This study confirms that high levels of rosetting are associated with severe malaria and reveals that the association between rosetting and severe malaria remains highly statistically significant after multivariate analysis that allows other factors such as patient parasitemia, hemoglobin (Hb) level, and ABO blood group to be taken into consideration. In addition, this study demonstrates, for the first time, that sub-categories of severe malaria all show high levels of rosetting, with no statistically significant difference in rosette frequencies between sub-categories ().
Early studies on rosetting and severe malaria focused on patients with strictly defined cerebral malaria and showed markedly higher rosette frequencies in P. falciparum
isolates from comatose patients compared with isolates from patients with uncomplicated malaria in West Africa (). 1,2
Studies in East Africa showed an association between rosetting and cerebral malaria in some, 3,4
but not all studies. 5,8,32
Furthermore, an association between rosetting and disease severity in mixed groups of severe malaria patients 3,6,7,9
or patients with severe malarial anemia 5,8
was noted. Considerable uncertainty remains regarding whether rosetting is associated with all clinical forms of severe malaria or only some specific syndromes. The current study aimed to clarify the relationship between rosetting and sub-categories of severe disease. We found high levels of rosetting in all sub-categories of severe malaria in Mali, including cerebral malaria (coma), severe malarial anemia, non-comatose neurological impairment (including prostration), repeated seizures without long-lasting neurological impairment, and in a mixed group of patients with symptoms or signs of renal failure or jaundice ().
Despite the fact that this is one of the largest studies of rosetting and severe malaria carried out to date, the sample size is still small in each sub-category of severe disease. This is particularly true for the heterogeneous renal failure/jaundice sub-category, as these clinical features are rare in African children with severe malaria. 28
Severe anemia is also relatively uncommon in this study, due to the age distribution and epidemiology of severe disease in the study area. 33
Neurological abnormalities are frequently seen in children with severe malaria, and all manifestations (unrousable coma, non-comatose impaired consciousness, and repeated seizures) were associated with high rosetting levels (). Ideally, larger studies should be carried out to confirm these results and to clarify the associations between rosetting and severe malaria syndromes under different transmission intensities. However, studies of rosetting and malaria severity are likely to remain problematic due to the logistical difficulties of collecting and culturing large numbers of P. falciparum
isolates from severely ill children. Parasite isolates collected from patients have to be cultured in vitro
for 12–36 hours before assessment of rosetting to allow development of ring-stage parasites (the only form found in peripheral blood) to the pigmented-trophozoite stage at which rosetting occurs. This process is time-consuming and requires facilities for cell culture and microscopy that limit the sites at which studies can be undertaken.
Despite the logistic difficulties and small sample sizes affecting many rosetting studies, a clear pattern emerges across sub-Saharan Africa. Children with uncomplicated malaria are infected with parasite isolates that form few rosettes ( and ). In contrast, most (although not all) children with severe malaria are infected with parasite isolates showing high rosetting levels. In the current study, more than half of the severe malaria cases had parasite isolates with rosette frequencies of 20% or higher. The rosette frequency threshold at which pathogenic effects occur in vivo
is unknown, and could be influenced by numerous factors such as the patient's overall parasite burden, and the size and strength of the rosettes. 34,35
Although the association between rosetting and severe malaria in sub-Saharan Africa is well-established, direct evidence that rosetting plays a causal role in malaria pathogenesis in humans is lacking. This is because experiments addressing the role of rosetting in pathogenesis cannot be performed in humans for ethical reasons, and there is no animal model that fully mimics the pathological and clinical features of falciparum malaria. There is, however, strong indirect evidence from human genetic epidemiological studies that supports a pathogenic role for rosetting. Human erythrocyte polymorphisms of rosetting receptors that reduce the ability of P. falciparum
to form rosettes, such as blood group O 4,14
and complement receptor 1 deficiency, 13
confer protection against severe malaria. 15,16,36,37
These polymorphisms have a specific effect on rosetting and do not influence total parasite burdens, 15,16
therefore their protective effect is compelling evidence that rosetting plays a causal role in pathogenesis. In addition, a plausible mechanism for a pathogenic effect of rosetting has been demonstrated. In an ex vivo
microvasculature model, rosetting parasites cause significantly greater obstruction to flow in small blood vessels that cytoadherent non-rosetting parasites. 10
In this model, rosettes were disrupted by high shear forces in the arterial side of the circulation, but in capillaries and post-capillary venules, rosetting parasitized erythrocytes bound to endothelial cells and uninfected erythrocytes simultaneously to occlude vessels and impair blood flow. Impairment of microvascular blood flow leading to hypoxia, ischemia, and metabolic disturbances is thought to be the fundamental cause of tissue damage and death in severe malaria. 11,12
Taking all the above data together, current evidence supports a direct role for rosetting in the pathogenesis of severe malaria.
One unexpected result that emerged from the multivariate analysis of rosetting was an interaction between ABO blood group and hemoglobin. Rosette frequency was negatively associated with hemoglobin level in isolates from patients with blood groups A and AB, whereas this negative association was not apparent in blood groups B and O (F3,117
= 4.88, P
= 0.003). Further investigation will be required to determine if this is a reproducible finding and to examine its biological significance. Previous work has shown that rosetting parasites show a preference for erythrocytes bearing either A or B blood group antigens, and form larger, stronger rosettes with cells of the preferred type compared with group O cells. 14,38
The preference for the A antigen is particularly common, 39
and direct binding of the parasite rosetting ligand PfEMP1 to the A antigen has been demonstrated. 40
The mechanism through which binding to A antigen (and rosetting in general) might lead to lower hemoglobin levels is unclear. One possibility is that parasite-induced damage of the uninfected erythrocytes in rosettes could occur, including the formation of oxidative products such as 4-hydroxynonenal, 41
that could lead to the phagocytic clearance of uninfected cells and so contribute to anemia. If rosettes in group A patients are larger (i.e., contain more uninfected erythrocytes per rosette than in B and O patients) this could lead to greater clearance of uninfected erythrocytes and account for the relationship with hemoglobin level seen here.
Recent research has identified compounds that reverse rosetting in vitro
and may have potential as adjunctive therapies for severe malaria. 20,21
The mortality rate of severe malaria is as high as 15–20%, even in patients who reach hospital and are treated with effective antimalarial drugs. Approximately 85% of severe malaria-related deaths in hospital occur in the first 24 hours after admission, before the parasite-killing effects of antimalarial drugs have time to act. 28
Therefore adjunctive therapies for severe malaria that target the underlying disease process are urgently needed. 17
Rosette-disrupting therapies that relieve or prevent microvascular obstruction have potential to ameliorate the symptoms of severe malaria. Heparin has been shown to reverse rosetting in a subset (one-third to one-half) of rosetting isolates, 18,19,42
and a heparin derivative reverses sequestration in an animal model. 20
Curdlan sulfate, a glycoconjugate drug that was initially developed as a possible AIDS therapy, 43
was shown to be an effective rosette-reversing agent against a range of clinical isolates 21
and is another potential candidate for severe malaria adjunctive therapy. Curdlan sulfate was shown to be safe for use in Thai adult patients with severe malaria, 44
however, rosetting is not associated with severe disease in this region (possibly due to differing pathogenic mechanisms related to low levels of malaria transmission and immunity, reviewed in reference 17
). Curdlan sulfate has not yet been tested for its effectiveness as an adjunctive therapy in the most appropriate patient population, i.e., children in sub-Saharan Africa. We have demonstrated that all clinical forms of severe malaria are associated with high levels of rosetting in a sub-Saharan African study, which suggests that all severe malaria syndromes in this region might benefit from rosette-disrupting therapies. Clinical trials in well-defined patient populations in parallel with further studies on parasite rosetting properties will be required to determine whether rosette-reversing therapies can reduce the high mortality rate of severe malaria.