correspondence Dr B.K. Das, Department of Internal Medicine, SCB Medical College, Cuttack – 753007, Orissa, India. Fax: 91-674-2301399; E-mail: firstname.lastname@example.org
Plasma glucose was assessed in 81 patients with severe falciparum malaria at the time of presentation along with tumour necrosis factor-α (TNF-α). The lowest plasma glucose value was 3.38 mmol/l and none of the patients had hypoglycaemia at admission. Plasma glucose values were not significantly lower in those with multiple organ dysfunction (MOD) than in patients with single organ dysfunction (cerebral malaria only) and in those who died compared with patients who survived. Conversely, TNF-α showed a good correlation with depth of coma and was significantly higher in patients who had MOD and those who died. There was no correlation between plasma glucose and TNF-α values.
Hypoglycaemia has often been described as a common complication of severe falciparum malaria. It has been implicated as a major cause of morbidity and mortality in children and pregnant women (1985Looareesuwan et al. 1985; 1988Taylor et al. 1988; 1989Grau et al. 1989). Most reports on these groups come from areas of stable endemicity such as Africa (1988Taylor et al. 1988; 1989Grau et al. 1989). This demographic profile is not seen in areas experiencing unstable endemicity or epidemic outbreaks, such as India. Here healthy adults constitute a major segment of the susceptible population. There are few reports describing hypoglycaemia as a complication from this region (1988, 1992Das et al. 1988, 1992).
There are several postulated mechanisms of disease-induced hypoglycaemia. One of them involves the release of tumour necrosis factor-α (TNF-α) (1987Clark et al. 1987). It has been observed that high levels of TNF-α are associated with increased morbidity and mortality in children with cerebral malaria (1989Grau et al. 1989) and few reports in adults with multiorgan failure. TNF-α causes hypoglycaemia in mice (1987Clark et al. 1987). It is also reported that high TNF-α levels were associated with hypoglycaemia but predicted fatal outcome independently of glucose concentration (1990Kwiatkowski et al. 1990). Hypoglycaemia is said to be associated with a high concentration of TNF-α (2000WHO 2000).
This prospective study was conducted on adult patients of cerebral malaria and multiorgan failure to evaluate the occurrence of hypoglycaemia at the time of presentation and observe an association if any, between TNF-α and plasma glucose. Here we provide evidence that hypoglycaemia is uncommon in acute severe falciparum infection in adults irrespective of severity. TNF-α levels are significantly high in patients of cerebral malaria, more with multiorgan failure than with single organ involvement (cerebral).
Patients and methods
Adult patients admitted to the Department of Internal Medicine at S.C.B. Medical College Hospital, Cuttack, with a short history of fever and unarousable coma (Glasgow Coma Score < 9) were screened for asexual stage Plasmodium falciparum in Giemsa-stained peripheral smears for the diagnosis of cerebral malaria and assessed clinically for other associated complications. Patients with other causes of encephalopathy such as trauma, toxins, metabolic causes and bacterial meningitis were excluded by clinical, biochemical and lumbar puncture examinations. Patients previously treated with intravenous fluids containing dextrose and/or quinine were excluded from the study. Depth of coma was evaluated using the modified Glasgow Coma Scale. On admission, blood samples were collected for biochemical investigations and sera separated and stored at −20 °C for TNF-α assay.
The Glucose Oxidase method (Qualigens, India) using a spectrophotometer (Systronics 106) was employed in the estimation of plasma glucose levels from fluoridated blood samples and the results expressed in mmol/l. For the TNF-α assay 2 μgm/ml of purified anti cytokine capture mAb (monoclonal Antibody) (Pharmingen, USA) in 0.1 m sodium bicarbonate pH 8.2 was added 50 μl to each well of the enzyme-linked immunosorbent assay (ELISA) plate (Costar, USA). The plate was covered and kept overnight at 4 °C. The plate was washed twice in phosphate-buffered saline (PBS)-Tween (0.05%), blocked with 200 μl/well PBS 10% fetal calf serum (FCS, Sigma Chemical Co., USA) for 2 h at room temperature. Then the plate was washed twice in PBS-Tween and 100 μl of neat undiluted serum was added to each well along with the standard (10 000 pg/ml downwards with double dilutions). The plate was incubated at 4 °C overnight and washed four times in PBS-Tween and 100 μl of diluted biotinylated anti cytokine detecting mAb (Pharmingen, USA) 2–3 μg/ml in PBS 10% FCS was added, incubated for 45 min at 37 °C and washed six times with PBS-Tween. Five hundred fold diluted avidin peroxidase (Sigma) was added 100 μl/well to the plate for 30 min at room temperature and the plate was washed eight times in PBS-Tween and the enzyme activity was measured by addition of o-phenylene diamine in citrate phosphate buffer pH 5.6 (1 mg/ml). The reaction was stopped with 8 n H2SO4 and the absorbance was measured at 492 nm by using a micro ELISA reader (Bio-Rad, USA). A standard curve was drawn from the optical density of the standards using the same log graph paper. Values were expressed in pg/ml of serum.
Comparative analyses between mean plasma glucose and TNF-α values of different subsets were calculated using the Wilcoxon–Mann–Whitney non-parametric test for unpaired samples. Coefficients of correlation were calculated using the Karl Pearson formula.
We studied 81 patients (64 males and 17 females), aged 16–72 years (mean = 35.6 years). All had been healthy before the onset of malaria. Thirty-one patients developed multiple organ dysfunction (MOD) and the remaining 50 had cerebral malaria (single organ dysfunction, SOD). Fourteen patients died during the course of treatment. Hypoglycaemia was defined as a plasma glucose level of less than 2.2 mmol/l. None of the patients were found to be hypoglycaemic. The lowest plasma glucose value was 3.38 mmol/l. The mean glucose level was 5.68 mmol/l (SD = ±1.08 mmol/l) (1Table 1). There was a wide variation in the TNF levels (22–800 pg/ml). The mean TNF-α value was 154.5 pg/ml (SD = ±171.23 pg/ml).
Table 1. Mean values of plasma glucose levels and TNF-α in different subsets of patients and their correlation
SOD, Single Organ Dysfunction; MOD, Multiple Organ Dysfunction.
Glucose levels did not vary significantly between patients with multiorgan failure and single organ involvement (P > 0.05) or between patients who survived and those who died (P > 0.05). Conversely, TNF-α levels showed a strong correlation with depth of coma (P < 0.01). It was significantly higher in patients having MOD (P < 0.05) and in those who died (P < 0.05). There was no correlation between plasma glucose and TNF-α values in all patients taken together (P > 0.05) (1Figure 1).
In complicated malaria, hypoglycaemia mainly occurs at the time of presentation (1996White 1996). In this study, the mean glucose level at admission was 5.68 mmol/l (SD = ±1.02 mmol/l). The lowest glucose value was 3.38 mmol/l. The absence of hypoglycaemia in this series of 81 patients suggests that it is uncommon in adults in this region. Hypoglycaemia was reported in five cases in a series of 46 patients of severe falciparum malaria from the western belt of Orissa, India, an area of high endemicity (1988Das et al. 1988). One of the five patients had hypoglycaemia at presentation (hepatic glycogen was depleted on liver biopsy) and in the rest it developed during quinine therapy. The low glycogen reserves in children and increased metabolic requirements in pregnancy have been postulated as the cause of higher incidence of hypoglycaemia in these groups of patients (1987White et al. 1987; 2000WHO 2000). Interestingly, a study on severe malaria in a paediatric population in this region also observed a low incidence of hypoglycaemia. The relatively uncommon episodes of hypoglycaemia in both adult and paediatric patients with severe malaria in this region could be as a result of factors in the hosts or parasites that need to be determined. All the patients in this series were nutritionally healthy adults, which could have contributed to the absence of hypoglycaemia attacks.
Though we failed to demonstrate any instance of hypoglycaemia, we looked for variation in plasma glucose levels with disease severity. Plasma glucose levels did not correlate with depth of coma. The variation in plasma glucose levels between patients having cerebral affection only (SOD) and in those with multiple organ involvement (MOD) was not significant. Similarly, there was no significant difference in glucose levels between patients who died and those who survived.
One of the factors implicated in the pathogenesis of hypoglycaemia is TNF-α (1987Clark et al. 1987). Therefore, we assessed the levels of TNF-α in various subsets of patients. Plasma glucose levels failed to correlate with TNF-α. However, TNF-α values correlated well with the depth of coma. It was significantly higher in patients having MOD and those who died. This is consistent with the observations of other workers in children (1989Grau et al. 1989; 1990Kwiatkowski et al. 1990) and confirms the role of TNF-α as a predictor of morbidity and mortality in adults. There are no reports to our knowledge on adult patients of falciparum malaria that compare plasma glucose and TNF-α in different subsets of patients with severe falciparum malaria. A study in Malawian children found that high concentrations of TNF were associated with hypoglycaemia (1989Grau et al. 1989). A later work in Gambian children found that high concentrations of TNF were associated with hypoglycaemia but predicted fatal outcome independently of glucose concentrations (1990Kwiatkowski et al. 1990). On the other hand, recent reports from experiments in animals provide evidence that TNF is associated with a rise rather than a fall in plasma glucose levels (1988Bagby et al. 1988; 1996Elased et al. 1996).
In conclusion, hypoglycaemia is an uncommon manifestation of severe falciparum malaria, at presentation, in adults, in this region. Plasma glucose is unaffected irrespective of severity of disease. TNF-α was significantly higher in patients with deep coma, and in those with MOD than in cerebral malaria patients and in those who died compared with those who survived.
We thank the Director of the Regional Medical Research Centre (ICMR), Bhubaneswar and Dr B. Ravindran, Deputy Director, Division of Immunology, RMRC for extending laboratory facilities. We also thank Dr P.K. Sahoo, Research Assistant, RMRC for his help in running the TNF-α assays.