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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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5 g/dL) but was not associated with hyperparasitemia (> 500,000 asexual forms/mm3). Additionally, pigmented PMNs/mm3 was associated with a fatal outcome in patients with severe malaria. This study validates the presence of malaria pigment in monocytes and neutrophils as a marker for disease severity, and demonstrates that pigmented neutrophils are associated with cerebral malaria and with death in children with severe malaria. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Hemozoin, also known as malaria pigment, is a product of hemoglobin digestion by Plasmodia.6 The existence of hemozoin has been recognized for centuries, but the complexities of its formation and its biologic and clinical relevance are incompletely understood. As part of parasite erythrocytic invasion, hemoglobin is proteolyzed releasing toxic heme. Due to the absence of heme oxygenase, Plasmodia are unable to cleave heme into open-chain tetrapyrrole to allow for cellular excretion.7 To detoxify soluble heme, a novel breakdown product known as hemozoin is created intracellularly. This digestive end-product of hemoglobin is sequestered in the P. falciparum digestive vacuole within infected red blood cells and released into host circulation during schizogeny. Hemozoin is composed of FeIII-porphyrin units linked by propionate oxygen-iron bonds into polymers accompanied by additional host and parasite nucleic acids and lipids.8,9 Once this insoluble polymer is released into host circulation, scavenger neutrophils and monocytes phagocytose the material. It is easily visible by light microscopy, appearing as a black, brown, or amber pigment or as a birefringent crystal under polarized light10,11 (Figures 1
and 2). Given that the typical half-life of a neutrophil is 6–8 hours and that of a monocyte is several days, the quantity and distribution of engulfed pigment within these phagocytic cells may reflect the chronology of a patient’s infection.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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). More than one clinical diagnosis for severe malaria was possible, but cerebral malaria and severe anemia were considered to be the primary defining features when they co-existed with other criteria. Each index case was age-, residence-, and ethnicity-matched to a case of uncomplicated malaria and a healthy control. Age categories were defined as 3–5 months, 6–11 months, 1 year, 2 years, 3–4 years, 5–6 years, 7–8 years, 9–10 years, 11–12 years, and 13–14 years. Residence was defined as one of eight distinct sectors of Bandiagara town or, in the case of children from outer villages, the specific village of origin. Uncomplicated malaria was defined as P. falciparum parasitemia and an axillary temperature 
37.5°C detected by active surveillance, or parasitemia and symptoms leading to treatment-seeking behavior in the absence of other clear cause of fever on passive surveillance. Matched uncomplicated malaria controls were enrolled from the population of children presenting to the daily Bandiagara Malaria Project clinic. Healthy controls were enrolled after traveling to the home of the child with severe malaria and following a standardized routine of exiting the front entrance of a compound and making consecutive left turns until another compound with an eligible control was identified. Children were enrolled as healthy controls if they were asymptomatic for acute illness, had no evidence or history of chronic illness, and if they were found to be aparasitemic upon examination. The study protocol was reviewed and approved by the University of Mali and the University of Maryland Institutional Review Boards. Written informed consent was obtained from parents or guardians of all study participants.
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Statistical analysis. Pooled analyses between clinical groups were made using a two-sided Student’s t-test for continuous variables with equal variance and a chi-square test for categorical variables using Stata version 7.0 (Stat Corp., College Station, TX) and SPSS version 10.0 (SPSS, Inc., Chicago, IL). For the purposes of analyzing differences between matched pairs, McNemar’s test was used to assess absolute pigment counts and pigment/mm3 using Stata version 7.0. A level of statistical significance (two-sided) was set at P < 0.05. A Kappa coefficient was used in assessing inter-observer variability in the reading of a 10% proportion of thin smears.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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. Fifteen of 172 patients with severe malaria (8.7%) died. Of those with severe malaria, 49% were hyperparasitemic, 45% had cerebral malaria, 16% were severely anemic, and 2.4% met other criteria. Patients meeting the criteria for both cerebral malaria and severe anemia (n = 15) were analyzed as a unique subset. Anemia was more common among those with a fatal outcome than in survivors (mean hemoglobin concentration = 5.9 g/dL versus 8.2 g/dL, respectively; P < 0.0001). Additionally, geometric mean parasite densities in children with severe malaria were significantly lower in those that died than in survivors (29,994 asexual forms/mm3 versus 174,428 asexual forms/mm3; P < 0.0001). When patients whose sole criterion for severe malaria was hyperparasitemia (mortality = 0.01%) were excluded, the geometric mean parasite density in survivors was 69,278 asexual forms/mm3 compared with 29,994 asexual forms/mm3 in those who died (P = 0.06). No significant differences were noted among study groups in age distribution, sex, or total WBC count.
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] = 0.88 for PMN agreement, = 0.77 for monocytes). Total hemozoin counts are summarized in Table 3. The results are depicted graphically in a scatter plot in Figure 3.
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Using the matched design (age, residence, and ethnicity) of the study population, we performed McNemar’s testing to assess differences between individually matched pairs. An increased amount of malaria pigment in phagocytic cells was more likely in severe malaria cases compared with uncomplicated malaria cases (pigmented PMNs: odds ratio [OR] = 5.6, 95% confidence interval [CI] = 2.83–12.31, P < 0.0001, pigmented monocytes: OR = 2.85, 95% CI = 1.38–5.85, P = 0.003). Within the severe malaria group, an average of 635 pigmented PMNs/mm3 were noted at admission in subjects who subsequently died due to severe disease, compared with an average of 320 pigmented PMNs/mm3 in the survivors (P = 0.02). A trend was noted, but did not achieve statistical significance, when the same analysis was performed evaluating pigment presence in monocytes in severe malaria survivors versus children who died of severe disease (407 cells/ mm3 versus 198 cells/mm3; P = 0.09).
To evaluate association between presence of pigment and disease syndrome, the severe malaria group was stratified into the four predominant presenting diagnoses: cerebral malaria (n = 77), severe anemia (n = 26), severe anemia and cerebral malaria (n = 15), and hyperparasitemia (n = 85). For subjects in both the cerebral malaria group and the combined cerebral malaria/severe anemia group, significantly more pigmented PMNs/mm3 were noted at presentation than in patients with severe malaria without cerebral symptoms (Table 4). No such association with clinical syndrome was found for monocytic pigment. When stratified by parasitemia (those with peripheral parasite densities 500,000 asexual forms/ mm3 and those with < 500,000) no significant differences were noted either in amount of pigmented PMNs or monocytes/ mm3. A higher concentration of monocyte pigment was noted in children with severe malaria who were not hyperparasitemic compared with those who were (290 cells/mm3 versus 143 cells/mm3; P = 0.04). Multivariate regression analysis found no significant association between pigmented monocytes/mm3 and peripheral parasite density (P = 0.11). Similarly, no association was noted between polymorphonuclear cell pigment and level of parasitemia (P = 0.68). A separate analysis was performed recalculating peripheral parasitemia based on the absolute WBC count determined on a thin smear rather than the standardized WBC count (7,500 cells/ mm3 in this study) that is typically used in field trials. Peripheral parasite density results were not significantly different than those calculated using the standard WBC count, and no association was noted between PMN pigment and level of parasitemia.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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Clinical evidence supports a role for hemozoin as an indicator for disease severity in both children and adults and for prognosis in adults.12–15 Additionally, malaria pigment itself may possess physiologic properties that contribute to the course of disease. Natural pigment has been demonstrated in vitro to induce production of both tumor necrosis factor-
and interleukin-1-ß; this effect is ameliorated by protease digestion, suggesting the role of uncharacterized proteins.18 Monocytic cell dysfunction has been demonstrated (inhibition of oxidative burst, inability to digest hemozoin, or repeatedly phagocytose).19,20
In the present study, we used a matched case-control design to explore hemozoin characteristics as it relates to severe malarial disease. Because of the broad range of peripheral WBC counts (1,700–44,500 cells/mm3), and the disparity between neutrophil and monocyte percentages on absolute differential assessment, we standardized hemozoin measurements among groups by assessing the total amount of malaria pigment/ mm3. We first analyzed groups by quantifying the percentage of pigmented monocyte and polymorphonuclear cells. Consistent with previous studies, differences were noted between patients with severe and uncomplicated malaria and aparasitemic controls. By analyzing the data as a calculated amount of pigment/mm3, a significantly higher amount of pigment for both neutrophils and monocytes was again observed between the severe malaria group and the matched uncomplicated malaria controls, as well as between the uncomplicated malaria group and matched healthy controls. These results validate the correlation of malaria pigment and disease severity.
Malaria pigment has been found to correlate with death in adults, but this has not been substantiated in children. We analyzed the role of hemozoin as a prognostic indicator of severe malaria. After stratifying the severe malaria cases into children who died versus those who survived, a significantly greater amount of pigmented PMNs/mm3 was observed in the group who died. These findings mark the first time that neutrophilic pigment presence has proven a prognostic indicator for severe malaria mortality in children. No difference was detected in pigmented monocytes/mm3.
To illuminate differences between categories of severe malaria, subjects were stratified into one of the three predominant admission diagnoses; cerebral malaria, severe anemia and hyperparasitemia. A subset analysis was performed on children with the combined diagnosis of cerebral malaria with severe anemia. Children with cerebral malaria had more pigmented PMNs/mm3 on admission. Conversely, more pigmented monocytes were noted in children with severe anemia. Of the 26 children with severe anemia, 15 had concomitant cerebral malaria. As in children with cerebral malaria alone, those with both diagnoses had more pigmented PMNs/ mm3 but no increase in pigmented monocyte/mm3.
In children with severe anemia as a sole diagnosis (n = 11), pigmented monocytes were significantly increased; this was also noted in children with severe anemia combined with other manifestations of severe malaria. It is possible that the low numbers of children with anemia as a sole diagnosis did not provide adequate power to detect an association between hemoglobin and pigmented PMNs/mm3. It is also possible that the pathophysiology of acute cerebral malaria and severe anemia may, in and of itself, have properties creating a milieu for increased pigment formation independent of either manifestation alone.
The reasons for variability in monocyte and neutrophil pigment in different disease manifestations of severe malaria remain unclear. The proportion of neutrophils and monocytes containing malaria pigment is affected by total parasite burden and synchronicity of the parasite life cycle, and the clearance kinetics of these pigmented cells may be inherently different. Cellular clearance kinetic studies performed by Day and others have shown peripheral pigment-containing neutrophil clearance times of 72 hours (range = 49–95 hours) and peripheral pigment-containing monocyte clearance of 216 hours (range = 180–240 hours). While clearance of pigmented monocytes appears to follow first-order kinetics, that of pigmented neutrophils departs from first-order kinetics, with increased rates of clearance at a lower cell density. The presence of pigmented neutrophils, with the rapid turnover of PMNs, may indicate a recent heavy parasitic burden and provide prognostic indications of disease, while longer-lived pigmented monocytes with longer clearance rates may reflect a more protracted infection or repeated infections.21
Malaria-induced anemia is multifactorial with hemolysis occurring more frequently in non-immune children and dyserythropoiesis occurring more often in regions with frequent and recurrent infections. The predominance of monocytic pigment in our anemic children may suggest that the profound anemia that occurred over a more protracted time period.22 The elevated pigmented polymorphonuclear cells in children with both cerebral malaria and severe anemia compared with those with cerebral malaria alone may relate to the acuity of the illness with cerebral manifestations coupled with acute hemolysis.21 Thus, we hypothesize that pigmented monocytes indicate a protracted or indolent infection, while the factors leading to severe disease and death may be more fulminant and reflected in pigmented neutrophils. Longitudinal studies with serial pigment analysis would be better suited to address these hypotheses.
The use of hyperparasitemia as a criterion for severe malaria is recommended by the World Health Organization, but the clinical course in children who present with this manifestation appears less acute. At our study site, children whose sole defining criterion for severe malaria was hyperparasitemia appear to fall somewhere between uncomplicated malaria and severe malaria in the spectrum of illness. We noted a lower peripheral parasite density in children who died compared with children that survived, although the statistical significance of this finding disappeared upon removing hyperparasitemia as a criterion for severe disease. Children with hyperparasitemia alone are presumably healthier children who were more likely to survive. It is possible that this group should be considered separately from those with more severe forms of malaria, which would require larger studies, probably including multiple sites.
Ongoing trials conducted through the Severe Malaria in African Children network have a large sample size at multiple study sites and should further illuminate the significance of malaria pigment in severe malaria, including hyperparasitemia. Linear regression models taking into account hyperparasitemia or peripheral parasite density demonstrated no association with either pigmented PMNs or monocytes. An inconsistency was noted in that monocyte presence did not appear to be associated with hyperparasitemia on regression analysis, although upon stratifying patients with peripheral parasitemias greater than and less than 500,000 asexual forms/mm3, increased monocyte pigment was observed in patients without severe disease. While the signficance of this finding is unclear, we have demonstrated that the degree of parasitemia is not associated with increased amount of pigment seen in leukocytes, consistent with our hypothesis that the presence of pigment reflects overall sequestered parasite burden.
In summary, in an age-, residence-, and ethnicity-matched case-control study, we have found malaria pigment/mm3 in neutrophils to be associated with disease mortality in Malian children three months to 14 years of age. We have validated previously reported associations between the amount of neutrophilic malaria pigment and disease severity, and have established an association with monocyte pigment when measured as pigment/mm3. In this study, we found that intra-neutrophilic pigment was associated with cerebral malaria and the combination of cerebral malaria and severe anemia, while intra-monocytic pigment is associated with severe anemia. These results do not appear to be biased by the degree of peripheral parasitemia. Based on these results, the assessment of intraleukocytic pigment appears to be valuable for more detailed characterization of children who present with P. falciparum parasitemia and clinical symptoms consistent with a malarial illness.
Received April 29, 2003. Accepted for publication July 1, 2003.
Acknowledgments: We thank the members of the Severe Malaria in African Children Clinical Network for their work in the genesis of this study and the protocol to which we adhered. We also thank Timothy Mnalemba (Blantyre Malaria Project, Blantyre, Malawi) for his patient teaching, which was invaluable in identifying pigment and applying the protocols.
Financial support. This study was supported by contract no. N01-AI-85346 from the National Institutes of Health.
Authors’ addresses. Kirsten E. Lyke and Christopher V. Plowe, Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, 685 West Baltimore Street, HSF 480, Baltimore, MD 21201, Telephone: 410-706-3082, Fax 410-706-6205, Email: cplowe{at}medicine.umaryland.edu. Dapa A. Diallo, Alassane Dicko, Abdoulaye Kone, Drissa Coulibaly, Ando Guindo, Yacouba Cissoko, Lansana Sangare, Seydou Coulibaly, Blaise Dakouo, and Ogobara K. Doumbo, Malaria Research and Training Center, Department of Epidemiology of Parasitic Diseases, Faculty of Medicine, Pharmacy and Dentistry, University of Mali, BP 1805 Point G, Bamako, Mali, Fax 223-2-8109. Terrie E. Taylor, Department of Internal Medicine, College of Osteopathic Medicine, Michigan State University, B315-C W. Fee Hall, East Lansing MI 48824, Fax 517-432-1062.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONREFERENCES |
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