The primate malarial parasite, Plasmodium knowlesi, is responsible for inducing malaria in its natural hosts, the long-tailed and pig-tailed macaques, and is transmitted by forest-dwelling mosquitoes belonging to the Anopheles leucosphyrus group. Human infection with this species was considered rare until a large focus of cases in the Kapit division of Sarawak state, Malaysian Borneo, occurred in 2004. Since then, cases have been described throughout Southeast Asia, and the organism is now recognized as the fifth species of Plasmodium to cause malaria in humans. People at risk of acquiring the infection are predominantly farmers, hunters, or camp workers who venture out into the forest. Occasional cases in travelers to Southeast Asia have also been described. To date, only one case of P. knowlesi infection has been reported in the UK, in a traveler returned from Brunei in 2006. This report describes the first case of P. knowlesi infection in a Scottish traveler following a trip to Borneo, Malaysia.
The first imported case of Plasmodium knowlesi in Scotland is described in a 33-year-old female with a travel history to Borneo. The patient ceased to take antimalarial prophylaxis after 4 days of her 10-day visit and presented with a history of fever, rigor, vomiting, and diarrhea after 13 days on her return to the UK. Malaria antigen detection using the Optimal-IT and Binax-NOW kits was negative. Unusual trophozoite-like structures were observed under microscopic examination and the identification of P. knowlesi performed by a nested polymerase chain reaction (PCR) gel-based approach was confirmed by using a PCR-sequencing assay.
A 33-year-old female student nurse of British nationality presented to the accident and emergency department (A & E) at the Western Infirmary, Glasgow in July 2012 with a 2-day history of fever, rigor, diarrhea, and vomiting after having returned 13 days before from a 10-day trip to Borneo. She had a past medical history of epilepsy and had previously been treated for malaria while traveling in Malawi in 2009. She was prescribed atovaquone/proguanil (Malarone) as antimalarial prophylaxis for the journey to Borneo which was taken for the first 4 days of her visit, but then discontinued owing to the development of nausea which was attributed to the medication. During her stay in Borneo she visited a primate sanctuary within a nature reserve which had jungle areas known to harbor Anopheles species, the mosquito vector of P. knowlesi.
A urine dipstick test was performed at the A & E, which revealed numerous erythrocytes. A stool sample was sent to the Scottish Parasite Diagnostic and Reference Laboratory (SPDRL) for microscopy examination of ova, cysts, and parasite, which was reported as negative, however the presence of Charcot–Leyden crystals indicated the presence of a parasitic infection. No urinary tract infection symptoms were described and the patient was not menstruating at the time. She was given IV fluids and antiemetics, and discharged with a plan for repeat urinalysis and for further parasite testing. Her fever settled 4 days after discharge, but the nausea and diarrhea persisted, resulting in the patient presenting to the infectious diseases department in Gartnavel General Hospital in Glasgow 13 days after the visit to A & E. Physical examination at presentation was unremarkable. Initial blood tests revealed a white blood cell count of 6.85 (normal adult reference range 3.4–11.0 × 109/L), an eosinophil count of 0.74 (normal 0.00–0.5 × 109/L), hemoglobin of 129 (normal 115–165 g/L), platelets of 241 (normal 140–450 × 109/L), a normal C-reactive protein of 0.2 (normal 0–8 mg/L), and normal urea and electrolytes. Blood samples (clotted blood and EDTA whole blood) were sent to the SPDRL for blood film analysis based on the initial presentation of fever, and serological tests were performed for a range of parasitic diseases namely, schistosomiasis, strongyloides, and filariasis in light of the raised eosinophilia count and foreign travel history. Serology test results were negative with the exception of the Schistosome ELISA which was weakly positive. As the serology test for schistosomiasis is IgG based, not IgM, it was not possible to conclude if this was a current infection. Results of three stools and a 24-h urine sample were negative.
The EDTA whole blood was negative for malaria antigens using the BinaxNOW kit (Alere Ltd., Cheshire, UK) and the Optimal-IT kit (TCS Biosciences, Buckingham, UK). However, on examination of the thin blood films, “unusual trophozoite-like structures” were observed, with less than 1% of cells infected. Because of the patient's travel history to Borneo, the possibility of a P. knowlesi infection was considered. Molecular detection of this parasite was performed using two methods. In the first method, DNA was extracted from 200 μL of the patient's blood using the QIAmp DNA mini kit (QIAGEN, Hilden, Germany). Amplification of the DNA was performed using nested assays to amplify the ssrRNA in the Plasmodium genome.[5-7] The outer round primers were genus-specific, rPLU1 and rPLU5 (Table 1), whereas the inner round primers were species-specific targeting either P. falciparum, P. vivax, P. ovale, P. malariae, or P. knowlesi (Table 1). Polymerase chain reaction (PCR) products were subjected to electrophoresis on a 1% agarose gel. The reactions for all PCR assays were negative except for the P. knowlesi assay which produced an amplicon of 153 base pairs. Further supporting evidence was provided by performing a second molecular method of detection on DNA extracted from blood spotted onto filter paper using a EZ1 BioRobot™ (QIAGEN, Germany) according to the manufacturer's instructions. An 850 base pair portion of the mitochondrial cytochrome b gene was amplified using nested PCR, the outer PCR using the primers DW2 and DW4, and the inner using NCYBINF and NCYBRINR (Table 1). Sequencing was performed on two independently amplified PCR products from separate DNA extractions of the same blood. Sequences were compared to homologous sequences from all available Plasmodium species, and a 100% match was achieved with P. knowlesi, and no other species.
|Genus specific||Singh et al.|
|rPLU5||CCT GTT GTT GCC TTA AAC TCC|
|rPLU1||TCA AAG ATT AAG CCA TGC AAG TGA|
|P. falciparum||Snounou et al.|
|FAL1||TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT|
|FAL2||ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC|
|P. knowlesi||Lee et al.|
|Pmk8||GTT AGC GAG AGC CAC AAA AAA GCG AAT|
|Pmk9||ACT CAA AGT AAC AAA ATC TTC CGT A|
|P. ovale||Snounou et al.|
|OVAL1||ATC TCT TTT GCT ATC TTT TTT TAG TAT TGG AGA|
|OVAL2||GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG|
|P. malariae||Snounou et al.|
|MAL1||ATA ACA TAG TTG TAC GTT AAG AAT AAC CGC|
|MAL2||AAA ATT CCC ATG CAT AAA AAA TTA TAC AAA|
|P. vivax||Snounou et al.|
|VIV1||CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC|
|VIV2||ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA|
|DW2||TAA TGC CTA GAC GTA TTC CTG ATT ATC CAG||Liu et al.|
|DW4||TGT TTG CTT GGG AGC TGT AAT CAT AAT GTG|
|NCYBINF||TAA GAG AAT TAT GGA GTG GAT GGT G||Liu et al.|
|NCYBRINR||CTT GTG GTA ATT GAC ATC CAA TCC|
The patient was treated as an outpatient with a 4-day course of chloroquine for the P. knowlesi infection, and with praziquantel for schistosomiasis. On follow-up, she remained well.
The initial findings of fever and eosinophilia count combined with the recent foreign travel history were suggestive of a potential parasite infection. Schistosome species and Plasmodium species are capable of inducing an eosinophilic response, and therefore may have contributed this. However, it was not possible to conclude when the Schistosome infection was acquired based on a positive ELISA result as the antibody test is IgG-based and determines if a patient has been previously exposed to Schistosome egg antigens rather than detecting current infection. To assess the presence, viability, and species of Schistosome ova, and to provide supporting evidence for a current infection, microscopy detection of stools/urine is performed. This is often insensitive; therefore a negative microscopy result does not rule out a current Schistosome infection and treatment is offered on the basis of a positive ELISA result. It is known that the patient had visited Malawi in 2009, a Schistosome endemic area, and therefore it cannot be ruled out that the patient acquired schistosomiasis during her earlier vacation.
The presentation of P. knowlesi infection varies from asymptomatic infection to severe/fatal disease with high parasitemia. Plasmodium knowlesi has a periodicity of 24 h, and tends to manifest itself between 3 and 14 days although longer incubation times have been noted in other imported cases.[9-11] Relapse of the disease is not a noted feature of a P. knowlesi infection. Although there are no distinctive features or clinical symptoms that assist in a specific diagnosis of P. knowlesi from any other malaria species, infection most typically presents with a nonspecific febrile illness; thrombocytopenia is common whereas anemia is rarely observed in adult cases on hospital admissions. In this particular case, the patient initially presented with fever within 13 days of her return which settled and the persistence of gastrointestinal disturbances is not uncommon in patients with malaria.
For this uncomplicated P. knowlesi infection, atovaquone/proguanil were used to successfully treat the patient. Other commonly used antimalarial drugs include chloroquine, chloroquine/primaquine, mefloquine, quinine/doxycycline which are known to be effective and, in addition, the artemisinin derivatives have shown faster clearance of the parasite. The optimal treatment for severe P. knowlesi infections, where infected red cells sequester in capillaries of the brain, heart, and kidneys, with high parasitemias, has not been determined.[12, 14] In terms of using antimalarial chemoprophylaxis, which the patient stopped prematurely, P. knowlesi is currently not considered when advising on suitable prophylaxis as guidance tends to focus on the risks of P. falciparum. It is interesting that the patient did not present with more severe clinical symptoms given the high replicative rate of this parasite, and perhaps having taken atovaquone/proguanil may have had an impact on this.
Clinically, P. knowlesi can be difficult to diagnose as accurate identification using microscopy is not always possible since early trophozoite stages are identical to those of P. falciparum, while mature blood stages resemble those of P. malariae. In addition, commercially available rapid diagnostic tests have poor sensitivity for P. knowlesi. Therefore, the only accurate means of diagnosis is by P. knowlesi-specific PCR assays that use nested-PCR, real-time PCR, or PCR combined with sequencing. The introduction of molecular methods has enabled the accurate diagnosis of P. knowlesi infections, and the enhanced recognition of this infection in travelers. This case highlights the need for a high index of suspicion of P. knowlesi infection in patients with a history of travel to Southeast Asia, and demonstrates that negative antigen testing in a febrile traveler from Malaysia does not rule out a malaria diagnosis. We recommend that in cases with a travel history to a P. knowlesi endemic region combined with a high index of suspicion of malaria, specialist molecular testing is performed in addition to routine examination of blood films and antigen testing.
This work was supported by the NHS Greater Glasgow and Clyde Endowment Award, UK and the Asia Africa Science Platform Program JSPS, Japan. We would like to thank Sarina Hokama, Japan, and Lisa Connelly, SPDRL, for their technical contributions.
Declaration of Interests
The authors state that they have no conflicts of interest.