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ABSTRACT |
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and inducible nitric oxide synthase (iNOS) were examined for associations with the incidence of symptomatic malaria in a cohort of 307 Ugandan children. After adjustment of incidence rates for age, water source, use of preventative measures, and proximity to mosquito breeding sites, glucose-6-phosphate dehydrogenase A- heterozygous females had a significantly higher incidence of malaria (incidence rate ratio [IRR] = 1.63, P = 0.03) and a trend towards higher parasite densities (37,100 versus 26,200 parasites/µL; P = 0.18) compared with wild-type children. Male hemizygotes had trends in the same direction. Heterozygotes for sickle hemoglobin had trends toward a lower incidence of malaria and lower parasite density at presentation. Heterozygotes for the iNOS promoter G954C polymorphism, but not other promoter polymorphisms, had a significantly lower incidence of malaria compared with wild-type children (IRR = 0.69, P = 0.05). Host polymorphisms appear to impact upon the incidence of uncomplicated malaria in Ugandan children. |
INTRODUCTION |
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(TNF-; G238A, G308A, and G376A) and inducible nitric oxide synthase (iNOS; G954C and C1173T) genes and severe malaria have been inconsistent.2–5
Compared with effects on severe malaria, the effect of host polymorphisms on the overall incidence of malaria has received little attention. This is an important omission because the vast majority of malaria episodes are uncomplicated, and these cases, although not immediately life-threatening, have important public health consequences. To better characterize the impact of key polymorphisms on the incidence of malaria, we examined the effects of polymorphisms in the ß-globin, G6PD, TNF-, and iNOS genes upon the incidence of malaria in a previously described cohort of Ugandan children who were followed for one year.6
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METHODS |
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Assessment of malaria incidence.
Upon enrollment, children were randomly assigned to receive sulfadoxine-pyrimethamine (SP), SP plus amodiaquine, or SP plus artesunate for treatment of all episodes of malaria that were identified during follow-up.6 Parents/guardians were instructed to bring their child to the clinic whenever they needed medical attention and to avoid using any drugs not administered or approved by a study physician. When a child presented with a history of fever (previous 48 hours) or a tympanic temperature 
38.0°C, a thick blood smear was prepared. Patients were diagnosed with symptomatic malaria if they had 1) a tympanic temperature 38.0°C and any parasitemia, 2) a history of fever and 500 asexual parasites/µL, or 3) severe malaria or danger signs and any parasitemia.6 Patients with uncomplicated malaria were treated with assigned regimens; those with severe malaria were treated with quinine. For recurrent episodes, molecular genotyping was performed based on polymorphisms in merozoite surface protein-2 to distinguish recrudescent (treatment failure) from new infections, as previously described.7 For the study of associations with genetic polymorphisms, malaria incidence density was based only on new infections and did not include episodes due to recrudescence (treatment failure).6 Time at risk for new infection was defined as the duration of study participation minus 14 days after each episode of malaria.
Assessment of host genetic polymorphisms.
Blood was collected on filter paper at enrollment and every time an episode of malaria was diagnosed. For genetic analysis, DNA was extracted from filter paper with Chelex (Bio-Rad Laboratories, Hercules, CA). Polymorphisms were detected using nested polymerase chain reaction (PCR) amplification followed by restriction endonuclease digestion (Table 1
). For each reaction, 1–2 µL of Chelex-extracted sample was incubated with Taq polymerase (Invitrogen, Carlsbad, CA), 200 µM dNTPs, and buffer (200 mM Tris, pH 8.4, 500 mM KCl). Primer concentrations and MgCl2 were optimized for each reaction. After first-round and nested PCRs, 5 µL of each reaction was digested with restriction endonucleases. Digestion products were subjected to electrophoresis on 2.5% NuSieve agarose (FMC Bioproducts, Rockland, ME) gels and visualized with ethidium bromide. Genotypes were assessed based on comparison of the sizes of reaction products and controls after digestion.
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Statistical methods. Associations between host polymorphisms and malaria incidence were estimated using a multivariate negative binomial regression model, controlling for clustering within households. In previous analysis in this same cohort of children, age, primary source of water, use of malaria preventative measures, and proximity to mosquito breeding sites were identified as independent predictors of malaria incidence.9,10 The final model included treatment regimen and all four known predictors of incidence. Geometric mean parasite densities were compared using generalized estimating equations with exchangeable correlation and robust standard errors to control for repeated measures within the same patient. A P value < 0.05 was considered statistically significant. Analysis was done using STATA statistical software version 8.0 (StataCorp., College Station, TX).
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RESULTS |
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G6PD A- heterozygous females, hemizygous males, and homozygous females comprised 12% (36 of 303), 8% (25 of 303), and < 1% (1 of 303) of our study population, respectively. Crude annual malaria incidence rates were 1.88 for G6PD wild-type, 2.09 for G6PD A- male hemizygotes, and 1.95 for female heterozygotes. After controlling for previously identified predictors of malaria incidence (age, water source, use of preventative measures, and proximity to mosquito breeding sites), a significantly higher malaria incidence rate ratio for heterozygous G6PD A- females compared with wild type individuals was noted (Table 2). In addition, heterozygous females had a trend toward higher geometric mean parasite densities upon malaria diagnosis compared with those lacking the G6PD A-mutations (37,100 parasites/µL versus 26,200 parasites/µL; P = 0.18). G6PD A- hemizygous males showed similar trends, with a higher incidence of malaria and higher parasite density upon diagnosis.
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). Only two children were heterozygous for hemoglobin C (A17T ß-globin mutation).
There were 55 (18%) iNOS G954C heterozygotes and no homozygotes in the cohort. The crude annual malaria incidence rates were 1.97 for wild-type and 1.73 for heterozygous children. With adjustment for known predictors of malaria incidence, G954C heterozygotes had a significantly lower malaria incidence rate ratio than wild-type children (Table 2). Parasite densities did not differ between the two groups.
The TNF- polymorphisms (G238A, G308A, and G376A) and the iNOS polymorphism C1173T did not show significant associations with either incidence or parasite density (Table 2). Of note, all TNF –376 heterozygotes and homozygotes were heterozygous or homozygous at the TNF –238 locus.
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DISCUSSION |
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G6PD A- heterozygous females and hemizygous males had a higher incidence of uncomplicated malaria and higher parasite densities when presenting with malaria compared with wild type children. Our results might be seen as surprising, since G6PD A- females (and in some studies hemizygous males) were protected against severe malaria.11 However, our results are consistent with two recent longitudinal studies in Gabon, in which G6PD A- females had significantly higher incidences of uncomplicated malaria.12,13 Some other studies have reported a protective effect of G6PD deficiency in uncomplicated malaria, but these studies were case-control or cross-sectional in design, and thus not best suited for this analysis.8 In addition, older studies often relied on phenotypic G6PD assessment, a method known to be inaccurate, particularly in heterozygotes.8
Our findings, in addition to two prior studies, now suggest that G6PD-deficient individuals have an increased incidence of malaria.12,13 How can we reconcile the findings that G6PD deficiency protects against severe malaria but is associated with an increased overall incidence of malaria? The protection afforded by G6PD deficiency against severe malaria has been attributed to a diminished ability of parasites to survive additional oxidative stress in deficient erythrocytes, a finding that has been confirmed in vitro, although differences in growth between parasites in wild-type and G6PD deficient erythrocytes have been modest.14 We hypothesize that increased malarial incidence renders G6PD-deficient individuals more immune, and thus better able to control malaria once it has progressed to clinical illness. Similarly, it was hypothesized that -thalassemia was selected in Vanuatu despite an increased incidence of malaria, as increased incidence protected against severe malaria.15 Our hypothesis agrees with prior suggestions that a G6PD deficiency has been maintained in humans due to the selective pressure of malaria, but suggests that it has been selected, at least in part, due to an increased predilection for clinical malaria in heterozygotes.
We also found that G6PD A- individuals had a trend towards higher parasitemias when they presented with malaria, although this finding differed from two other studies, and so must be interpreted with caution.12,13 Nonetheless, it offers support for the hypothesis that higher rates of malaria in G6PD deficient children lead to higher levels of immunity, thus requiring greater parasitemias for the expression of clinical illness.
We also identified a lower incidence of malaria in heterozygotes for the iNOS G954C mutation (which led to higher iNOS activity in some studies) compared with wild-type children.16,17 A study in Gabon found that the –954C allele was correlated with both higher baseline iNOS activity and protection, with a delay in the development of clinical malaria after prior treatment.17 Other studies in Tanzania and Gabon found no association between G954C and cerebral malaria or the incidence of uncomplicated malaria, although the latter study did not include younger children most likely to benefit from protection afforded by higher levels of nitric oxide. Our result suggests a protective role for this mutation against malaria in Ugandan children.
Significant associations were not identified between malarial incidence and the other polymorphisms studied, although trends toward protection in sickle hemoglobin heterozygotes were consistent with other studies showing protection against severe malaria and trends toward protection against malarial incidence.1 Differences in the associations seen in this cohort and others may relate to variations in the impact of these polymorphisms upon severe compared with uncomplicated malaria, or may relate to the interplay of other polymorphisms not studied in this cohort. Additional longitudinal studies with larger sample sizes will be needed to more fully evaluate associations between these host polymorphisms and the incidence of malaria.
Received April 9, 2004. Accepted for publication July 9, 2004.
Acknowledgments: We thank the clinical study team of Adithya Cattamanchi, Moses R. Kamya, Sarah Staedke, Anne Gasasira, Denise Njama, B. M. Karakire, Marx Dongo, Sam Nsobya, Moses Kiggundu, Christopher Bongole, Regina Nakafero, Bridget K. Nzarubara, Pauline Byakika, and Sarah Kibirango; the community leaders from the Kawempe Division of Kampala; and the study participants and their parents/guardians for their contributions to the study.
Financial support: This study was supported by the National Institutes of Health (grants UO1AI52142 and T32AI07641).
Disclosure: The authors have no conflicting interests to declare. Authors’ address: Sunil Parikh, Grant Dorsey, and Philip J. Rosenthal, Division of Infectious Diseases, Department of Medicine, San Francisco General Hospital, University of California, Box 0811, San Francisco, CA 94110, Telephone: 415-206-8687, Fax: 415-648-8425, E-mails: sunil{at}itsa.ucsf.edu, grantd{at}itsa.ucsf.edu, and rosnthl{at}itsa.ucsf.edu
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REFERENCES |
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promoter region is associated with susceptibility to cerebral malaria. Nature 371: 508–511.[Medline]
-308 is associated with shorter intervals of Plasmodium falciparum reinfections. Tissue Antigens 59: 287–292.[Web of Science][Medline]
-thalassemic children. Nature 383: 522–525.[Medline]
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