MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY DOAN HUU THIEN

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1 MINISTRY OF EDUCATION AND TRAINING MINISTRY OF HEALTH NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY DOAN HUU THIEN EPIDEMIOLOGICAL, CLINICAL CHARACTERISTICS AND MOLECULAR BIOLOGY OF DENGUE VIRUS FROM DENGUE HEMORRHAGIC PATIENTS AT DAK LAK GENERAL HOSPITAL Major: Epidemiology Serial number: SUMMARY OF MEDICAL PHD THESIS HANOI

2 THIS STUDY HAS BEEN CONDUCTED AT NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY SCIENTIFIC ADVISORS: 1. Prof. Phan Thi Nga 2. Prof. Dang Tuan Dat Critic 1: Critic 2: Critic 3: The dissertation shall be defended at Thesis evaluation committee, National Institute of Hygiene and Epidemiology at h day month The dissertation can be found at: 1. National library 2. Library of National Institute of Hygiene and Epidemiology

3 RELATED PUBLICATIONS 1. Doan Huu Thien, Nguyen Hai, Tran Viet Anh, Nguyen Duc Hanh, Phan Thi Tuyet Nga, Dang Tuan Dat, Bui Manh Tuan, Ngo Phi Phuong, Bui Minh Trang, Phan Thi Nga. Epidemiological characterizations and related factors of dengue hemorrhagic fever patient in Dak Lak, Journal of Preventive Medicine, 2017, vol.27, no.7: Doan Huu Thien, Nguyen Hai, Tran Viet Anh, Nguyen Duc Hanh, Phan Thi Tuyet Nga, Dang Tuan Dat, Bui Manh Tuan, Nguyen Ngo Phuong, Bui Minh Trang, Phan Thi Nga. Analysis several clinical. subclinicalcharacterizations of dengue patient associated with dengue virus type in Dak Lak general hospital, Journal of Preventive Medicine, 2017, vol.27, no.7: Doan Huu Thien, Do Phuong Loan, Bui Minh Trang, Phan Thi Tuyet Nga, Le Van Tuan, Dang Thi Thu Thao, Mai Thi Hien, Nguyen Thanh Luan, Phan Thi Nga. Molecular epidemiological biology of dengue virus in Dak Lak, Central Highland, Journal of Preventive Medicine, 2017, vol.27, no.3:

4 1 Introduction Dengue hemorrhagic fever (DHF) is one of the most infectious and fatal disease. Dengue virus has 4 serotypes, all of them can cause disease, and severe clinical cases usually associate with serotype 2. In Vietnam, number of new cases and deaths related to dengue has increased sharply. Statistics from Ministry of health during shows Central highland (Tay Nguyen) has the highest DHF prevalence in the country, in which area the disease is relatively prevalent in Dak Lak province. Based on these facts, we conduct the study: Epidemiological, clinical characteristics and molecular biology of dengue virus from dengue hemorrhagic patients at Dak Lak general hospital, with 3 objectives: 1. Describe epidemiological characteristics and determinants of dengue hemorrhagic fever in Dak Lak provine, Describe clinical characteristics and determinants of dengue hemorrhagic fever in Dak Lak provine, Identify molecular biology features of the types of dengue virus circulated in Dak Lak province, New scientific findings and practicality of the study 1. The first study describes epidemiological characteristics and determinants of dengue hemorrhagic fever within a long period , providing an epidemiological overview dengue hemorrhagic fever in Dak Lak. 2. The first study provides association between DHF clinical symptoms and virus types. The study shows petechiae and gum/teeth/subconjunctival hemorrhage are most seen DENV-3 patients, less common in DHF type nucleotide sequences of of C-PrM region were granted accession number in Genbank. The study has identified a new group of 5 DENV-2 isolates from patients in Dak Lak, which is significantly different from 5 previously characterized genotypes, does not belong to published genotypes, and possibly represents a new genotype/subgenotype. The study does not not only identifies circulating types of dengue virus in Dak Lak, it also helps finding the genotype origins of types of dengue virus circulate in the province and enriches molecular biology database of dengue virus in Central Highland (Tay Nguyen) as well as in Vietnam.

5 2 THESIS STRUCTURE The thesis consists of 109 pages, 17 tables and 27 figures. 2 pages Introduction, 33 pages Overview, 17 pages Subjects and Research methods, 27 pages Results, 27 pages Discussion; 2 pages Conclusion and 1 page Recommendations Chapter 1: OVERVIEW 1.1. Epidemiology of dengue hemorrhagic fever Cause of disease Dengue hemorrhagic fever (DHF) is caused by dengue virus, of the family Flaviviridae, genusflavivirus, including 4 serotypes, and there are several genotypes in each serotype. The disease is transmitted by Aedes aegypti and Aedes albopictus mosquitoes Transmission route DHF is primarily transmitted by Aedes aegypt mosquitos and by Aedes albopictus mosquitos as well Predisposition Everyone, including people previously have DHF, can be infected with dengue virus and contracted the disease Epidemiological characteristics of dengue hemorrhagic fever in the world DHF is highly contagious. Initially, the disease emerged in areas with high population density, then gradually it appeared in lesser populous such as countryside and mountainous region Epidemiological characteristics of dengue hemorrhagic fever in Vietnam Current situation of dengue hemorrhagic fever in Vietnam Figure 1.1. Number of DHF cases in Vietnam per

6 3 In the past, DHF circulated mostly in central and southern provinces. In , central highland had highest DHF prevalence in the country, Dak Lak province has the highest DHF prevalence in central highland Predisposition DHF is seen in at all age group but mostly in adults in the Northern region and children in Southern region Transmission season The disease appears year round in Southern and Central regions, the disease is commonly seen in Northern and Central highland from April to November. Endemics is usually recorded from August to November and peaks in October. Figure 1.2. DHF cases monthly in Vietnam Recently, DHF progresses unusually, high incidences are recorded in end-of-year month. In 2015, number of cases increased 11.5% compare to , and mostly in October, November and December. In 2015 and early 2016, DHF incidences elevated countrywide and almost did not follow previous disease pattern Characteristics of dengue hemorrhagic fever in Central highland From 1983 to 2004, in 3 Central highland provinces, DHF became endemic in several year, between those years DHF still occurred ocassionally.

7 4 Figure 1.3. DHF prevalence of 4 Central highland provinces, In period, new DHF cases were recorded every year in 4 Central highland provinces. Especially in 2010 and 2013, disease prevalence were very high (297,67/ and 293,52/ ). In 2013, Dak Lak province had highest prevalence. Figure 1.4. DHF prevalence in Dak Lak province and Central highland region 1.4. Clinical characteristics of dengue hemorrhagic fever and association to types of dengue virus Clinical characteristics of dengue hemorrhagic fever DHF has typical symptoms including, fever, hemorrhage and plasma leakage. The disease can lead to hypovolemic shock, blood clotting disorder, organ failure and even death. Clinical signs of DHF are diverse, disease progresses rapidly from mild to severe. World Health Organization classifies DHF in 3 categories: dengue hemorrhagic fever without warning sign, dengue hemorrhagic fever with warning sign, and severe dengue hemorrhagic fever.

8 Clinical presentation and types of virus All 4 of dengue virus can cause disease but severe/fatal cases usually relates to type DENV-2. It is hypothesized that DENV-2 is more virulent and more capable to cause shock than other types. DENV-1 and DENV-3 infections usually cause uncomplicated fever in adults, occasionally severe hemorrhage Molecular epidemiology of dengue virus All 4 types of dengue virus are circulating in Asia, Pacific region, Africa and America. Figure 1.5. Types of dengue virus in Vietnam, In Vietnam virological surveillance before 1970 showed only a single type of dengue virus caused an endemic. In the last decades, a DHF endemic can have 2 to 4 types of dengue virus co-circulate, but with different dominant type of virus. Chapter 2 Subjects and research methods 2.1. Research subjects Subject of objective 1: Patients clinically diagnosed with DHF at Dak Lak General hospital. Diagnosis criteria: according to QĐ no.1499 dated 17/5/2011 of Ministry of Health Subject of objective 2: Medical records are used to investigate clinical symptoms of DHF patients based on virus types: Subject of objective 3: Part of C-PrM region of dengue virus isolated from DHF patient in Dak Lak province

9 Time frame and location of the research - Time and location: Retrospective study , monitoring study ; at Dak Lak General hospital Study design of objectives Cross-sectional study combined with retrospective study ( ) and anterospective study ( ) Research method of objective Study design: Descriptive epidemiological study Selecting patient with suspected DHF signs at Dak Lak General hospital, sample size: 1675 patients Process of data collection a) Selecting patient with suspected DHF indication *From period: Review list of all clinically diagnosed DHF cases 1 year old; live or traveled from dengue endemic area within 14 days, continued fever from 2-7 days and show at least 2 of these signs: hemorrhage at different severity; headache, loss of appetite, nauseous; skin rash, myalgia, arthralgia, pain behind eyes; lethargy; pain in liver area or pain when pressed on liver area. * From period: Select all cases clinically diagnosed with DHF treated at Dak Lak General hospital. b) Selecting patients diagnosed with DHF Selecting patients diagnosed with DHF by one of the following methods MAC-ELISA, RT-PCR or virus isolation: 694 DHF (107 retrospect cases in and 587 follow up monitoring cases in ). c) Collect data on epidemiological characteristics of all cases positive with DHF. Record information using medical record research form (Appendix 1-survey form on epidemiology and clinical symptoms of DHF)

10 Pathology collection: From period, select 10% patients, collect blood sample of each patient once. From period, blood sample of each patient is collected twice on: First collection: hospital admission Second collection: before discharge or 7-10 days after disease onset Diagnostic method:mac-elisa or Multiplex RT-PCR Research method of objective Study design:cross-sectional descriptive study using retrospect stastistics ( ) and follow up monitoring ( ) Sample size: All of 276 serotyped sample in period. Selecting sample: all cases with positive diagnosis and serotyped by Multiplex RT-PCR. Number of DHF patients of type 1, type 2, type 3 and type 4 are 3, 63, 50 and 100, respectively. Medical records were selected to investigate the relationship between clinical symptoms and virus types Research technique - Investigate clinical characteristics and related factor according to survey form. - Typing virus by Multiplex RT-PCR Data collection Collect data using form in Appendix 2 (Retrospect clinical investigation form) Research method of objective Study design: Descriptive study Sample size: 37 dengue virus specimen isolated from patient in Dak Lak province (12 DENV-1 ; 5 DENV-2; 7 DENV-3 and 13 DENV-4 specimen).

11 Subject of the study: A part of C-PrM region in dengue genome of 37 dengue virus isolates in the study. Aligning the obtained sequences with a part of of C-PrM region in dengue genome of 43 DENV-1; 51 DENV-2; 62 DENV-3 and 63 DENV-4 strains on GenBank and 19 dengue strains previously collected in Central highland from Research technique Typing dengue virus by direct Sanger sequencing Construct phylogenetic tree Nucleotide sequences of the isolates are sorted by MUSCLE software integrated in MEGA6.06. Phylogenetic tree is built by Maximum Likelihood algorithm with 1000 times repeatability using appropriate model (K2 + G: Kimura parameter and Gamma distribution) in MEGA Parameters of the research Parameter of objective Epidemiological characteristics of dengue hemorrhagic fever Epidemiological characteristics of DHF each year; epidemiological distribution on gender, age group, locality, season; and preexposure history Determinants: Living condition, anti-mosquitos bite and anti-larvae practice Parameters on clinical characteristics and association to virus types - Identify clinical characteristics of DHF in children and adults related to virus type. - Identify clinical symptoms in admission day, hemorrhagic symptoms related to virus type, several test parameters related to DHF, disease severity to virus type, age group and average treatment duration Characteristics and association between genotypes of dengue virus - Genotyping each dengue isolate by sequencing. - Construct phylogenetic tree describing the relationship of dengue virus types isolated in Dak Lak, aligning nucleotide sequence of

12 9 isolates with known dengue sequences in Central highland, Compare isolated genotypes in Dak Lak with some dengue strains found in Vietnam and the world Methods to limit error Errors are eliminated by: - Rigorous study design, working forms are prepare thoroughly with details. Diagnostic techniques are performed regularly in nationally or regionally recognized laboratories of Central highland or National Institute of Hygiene and Epidemiology - Maximum use of statistical algorithm to remove random error. - Use of bioinformatic software to compare/analyze nucleotide sequence of dengue isolates in Dak Lak and dengue strains in Vietnam and in the world. - Data collectors are specialized doctors with experience in communicable diseases, organized training on patient monitoring, frequent investigation and monitoring. - Sufficiently larger sample size Statistics analysis Enter and analyze data by MS Excel, EpiInfo and STATA; using Meggas, Bioedit, etc. to build phylogenetic tree, identify the origin of dengue virus types in Dak Lak. Chapter 3 Study results 3.1. Description of epidemiological characteristics and determinants Epidemiological characteristics In 1675 cases clinically diagnosed with DHF, there are 694 cases positive with DHF.

13 Số trường hợp 10 Figure 3.1. Epidemiological characteristics Parameters Number of clinical DHF Number of confirmed DHF (+) n % n % Gender Male Female Age group < Locality Urban Countryside No difference is found between male and female, p>0,05. DHF rate in children <5 years old and 5-9 years old is very low (<1%). In contrast, DHF rate in people 15 years old is 96,97%. DHF rate in countryside is higher than urban area, 59,94% and 40,06% respectively Figure 3.1. DHF distribution in monthly DHF is seen year round, peaks differently between years. From period, endemics peak in June, July and October (34.57% total cases); in 2015, endemic peaks in Octorber, November (33.83% Tháng

14 11 total cases); in 2016 endemic peaks in May and June (56.91 % total cases). Figure 3.2. Preexposure history to dengue virus Pre-exposure DHF cases (%) non-dhf (%) OR (CI 95%) Travel out of residence area 2 weeks before disease onset Yes No 12 (52.17) 575 (70.99) 11 (47.83) 235 (29.01) 0.44 ( ) Direct contact with dengue patients 2 weeks before disease onset Yes No 118 (79.73) 469 (68.47) 30 (20.27) 216 (31.53) 1,81 ( ) Patients travel out of residence area have DHF risk equals to 0.44 of patients who did not travel out of residence area. Direct contact with DHF patients increases DHF rate by 1.81 times of patients who did not have direct contact (CI 95%) Determinants Analysis results show: Patients without well built house has 1.28 times more DHF risk than patients with proper housing, impoverished family has 2.11 times more risk than non-impoverished family; anti-mosquitos practice is poor (chemically treated mosquitos net, using anti-mosquitos incense and spray). The groups which did not use anti-mosquitos incense and spary has 2.11 and 1.93 times more risk than users. Patients who did not apply common anti-mosquitos practice has 1.18 DHF risks of user.

15 Results of clinical characteristics and association with types of dengue virus Table 3.3. DHF patient distribution Parameters n = 276 % DENV DENV Virus type DENV DENV Total Male and female ratio is equivalent; group with highest risk is above 15 years old (88.04%). DHF due to DENV-4 is highest (36.23%), lowest is DENV-3 (18.12%), DENV-1 and DENV-2 are similar (22.83%). Myalgia is most common is DHF patients due to DENV-4 (90%), more common in 15 years old than in <15 years old group. 50% DHF patients has positive Lacet test, more common in 15 years old than in <15 years old group. Figure 3.2. Symptoms on day of admission and virus typing Myalgia is least seen in DHF patient with DENV-3 (56%), other types is 68-90%, significantly different, p <0.001.

16 13 Table 3.4. Hemorrhagic symptoms on admission and virus type Type DENV- DENV- DENV- DENV- N= p Symptoms n (%) n (%) n (%) n (%) n (%) Subcutaneous <15 4/13 1/1 8/14 0/5 13/33 hemorrhage (30.77) (100) (57.14) (0) (39.39) > /50 18/62 17/36 22/95 73/243 (32) (29.03) (47.22) (23.16) (30.04) >0.05 Petechia, <15 4/13 0/1 (0) 6/14 3/5 13/33 (30.77) (42.86) (60) (39.39) > /50 23/62 20/36 33/95 94/243 >0.05 tuổi (36) (37.01) (55.56) (34.74) (38.7) Gum/teeth/ <15 3/13 0/1 8/14 1/5 12/33 conjunctival tuổi (23.08) (0) (57.14) (20) (36.36) > /50 17/62 12/36 17/95 54/243 tuổi (16) (27.42) (33.33) (17.89) (22.22) >0.05 Hematuria <15 0/13 0/1 0/14 0/5 0/33 -- tuổi (0) (0) (0) (0) 15 4/50 1/62 0/36 2/95 7/243 >0.05 tuổi (8) (1,61) (0) (2,11) (2.88) Blood in <15 1/13 0/1 4/14 0/5 5/33 vomit/stool (7,69) (0) (28,57) (0) (15.15) > /50 5/62 5/36 9/95 22/243 >0.05 (6) (8,06) (13,89) (9,47) (9.05) Pain <15 1/13 0/1 3/14 0/5 4/3 (7.69) (0) (21.43) (0) (12.12) >0, /50 5/62 5/36 3/95 15/243 >0,05 (4) (8.06) (13.89) (3.16) (6.17) Subcutaneous hemorrhage and petechiae and gum/teeth/conjunctival bleeding is most seen in DENV-3 patients(50% and 33,33%). Hematuria and vomiting blood is rare. Clinical symptoms on admission between 15 years old and <15 years old groups are not statistically different. Pain in liver area is rare, rate of liver area is highest in DENV-3 patients. No brain hemorrhage was recorded. Hemorrhage is less seen in DENV-4 cases, both in 15 years old and <15 years old groups, at 22% and 18% respectively. The difference is statistically different, p<0,007.

17 ,25 71, ,81 9, , , ,19 66, Figure 3.3. Hypovolaemic sign in DHF patients Rate of patients with elevated red blood cell count is % in all age groups. Rate of patients with increased hematocrit more than 42% is from %. Patients with lower white blood cell counts than 4000/mm 3 is 66-71,43%, no significantly different between 15 years old and <15 years old groups. Table 3.5. Severity of DHF patients, virus type and age group Virus type DENV-1 DENV-2 DENV-3 DENV-4 p n = 63 n = 63 n = 50 n = 100 Symptoms n (%) n (%) n (%) n (%) DHF with out < 15 years old 15 years old warning signs DHF with warning signs Severe DHF DHF with out warning signs DHF with warning signs Severe DHF DEN-1 DEN-2 DEN-3 DEN-4 (46.15) (0.0) 4 0 (30.77) (0.0) 3 1 (23.08) (100.0) 5/33 (15.15%) (92.0) (93.55) 3 3 (6.0) (4.84) 1 1 (2.0) (1.61) 6/243 (2.47%) (71.43) 1 (7.14) 3 (21.43) 35 (97.22) 0 (0.0) 1 (2.78) (80.0) 0 (0.0) 1 (20.0) 87 (91.58) 5 (5.26) 3 (3.16) >0.05 >0.05

18 15 Most patients have uncomplicated DHF(>82%). 15 years old has lesser severity than <15 years old group. Table 3.6. Treatment duration, virus type and disease outcome Criteria Virus type DENV-1 n = 63 DENV-2 n = 63 DENV-3 n = 50 DENV-4 n = 100 n (%) n (%) n (%) n (%) p Days hospitalz ed < 3 days 22 (34.92) 32 (50.79) 22 (44.0) 33 (33.0) 3-7 days 39 (61.90) 30 (47.62) 25 (50.0) 61 (61.0) > 7 days 2 (3.17) 1 (1.59) 3 (6.0) 6 (6.0) Normal 4.25± ± ± ±1.63 >0.05 Outcome Recovered Unknown 63 (100.0) 62 (98.41) 49 (98.0) 97 (97.0) 0 (0.0) 1 (1.59) 1 (2.0) 3 (3.0) Average treatment is in 4 days, rate of more than 7 days hospitalzed is very low(6%). Recovery rate is very high (>97%), No fatality Results of molecular epidemiology study of dengue virus circulate in Dak Lak There are 37 strains of dengue virus isolated in Dak Lak, including: 12 DENV-1; 5 DENV-2; 7 DENV- 3 and 13 DENV-4 strains. All 12 DENV-1 belong to genotype 1 (GI) same group with known DENV-1 GI genotypes in Central highland and Vietnam.

19 16 Table 3.7. Similarity of nucleotide sequences of DENV-1 in Dak Lak and other strains Other strains Similarity Average (%) Highest to lowest similar nucleotide (%) GI strains in Dak Lak GI strains in Vietnam GI strains in Vietnam-in the world GI strains in Vietnam GII strains GI strains in Vietnam GIII strains GI strains in Vietnam GIV strains GI strains in Vietnam GV strains Nucleotide sequences between DENV-1 GI circulate in Dak Lak in and other strains in Central highland isolated at the same time and other strains in Vietnam are 97.8% similar (95.9% 100%). Comparing Vietnam strains and GI strains in other country in the region resembles 97.9% ( %) and other genotype is between 91.6%-92.2%. DENV-2 has 5 genotypes but strains isolated in the study do not belong to any of 5 genotypes mentioned above, in this study those new strains are named Asian-III.

20 17 Table 3.8. Similarity of nucleotide sequences of DENV-2 in Dak Lak and other strains Other strains Similarity Average (%) Highest to lowest similar nucleotide (%) Strains in Central highland Strains in Central highland and other strains in Vietnam Strains in Vietnam-other strains in the group Strains in Vietnam Asian-I strains Strains in Vietnam Asian-II strains Strains in Vietnam Asian- American Strains in Vietnam American strains Strains in Vietnam Cosmopolitan-I Strains in Vietnam Cosmopolitan-II Strains in Vietnam Cosmopolitan-III

21 18 DENV-2 isolated in Dak Lak in are relatively distant to 5 known genotype, similarity is between 90,1%-96%, even though several DENV-2 in Vietnam in 2006 belong to Cosmopolitan- III. All 7 DENV-3 isolated in Dak Lak belong to genotype II and closely related to circulating DENV-3 circulating in Vietnam. DENV-4 is divided in 4 genotypes (GI GIII and GS) in which GI genotype is divided in two subtypes GI-A and GI-B. 13 DENV-4 isolates in Dak Lak in belong to GI-A subtype. Table 3.9. Similarity of nucleotide sequences of DENV-4 in Dak Lak and other strains Other strains Similarity Average (%) Highest to lowest similar nucleotide (%) Strains in Dak Lak group Strains in Central highland group 1 and group 2 Strains in Central highland group 1 other strains in the group Analysis results show strains in the same genotype has similarity of 98.9% ( %). Similarity between two groups are 94.7% ( %). Chapter 4 Discussion 4.1. Epidemiological characteristics and determinants of dengue hemorrhagic fever in Dak Lak province, DHF 2016 endemic had 13,324 DHF, comparing 2010 and 2013 endemic, Central highland has more than 13,000 new cases, proving Dak Lak is a DHF hotspot in Central highland. New cases mostly belongs to 15 years old group (96,97%), very few new cases of <5 and 5-9 years old, <1%, similar to

22 19 and study. Meanwhile, DHF is mostly seen in adults in the Northern and children in Southern area. In Dak Lak province, in , DHF occurred year round, months with highest incidence is varied year by year. It means DHF in Dak Lak does not retain its pattern and become severely endemic area. This could be the sign of new DHF emergence in Dak Lak and Vietnam as warned, 21st Century is marking the comeback of DHF endemic. 59,94% of cases is in the countryside. Results of this study, similar to disease surveillance report of Ministry of Health in recent years, DHF progress unusually, number of DHF cases increase nationwide and almost does not follow previous disease pattern. Rate of direct contact history with DHF patients within 2 weeks priot to disease onset is 1.81 times higher than no-contact of DHF patients. It means DHF patients in viremic period could be source of infection. Therefore it could be potential risk for endemic outbreak in Dak Lak. No practice against mosquitos, especially no anti-larvae practice is applied have a very high rate. A considerate number of families use unlid water container. This could be the cause of DHF outbreak and widespread. This results is similar to other epidemiological studies in Vietnam. Accordingly, endemic can break out due to several factor: over population, climate change, rapid urbanization, increase mobility, migration, water storage habit, sleeping without mosquito net, poor hygiene, etc. Therefore, it is needed to continue raising public awareness to prevent DHF, no Aedes larvae, no DHF meanwhile waiting for new breakthrough in preventive vaccine development Clinical characteristics of dengue hemorrhagic fever and association to types of dengue virus in Dak Lak province,

23 20 Rate of DHF is not different between male and female, but clearly different between age groups. Most DHF patients are 15 years old, accounts for 88.04%, only 11,96% cases <15 years old. Other results from Southern Vietnam or Thailand show more than 80% DHF cases is in children <15 years old. In 276 DHF cases, DHF due to DENV- 4 is highest, lowest is DENV- 3 (18.12%). Fever and headache is most common symptoms (> 90%). Comparing to other study of Hospital 103 authors, headache is seen in 88.7% DHF patients. Most DHF patients in Dak Lak General hospital belongs to DHF with and without warning signs, no severe DHF is recorded. Comparing study on 294 patients with DENV-2 and 91 patients with DENV-3 receiving treatment in 2 hospitals in Taiwan from 1998 to 2007 shows: DENV-3 causes more severe clinical cases, myalgia, rash, higher fever than DENV-2.DENV-2 infection is usually secondary, meanwhile DENV-3 is usually primary infection. Mean hospitalized days is about 4 in all groups, recovery rate is over 97%, no fatality. In this study, even though number of cases in <15 years old is very low but clinical manifestation is much higher than 15 years old, 15.15% and 2.47% accordingly. Rate of white blood cell count reduction below 4000/mm 3 is 66-71,43%. Rate of patients with hematocrit elevation above 42% is from %. Elevated hematocrit is a valuable diagnostic parameter recognized by WHO. This results is suitable to previous studies of Vietnamese and overseas authors. Highest platelet reduction rate in DHF patients is in DENV-2 infection, lowest in DENV-4 patients with 57% but the difference is statistically insignificant. This results is compatible to study results of to previous studies of Vietnamese and overseas authors, in early stage of DHF, white blood cell

24 21 count is initially reduced then recovered, rate of patient with elevated hematocrit more than 42% is 48%, reduced platelet is seen in all severe DHF cases. Based on WHO classification, we encountered mostly DHF without warning signs, no severe DHF case is recorded. In Thailand, DHF without warning signs rate is 40.9%; DHF with warning signs rate is 43%; severe DHF is 10,8% Molecular biology characteristics of dengue virus isolates from dengue hemorrhagic fever patients in Dak Lak province, The C-PrM region is selected to design several primers to type dengue virus by Multiplex RT-PCR, is the target region in moleculer epidemiological analysis of dengue virus. Genotyping dengue virus isolated from patients in Dak Lak is performed after analyzing a nucleotide sequence 511bp long in C-PrM region. DHF Endemic in Dak Lak is caused by 4 types of dengue virus but dominant role is shifted yearly. In period, DENV-1 và DENV-2 is more prominent. It is the evidence for the need of a preventive strategy to control continuous emergences of DHF in Dak Lak as well as Vietnam. Moreover, transmission vector surveillance in several Central highland provinces, reports Ae.aegypti presence at surveyed areas and isolated DENV-2 from this species in This study identifies types of dengue virus circulate in Dak Lak, and help tracing the origin of circulating genotypes in this province, and provide more data for molecular epidemiology in Central highland region and Vietnam. This study found all DENV-2 strains in Dak Lak do not belong to 5 known genotypes identified previously, and form a new group with 93.1% difference with other genotypes. This results prove the need of further study to sequence entire genome of these strains to

25 22 identify new genotype/subgenotype in Vietnam. Besides, appropriate software is required to research spot mutation of amino acid and biological clock to predict evolution trend and virulence of DENV-2 isolated in Dak Lak. DENV-2 is originated from South East Asia and India subcontinent used to be the cause of DHF with dengue shock syndrome in primary infection. All DENV-3 strains isolated in the study belongs to genotype II and closely related to genetics of DENV-3 strains circulate in Vietnam. For DENV-4, the similarity between DENV-4 strains, genotype I-A group 2 in Dak Lak is 98.9%; the similarity between 2 groups is 94.7% ( ). The strains are most closely related to DENV-4 group 2 in Singapore-2011 with 9.6% nucleotide difference. Analysis results shows DENV-4 strains in Vietnam could be domestic strains, circulate and evolve independently with other strains in genotype GI-A. DENV-4 strain isolated in Gia Lai province, 2010, is closely related to other strains in the region (Thailand and Cambodia), this could be an invading virus strain from border of Gia Lai province and Cambodia. All DENV-4 strains in Dak Lak belong to GI-A group. In phylogenetic tree, there is a strain isolated in Gia Lai different to Dak Lak strains, this could be an invading strain coming from Le Thanh international border gate, between Gia Lai and Cambodia (isolated in 2010), and a group could be domestic strains evolved from a strain isolated in Dak Lak in 2010 and other province in Central highland from , suitable to study results of DENV-4 isolated Southern patients

26 23 Conclusion 1. Epidemiological characteristics and contributing factors of dengue hemorrhagic fever in Dak lak province -Epidemiological characteristics +DHF is commonly contracted by patient cohort 15 years old (96,93%), the chance of acquiring DHF of 1-9 years old cohort is very low (<1%). +DHF occurs all year round but most often in May-June (56,91%). +Pre-exposure to virus 2 weeks before disease onset has 1.81 times more risk of DHF than patients with no pre-exposure - Contributing factor to dengue hemorrhagic fever +Patient without well built housing has 1.28 times more risk of DHF than patient with proper housing, impoverished family has 2.11 times more risk of DHF than non impoverished family. +Non users of mosquitos incense and chemical spray has respectively 2.11 and 1.93 times more risk of DHF than users. +Non users of common anti-mosquitos practice have 1.18 times more risk of DHF than user. 2. Clinical characteristics of dengue hemorrhagic fever and association to types of dengue virus in Dak Lak province, Over 90% (94.9%) DHF cases infected with different strains are uncomplicated DHF, most common symptoms are fever and headache (>90%). - Subcutaneous hemorrhage and hemorrhage in gum/teeth/conjunctiva is most seen in DHF type 3 patients (50% and 33,33%), less common in DHF type 4 and in patients <15 years old and 15 years old (22% and 18%). - The platelet count reduction is highest in DHF type 2 patients (76,19%), and lesser in DHF type 4 (57 %).

27 24 - The severity of DHF is higher in patient cohort <15 years old (15,15 %), higher than 15 years old cohort (2,47 %). - More than 97% DHF patients recovered, mean hospitalized time is 4 days and no fatality. 3. Molecular biology of dengue virus isolates from dengue hemorrhagic fever patients in Dak Lak province, nucleotide sequences of C-PrM region were granted accession number in Genbank strains of dengue virus type 1 belongs to genotype 1, similar to strains found in Vietnam; 5 unidentified strains of dengue virus type 2 did not resemble published genotype, possibly belonged to a new genotype/sub-genotype; 7 strains of dengue virus type 3 belongs to genotype 2, in the same circulating type in Vietnam; 13 strains of dengue type 4 in genotype 1 GI-A. Recommendation 1. Continue informing the public on disease prevention No Aedes larvae, no DHF. 2. Raise community awareness about signs of DHF for early disease diagnosis. 3. Complete sequencing the genome of 11 strains of dengue virus type 2 isolated in Dak Lak province in to find proof of a new sub-genotype/genotype.