Fatal human knowlesi malaria has hitherto not been reported at post-mortem, so this report may increase our understanding of severe and fatal malaria whatever species cause these syndromes. Interestingly, vivax malaria is also becoming recognized as causing severe and sometimes fatal infection in a significant proportion of individuals, suggesting re-evaluation of the dogma that falciparum infection may be the only important cause of fatal disease [19–21].
The WHO classification for severe falciparum malaria would have included this individual because he had several qualifying features including hyperparasitaemia (~10% infected erythrocytes), renal impairment, jaundice and ARDS, although coma was not prominent in the history [3, 4, 22]. Commonly, dengue haemorrhagic fever can cause some of these signs, but was excluded by investigations. Co-infection of knowlesi with falciparum was also excluded by specific PCR and immunohistochemical examination.
The beefy appearance of lung tissue in this case is consistent with respiratory distress syndrome, but could not be confirmed histologically. Changes in the kidney of some recovering areas of acute tubular injury are consistent with the observation of renal impairment reported in other severe cases of knowlesi malaria and an underlying process of acute tubular necrosis related to systemic shock. There was disorganized white pulp in spleen perhaps as part of a wider stress response .
Despite the absence of antecedent established coma, cerebral pathology in this case is very similar to that observed in fatal falciparum infections, confirming that it was important to exclude this co-infection. Although there was obvious accumulation/sequestration of infected erythrocytes in capillaries and venules, there were also some differences between these appearances and those seen with fatal falciparum infections. For example, there was no platelet clumping, no notable thrombi, and uninfected erythrocytes were also interspersed with infected cells. Neither was up-regulation of ICAM-1 expression detected in endothelial cells, suggesting that the mechanism of sequestration/accumulation of infected erythrocytes in knowlesi infections needs further investigation. In falciparum cerebral malaria, display of ICAM-1 by up regulated endothelial cells mediates adhesion to parasitized red cells .
Hyperparasitaemia is a marker of severe falciparum malaria. It is also apparent in severe knowlesi infections , but is less commonly observed with severe vivax malaria. The 24 hr replicative cycle of asexual knowlesi may contribute to the rapidity with which hyperparasitaemia and clinical complications ensue. This shorter cycle compared with falciparum and vivax infections may overcome the reduced multiplicative capacity associated with the fewer merozoites generated by P. knowlesi (up to 16 per mature schizont) compared with P. falciparum (up to 36). Although up to 24 merozoites per schizont have been observed in Plasmodium vivax, parasitaemia is restricted to reticulocytes by limitation of invasion pathways in this species, rather than merozoite number.
Hyperparasitaemia in falciparum malaria is often explained by protection of late stage parasites from the filtering action of the spleen. Unlike P. falciparum infections, all asexual developmental stages are seen in the peripheral circulation of knowlesi-infected patients . Hyperparasitaemia in knowlesi malaria may not be modulated by splenic clearance in a similar manner as suggested for other non-sequestering human infections.
As with many fatal cases of falciparum malaria, malaria pigment was evident in blood films and was present in circulating leucocytes (~40%), tissue sections and organ specific macrophages. Reticuloendothelial changes in both liver and spleen were associated with pigment, accumulation of red cells and laden macrophages, and some inflammation in liver portal tracts was observed. Circulating pigment has been variably implicated as an indicator of poor prognosis for falciparum malaria [23–25] and there is evidence to support pigment-induced immuno-suppression, particularly of pigment-laden macrophages and monocytes [26–28]. It may be that the associations between parasitaemia, pigmentaemia and disease severity are more quantifiable in knowlesi compared with falciparum infections, where pigmented parasites are sequestered from peripheral blood samples and, therefore, unreliably quantifiable. In vivax malaria, pigment in parasites often appears more dispersed and it is rarely reported as a correlate of disease .
Among the malaria parasites of humans, cytoadherence is purportedly unique to P. falciparum, resulting in sequestration of all but early trophozoite stages in falciparum malaria. Cytoadherence is often implicated in malaria pathology and is mediated by the expression of variable var gene products (of the PfEMP-1 family) at the surface of infected host red blood cells with concomitant expression of post-capillary endothelial cell receptors [15, 30–32]. Although not easily quantifiable, partial sequestration was observed in this fatal case of knowlesi malaria as evidenced by an abundance of large pigment bearing parasites on the blood film obtained post-mortem and as accumulations in the microvasculature. We hypothesize that partial sequestration may be due to P. knowlesi infected cell agglutination mediated by variant surface antigens of P. knowlesi SICA var genes (orthologues of the PfEMP-1 family) [33, 34]. Importantly, in this fatal case of P. knowlesi, up-regulation of ICAM-1 was not detected, nor was there evidence for cytoadherence as parasitized cells were not marginalized and were interspersed with uninfected erythrocytes in smaller vessels.
Cerebral malaria and severe acute anaemia are often peculiar to falciparum infection while organ, respiratory and metabolic dysfunction are common with severe knowlesi, vivax and other forms of severe falciparum infection [3, 4, 19, 35–40]. Although late stages of parasites may be visible in the blood in falciparum infections, they are rare and associated with a poor prognosis. Testing the relative contribution of virulence factors to the development of severe malaria including, cytoadherence, hyperparasitaemia, cerebral malaria, organ failure, metabolic and respiratory dysfunction and anaemia was previously difficult without comparative information from severe malaria caused by another species [16, 41–44]. Severe and fatal cases of knowlesi malaria will add much needed perspective to what is known of malaria pathophysiology.
The need to develop knowlesi-specific diagnosis, treatment and management guidelines is urgent. A recent prospective clinical study in Sarawak Malaysian Borneo revealed that approximately 1:10 patients infected with P. knowlesi present with or develop severe symptoms and 1-2% of cases are fatal . Complications in survivors included ARDS, liver or renal dysfunction, hypotension with or without parasitaemia >100,000/uL. Complications in all fatal knowlesi cases had either clinical or laboratory evidence of abdominal pain, combined hepatorenal dysfunction and hyperparasitaemia [3, 4]. There is a prospect that P. knowlesi may emerge as a human pathogen beyond its current zoonotic manifestations [11, 12], specific surveillance, control and clinical guidelines are necessary to contain potential larger outbreaks.