广东家庭聚集性登革3型病例特征及其病毒株全基因组序列分析
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摘要
登革热是由登革病毒通过蚊虫叮咬传播引起的急性传染性疾病。其病毒可分为四个血清型(DENV-1、DENV-2、DENV-3、DENV-4)。目前登革热已在热带及亚热带地区超过100多个国家流行,威胁到全球超过40%人口的健康。据WHO估计,目前全球每年有约1亿登革热患者,其中有登革出血热及登革休克综合症(DHF/DSS)患者约50万,死亡人数25000人。在我国,登革热主要在沿海地区流行,其中以广东省最为严重。自1978年以来,广东几乎每年都有登革热的流行,且四型登革病毒都有过流行,但广东登革热的研究目前主要集中在DENV-1和DENV-2,而对DENV-3及DENV-4,目前的研究资料都很少。而近十年来,广东主要还是以登革1型病毒流行为主,未有其它型DENV感染病例的报道。因此,密切关注临床登革热病例、及时获取早期样本、分离野生型登革毒株、研究分析其临床和病原学特征,对于全面了解广东登革热的流行形势及特点、病原体的来源及防治等均具有重要意义。
研究目的:分析2009年8月发生于广东省广州市的一起家庭聚集性的3例登革热病例的临床和实验室检查特征,从患者血清中分离病毒株并进行全基因组序列测定及分析,了解2009广东登革病毒流行特点及病原体基因组特征,以明确广东登革热的分子流行病学趋势和特点。
研究方法:
1.收集三例登革热患者的临床及实验室资料,并用Excell表进行统计分析;采用胶体金法检测三名临床登革热病例发病期和恢复期血清登革IgM、IgG抗体,并用ELISA细胞因子检测试剂盒检测三名登革患者发病期及恢复期血清IL-6、IL-10、IFN-γ、sTNFR1(可溶性肿瘤坏死因子受体-1)、TNFα细胞因子的水平;
2.采用RT-PCR法扩增血清登革病毒C-PrM基因区序列,并对PCR产物进行电泳及测序分析;
3.用C6/36细胞接种感染者血清,以分离登革病毒株;
4.用PCR DESIGN和DNAclub软件设计扩增登革病毒全基因组序列的11对PCR引物,经RT-PCR扩增和T载体克隆后进行测序,并对测序结果进行生物信息学分析。
研究结果:
1.三名患者发病前均有蚊虫叮咬史,2周内没有外出史,临床特征有发热头痛,颜面潮红,疲乏,关节肌肉痛,皮疹,束臂实验阳性,白细胞减少;3名患者急性期血清登革IgM抗体均为阳性,恢复期血清登革IgG抗体均为阳性;符合登革热的临床诊断;血清5种细胞因子发现,三名患者急性期血清IL-10、sTNFR1、IFN-γ细胞因子水平高于健康对照组和恢复期;
2.三名患者血清RT-PCR产物经电泳分析后均出现大小为290bp左右的特异性片段,PCR产物经测序鉴定,证实均为登革3型病毒感染,且三例患者血清病毒序列完全一致;
3.用C6/36细胞成功从其中1名患者急性期血清中分离出登革病毒株;经全基因组序列测定,广东登革3型病毒株全长10707bp,进化树显示该病毒株为登革3型病毒亚型Ⅲ毒株;
4.与目前测定的最古老的登革3型病毒亚型Ⅲ毒株相比,广东2009年登革3型病毒株发生了碱基变异377个,氨基酸变异39个。
结论:
1.2009年广东一起家庭聚集性的3例登革热病例均为登革3型病毒首次感染,这是近十几年来广东首次发生本地家庭集聚性登革3型病例。
2.这是广东省首次成功培养分离出登革3型病毒株并完成了全基因组序列的测定,进化树分析确定本次流行株为登革3型病毒亚型Ⅲ毒株。该病毒株共发生碱基变异377个,氨基酸变异39个。
Dengue fever (DF)is an acute infectious disease caused by dengue virus(DENV) infection, which is transmitted through the bite of infected mosquito .There are four serotypes of dengue virus(DENV-1、DENV-2、DENV-3、DENV-4).DF is now endemic in more than 100 countries in tropical and subtropical areas, and threatens the health of approximately 40% of the world’s population. The World Health Organization estimates that there may be around 100 million cases of dengue virus infection worldwide every year, resulting in approximately 500,000 cases of DHF/DSS and 25,000 deaths each year . Dengue mainly prevalent along the coast areas in China, while Guangdong province is the most affected area.Since 1978, there are dengue cases reported almost every year,all four serotypes of dengue virus have epidemics in Guangdong .But the research of DF in Guangdong was major in DENV-1 and DENV-2, however, little is known about DENV-3 and DENV-4. In the last ten years , the dengue endemic in Guangdong was mostly caused by DENV-1 ,no other serotype of DENV infected case was found . So we must pay close attention to clinical DF case, get the early specimen on time and isolate the wild strain of dengue virus, research and analyze the character of its clinic and pathology. All these are significant to understand the epidemic situation and its character, source of pathogen and control and prevention of DF in Guangdong
Aim of this research:
Analyze the clinical and laboratory characteristics of 3 cases of a family-clustered dengue fever in city of Guangzhou in Guangdong province August 2009 . Isolate dengue virus strain in serum from the patients, then sequence and analyze the whole genome of the virus, to find out the characters of dengue virus epidemic and the genome of the pathogen in Guangdong in 2009, so as to confirm the characteristics of molecular epidemiology of dengue fever in Guangdong .
Research Methods:
1. Collect clinical and laboratory data of the 3 dengue fever patients, statistical analyze the data by excel; dengue virus specific IgM and IgG antibody were detected by Collidal gold method in patients’serum collected in acute phase and convalescence phase; cytokine levels of IL-6、IL-10、IFN-γ、sTNFRI、TNFαwere detected by ELISA in patients’serum collected in acute phase and convalescence phase.
2. Amplify the C-PrM gene in dengue virus by RT-PCR, electrophoresis and sequence analyze the PCR product.
3. Inoculate cell C6/36 with serum from patients to isolate the strain of dengue virus.
4. Through software of DNA DESIGN and DNAclub, design 11 pairs of primer which could amplify the whole genome sequence of the virus, amplify by PCR and connect it with T clone vector before sequencing, bioinformatics analysis is made after we get the sequence of the genome.
Results:
1. All the patients were bitten by mosquito before onset of disease, have not went out in the last two weeks, and the characteristics of clinics included fever, headache, flushing, lassitude, arthalgia, myalgia, rash, tourniquet test positive and leucopenia, Serum dengue virus specific antibody tests indicate: dengue IgM antibody are positive in acute phase , while IgG antibody are positive in convalescence phase , those consistent with diagnostic criteria of dengue fever. By detecting the levels of IL-6、IL-10、IFN-γ、sTNFR1 and TNFαcytokines expression in the 3 patients in acute phase and convalescence phase,we find the IL-10、sTNFR1 and IFN-γlevel of the DF patients in acute phase are higher than the health controls.
2. Specific fragment about 290bp appear after electrophoresis using PCR products from the serum of the three patients. They all are DENV-3 which are confirmed by sequencing the products, and their genome are of 100% similarity.
3. Through cell C6/36,one dengue virus strain is Succeed isolated from acute stage’s serum of one patient.The length of the whole genome of DENV-3 strain in Guangdong is 10707bp after sequenced , and DENV-3 genotype III is identified while it is analyzed by phylogenetic tree analysis.
4.Compared to the oldest DENV-3 genotype III strain,there are 377 nucleotide mutation and 39 amino acids diversity happens in dengue virus type 3 strain of Guangdong.
Conclusions:
1 All the 3 patients are primary DENV-3 infection, and this is the first time to discover endemic of DENV-3 in GD in the last 10 years.
2. Succeed in cultivating and isolating DENV-3 and sequencing the whole genome of it, identify the epidemic strain this time is DENV-3 genotype III.
3. Compared to the oldest DENV-3 genotype III strain,there are 377 nucleotide mutation and 39 amino acids diversity happens in dengue virus type 3 strain of Guangdong
研究目的:分析2009年8月发生于广东省广州市的一起家庭聚集性的3例登革热病例的临床和实验室检查特征,从患者血清中分离病毒株并进行全基因组序列测定及分析,了解2009广东登革病毒流行特点及病原体基因组特征,以明确广东登革热的分子流行病学趋势和特点。
研究方法:
1.收集三例登革热患者的临床及实验室资料,并用Excell表进行统计分析;采用胶体金法检测三名临床登革热病例发病期和恢复期血清登革IgM、IgG抗体,并用ELISA细胞因子检测试剂盒检测三名登革患者发病期及恢复期血清IL-6、IL-10、IFN-γ、sTNFR1(可溶性肿瘤坏死因子受体-1)、TNFα细胞因子的水平;
2.采用RT-PCR法扩增血清登革病毒C-PrM基因区序列,并对PCR产物进行电泳及测序分析;
3.用C6/36细胞接种感染者血清,以分离登革病毒株;
4.用PCR DESIGN和DNAclub软件设计扩增登革病毒全基因组序列的11对PCR引物,经RT-PCR扩增和T载体克隆后进行测序,并对测序结果进行生物信息学分析。
研究结果:
1.三名患者发病前均有蚊虫叮咬史,2周内没有外出史,临床特征有发热头痛,颜面潮红,疲乏,关节肌肉痛,皮疹,束臂实验阳性,白细胞减少;3名患者急性期血清登革IgM抗体均为阳性,恢复期血清登革IgG抗体均为阳性;符合登革热的临床诊断;血清5种细胞因子发现,三名患者急性期血清IL-10、sTNFR1、IFN-γ细胞因子水平高于健康对照组和恢复期;
2.三名患者血清RT-PCR产物经电泳分析后均出现大小为290bp左右的特异性片段,PCR产物经测序鉴定,证实均为登革3型病毒感染,且三例患者血清病毒序列完全一致;
3.用C6/36细胞成功从其中1名患者急性期血清中分离出登革病毒株;经全基因组序列测定,广东登革3型病毒株全长10707bp,进化树显示该病毒株为登革3型病毒亚型Ⅲ毒株;
4.与目前测定的最古老的登革3型病毒亚型Ⅲ毒株相比,广东2009年登革3型病毒株发生了碱基变异377个,氨基酸变异39个。
结论:
1.2009年广东一起家庭聚集性的3例登革热病例均为登革3型病毒首次感染,这是近十几年来广东首次发生本地家庭集聚性登革3型病例。
2.这是广东省首次成功培养分离出登革3型病毒株并完成了全基因组序列的测定,进化树分析确定本次流行株为登革3型病毒亚型Ⅲ毒株。该病毒株共发生碱基变异377个,氨基酸变异39个。
Dengue fever (DF)is an acute infectious disease caused by dengue virus(DENV) infection, which is transmitted through the bite of infected mosquito .There are four serotypes of dengue virus(DENV-1、DENV-2、DENV-3、DENV-4).DF is now endemic in more than 100 countries in tropical and subtropical areas, and threatens the health of approximately 40% of the world’s population. The World Health Organization estimates that there may be around 100 million cases of dengue virus infection worldwide every year, resulting in approximately 500,000 cases of DHF/DSS and 25,000 deaths each year . Dengue mainly prevalent along the coast areas in China, while Guangdong province is the most affected area.Since 1978, there are dengue cases reported almost every year,all four serotypes of dengue virus have epidemics in Guangdong .But the research of DF in Guangdong was major in DENV-1 and DENV-2, however, little is known about DENV-3 and DENV-4. In the last ten years , the dengue endemic in Guangdong was mostly caused by DENV-1 ,no other serotype of DENV infected case was found . So we must pay close attention to clinical DF case, get the early specimen on time and isolate the wild strain of dengue virus, research and analyze the character of its clinic and pathology. All these are significant to understand the epidemic situation and its character, source of pathogen and control and prevention of DF in Guangdong
Aim of this research:
Analyze the clinical and laboratory characteristics of 3 cases of a family-clustered dengue fever in city of Guangzhou in Guangdong province August 2009 . Isolate dengue virus strain in serum from the patients, then sequence and analyze the whole genome of the virus, to find out the characters of dengue virus epidemic and the genome of the pathogen in Guangdong in 2009, so as to confirm the characteristics of molecular epidemiology of dengue fever in Guangdong .
Research Methods:
1. Collect clinical and laboratory data of the 3 dengue fever patients, statistical analyze the data by excel; dengue virus specific IgM and IgG antibody were detected by Collidal gold method in patients’serum collected in acute phase and convalescence phase; cytokine levels of IL-6、IL-10、IFN-γ、sTNFRI、TNFαwere detected by ELISA in patients’serum collected in acute phase and convalescence phase.
2. Amplify the C-PrM gene in dengue virus by RT-PCR, electrophoresis and sequence analyze the PCR product.
3. Inoculate cell C6/36 with serum from patients to isolate the strain of dengue virus.
4. Through software of DNA DESIGN and DNAclub, design 11 pairs of primer which could amplify the whole genome sequence of the virus, amplify by PCR and connect it with T clone vector before sequencing, bioinformatics analysis is made after we get the sequence of the genome.
Results:
1. All the patients were bitten by mosquito before onset of disease, have not went out in the last two weeks, and the characteristics of clinics included fever, headache, flushing, lassitude, arthalgia, myalgia, rash, tourniquet test positive and leucopenia, Serum dengue virus specific antibody tests indicate: dengue IgM antibody are positive in acute phase , while IgG antibody are positive in convalescence phase , those consistent with diagnostic criteria of dengue fever. By detecting the levels of IL-6、IL-10、IFN-γ、sTNFR1 and TNFαcytokines expression in the 3 patients in acute phase and convalescence phase,we find the IL-10、sTNFR1 and IFN-γlevel of the DF patients in acute phase are higher than the health controls.
2. Specific fragment about 290bp appear after electrophoresis using PCR products from the serum of the three patients. They all are DENV-3 which are confirmed by sequencing the products, and their genome are of 100% similarity.
3. Through cell C6/36,one dengue virus strain is Succeed isolated from acute stage’s serum of one patient.The length of the whole genome of DENV-3 strain in Guangdong is 10707bp after sequenced , and DENV-3 genotype III is identified while it is analyzed by phylogenetic tree analysis.
4.Compared to the oldest DENV-3 genotype III strain,there are 377 nucleotide mutation and 39 amino acids diversity happens in dengue virus type 3 strain of Guangdong.
Conclusions:
1 All the 3 patients are primary DENV-3 infection, and this is the first time to discover endemic of DENV-3 in GD in the last 10 years.
2. Succeed in cultivating and isolating DENV-3 and sequencing the whole genome of it, identify the epidemic strain this time is DENV-3 genotype III.
3. Compared to the oldest DENV-3 genotype III strain,there are 377 nucleotide mutation and 39 amino acids diversity happens in dengue virus type 3 strain of Guangdong
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[48]于曼,韩剑峰,陈水平. 2006年广州登革热病原体的分离鉴定及其生物学性质的观察[J].生物技术通讯, 2008, 19(6): 840-842.
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[50] Chuang VW, Wong TY, Leung YH,et al . Review of dengue fever cases in Hong Kong during 1998 to 2005[J]. Hong Kong Med J, 2008; 14(3): 170-177
[51] Chwan-Chuen King, Day-Yu Chao, Li-Jung Chien,et al .Comparative analysis of full genomic sequences among different genotypes of dengue virus type 3 [J]. Virol J, 2008; 5: 63
[52] Munoz-Jordan JL, Sanchez-Burgos GG, Laurent-Rolle M, Garcia-Sas- tre A: Inhibition of interferon signaling by dengue virus [J]. Proc Natl Acad Sci, 2003; 100:14333-14338.
[53] Munoz-Jordan JL, Laurent-Rolle M, Ashour J,et al : Inhibition of alpha/beta interferon signaling by the NS4B protein of flaviviruses [J]. J Virol, 2005; 79:8004-8013.
1 Teichmann D, G?bels K, Niedrig M. Dengue virus infection in travellers returning to Berlin,Germany:clinical,laboratory,and diagnostic aspects. Acta Trop. 2004 , 90(1): 87-95.
2 Díaz-Quijano FA, Villar-Centeno LA, Martínez-Vega RA. Complications associated to severe thrombocytopenia in patients with dengue. Rev Med Chil. 2006, 134(2):167-173.
3 Su DH, Bacsal K, Chee SP,et al. Prevalence of dengue maculopathy in patients hospitalized for dengue fever. Ophthalmology. 2007, 114(9): 1743-1747.
4 Bacsal KE, Chee SP, Cheng CL,et al. Dengue-associated maculopathy. Arch Ophthalmol. 2007, 125(4):501-510.
5 Domingues RB, Kuster GW, Onuki-Castro FL, et al. Involvement of the central nervous system in patients with dengue virus infection. J Neurol Sci.2008, 267(1-2): 36-40.
6 Lum LC, Lam SK, Choy YS, et al. Dengue encephalitis:a true entity?. Am J Trop Med Hyg. 1996, 54(3): 256-259.
7 Wiwanitkit V. Liver dysfunction in Dengue infection: an analysis of the previously published Thai cases. J Ayub Med Coll Abbottabad. 2007, 19(1):10-12.
8 Agarwal R, Kapoor S, Nagar R, et al. A clinical study of the patients with dengue hemorrhagic fever during the epidemic of 1996 at Lucknow,India. Southeast Asian J Trop Med Public Health. 1999, 30(4): 735-740.
9 Wichmann O, Gascon J, Schunk M, et al. Severe dengue virus infection in travelers: risk actors and laboratory indicators. J Infect Dis. 2007, 195: 1089–1096
10 Ramos MM, Argüello DF, Luxemburger C, et al. Epidemiological and clinical observations on patients with dengue in Puerto Rico:results from the first year of enhanced surveillance-June 2005-May 2006. Am J Trop Med Hyg. 2008 , 79(1): 123-127.
11 Thomas L, Verlaeten O, CabiéA, et al. Influence of the dengue serotype, previous dengue infection,and plasma viral load on clinical presentation and outcome during a dengue-2 and dengue-4 co-epidemic.Am J Trop Med Hyg. 2008, 78(6): 990-998.
12 Cordeiro MT, Silva AM,Brito CA, Nascimento EJ.et.al. Characterization of a dengue patient cohort in Recife,Brazil. Am J Trop Med Hyg. 2007, 77(6): 1128-1134.
13 Fuchun Zhang, Xiaoping Tang, Xuchu Hu,et al . A clinical, epidemiological and virological study of a dengue fever outbreak in Guangzhou,china 2002-2006. Dengue Bulletin.2007,31:10-18
14 Salgado DM, Rodríguez JA, Garzón M,et al. Clinical and epidemiological characterisation of dengue haemorrhagic fever in Neiva, Colombia,2004 Rev Salud Publica (Bogota). 2007, 9(1): 53-63.
15 Rocha LA, Tauil PL. Dengue in children: clinical and epidemiological characteristics, Manaus,State of Amazonas,2006 and 2007. Rev Soc Bras Med Trop. 2009, 42(1): 18-22.
16 García-Rivera EJ, Rigau-Pérez JG. Dengue severity in the elderly in Puerto Rico.Rev Panam Salud Publica. 2003, 13(6): 362-368.
17 Wang WK, Chen HL, Yang CF,et al. Slower rates of clearance of viral load and virus-containing immune complexes in patients with dengue hemorrhagic fever. Clin Infect Dis, 2006, 43: 1023–1030
18 Bozza FA, Cruz OG, Zagne SM, et al. Multiplex cytokine profile from dengue patients:MIP-1beta and IFN-gamma as predictive factors for severity. BMC Infect Dis, 2008, 8: 86.
19 Suvarna JC, Rane PP. Serum lipid profile:a predictor of clinical outcome in dengue infection. Trop Med Int Health. 2009, 14(5): 576-585.
20 Bouldouyre MA, Baumann F, Berlioz-Arthaud A. Factors of severity at admission during an epidemic of dengue 1 in New Caledonia(South Pacific) in 2003. Scand J Infect Dis, 2006, 38(8): 675-681.
21 Binh PT, Matheus S, Huong VT, et.al.Early clinical and biological features of severe clinical manifestations of dengue in Vietnamese adults. J Clin Virol.2009, 45(4): 276-280.
22 Phuong CX, Nhan NT, Kneen R,A. Clinical diagnosis and assessment of severity of confirmed dengue infections in vietnamese children: is the world health organization classification system helpful? Am J Trop Med Hyg. 2004, 70(2): 172-179.
23 Bandyopadhyay S, Lum LC, Kroeger A. Classifying dengue: a review of the difficulties in using the WHO case classification for dengue haemorrhagic fever. Trop Med Int Health, 2006, 11(8): 1238-1255.
24 Setiati TE, Mairuhu AT, Koraka P, et al. Dengue disease severity in Indonesian children:an evaluation of the World Health Organization classification system. BMC Infect Dis, 2007, 7: 22.
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1 Teichmann D, G?bels K, Niedrig M. Dengue virus infection in travellers returning to Berlin,Germany:clinical,laboratory,and diagnostic aspects. Acta Trop. 2004 , 90(1): 87-95.
2 Díaz-Quijano FA, Villar-Centeno LA, Martínez-Vega RA. Complications associated to severe thrombocytopenia in patients with dengue. Rev Med Chil. 2006, 134(2):167-173.
3 Su DH, Bacsal K, Chee SP,et al. Prevalence of dengue maculopathy in patients hospitalized for dengue fever. Ophthalmology. 2007, 114(9): 1743-1747.
4 Bacsal KE, Chee SP, Cheng CL,et al. Dengue-associated maculopathy. Arch Ophthalmol. 2007, 125(4):501-510.
5 Domingues RB, Kuster GW, Onuki-Castro FL, et al. Involvement of the central nervous system in patients with dengue virus infection. J Neurol Sci.2008, 267(1-2): 36-40.
6 Lum LC, Lam SK, Choy YS, et al. Dengue encephalitis:a true entity?. Am J Trop Med Hyg. 1996, 54(3): 256-259.
7 Wiwanitkit V. Liver dysfunction in Dengue infection: an analysis of the previously published Thai cases. J Ayub Med Coll Abbottabad. 2007, 19(1):10-12.
8 Agarwal R, Kapoor S, Nagar R, et al. A clinical study of the patients with dengue hemorrhagic fever during the epidemic of 1996 at Lucknow,India. Southeast Asian J Trop Med Public Health. 1999, 30(4): 735-740.
9 Wichmann O, Gascon J, Schunk M, et al. Severe dengue virus infection in travelers: risk actors and laboratory indicators. J Infect Dis. 2007, 195: 1089–1096
10 Ramos MM, Argüello DF, Luxemburger C, et al. Epidemiological and clinical observations on patients with dengue in Puerto Rico:results from the first year of enhanced surveillance-June 2005-May 2006. Am J Trop Med Hyg. 2008 , 79(1): 123-127.
11 Thomas L, Verlaeten O, CabiéA, et al. Influence of the dengue serotype, previous dengue infection,and plasma viral load on clinical presentation and outcome during a dengue-2 and dengue-4 co-epidemic.Am J Trop Med Hyg. 2008, 78(6): 990-998.
12 Cordeiro MT, Silva AM,Brito CA, Nascimento EJ.et.al. Characterization of a dengue patient cohort in Recife,Brazil. Am J Trop Med Hyg. 2007, 77(6): 1128-1134.
13 Fuchun Zhang, Xiaoping Tang, Xuchu Hu,et al . A clinical, epidemiological and virological study of a dengue fever outbreak in Guangzhou,china 2002-2006. Dengue Bulletin.2007,31:10-18
14 Salgado DM, Rodríguez JA, Garzón M,et al. Clinical and epidemiological characterisation of dengue haemorrhagic fever in Neiva, Colombia,2004 Rev Salud Publica (Bogota). 2007, 9(1): 53-63.
15 Rocha LA, Tauil PL. Dengue in children: clinical and epidemiological characteristics, Manaus,State of Amazonas,2006 and 2007. Rev Soc Bras Med Trop. 2009, 42(1): 18-22.
16 García-Rivera EJ, Rigau-Pérez JG. Dengue severity in the elderly in Puerto Rico.Rev Panam Salud Publica. 2003, 13(6): 362-368.
17 Wang WK, Chen HL, Yang CF,et al. Slower rates of clearance of viral load and virus-containing immune complexes in patients with dengue hemorrhagic fever. Clin Infect Dis, 2006, 43: 1023–1030
18 Bozza FA, Cruz OG, Zagne SM, et al. Multiplex cytokine profile from dengue patients:MIP-1beta and IFN-gamma as predictive factors for severity. BMC Infect Dis, 2008, 8: 86.
19 Suvarna JC, Rane PP. Serum lipid profile:a predictor of clinical outcome in dengue infection. Trop Med Int Health. 2009, 14(5): 576-585.
20 Bouldouyre MA, Baumann F, Berlioz-Arthaud A. Factors of severity at admission during an epidemic of dengue 1 in New Caledonia(South Pacific) in 2003. Scand J Infect Dis, 2006, 38(8): 675-681.
21 Binh PT, Matheus S, Huong VT, et.al.Early clinical and biological features of severe clinical manifestations of dengue in Vietnamese adults. J Clin Virol.2009, 45(4): 276-280.
22 Phuong CX, Nhan NT, Kneen R,A. Clinical diagnosis and assessment of severity of confirmed dengue infections in vietnamese children: is the world health organization classification system helpful? Am J Trop Med Hyg. 2004, 70(2): 172-179.
23 Bandyopadhyay S, Lum LC, Kroeger A. Classifying dengue: a review of the difficulties in using the WHO case classification for dengue haemorrhagic fever. Trop Med Int Health, 2006, 11(8): 1238-1255.
24 Setiati TE, Mairuhu AT, Koraka P, et al. Dengue disease severity in Indonesian children:an evaluation of the World Health Organization classification system. BMC Infect Dis, 2007, 7: 22.