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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.

malaria journal

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