Adapting Novel Molecular Diagnostic Methods for the Detection of Plasmodium knowlesi in Sarawak, Malaysia

Abstract

Background: Recent epidemiological studies demonstrate that the prevalence of the fifth major human malaria parasite, Plasmodium knowlesi (monkey malaria), is often underestimated and misdiagnosed with standard microscopy blood film. We sought to adapt and compare a new simple molecular diagnostic method for P. knowlesi with the gold standard nested molecular assay and microscopy blood film in P. knowlesi hotspot areas in Sarawak, Malaysia. In addition, we analyzed the statistical association between P. knowlesi positive test results and demographic and behavioral/occupational risk factors.Methods: The study was conducted at Sibu, Kapit and Sarikei Hospitals in Sarawak, Malaysia. 115 blood samples were collected from malaria suspected patients seeking treatment at these hospitals. Samples were analyzed by microscopy, Nested polymerase chain reaction (PCR) and single-step PCR. Sensitivity, specificity, and practical value of the new single-step PCR assay was calculated. Bivariate and multivariate regression was conducted to test the possible risk factors for the detection of P. knowlesi.Results: Single-step PCR showed low sensitivity (51.92%, 95%CI 37.63 - 65.99%) compared to nested PCR and 46.03% (95%CI 33.39 - 59.06%) compared to microscopy. When compared to nested PCR, microscopy had a false positive rate of 20.6%. However, it only missed 2 cases of P. knowlesi. The mean age in the study population was 40.35. Patients enrolled at Kapit hospital had higher odds ratio for positive P. knowlesi PCR results (adjusted OR = 4.46, 95%CI 1.16 – 11.51). Age above 21 years (adjusted OR = 6.28, 95%CI 1.53 – 25.64), male gender (adjusted OR = 2.46, 95%CI 0.91 – 6.65) and living near a vegetation (Plantation, forest, fruit trees or wet rice paddy) (adjusted OR = 5.96, 95%CI 1.11 – 31.83) were associated with increased risk for P. knowlesi infection.Conclusions: Data from this study showed that single-step PCR has a low sensitivity and thus, it is not a suitable alternative for accurate detection of P. knowlesi. Further studies are required for assessment and development of other diagnostic assays or new primer sets. Multivariate analysis revealed that adult men over the age of 21 who live near agricultural areas have the highest risk for P. knowlesi malaria infection. Large- scale descriptive studies of both non-human hosts and vectors would greatly influence prevention and control strategies of this zoonotic disease.