Inflammatory molecules expression pattern for identifying pathogen species in febrile patient serum
- Authors:
- Published online on: May 10, 2016 https://doi.org/10.3892/etm.2016.3323
- Pages: 312-318
Abstract
Introduction
Fever is among the most common presenting symptoms in emergency department hospital cases; however, fever may be a symptom of numerous diseases (1). Pathogen infection is one of the most common causes of induced fever. In addition to the basic history assessment and physical examination of patients, emergency physicians typically require more information, such as laboratory test results and imaging examination, to diagnose the causes of fever-associated diseases (2).
In clinical settings, certain laboratory tests have been used to facilitate the diagnosis of severe bacterial infection, such as white blood cell (WBC) count, C-reactive protein (CRP) and procalcitonin (3,4). As a host immune system recognizes pathogens and induces production of inflammatory molecules to resist pathogens, numerous cytokines and chemokines are used as biomarkers in laboratory research for the diagnosis and prognostic prediction of infectious diseases (5–13). For example, studies have shown that inflammatory cytokines levels are altered as a result of a variety of infections, including interleukin (IL)-1, IL-6, IL-10, IL-13, IL-18 and tumor necrosis factor-alpha (TNF-α) (5–8). Elevating plasma concentration of cytokines such as IL-6 and IL-10 may serve as a diagnostic marker of sepsis and is correlated with severity and survival of sepsis patients (9,10). In addition, certain chemokines, such as Regulated on Activation, Normal T cell Expressed and Secreted (RANTES), interferon-gamma-inducible protein 10 (IP-10) and CXCL8/IL-8, are associated with sepsis (11–13). These markers may provide important information for the early diagnosis of diseases.
Immune system recognizes different pathogens with particular characteristics, such as lipopolysaccharides on gram-negative bacteria, peptidoglycan on gram-positive bacteria and single-stranded and double-stranded RNA in RNA viruses, using pattern-recognition receptors (14). Each characteristic triggers distinct downstream pathways and the production of inflammatory molecules (15). Since different pathogens induce different immune responses, we aimed to determine whether the pathogen species could be identified by patterns of serum inflammatory molecules. In the present study, we examined whether bacterial and viral infections, gram type of bacteria, different species of bacteria, or different types of virus could be characterized by measuring a variety of cytokines and chemokines using a cytometric bead array. The expression pattern of seven cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ) and five kinds of chemokines [IL-8, RANTES, monokine induced by interferon-γ (MIG), monocyte chemoattractant protein 1 (MCP-1) and IP-10] were detected in fever patients.
Materials and methods
Patient enrollment
This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital. Patients visiting the Emergency Department of Kaohsiung Medical University Hospital (Taiwan, China) with fever or high body temperature (tympanic temperature, >38.3°C) were sequentially enrolled between May 2013 and October 2014. Patients received treatments and examinations according to emergency physicians' evaluation. If patients agreed and provided informed consent, an extra 10 ml blood would be collected to undergo cytokine test. Serum was separated by centrifugation at 1,600 × g for 20 min at 4°C, divided in aliquots and immediately frozen (−80°C) until the time of the assay. Clinical information including the patients' age, gender, culture result, laboratory data at the Emergency Department and diagnosis were collected by a research nurse.
Cytometric bead array
Cytokines and chemokines from the sepsis patients were analyzed using the Cytometric Bead Array for human Th1/Th2/Th17 Cytokine kit (cat no. 560484; including IL-2, IL-4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ) and a Human Chemokine kit (cat no. 552990; including IL-8, RANTES, MIG, MCP-1, and IP-10; BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Data was acquired using a BD Accuri C6 flow cytometer (BD Biosciences) and the data were analyzed using FCAP Array™ version 3.0.1 Software (BD Biosciences) according to a standard concentration curve.
Statistical analysis
Differences between two independent groups were analyzed by Mann-Whitney U test. Comparisons between three groups were via use of the Kruskal-Wallis test with Dunn's multiple comparison test. All calculations were performed using GraphPad Prism version 5.03 (GraphPad Software, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Laboratory data
Enrolled febrile patients were divided into two groups: Clinically suspected viral infection and bacterial infection, according to initial diagnosis. C-reactive protein (CRP) level and white blood cell (WBC) count are crucial indicators of sepsis (1,2). High levels of CRP (12.81±17.78 vs. 124.91±90.44 mg/l; P<0.0001) and WBC counts (4.93±2.5×103 vs. 7.84±5.28×103 cells/µl; P<0.0001) were detected in patients who were diagnosed as having bacterial infection compared with patients who were diagnosed with viral infection (Table I). Compared to viral infection, significantly higher levels of blood urea nitrogen (BUN), blood glucose, creatinine (Cr), platelet and significantly lower levels of hemoglobin (Hb) were observed in bacterial infection patients.
Association between viral and bacterial infection and pattern of inflammatory molecule expression
We further examined the inflammatory molecules in patients serum (Table II). Patients with suspected bacterial infection exhibited significantly higher levels of IL-6 (63.23±265 vs. 2,533±6,559 pg/ml, P<0.0001), IL-10 (14.27±16.75 vs. 205.11±741.85 pg/ml, P=0.0101), IL-17A (18.66±54.93 vs. 34.58±63.99 pg/ml, P=0.0162), TNF-α (0.52±1.37 vs. 19±100.22 pg/ml, P=0.0007), IL-8 (30.31±38.01 vs. 1,249±6,944 pg/ml, P<0.0001) and MIG (660.71±661 vs. 4,533±14,580 pg/ml, P=0.0367). The present results were comparable to those of previous reports (5–8).
Currently, bacteria identification is primarily dependent on culture diagnosis in hospitals. However, studies have shown that culture-negative patients account for 28–48% of severe sepsis patients in North American, French and Canadian intensive care units (16–18). There were ~40% of culture-negative cases in a pan-European study and 49% of culture-negative cases in sepsis patients in the United States (19,20). These studies suggest that methods development for pathogen species identification is a critical issue. In the present study, there were 47 culture-positive patients and 37 culture-negative patients. We investigated whether different patterns of inflammatory molecules could be observed between viral infection group, culture-negative group and culture-positive group, particularly between viral infection and culture-negative group (Fig. 1). There was a statistically significant difference in IL-6, IL-8 and IP-10 levels between the viral infection and culture-negative groups or viral infection and culture-positive groups (Fig. 1). Similar results were observed in serum CRP level and WBC count (Fig. 2). Notably, the IL-2 and MCP-1 levels in the culture-positive group were higher than those in culture-negative group. However, neither molecule could not distinguish viral infection from bacterial infection (Table II). The results suggest that IL-6, IL-8 and IP-10 may be suitable for distinguishing viral infection from bacterial infection, even though bacterial culture is negative.
Association between species of bacteria and pattern of inflammatory molecule expression
Although serum CRP level and WBC counts were markers of bacterial infection, they showed no significant difference between gram-negative and gram-positive groups in the culture-positive group in the present study (data not shown). IL-8 level in gram-negative bacteria infection was higher compared with gram-positive bacteria infection group among the 47 culture-positive patients though IL-6 and IP-10 level did not show statistical difference (Table III). According to the result of bacterial culture, serum from Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus infected patients were selected. However, the results might indicate that these molecules were not sufficient to identify the species of bacteria (Table IV).
Association between virus type and pattern of inflammatory molecule expression
In the viral infection group, influenza virus or dengue virus-infected patients were enrolled after initial diagnosis. IL-6 level in influenza virus infection group is higher compared with the dengue virus infection group. However, the IL-6 level in influenza virus group was significantly lower (P<0.05) compared with the bacterial infection group (Table II). By contrast, serum from dengue virus infected patients contained significantly higher levels of IP-10 compared with serum from influenza virus infected patients (Table V). These results suggested that IP-10 levels may be used to distinguish influenza virus infection from dengue virus infection.
Discussion
Clinical laboratory data, including WBC count and CRP level, is rapid, sensitive and specific method for detecting bacterial infection (3,4). Platelet, BUN and Cr levels exhibited significant differences between suspected viral infection and bacterial infection patients. As patients with dengue fever, which affected platelet level, were recruited (21), platelet level was lower in viral infection patients than in bacterial infection patients. Anemia, hyperglycemia and elevated BUN and creatinine have been shown to be associated with bacterial infection (22,23); however, this may be due to patients selected in the clinically suspected bacterial infection group being older than patients in a viral infection group. IL-6, IP-10 and IL-8 may be potential markers for distinguishing viral infection from bacterial infection, although bacterial culture was negative. Furthermore, IP-10 level was highly correlated with dengue virus infection.
Culture-negative state is a commonly observed in patients diagnosed with sepsis (11–15). In the present study, serum CRP levels and WBC counts were sensitive and reliable indexes for distinguishing bacterial infection from viral infection. Numerous serum molecules, including IL-6, IL-8, IL-10 and TNF-α, have been reported to be markers of sepsis (5–7,9–11,24,25). Similar to the results of serum CRP level and WBC counts, significantly elevated concentrations of IL-6 and IL-8 were observed in fever patients in the bacterial infection group, regardless of culture-positive or culture-negative status. IL-10 and TNF-α were observed to have significant differences between viral and bacterial infection modalities. However, differences in IL-10 and TNF-α were observed between culture-positive and viral infection groups, but not between culture-negative and viral infection groups. The results suggested that IL-6 and IL-8 were sensitive and specific markers of bacterial infection.
Accurate and rapid identification of bacterial species improve clinical outcome (26). However, blood culture typically requires 1–3 days to become positive for detection of bacteria. Previous reports suggest that microarray or quantitative polymerase chain reaction analyses may be a faster and sensitive method for clinical identification of bacterial infection (27). It is reported that procalcitonin, but not CRP and WBC counts, is associated with gram-negative bacteria (28). Due to the different components of the cell walls of gram-positive and gram-negative bacteria, different patterns of immune responses may be induced in vitro (29,30). Therefore, we hypothesized that inflammatory molecules in the serum may provide information for identifying bacterial species. Gram-negative bacteria induce higher levels of IL-6, IL-10 and IL-8 compared with gram-positive bacteria in human monocytes (29–31). To the best of our knowledge, there are few studies reporting whether cytokines or chemokines could be markers to distinguish infection of gram-positive or gram-negative bacteria in patient serum. The present results revealed that IL-8 was the only molecule which exhibited a significant difference in expression between gram-negative and gram-positive bacteria. In the present study, bacterial species could not be identified through the pattern of inflammatory molecules. These results imply that serum inflammatory molecule levels alone were insufficient data for identifying bacterial species. As different species of bacteria have common characteristics (such as lipopolysaccharide on gram-negative bacteria), a similar pattern of inflammatory molecules were produced by innate immune system (32). However, in order to identify bacterial species on the basis of the pattern of serum molecule expression levels, the present findings suggest that the detection of more molecules is necessary.
In the present study, IP-10 level in the viral infection group was found to be higher than in the bacterial infection group. Previous studies have indicated that IP-10 level is associated with viral infection, including hepatitis C virus (HCV) infection and HCV-human immunodeficiency virus co-infection (33–35). Dengue virus infection is associated with higher concentrations of IP-10 in patient serum compared with healthy individuals (36). Furthermore, IP-10 is induced by viral and bacterial infection. Higher levels of IP-10 may be detected in sepsis, severe sepsis and septic shock groups compared with systemic inflammatory response syndrome (37). High levels of IP-10 have previously been detected in preterm infants with bacterial infection (12). Although bacterial and viral infection induce IP-10 level, the highest levels of IP-10 in the present study were observed among influenza virus, culture-negative and culture-positive groups (data not shown). IP-10 is a ligand for CXCR3 chemokine receptor, and expression of IP-10 recruits CXCR3 expressing NK and T cells (38). CXCR3 and IP-10 defective mice exhibited a lower survival rate compared with wild type mice in a dengue infection model (39). The CXCR3-IP-10 pathway may be essential for clearance of dengue virus, which may explain why dengue virus induced the highest level of IP-10 expression in the present study.
In summary, bacterial infection was associated with the production of IL-6, IL-8 and IP-10. In addition, an increased IL-8 level was associated with gram-negative bacteria and a high IP-10 level was associated with dengue virus infection. Although the pattern of inflammatory molecules alone could not be used to identify bacteria species, these molecules may provide useful information for diagnosis and clinical treatment.
Acknowledgements
The present study was supported by grants from the Ministry of Science and Technology (grant no. 104-2320-B-037-014-MY3) and the Kaohsiung Medical University Hospital Research Foundation (grant no. KMUH101-1M65).
References
Fernández Lopez A, Luaces Cubells C, García García JJ and Fernández Pou J: Spanish Society of Pediatric Emergencies: Procalcitonin in pediatric emergency departments for the early diagnosis of invasive bacterial infections in febrile infants: Results of a multicenter study and utility of a rapid qualitative test for this marker. Pediatr Infect Dis J. 22:895–903. 2003. View Article : Google Scholar : PubMed/NCBI | |
Fitch MT and van de Beek D: Emergency diagnosis and treatment of adult meningitis. Lancet Infect Dis. 7:191–200. 2007. View Article : Google Scholar : PubMed/NCBI | |
Andreola B, Bressan S, Callegaro S, Liverani A, Plebani M and Da Dalt L: Procalcitonin and C-reactive protein as diagnostic markers of severe bacterial infections in febrile infants and children in the emergency department. Pediatr Infect Dis J. 26:672–677. 2007. View Article : Google Scholar : PubMed/NCBI | |
Hatherill M, Tibby SM, Sykes K, Turner C and Murdoch IA: Diagnostic markers of infection: Comparison of procalcitonin with C reactive protein and leucocyte count. Arch Dis Child. 81:417–421. 1999. View Article : Google Scholar : PubMed/NCBI | |
van der Poll T, de Waal Malefyt R, Coyle SM and Lowry SF: Antiinflammatory cytokine responses during clinical sepsis and experimental endotoxemia: Sequential measurements of plasma soluble interleukin (IL)-1 receptor type II, IL-10, and IL-13. J Infect Dis. 175:118–122. 1997. View Article : Google Scholar : PubMed/NCBI | |
Damas P, Reuter A, Gysen P, Demonty J, Lamy M and Franchimont P: Tumor necrosis factor and interleukin-1 serum levels during severe sepsis in humans. Crit Care Med. 17:975–978. 1989. View Article : Google Scholar : PubMed/NCBI | |
Damas P, Ledoux D, Nys M, Vrindts Y, De Groote D, Franchimont P and Lamy M: Cytokine serum level during severe sepsis in human Il-6 as a marker of severity. Ann Surg. 215:356–362. 1992. View Article : Google Scholar : PubMed/NCBI | |
Grobmyer SR, Lin E, Lowry SF, Rivadeneira DE, Potter S, Barie PS and Nathan CF: Elevation of IL-18 in human sepsis. J Clin Immunol. 20:212–215. 2000. View Article : Google Scholar : PubMed/NCBI | |
Patel RT, Deen KI, Youngs D, Warwick J and Keighley MR: Interleukin-6 is a prognostic indicator of outcome in severe intra-abdominal sepsis. Br J Surg. 81:1306–1308. 1994. View Article : Google Scholar : PubMed/NCBI | |
Casey LC, Balk RA and Bone RC: Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med. 119:771–778. 1993. View Article : Google Scholar : PubMed/NCBI | |
Kurt AN, Aygun AD, Godekmerdan A, Kurt A, Dogan Y and Yilmaz E: Serum IL-1beta, IL-6, IL-8, and TNF-alpha levels in early diagnosis and management of neonatal sepsis. Mediators Inflamm. 2007:313972007. View Article : Google Scholar : PubMed/NCBI | |
Ng PC, Li K, Chui KM, Leung TF, Wong RP, Chu WC, Wong E and Fok TF: IP-10 is an early diagnostic marker for identification of late-onset bacterial infection in preterm infants. Pediatr Res. 61:93–98. 2007. View Article : Google Scholar : PubMed/NCBI | |
Ng PC, Li K, Leung TF, Wong RP, Li G, Chui KM, Wong E, Cheng FW and Fok TF: Early prediction of sepsis-induced disseminated intravascular coagulation with interleukin-10, interleukin-6, and RANTES in preterm infants. Clin Chem. 52:1181–1189. 2006. View Article : Google Scholar : PubMed/NCBI | |
Akira S, Uematsu S and Takeuchi O: Pathogen recognition and innate immunity. Cell. 124:783–801. 2006. View Article : Google Scholar : PubMed/NCBI | |
O'Shea JJ and Murray PJ: Cytokine signaling modules in inflammatory responses. Immunity. 28:477–487. 2008. View Article : Google Scholar : PubMed/NCBI | |
Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, Suppes R, Feinstein D, Zanotti S, Taiberg L, et al: Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med. 34:1589–1596. 2006. View Article : Google Scholar : PubMed/NCBI | |
Martin CM, Priestap F, Fisher H, Fowler RA, Heyland DK, Keenan SP, Longo CJ, Morrison T, Bentley D and Antman N: STAR Registry Investigators: A prospective, observational registry of patients with severe sepsis: The Canadian sepsis treatment and response registry. Crit Care Med. 37:81–88. 2009. View Article : Google Scholar : PubMed/NCBI | |
Brun-Buisson C, Meshaka P, Pinton P and Vallet B: EPISEPSIS Study Group: EPISEPSIS: A reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units. Intensive Care Med. 30:580–588. 2004. View Article : Google Scholar : PubMed/NCBI | |
Martin GS, Mannino DM, Eaton S and Moss M: The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med. 348:1546–1554. 2003. View Article : Google Scholar : PubMed/NCBI | |
Vincent JL, Sakr Y, Sprung CL, Ranieri VM, Reinhart K, Gerlach H, Moreno R, Carlet J, Le Gall JR and Payen D: Sepsis Occurrence in Acutely Ill Patients Investigators: Sepsis in European intensive care units: Results of the SOAP study. Crit Care Med. 34:344–353. 2006. View Article : Google Scholar : PubMed/NCBI | |
Hottz ED, Medeiros-de-Moraes IM, Vieira-de-Abreu A, de Assis EF, Vals-de-Souza R, Castro-Faria-Neto HC, Weyrich AS, Zimmerman GA, Bozza FA and Bozza PT: Platelet activation and apoptosis modulate monocyte inflammatory responses in dengue. J Immunol. 193:1864–1872. 2014. View Article : Google Scholar : PubMed/NCBI | |
Van Cromphaut SJ, Vanhorebeek I and Van den Berghe G: Glucose metabolism and insulin resistance in sepsis. Curr Pharm Des. 14:1887–1899. 2008. View Article : Google Scholar : PubMed/NCBI | |
Yegenaga I, Hoste E, Van Biesen W, Vanholder R, Benoit D, Kantarci G, Dhondt A, Colardyn F and Lameire N: Clinical characteristics of patients developing ARF due to sepsis/systemic inflammatory response syndrome: Results of a prospective study. Am J Kidney Dis. 43:817–824. 2004. View Article : Google Scholar : PubMed/NCBI | |
Livaditi O, Kotanidou A, Psarra A, Dimopoulou I, Sotiropoulou C, Augustatou K, Papasteriades C, Armaganidis A, Roussos C, Orfanos SE and Douzinas EE: Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis. Cytokine. 36:283–290. 2006. View Article : Google Scholar : PubMed/NCBI | |
Wunder C, Eichelbrönner O and Roewer N: Are IL-6, IL-10 and PCT plasma concentrations reliable for outcome prediction in severe sepsis? A comparison with APACHE III and SAPS II. Inflamm Res. 53:158–163. 2004. View Article : Google Scholar : PubMed/NCBI | |
Andrade SS, Bispo PJ and Gales AC: Advances in the microbiological diagnosis of sepsis. Shock. 30(Suppl 1): S41–S46. 2008. View Article : Google Scholar | |
Tissari P, Zumla A, Tarkka E, Mero S, Savolainen L, Vaara M, Aittakorpi A, Laakso S, Lindfors M, Piiparinen H, et al: Accurate and rapid identification of bacterial species from positive blood cultures with a DNA-based microarray platform: An observational study. Lancet. 375:224–230. 2010. View Article : Google Scholar : PubMed/NCBI | |
Charles PE, Ladoire S, Aho S, Quenot JP, Doise JM, Prin S, Olsson NO and Blettery B: Serum procalcitonin elevation in critically ill patients at the onset of bacteremia caused by either gram negative or gram positive bacteria. BMC Infect Dis. 8:382008. View Article : Google Scholar : PubMed/NCBI | |
Skovbjerg S, Martner A, Hynsjö L, Hessle C, Olsen I, Dewhirst FE, Tham W and Wold AE: Gram-positive and gram-negative bacteria induce different patterns of cytokine production in human mononuclear cells irrespective of taxonomic relatedness. J Interferon Cytokine Res. 30:23–32. 2010. View Article : Google Scholar : PubMed/NCBI | |
Hessle CC, Andersson B and Wold AE: Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes. Cytokine. 30:311–318. 2005. View Article : Google Scholar : PubMed/NCBI | |
Engel A, Mack E, Kern P and Kern WV: An analysis of interleukin-8, interleukin-6 and C-reactive protein serum concentrations to predict fever, gram-negative bacteremia and complicated infection in neutropenic cancer patients. Infection. 26:213–221. 1998. View Article : Google Scholar : PubMed/NCBI | |
Feezor RJ, Oberholzer C, Baker HV, Novick D, Rubinstein M, Moldawer LL, Pribble J, Souza S, Dinarello CA, Ertel W and Oberholzer A: Molecular characterization of the acute inflammatory response to infections with gram-negative versus gram-positive bacteria. Infect Immun. 71:5803–5813. 2003. View Article : Google Scholar : PubMed/NCBI | |
Berres ML, Trautwein C, Schmeding M, Eurich D, Tacke F, Bahra M, Neuhaus P, Neumann UP and Wasmuth HE: Serum chemokine CXC ligand 10 (CXCL10) predicts fibrosis progression after liver transplantation for hepatitis C infection. Hepatology. 53:596–603. 2011. View Article : Google Scholar : PubMed/NCBI | |
Zeremski M, Markatou M, Brown QB, Dorante G, Cunningham-Rundles S and Talal AH: Interferon gamma-inducible protein 10: A predictive marker of successful treatment response in hepatitis C virus/HIV-coinfected patients. J Acquir Immune Defic Syndr. 45:262–268. 2007.PubMed/NCBI | |
Reiberger T, Aberle JH, Kundi M, Kohrgruber N, Rieger A, Gangl A, Holzmann H and Peck-Radosavljevic M: IP-10 correlates with hepatitis C viral load, hepatic inflammation and fibrosis and predicts hepatitis C virus relapse or non-response in HIV-HCV coinfection. Antivir Ther. 13:969–976. 2008.PubMed/NCBI | |
Conroy AL, Gélvez M, Hawkes M, Rajwans N, Liles WC, Villar-Centeno LA and Kain KC: Host biomarkers distinguish dengue from leptospirosis in Colombia: A case-control study. BMC Infect Dis. 14:352014. View Article : Google Scholar : PubMed/NCBI | |
Punyadeera C, Schneider EM, Schaffer D, Hsu HY, Joos TO, Kriebel F, Weiss M and Verhaegh WF: A biomarker panel to discriminate between systemic inflammatory response syndrome and sepsis and sepsis severity. J Emerg Trauma Shock. 3:26–35. 2010. View Article : Google Scholar : PubMed/NCBI | |
Loetscher M, Gerber B, Loetscher P, Jones SA, Piali L, Clark-Lewis I, Baggiolini M and Moser B: Chemokine receptor specific for IP10 and Mig: Structure, function, and expression in activated T-lymphocytes. J Exp Med. 184:963–969. 1996. View Article : Google Scholar : PubMed/NCBI | |
Hsieh MF, Lai SL, Chen JP, Sung JM, Lin YL, Wu-Hsieh BA, Gerard C, Luster A and Liao F: Both CXCR3 and CXCL10/IFN-inducible protein 10 are required for resistance to primary infection by dengue virus. J Immunol. 177:1855–1863. 2006. View Article : Google Scholar : PubMed/NCBI |