This study highlights the difficulties of microscopic diagnosis of Plasmodium species in an area where P. falciparum, P. vivax and P. knowlesi all commonly occur. Misdiagnosis of P. knowlesi as both P. vivax and P. falciparum, and vice versa, is common. Only 72% of patients with PCR-confirmed P. knowlesi received an accurate diagnosis by routine or by cross-check microscopy, and correlation between the microscopic methods was poor, with even fewer patients receiving an accurate diagnosis of P. knowlesi by both methods. These findings occurred despite considering P. knowlesi and P. malariae as a single group, and so were not a consequence of the well described near impossibility of distinguishing these two species. Rather, patients with PCR-confirmed P. knowlesi were commonly misdiagnosed as having either P. falciparum or P. vivax malaria, with misdiagnosis of P. vivax as “P. malariae/P. knowlesi” also common.
The difficulty with distinguishing P. knowlesi from P. falciparum by microscopy has been previously described, due to similarities between the young rings of P. knowlesi and ring forms of P. falciparum, including double chromatin dots, multiple-infected erythrocytes and applique forms [22, 23]. In a previous study in Sarawak, 11/216 (5%) patients diagnosed by microscopy as “P. malariae” were actually P. falciparum by PCR, and 33/312 (11%) microscopy-diagnosed P. falciparum cases were P. knowlesi by PCR . In this previous study and in the current study, however, the difficulty differentiating P. knowlesi and P. vivax was also notable. In the current study 30% of patients with PCR-confirmed P. vivax were misdiagnosed by routine microscopy as “P. malariae/P. knowlesi”, with four patients subsequently re-admitted with presumed vivax relapses due to lack of primaquine treatment. In the Sarawak study, 43 of 440 (10%) patients with PCR-confirmed P. vivax malaria were misdiagnosed as “P. malariae” . In a series of malaria deaths in Sabah, one of six fatal cases of P. knowlesi was misdiagnosed as P. vivax by microscopy and a fatal case of P. vivax was misdiagnosed as “P. malariae” .
This frequent misdiagnosis of P. vivax as “P. malariae/P. knowlesi” has significant implications for malaria control, as failure to administer anti-hypnozoite treatment may lead to increased transmission and may hamper efforts to eliminate vivax malaria in regions where P. knowlesi is common. These findings support the current Sabah Ministry of Health policy for the performance of reference centre PCR on all patients with a microscopic diagnosis of “P. malariae/P. knowlesi” to enable administration of primaquine to those found to have misdiagnosed P. vivax. In knowlesi-endemic areas, where logistically possible, PCR should also be performed on at least a proportion of slides diagnosed as P. falciparum or P. vivax, to allow monitoring of the accuracy of microscopic diagnoses at different clinical sites, and to identify areas where additional training of microscopists may be required. Given the inaccuracies of microscopic diagnosis, performance of PCR is also essential to maintain accurate surveillance, particularly monitoring the emergence of P. knowlesi.
The inaccuracy of microscopy for differentiating Plasmodium species creates difficulties with basing treatment decisions on microscopic results. The 2008 Malaysian Ministry of Health malaria treatment guidelines recommend chloroquine for uncomplicated P. malariae/P. knowlesi malaria; chloroquine plus primaquine for uncomplicated P. vivax malaria; artemisinin combination treatment (ACT; artesunate/mefloquine [Artequine®] or artemether/lumefantrine [Riamet®]) for uncomplicated P. falciparum malaria; and intravenous artesunate, or intravenous quinine plus oral doxycycline, for severe P. falciparum or severe P. malariae/P. knowlesi malaria . No recommendations are given for treatment of severe vivax malaria. At Queen Elizabeth Hospital and referral hospitals in its catchment area, updated treatment guidelines recommend ACT for uncomplicated P. falciparum or P. malariae/P. knowlesi malaria; chloroquine plus primaquine, or ACT, for uncomplicated P. vivax malaria; and intravenous artesunate (followed by oral therapy as above) for severe malaria from any species .
Given the different treatment recommendations for each species in both of these guidelines, inappropriate treatment decisions may be made as a result of incorrect microscopic diagnoses. Misdiagnosis of P. falciparum as P. knowlesi would, under current national guidelines, result in administration of chloroquine for P. falciparum. Given widespread resistance of P. falciparum to chloroquine, this would lead to increased risk of complications and/or fatal outcome, as well as increased transmission. Patients with severe P. knowlesi malaria misdiagnosed as P. vivax may fail to receive immediate parenteral treatment if national guidelines are followed, and this scenario has been previously associated with fatal outcomes . Even if the updated hospital guidelines are followed, misdiagnosis of severe P. knowlesi as P. vivax may still lead to treatment with oral chloroquine if signs of severity are missed, potentially leading to adverse outcomes given the slower parasite clearance associated with chloroquine as compared to oral ACT .
These results therefore support the argument for a unified treatment strategy of ACT for uncomplicated malaria from all Plasmodium species in knowlesi-endemic areas, an approach increasingly recommended for regions co-endemic for P. falciparum and P. vivax. These data also support the 2012 WHO recommendation for intravenous artesunate to be given to any patient meeting severe malaria criteria . Even if signs of severity are overlooked among patients with knowlesi malaria, treatment with oral ACT may ensure more rapid parasite clearance and may lead to improved outcomes compared to treatment with oral chloroquine , although the optimal oral agent for uncomplicated P.knowlesi remains undetermined.
An additional advantage of this unified treatment approach would be the avoidance of inadvertent use of chloroquine for P. falciparum misdiagnosed as another species. Furthermore, chloroquine-resistant P. vivax is an increasing problem throughout Southeast Asia , and has been previously documented in Sabah  and Peninsular Malaysia [33–35]. Use of chloroquine for P. vivax malaria may therefore be associated with treatment failures, and may potentiate spread of chloroquine resistance.
Finally, this study highlights the need to develop rapid diagnostic tests (RDTs) that have the ability to distinguish between Plasmodium species, in order that reliable results can be obtained more quickly and cheaply than is possible with PCR. Although histidine-rich protein 2 (HRP2)-based RDTs are able to diagnose P. falciparum, RDTs that distinguish P. knowlesi from P. vivax are not yet available. Limited data suggest that P. knowlesi cross-reacts with both P. falciparum and P. vivax-specific pLDH [36–39], and RDTs that combine these antigens with HRP2 may therefore allow differentiation between P. vivax, P. falciparum and P. knowlesi mono-infections. However, while a pLDH RDT has shown high sensitivity for the diagnosis of severe malaria from all three of these species, neither pLDH- or aldolase-based RDTs have demonstrated sufficiently high sensitivity for uncomplicated P. knowlesi. Prospective evaluation of more sensitive RDTs in knowlesi-endemic areas is needed.
This study has found that microscopy does not reliably distinguish between P. falciparum, P. vivax and P. knowlesi in areas co-endemic for all three species, with misdiagnosis of P. vivax and P. knowlesi particularly common. In P. knowlesi-endemic areas these limitations of microscopic diagnosis must be considered when developing strategies to monitor the prevalence of the different malaria species, and when developing treatment guidelines.