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Background   Plasmodium knowlesi  is recognized as the fifth  Plasmodium  species causing malaria in humans. It is morphologically similar to the human malaria parasite  Plasmodium malariae , so molecular detection should be used to clearly discriminate between these  Plasmodium  species. This study aimed to quantify the rate at which  P. knowlesi  is misidentified as  P. malariae  by microscopy in endemic and non-endemic areas.    Methods  The protocol of this systematic review was registered in the PROSPERO International Prospective Register of Systematic Reviews (ID = CRD42020204770). Studies reporting the misidentification of  P. knowlesi  as  P. malariae  by microscopy and confirmation of this by molecular methods in MEDLINE, Web of Science and Scopus were reviewed. The risk of bias in the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). The pooled prevalence and 95% confidence interval (CI) of the misidentification of  P. knowlesi  as  P. malariae  by microscopy were estimated using a random effects model. Subgroup analysis of the study sites was performed to demonstrate any differences in the misidentification rates in different areas. Heterogeneity across the included studies was assessed and quantified using Cochran’s Q and I 2  statistics, respectively. Publication bias in the included studies was assessed using the funnel plot, Egger’s test and contour-enhanced funnel plot.    Results  Among 375 reviewed studies, 11 studies with a total of 1569 confirmed  P. knowlesi  cases in humans were included. Overall, the pooled prevalence of the misidentification of  P. knowlesi  as  P. malariae  by microscopy was estimated at 57% (95% CI 37–77%, I 2 : 99.3%). Subgroup analysis demonstrated the highest rate of misidentification in Sawarak, Malaysia (87%, 95% CI 83–90%, I 2 : 95%), followed by Sabah, Malaysia (85%, 95% CI 79–92%, I 2 : 85.1%), Indonesia (16%, 95% CI 6–38%), and then Thailand (4%, 95% CI 2–9%, I 2 : 95%).    Conclusion  Although the World Health Organization (WHO) recommends that all  P. malariae -positive diagnoses made by microscopy in  P. knowlesi  endemic areas be reported as  P. malariae / P. knowlesi  malaria, the possibility of microscopists misidentifying  P. knowlesi  as  P. malariae  is a diagnostic challenge. The use of molecular techniques in cases with  malariae -like  Plasmodium  with high parasite density as determined by microscopy could help identify human  P. knowlesi  cases in non-endemic countries.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12936-021-03714-1.

Quantification of the misidentification of Plasmodium knowlesi as Plasmodium malariae by microscopy: an analysis of 1569 P. knowlesi cases