DOI: https://doi.org/10.21203/rs.3.rs-2491278/v1
Plasmodium knowlesi has been reported as an emerging infection throughout the Southeast Asian region, especially in the Malaysian state of Sabah, where it accounts for the majority of the malaria cases reported. We present a case of P. knowlesiinfection in a Danish woman returning from a short trip to Malaysian Borneo. The patient spent 14 days in forested areas of Sabah and fell ill after returning to Denmark in November 2022. Microscopy of blood smears revealed 0.8% infected erythrocytes, but due to the atypical morphological presentation, a conclusive species identification was made by molecular methods. The patient was treated successfully with artemisinin-based combination therapy. Plasmodium knowlesi is a potentially fatal infection, and taking the increasing travel activity into consideration after the coronavirus disease 2019 (COVID-19) pandemic, P. knowlesishould be a differential diagnosis in patients with travel-associated illness returning from highly endemic Southeast Asian areas.
The simian parasite Plasmodium knowlesi with a natural reservoir in long-tailed macaques, pig-tailed macaques and banded leaf monkeysis considered the fifth Plasmodium species causing malaria in humans (1,2). P. knowlesi cases have been reported from almost every country in Southeast Asia, but the Malaysian states of Sarawak and Sabah have the highest incidence in the region (3), where it is an emerging challenge in efforts to eliminate malaria (4). No indigenous cases of Plasmodium falciparum, Plasmodium vivax or Plasmodium malariae have been recorded in Malaysia since 2017, and in 2020, the country reached the World Health Organization Global Technical Strategy for Malaria 2016-2030 goal by interrupting local malaria transmission (5). Although Malaysia has successfully eliminated indigenous transmission of the previously three common human malaria species in the region, infections due to P. knowlesi are on the rise (6).
Plasmodium knowlesi has a short 24-h erythrocytic replication cycle, and most cases are mild with low parasitaemia (7). However, in highly endemic areas, severe P. knowlesi infections with fatal outcomes have been observed (8). Since the morphology of early- and late-stage trophozoites of P. knowlesi resembles that of P. falciparum and P. malariae,respectively (9), microscopic examination of blood smears can lead to misdiagnosis, especially in non-endemic areas (10). In Scandinavia, the most recent P. knowlesi casewas reported more than 15 years ago (11). The low number of P. knowlesi cases reported from European countries may be a matter of misdiagnosis rather than a low infection rate.
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)
Ethics approval and consent to participate: Not applicable
Consent for publication: Written informed consent was obtained from the patient for publication of this case report
Availability of data and materials: Not applicable Competing interests: The authors declare that they have no competing interests
Funding: The study received no external funding.
Authors’ contribution: HA, MK and CRS drafted the manuscript. AP and MMJ obtained a detailed travel history and treated the patient. HA, RBD and MK participated in primary microscopy and follow up. CRS and HVN were responsible for molecular detection, molecular characterization and analysis of sequence data. All authors read and approved the final manuscript.
Acknowledgement We wish to thank Ermina K. Mrgan for her excellent assistance.