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      Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia

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          Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection.


          Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit ( Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs.


          The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9–100), P. falciparum (95% CI 83.2–100), and P. vivax (95% CI 29.2–100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL.


          The Eiken Loopamp™ MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.

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          Most cited references 17

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          High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction.

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            Clinical and laboratory features of human Plasmodium knowlesi infection.

            Plasmodium knowlesi is increasingly recognized as a cause of human malaria in Southeast Asia but there are no detailed prospective clinical studies of naturally acquired infections. In a systematic study of the presentation and course of patients with acute P. knowlesi infection, clinical and laboratory data were collected from previously untreated, nonpregnant adults admitted to the hospital with polymerase chain reaction-confirmed acute malaria at Kapit Hospital (Sarawak, Malaysia) from July 2006 through February 2008. Of 152 patients recruited, 107 (70%) had P. knowlesi infection, 24 (16%) had Plasmodium falciparum infection, and 21 (14%) had Plasmodium vivax. Patients with P. knowlesi infection presented with a nonspecific febrile illness, had a baseline median parasitemia value at hospital admission of 1387 parasites/microL (interquartile range, 6-222,570 parasites/microL), and all were thrombocytopenic at hospital admission or on the following day. Most (93.5%) of the patients with P. knowlesi infection had uncomplicated malaria that responded to chloroquine and primaquine treatment. Based on World Health Organization criteria for falciparum malaria, 7 patients with P. knowlesi infection (6.5%) had severe infections at hospital admission. The most frequent complication was respiratory distress, which was present at hospital admission in 4 patients and developed after admission in an additional 3 patients. P. knowlesi parasitemia at hospital admission was an independent determinant of respiratory distress, as were serum creatinine level, serum bilirubin, and platelet count at admission (p < .002 for each). Two patients with knowlesi malaria died, representing a case fatality rate of 1.8% (95% confidence interval, 0.2%-6.6%). Knowlesi malaria causes a wide spectrum of disease. Most cases are uncomplicated and respond promptly to treatment, but approximately 1 in 10 patients develop potentially fatal complications.
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              A prospective comparative study of knowlesi, falciparum, and vivax malaria in Sabah, Malaysia: high proportion with severe disease from Plasmodium knowlesi and Plasmodium vivax but no mortality with early referral and artesunate therapy.

              Plasmodium knowlesi commonly causes severe malaria in Malaysian Borneo, with high case-fatality rates reported. We compared risk, spectrum, and outcome of severe disease from P. knowlesi, Plasmodium falciparum, and Plasmodium vivax and outcomes following introduction of protocols for early referral and intravenous artesunate for all severe malaria. From September 2010 to October 2011 we prospectively assessed nonpregnant patients aged ≥12 years admitted to Queen Elizabeth Hospital (QEH), Sabah, with polymerase chain reaction-confirmed Plasmodium monoinfection. Standardized referral and prereferral intravenous artesunate were instituted at district hospitals. Severe malaria occurred in 38 of 130 (29%) patients with P. knowlesi, 13 of 122 (11%) with P. falciparum, and 7 of 43 (16%) with P. vivax. The commonest severity criteria in knowlesi malaria included parasitemia >100 000/µL (n = 18), jaundice (n = 20), respiratory distress (n = 14), hypotension (n = 13), and acute kidney injury (n = 9). On multivariate analysis, P. knowlesi was associated with a 2.96-fold (95% confidence interval, 1.19-7.38-fold) greater risk of severity than P. falciparum (P = .020); only parasitemia and schizontemia >10% independently predicted knowlesi severity. Risk of severe knowlesi malaria increased 11-fold with parasitemia >20 000/µL, and 28-fold with parasitemia >100 000/µL. Nearly all (92%) knowlesi malaria patients received oral artemisinin therapy; 36 of 38 (95%) and 39 of 92 (42%) with severe and nonsevere disease, respectively, also received ≥1 dose of intravenous artesunate. No deaths occurred from any species. Plasmodium knowlesi is the commonest cause of severe malaria at QEH, with parasitemia the major risk factor for severity. Early referral and treatment with artesunate was highly effective for severe malaria from all species and associated with zero mortality.

                Author and article information

                Malar J
                Malar. J
                Malaria Journal
                BioMed Central (London )
                13 January 2017
                13 January 2017
                : 16
                [1 ]Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, NT Australia
                [2 ]Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah Malaysia
                [3 ]Jesselton Medical Centre, Kota Kinabalu, Sabah Malaysia
                [4 ]Clinical Research Centre, Queen Elizabeth Hospital, Kota Kinabalu, Sabah Malaysia
                [5 ]Global Good Fund/Intellectual Ventures Laboratory, Bellevue, WA USA
                [6 ]FIND, Geneva, Switzerland
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

                Funded by: Ministry of Health, Malaysia
                Award ID: BP00500420
                Funded by: Asia Pacific Malaria Elimination Network
                Award ID: 108-07
                Funded by: National Health and Medical Research Council (AU)
                Award ID: 1037304
                Award ID: 1045156
                Award ID: 1042072
                Award ID: 1074795
                Award Recipient :
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                © The Author(s) 2017

                Infectious disease & Microbiology

                plasmodium knowlesi, lamp, malaria, diagnosis, rdt, pcr


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