An Immunohistochemical Study of the Pathology of Fatal Malaria: Evidence for Widespread Endothelial Activation and a Potential Role for Intercellular Adhesion Molecule-1 in Cerebral Sequestration

The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P.falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.     

Erythrocyte survival in severe falciparum malaria.

Erythrocyte survival was studied in 17 Thai patients (10 males, 7 females; aged 13-57 years) with severe falciparum malaria. To ensure radioisotopic labelling of cells before bone marrow recovery and survival analysis under near-steady state conditions, 51Cr labelling of autologous erythrocytes was performed at the time of admission (0 h) and calculation of mean cell lifespan (MCL) was based on semilogarithmic plots of corrected counts from 60 h onwards. Five patients received blood transfusions, all within 48 h of admission. The overall mean (+/- S.D.) MCL was short (44.1 +/- 21.7 days). Nontransfused patients had similar MCL values (43.6 +/- 20.4) to those of transfused patients (45.5 +/- 27.3 days, p greater than 0.8). Patients with and without palpable splenomegaly had MCL values which were not significantly different (54.1 +/- 28.8 vs. 37.2 +/- 12.3 days respectively, p greater than 0.1). There was no association between admission haematocrit or peripheral parasitaemia and MCL (p greater than 0.2 in each case), but there was an inverse correlation between total serum bilirubin and MCL (r = -0.49, p less than 0.025). There is accelerated destruction of non-parasitised erythrocytes in severe malaria resulting in a mean MCL that is half that found previously in healthy Thai volunteers (89.6 +/- 13.1 days, p less than 0.001) and significantly shorter than that reported previously in Thai patients with uncomplicated P. falciparum infections studied after parasite clearance (56.8 +/- 10.2 days, p less than 0.05).     

In vitro efficacy of antimalarial drugs against Plasmodium vivax on the western border of Thailand

The susceptibility of 20 isolates of Plasmodium vivax on the Thailand-Myanmar border to seven antimalarial drugs was evaluated using the schizont maturation inhibition technique. The geometric mean 50% inhibition concentration (IC(50)) values were quinine = 308 ng/mL, amodiaquine =14 ng/mL, chloroquine =50 ng/mL, mefloquine = 127 ng/mL, sulfadoxine/pyrimethamine (80:1) = 800/10 ng/mL, pyrimethamine = 8 ng/mL, and artesunate = 0.5 ng/mL. Compared with P. falciparum in this area, P. vivax was more sensitive to chloroquine and artesunate, equally sensitive to quinine, and more resistant to mefloquine.     

Activation of the coagulation cascade in falciparum malaria

The incidence and progression of coagulation abnormalities were studied in 52 patients with acute falciparum malaria. The patients were prospectively divided into 3 groups; severe (parasitaemia greater than or equal to 5% or vital organ dysfunction), 12 patients; moderate (parasitaemia 1%- less than 5% without complications), 16 patients; and mild (parasitaemia less than 1%), 24 patients. No case died or developed clinical evidence of disseminated intravascular coagulation. Conventional indices of coagulation (prothrombin time, partial thromboplastin time, fibrinogen, fibrin degradation products) were usually within the normal range but reduced plasma concentrations of antithrombin III (AT-III) levels were noted in all groups, and the incidence was significantly higher in patients with severe and moderate malaria (83% and 81%) compared with the mild group (37%; P less than 0.005). Depletion of AT-III was associated with thrombocytopenia, decreased AT-III activity and elevated plasma concentrations of thrombin-antithrombin III complexes (P less than 0.01), confirming activation of the coagulation cascade and increased clotting factor consumption. AT-III levels returned to normal coincident with clinical improvement. Activation of coagulation is a common and sensitive measure of disease activity in acute falciparum malaria. It is not a specific feature, nor is there evidence to suggest it has a primary pathological role in severe infections.     

Therapeutic responses to antibacterial drugs in vivax malaria.

Some antibacterial drugs have antimalarial activity that can be exploited for the prevention or treatment of malaria. Monotherapy with tetracycline, doxycycline, clindamycin or azithromycin was assessed in 1995-98 in 92 adult patients in Thailand with Plasmodium vivax malaria. All patients recovered following treatment and the early therapeutic responses were similar among the 4 groups. The overall median fever clearance time was 57 h and the mean (SD) overall time to parasite clearance was 134 (48) h. Of 66 patients who completed a 28-day follow-up, reappearances of vivax infection occurred in 27 patients (41%) from all groups; delayed appearances of falciparum malaria occurred in 6 patients (9%), only from the azithromycin group. The overall mean (SD) time to reappearance of P. vivax was 23 (5) days and time taken for detection of falciparum malaria was 13 (4) days after starting treatment for vivax malaria. The 28-day cumulative cure rates of clindamycin (n = 12), tetracycline (n = 18) and doxycycline (n = 18) groups were similar (P > or = 0.14) and all were significantly higher compared to the azithromycin group (n = 18; P < or = 0.04). The intervals until vivax reappearance were also significantly shorter in the azithromycin group [mean (SD) = 21 (6) vs 25 (3) days, P < 0.05] suggesting that some of these were recrudescences. The apparent success rate (no subsequent appearances of either vivax or falciparum infection) was significantly lower for the azithromycin group (11%) compared to the other groups (34-78%; P < 0.01). In current antibacterial treatment regimens, short-course azithromycin has inferior antimalarial activity compared to clindamycin or the tetracyclines.     

Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae

Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).     

Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand

The dhps sequences of 55 Plasmodium vivax isolates (39 from Thailand and 16 from elsewhere) revealed mutant Pvdhps at codons 383 and/or 553 (A --> G) in 33 isolates, all from Thailand. Mutations of Pvdhps and Pvdhfr were correlated. Multiple mutations were associated with high-grade sulfadoxine-pyrimethamine resistance.     

Relapses of Plasmodium vivax infection usually result from activation of heterologous hypnozoites

BACKGROUND: Relapses originating from hypnozoites are characteristic of Plasmodium vivax infections. Thus, reappearance of parasitemia after treatment can result from relapse, recrudescence, or reinfection. It has been assumed that parasites causing relapse would be a subset of the parasites that caused the primary infection. METHODS: Paired samples were collected before initiation of antimalarial treatment and at recurrence of parasitemia from 149 patients with vivax malaria in Thailand (n=36), where reinfection could be excluded, and during field studies in Myanmar (n=75) and India (n=38). RESULTS: Combined genetic data from 2 genotyping approaches showed that novel P. vivax populations were present in the majority of patients with recurrent infection (107 [72%] of 149 patients overall [78% of patients in Thailand, 75% of patients in Myanmar {Burma}, and 63% of patients in India]). In 61% of the Thai and Burmese patients and in 55% of the Indian patients, the recurrent infections contained none of the parasite genotypes that caused the acute infection. CONCLUSIONS: The P. vivax populations emerging from hypnozoites commonly differ from the populations that caused the acute episode. Activation of heterologous hypnozoite populations is the most common cause of first relapse in patients with vivax malaria.