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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumor necrosis factor-
) and serum levels of antibodies to dengue virus were elevated in the same range in male and female infants with DHF/DSS, as well as in infants with and without malnutrition. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A preponderance of DHF/DSS in Thai females 
4 years old has been documented.6 Rates of infection with dengue (DEN) virus among boys and girls have been shown to be identical in countries endemic for DHF/DSS.6 The integrity and strength of the cell-mediated immune response should be correlated with the severity of DHF/DSS.7 It is known that malnutrition suppresses cellular immune responses. Anto and others8 and Thiasykorn and Nimmannitya9 have shown that even mild degrees of protein-calorie malnutrition serve to spare children from severe DHF/DSS. The immunopathogenesis of dengue virus infection, with an emphasis on the immune deviation, autoantibodies, and cytokine production has been proposed.10 In our previous report as part of study project of DHF in infants, we demonstrated the production of high levels of both proinflammatory cytokines (interferon- [IFN-] and tumor necrosis factor- [TNF-]) and anti-inflammatory cytokines (interleukin-10 [IL-10] and IL-6) in infants with DHF/DSS that was consistent with infection-enhancement due to acquired maternal antibody.11 Primary dengue virus infections in infants less than one year of age comprise a unique subgroup of patients with DHF/DSS.5 Here we further characterize attributes of the DHF syndrome observed in infants less than one year of age experiencing predominantly primary dengue virus infections.
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MATERIALS AND METHODS |
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Assessment of nutritional status of DHF patients and healthy control infants. Epi-Info 2000 1.1 software (Centers for Disease Control and Prevention, Atlanta, GA) was used to calculate the anthropometric indices such as weight-for-age (WA), height-for-age (HA), and weight-for-height (WH) of infants with DHF and healthy control infants. These nutritional measurements were converted to z-scores (weight-forage Z-score [WAZ]; height-for-age Z-score [HAZ]; and weight-for-height Z-score [WHZ]), also referred to as SD units, based on the National Center for Health Statistics/World Health Organization reference. The Z-score cutoff point to classify low anthropometric levels is 2 SD units below the reference median for the three indices. The cutoff for very low anthropometric levels is more than 3 SD units below the median.
Sample collection. Paired blood samples were collected from each patient in the study: one acute-phase and one convalescent-phase. An acute-phase blood sample (2–3 mL) was obtained at admission (days 3–7 after the onset of fever). A convalescent-phase blood sample (2–3 mL) was obtained in the convalescent phase (days 8–19 after the onset of fever). Sera were separated as quickly as possible and stored at –70°C until used.
Assessment of immune response of the patients. Immune response of the patients was assessed by determining the capacity to produce cytokines, IgM antibody to dengue virus, and/or NS1 serotype-specific IgG antibodies.
Capture IgM and IgG ELISAs.
Capture IgM and IgG ELISAs using diluted pooled virus antigens from culture supernatants of DEN 1-, DEN 2-, DEN 3-, DEN 4-, and Japanese encephalitis (JE) virus-infected Vero cells as antigens were performed to measure the IgM and IgG antibodies from paired sera of 118 infants at the Center for Disease Control in Taipei, Taiwan. The optical densities of culture supernatants of Vero cells with and without dengue virus infection were designated the test absorbance and negative control value, respectively, for each sample in the ELISA. Positivity was determined by comparison with individual negative controls. A positive sample had a ratio of test absorbance to negative control 2.0, and a negative sample had a ratio < 2.0. For serum samples with positive results in the capture IgM and IgG ELISAs, a ratio of IgM to IgG 1.2 was defined as a primary dengue virus infection, and a ratio < 1.2 was defined as a secondary dengue virus infection.12 A capture IgM ELISA using diluted dengue or JE virus antigens from infected suckling mouse brain extracted by the sucrose-acetone method and monoclonal antibody SLE 6B6C-1/HRP conjugate was performed to measure the IgM antibodies in 154 infants following the protocol of the Centers for Disease Control and Prevention (Fort Collins, CO) at the Pasteur Institute in Ho Chi Minh City.13
NS1 serotype-specific IgG ELISA. An NS1 serotype-specific IgG ELISA using diluted NS1-containing culture supernatants of DEN 1-, DEN 2-, DEN 3-, DEN 4-, or JE virus-infected Vero cells as antigens was performed to measure the NS1-specific IgG antibody from the sera of the patients. The enzyme activity was developed and the optical density was determined.12
Cytokine assays.
The plasma levels of six cytokines (IFN-, TNF-, IL-10, IL-6, IL-4, and IL-2) of the patients were measured simultaneously by the BD Human Th1/Th2 Cytokine Cytometric Bead Array Kit-II (BD Biosciences, Pharmigen, CA) in a 50-µL sample according to the instructions of the manufacturer.11
Data and statistical analysis. Analysis of variance was used to compare the statistical significance of differences in normally distributed data, while the Kruskal-Wallis test for two groups was used if the variances in the samples differed. Statistical analyses were performed with Epi-Info 2000 version 1.1 software. Differences with P values < 0.05 were considered significant.
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RESULTS |
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Hemoconcentration, as shown by a 20% increase in the hematocrit in reference to a convalescent value, was observed in 224 (91.4%) patients. The remaining 21 (8.5%) patients had a 10–19% increase in the hematocrit. Thrombocytopenia (platelet count 
100 x 103/mm3) was found in 230 (93.8%); the remaining 15 (6.1%) had platelet counts of 104–190 x 103/mm3.
Association between sex, nutritional status, and the severity of DHF in infants.
The male/female ratio for all DHF infants was 138:107 (1.29:1); for DSS cases, it was 40:23 (1.73: 1). Among 533 healthy control infants, the male/female ratio was 276:257 (1.07:1). The distribution of sex in infants with DHF or DSS in the study is not different from that of healthy control subjects (odds ratio [OR] = 1.20, 95% confidence interval [CI] = 0.88–1.65, P = 0.2) (Table 1
). There was no sex bias associated with prolonged shock, GI bleeding, respiratory failure, or encephalopathy (Table 2).
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–3). Height was measured in 218 of 245 infants. When assessed by HA, 17 (7.7%) infants had malnutrition (stunting) on admission, including 14 with moderate malnutrition and 3 with severe malnutrition. When assessed by WH, 31 (14.2%) infants were malnourished (wasting): 25 with moderate malnutrition and 6 with severe malnutrition. Among 533 control infants, 62 (11.6%), 6 (1.1%), and 121 (22.7%) infants had malnutrition as assessed by WA, WH, and HA, respectively. Compared with healthy controls, malnourished infants, as assessed by WA and HA, occurred less frequently among DHF cases (6.9% versus 11.6%; P = 0.03; 7.7% versus 22.7%; P < 0.001, respectively). In contrast, infants with DHF had a higher percentage of malnutrition (wasting) as assessed by WH (14.2% versus 1.1%; P < 0.001). With smaller numbers, no statistically significant differences were observed in the distribution of various parameters of undernutrition in the DSS and nonshock DHF group (Table 1
).
Further analysis indicated that there was no association between malnutrition status as assessed by WA and severe complications (prolonged shock, GI bleeding, respiratory failure, and encephalopathy) in DHF infants (Table 2).
Association between sex, nutritional status, and production of cytokines and antibodies to dengue virus in infants with DHF/DSS.
Serum concentrations of IFN-, TNF-, IL-10, and IL-6 measured at one interval during the acute stage of illness in 62 infants with DHF/DSS were higher than those in control group.11 No significant differences were observed in serum levels of IFN-, TNF-, IL-10, and IL-6 in male DHF/DSS patients compared with female DHF/DSS patients (Table 3).
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, TNF-, IL-10, and IL-6 in infants with DHF/DSS with or without malnutrition as assessed by WA (Table 3). However, except for TNF-, the serum levels of IFN-, IL-10, and IL-6 were not different between infants with and without malnutrition. Further analysis also showed that the titers of IgM antibody to dengue virus and NS1 serotype-specific IgG antibodies in infants with DHF/DSS with or without malnutrition as assessed by WA were not significantly different (Table 4). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The results of this study also showed that there was no relationhip between sex and severe complications of DHF, such as prolonged shock, GI bleeding, respiratory failure, and encephalopathy, in infants. Assessment of the nutritional status of infants with DHF/DSS in the present study showed that only 6.9%, 14.2%, and 7.7% of the patients were malnourished as assessed by WA, HA, and WH, respectively. Infants with DHF had lower percentage of malnutrition as assessed by WA and HA (6.9% versus 11.6%; P = 0.03; 7.7% versus 22.7%; P < 0.001, respectively) compared with the healthy control subjects. The results of two surveys (500 patients for each survey) to assess the nutritional status of inpatients of other diseases at Children’s Hospital No.1 in Ho Chi Minh City in 2001 and 2003 conducted by the Department of Clinical Nutrition showed that 31.4% and 29.4% of these patients had malnutrition as assessed by WA, respectively, while 24.6–34.9% of the infant group had malnutrition (Hoa NT and others, unpublished data). In humans, moderate/severe malnutrition is associated with a significant reduction in cell-mediated immunity, as indicated by a reduced number of CD4+ T helper cells, and a lower CD4+/CD8+ratio. There is also a reduction in the production of secretory IgA antibody and various complement components (C3, C4, and factor B) and impairment of phagocytosis. The production of certain cytokines such as IL-2 and TNF is also decreased.16 Anto and others8 and Thiasykorn and Nimmannitya9 have shown that even mild degrees of protein-calorie malnutrition are in children more than one year of age with severe DHF/DSS compared with controls. This suggests that malnutrition blunts the severity of secondary dengue infections by possibly reducing T cell activities. Here we show a similar effect on the severity of dengue infections in infants, with the difference being that nearly all these infants were experiencing a primary immune response. In this immunologic setting, cytokine production is not enhanced by a memory T cell phenomenon.
Nutritional status did not convey absolute protection from severe clinical dengue disease. Infants with moderate malnutrition (WA) and DHF/DSS had serum levels of cytokines (IFN-, TNF-, IL-10, and IL-6), and antibodies to dengue virus that were not different from infants with DHF/DSS without malnutrition.
The present study showed that there was no association between sex, nutritional status, and the severity of DHF/DSS in infants with a primary type dengue antibody response (nonshock DHF versus DSS). The production of cytokines and antibodies to dengue virus was not different between male and female infants with DHF, as well as between infants with and without malnutrition.
Received September 18, 2004. Accepted for publication November 18, 2004.
Acknowledgments: We thank Dr. Tran Tan Tram (Children’s Hospital No. 1, Ho Chi Minh City, Vietnam) and Dr. Chung-Ming Chang (National Health Research Institutes, Taipei, Taiwan) for help and support during the course of this study. We also thank Dr. Hoang Le Phuc for help with data analyses, and the doctors and nurses of the Department of Dengue Hemorrhagic Fever, Children’s Hospital No. 1-Ho Chi Minh City for providing excellent patient care.
Financial support: This work was partially supported by grant NHRI-CN-CL9303P from the National Health Research Institutes (Taiwan).
Disclosure: None of the authors have commercial or other associations that might pose a conflict of interest.
Authors’ addresses: Nguyen Thanh Hung, Nguyen Trong Lan, Department of Dengue Hemorrhagic Fever, Children’s Hospital No.1, 341 Su Van Hanh Street, District 10, Ho Chi Minh City, Vietnam, Telephone: 84-8- 927-1119 or 84-8- 927-1156, Fax: 84-8-927-0053, E-mail: hungdhf{at}hcm.fpt.vn. Huan Yao-Lei, Yee-Shin Lin, Le Bich Lien, Kao-Jean Huang, and Chiou-Feng Lin, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China. Do Quang Ha and Vu Thi Que Huong, Arbovirus Laboratory, Pasteur Institute, Ho Chi Minh City, Vietnam. Lam Thi My, Department of Pediatrics, University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam. Trai-Ming Yeh, Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China. Jyh-Hsiung Huang, Division of Research and Diagnosis, Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China. Scott B. Halstead, Uniformed Services University of the Health Sciences, Bethesda, MD 20815, E-mail: halsteads{at}erols.com.
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REFERENCES |
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