophylaxis. The patient starting feeling ill 11 days after returning to Denmark. At admission, the patient was febrile (38 ºC) and hypotensive (96/59 mmHg) with normal respiratory parameters. Physical examination revealed nystagmus and inability to perform a neurological coordination test. On suspicion of viral encephalitis, a lumbar puncture was performed, which revealed a normal cell count, and normal protein and glucose levels. Blood testing revealed a normal haemoglobin concentration (11.6 g/L), thrombocytopenia (50 × 109/L), lymphocytopenia (0.5 x 109/L), and an elevated lactate dehydrogenase level (270 U/L). Liver enzymes, bilirubin and creatinine levels were within the normal ranges. Based on the patient’s travel history and symptomology, malaria was a differential diagnosis. Microscopy of Giemsa-stained blood smears revealed Plasmodium parasites with 0.8% infected erythrocytes. The morphological characteristics were atypical for Plasmodium species infecting humans, resembling both P. falciparum (Figure 1, B) and P. malariae (Figure 1, F). All developmental stages except for mature schizonts of P. knowlesi were observed (Figure 1, A-H). Erythrocytes were not enlarged, and multiple ring forms within a single erythrocyte, double chromatin and accole forms were seen. Stippling was absent, although dots were observed in a small number of infected erythrocytes. Both delicate, and more compact/dense trophozoite rings with various forms were seen. Brown/yellow malaria pigment was present in developmental stages from mature trophozoites to gametocytes. Blood was analysed by Loop-mediated isothermal amplification (LAMP) and was positive for Plasmodium DNA. The Rapid Diagnostic Test (First Response® Combo Malaria Ag (pLDH/HRP2) card test) was positive for the pan-malaria antigen but negative for the P. falciparum specific antigen. The patient was given one dose of intravenous artesunate 2.4 mg/kg followed by a three-day course of artemether-lumefantrine (20 + 120 mg). Parasitaemia decreased to <0.01% after just one dose of artesunate, and the patient recovered rapidly. Two days later, she was discharged with normal haematological parameters and negative blood and cerebrospinal fluid cultures.
Due to the patient’s travel history and atypical morphological presentation of the parasites in the blood smears, P. knowlesi was suspected. Since a conclusive Plasmodium species identification was not possible by microscopy alone, an EDTA blood sample was submitted to DNA extraction and in-house real-time PCR for detection of P. falciparum, P. vivax, P. ovale, P. malariae, and P.knowlesi. The sample produced a strong signal (cycle threshold, 27) for P. knowlesi. In order to characterize the strain and rule out mixed infection with another Plasmodium species not included in the real-time PCR assay, the DNA was also submitted to metabarcoding similarly to a previous study (12), which confirmed mono-infection by P. knowlesi. Two consensus sequences were produced from the BION DNA sequence output and subjected to phylogenetic analysis with reference sequences from the NCBI Database (Figure 2). One sequence clustered with 100% bootstrap support with reference P. knowlesi SSU rDNA sequences of the S-type; the other clustered with 100% bootstrap support with reference P. knowlesi SSU rDNA sequences of the A-type. The two types of rDNA sequences were obtained by two different primer sets (the G6 and the G3 primers, respectively)(13)