Limitations of microscopy to differentiate Plasmodium species in a region co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi

Background: In areas co-endemic for multiple Plasmodium species, correct diagnosis is crucial for appropriate treatment and surveillance. Species misidentification by microscopy has been reported in areas co-endemic for vivax and falciparum malaria, and may be more frequent in regions where Plasmodium knowlesi also commonly occurs. Methods: This prospective study in Sabah, Malaysia, evaluated the accuracy of routine district and referral hospital-based microscopy, and microscopy performed by an experienced research microscopist, for the diagnosis of PCR-confirmed Plasmodium falciparum, P. knowlesi, and Plasmodium vivax malaria. Results: A total of 304 patients with PCR-confirmed Plasmodium infection were enrolled, including 130 with P. knowlesi, 122 with P. falciparum, 43 with P. vivax, one with Plasmodium malariae and eight with mixed species infections. Among patients with P. knowlesi mono-infection, routine and cross-check microscopy both identified 94 (72%) patients as "P. malariae/P. knowlesi"; 17 (13%) and 28 (22%) respectively were identified as P. falciparum, and 13 (10%) and two (1.5%) as P. vivax. Among patients with PCR-confirmed P. falciparum, routine and cross-check microscopy identified 110/122 (90%) and 112/118 (95%) patients respectively as P. falciparum, and 8/122 (6.6%) and 5/118 (4.2%) as "P. malariae/P. knowlesi". Among those with P. vivax, 23/43 (53%) and 34/40 (85%) were correctly diagnosed by routine and cross-check microscopy respectively, while 13/43 (30%) and 3/40 (7.5%) patients were diagnosed as "P. malariae/P. knowlesi". Four of 13 patients with PCR-confirmed P. vivax and misdiagnosed by routine microscopy as "P. malariae/P. knowlesi" were subsequently re-admitted with P. vivax malaria. Conclusions: Microscopy does not reliably distinguish between P. falciparum, P. vivax and P. knowlesi in a region where all three species frequently occur. Misdiagnosis of P. knowlesi as both P. vivax and P. falciparum, and vice versa, is common, potentially leading to inappropriate treatment, including chloroquine therapy for P. falciparum and a lack of anti-relapse therapy for P. vivax. The limitations of microscopy in P. knowlesi-endemic areas supports the use of unified blood-stage treatment strategies for all Plasmodium species, the development of accurate rapid diagnostic tests suitable for all species, and the use of PCR-confirmation for accurate surveillance.