2001~2003年台灣南部地區登革熱/登革出血熱的流行病學探討

Abstract

The largest epidemic of dengue hemorrhagic fever (DHF) with the highest case numbers and case fatality rate (CFR：8.7%) of DHF caused by dengue virus serotype 2 (DEN-2) exploded in southern Taiwan in 2002, since 1942. We investigated the molecular changes of viral factors plus host variables in this epidemic in KaoHsiung City/County and Pingtung City/County during 2001-2003 by involving three parts: (1) to understand the epidemiological, virological and serological data on DHF versus dengue fever (DF) cases which were defined by dengue team scholars through field investigations, and (2) to elucidate the molecular changes in nucleotide and amino acid sequences of complete envelope (E) gene (1485 nucleotides) of DEN-2 by collecting isolates obtained from 2001-2003 compared with those from past epidemics in Taiwan and other countries from GenBank through phylogenetic and qualitative analyses, and (3) to search for the relationship between the diversity of quasispecies in E gene in DF versus DHF patients with or without different underlying diseases. The most DHF cases were emerged from the peak of the epidemic curve (35th-44th wk). By univariate analysis of several risk factors for DHF vs. DF cases, our results indicated that : (1)the percentage of DHF cases in ChienJen District of KaoHsiun City (initial epi-center) increased significantly from the second period [7.05%(28/397)] to the third period [9.7%(14/144)] and the fourth period [10.9%(43/394)] (p=0.0003) besides the initial epidemic period; (2) DHF cases occurred more frequently in 40~60 y/o adults [44.79% (118/295)，p=0.008], in patients with chronic diseases such as asthma [3.04% (7/230) vs. 1.12% (8/715)，p=0.04], kidney disease [6.52% (15/230) vs. 3.08% (22/715)，p=0.01], hypertension [27.83% (64/230) vs. 18.23% (131/715)，p=0.001], HBV infection [6.52% (15/230) vs. 3.36% (24/715)，p=0.04], diabetes mellitus [22.17% (51/230) vs. 14.4% (103/715)，p=0.005], gastric ulcer [10.43% (24/230) vs. 4.2% (30/715)，p=0.0004]; and (3) DHF had significant association with the patient’s self-described past dengue history [18.29% (47/257) vs. 12.16% (300/2467)，p=0.005] but smaller-scale serological data did not show such an association with past dengue virus infection [93.38% (127/136) vs. 88.38% (1148/1299)，p=0.08]. Molecular epidemiologic study to determine the role of viral evolution in emerging a large-scale epidemic of DHF during 2001-2003 was to analyze the complete sequences of E gene of 38 DEN-2 isolates obtained from epidemics in 1981, 1987, 1997, and 2001-2003 in Taiwan. The maximum-likelihood phylogenetic tree analysis revealed that Taiwan’s DEN-2 isolates fell into 2 clusters (diversity 0~8.7%). The 2001-2003’s DEN-2 isolates, which belonged to the cosmopolitan genotype, showed 99.1%~100% sequence identity and 8, 20 and 2 DEN-2 isolates of 2001, 2002 and 2003 had 99.6-100%, 99.5-100% and.99.7% homology, respectively. The 1981’s DEN-2 isolate had 95.3% nt homology with 1987’s and such an identity dropped to 94.4% in 1997, and declined to 92.6% in 2001-2003. The nucleotide sequences of E gene at positions of 137, 291, 1128 of 2001’s eight DEN-2 isolates had consistent changes from C, A, T to T (only one remained as C), T, C in 2002-2003’s twenty-two isolates whereas the amino acid at position 46 was consistently changed from Thr in 2001 to Ile in 2002 as the epidemic became longer. Tungkang’s DEN-2 isolates showed geographic differences from Kaoshoung’s DEN-2 isolates in both 2001 and 2002, implying different environmental factors or selective pressures affecting various evolution rate or directions of DEN-2. Among 4 imported DEN-2 isolates, the one from the Philippines (Cosmopolitan genotype) had the highest homology (98.9-99%) with Taiwan’s indigenous DEN-2 isolates in 2001-2003. There were no molecular signatures of distinct lineage for those isolates from DHF vs. DF or dengue cases with or without specific underlying diseases. To understand whether quasispecies of DEN-2 would have more variation in those dengue patients with underlying diseases (diabetes, hypertension etc.), 7 plasma and 1 peripheral blood mononuclear samples of 4 DF and 4 DHF patients with clear epidemiological characteristics and medical history were collected. The PCR product from E region of DEN-2 isolates from blood in acute phase were ligased with the T/A cloning vector, and PCRⅡ-TOPO was used to transform to E. coli TOP 10 competent cells. About 12~20 clones were randomly selected, completely E gene sequenced and aligned, p-distance was used in analyzing sequence diversity. Our results showed that there was no relationship between DHF and more diversity of quasispecies at E region but those dengue patients had underlying diseases demonstrated higher diversity (p=0.072, Mann-Whitney test). In summary, the Cosomopolitan genotype of DEN-2 came into Taiwan in 2001 and increased its diversity in 2002-2003 plus geographical variation taken together had facilitated to emerge more variants of DEN-2. Furthermore, the rapid infection of large human populations including patients with underlying diseases increased the dimension of variability of DEN-2. Therefore, diversified DEN-2 strains through a series of human-mosquito-human transmission chains interacting with host and environmental factors might emerge the phenotype of DEN-2 with more virulence or higher epidemic potentials. Therefore, future studies on pathogenesis of DHF need to emphasize the interactions between virus evolution and alternating hosts through transmission chains.