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清明掃墓, 別來無恙 " 恙蟲病之診療及實例探討台北榮民總醫院感染科王永衛醫師 1 課程大綱恙蟲病 (Scrub typhus) 實例恙蟲病感染流行病學恙蟲病感染的病理機轉恙蟲病的診斷理學檢查及實驗室檢查恙蟲病及不明原因發燒 (Fever cause unknown) 的鑑別診斷恙蟲病感染抗生素治療 2

病史 43 歲家庭主婦過去並無特殊病史住新北市淡水區, 無特殊旅遊史, 主訴一週前有頭痛發燒寒顫肌肉酸痛及食慾不振等, 在診所治療, 打針吃藥一直沒退燒, 三天後出現皮疹, 因為病情未改善, 到台北榮民總醫院急診室進一步檢查及治療急診室血壓 104/64mmHg, 體溫過高達 39.5 C, 心跳每分鐘 102 次進一步詢問病史, 家裡無養寵物, 及近一個月內無出國旅遊史最近因發燒, 胃口不佳倦怠和虛弱發燒和合併寒顫, 有嚴重頭痛及肌肉酸痛情形, 並有些咳嗽流鼻水及喉嚨痛 3 病史理學檢查出現急性病態意識清楚, 血壓 104/64mmHg 偏低, 體溫過高達 39.5 C, 呼吸稍快每分鐘 20 次, 心跳每分鐘 102 心悸皮膚無皮疹及瘀斑發燒前兩週曾到宜蘭控窯, 也經常在家附近 ( 淡水興福竂 ) 爬山在腋下有一個 0.5x0.5 公分大小之傷口 4

實驗室檢查結果檢查項目檢查結果 W.B.C(/cumm) 5300 D/C % R.B.C(X 10 6 /cumm) 3.94 Hemoglobin(gm/dl) 12.5 Ht. (%) 36.7 MCV(fl) 77.0 Platelet (/cumm) 102000 PT/APTT 11.2/33.8 Band Segment Lymphocyte Monocyte Baso - 81.5 16.1 2.1 0 5 實驗室檢查結果檢查項目檢查結果 Glucose(mg/dl) 95 BUN(mg/dl) 8 Creatinie(mg/dl) 0.68 Albumin(g/dl) - NA(meq/l) 134 K(meq/l) 3.8 Total Bilirubin - Direct Bilirubin 0.55 ALP(IU/L) - γgt (IU/L) 23 ALT(GPT) (IU/L) 165 AST(GOP) (IU/L) 119 LDH(IU/L) 323 C.R.P 4.76 6

微生物學檢查結果細菌培養尿液培養及血液培養皆無分離出任何微生物微生物及免疫檢查檢查結果 HBsAg (EIA) positive Anti-HBs negative HBeAg(EIA) negative Anti-HBC IgM negative Anti-HCV negative CMV Ab IgG positive CMV Ab IgM negative Mycoplasma Ab 40x negative Cold Hemagglutin 4x negative Chlamydia pneumoniae IgA positive Chlamydia pneumoniae IgM negative Widal test 20x negative Weil-Felix test 20x negative Ferritin 137.53 TIBC 315 Fe 8 Rheumatoid factor <10 Anti-Ds DNA 4.7(negative) 7 胸部 X 光檢查正常腹部 X 光檢查結果正常腹部超音波結果正常影像學檢查結果 8

9 病史實驗室檢查發現 thrombocytopenia, 而且肝功能不正常, 經仔細詢問病史後, 發現病患發病約 2 週前到宜蘭控窯, 也經常在家附近 ( 淡水興福竂 ) 爬山病史應和此次發病有關聯, 再重新做理學檢查, 於病患的右腋下有一個 0.5X0.5 公分的結痂傷口, 判斷為焦痂綜合病患的臨床的表徵及檢查, 初步診斷為叢林性斑疹傷寒 ( 恙蟲病 ), 抽血送疾病管制局檢驗恙蟲病抗體給予口服 Doxycyclin 100mg 每 12 小時一次, 病患在第三天後逐漸退燒, 臨床症狀頭頸疼痛大為進步, 在口服 7 日 Doxycyclin 後病患完全退燒出院恙蟲病的血清抗體試驗 (IFA) 為陽性, 最後診斷為恙蟲病 10

Rickettsial Diseases 立克次體病 11 Rickettsial Diseases 立克次體病 Rickettsiae 屬絕對細胞內寄生的革蘭氏陰性立克次體科細菌 Usually transmitted by tick, mite, flea, or louse vectors. Except in the case of louse-borne typhus, humans are incidental hosts. 12 行政院衛生署疾病管制局人畜共通傳染病臨床指引 A Clinical Guide to Zoonoses

Rickettsial Diseases 立克次體病依據 16S rrna 序列立克次體亦可明顯分為 3 群, 1. 斑點熱群 (spotted fever group rickettsia;sfg) 立克次體, 為立克次體中依抗原性分為 3 群中最複雜的一群, 種類也最多 2. 斑疹傷寒群 (typhus group;tg) 3. 恙蟲病 ( 又稱叢林型斑疹傷寒 ) 13 行政院衛生署疾病管制局人畜共通傳染病臨床指引 A Clinical Guide to Zoonoses 恙蟲病 (Scrub Typhus) 恙蟲病 (Tsutsugamushi disease,tsutsu 是惡疾之意, 而 mushi 是指恙蟲 ) 又名叢林型斑疹傷寒 Scrub typhus 病原 : 恙蟲立克次體 Orientiatsutsugamushi 病媒 : 恙蟎, 屬於蛛形綱 (Arachnida), 幼螨 (chigger) 微小約 0.2~0.3 mm, 肉眼幾乎看不見台灣主要的恙蟲病媒以地里恙螨 (Leptotrombidiumdeliense) 為主疾病分類屬第四類法定傳染病通報期限於 1 週內進行通報 14

分佈區域 : 恙蟲病三角亞洲及大洋洲流行的感染症易感族群在恙蟲流行地區特別是草叢活動者流行季節 4 月份病例數開始增加流行區域以花蓮縣台東縣南投縣及離島地區 ( 金門縣澎湖縣 ) 病例數較多恙蟲病流行病學特徵 15 Map shows the geographic areas where scrub typhus is endemic (black) 流行病學 Epidemiology 分布於中亞東亞及東南亞 ; 從西伯利亞東南部日本北部至澳洲北部以及新赫布里, 更西邊至巴基斯坦, 甚至海拔 10,000 英呎高度之喜馬拉雅山上都有此病的分布 2007 by Radiological Society of North America Jeong Y J et al. Radiographics 2007;27:161-172 16

Schematic representation of the phylogenetic relationships among major clades of Orientia tsutsugamushi as represented by the DNA sequences of the 56-kDa surface antigen gene. Scrub Typhus: The Geographic Distribution of Phenotypic and Genotypic Variants of Orientia tsutsugamushi 血清型別除了有日本的 Kato 新幾內亞的 Karp 與緬甸的 Gilliam 三種標準株外, 各地還有 Kuroki Kawasaki 等超過三十種不同血清型之分布 Kelly D J et al. Clin Infect Dis. 2009;48:S203-S230 2009 by the Infectious Diseases Society of America 17 Phylogenetic tree of O. tsutsugamushi based on the nucleotide sequences of the 56-kDa cell surface antigen gene. KARP-related, Gilliam, TA763, JG, JG-v, and KATO-related in Taiwan. Lin P et al. Appl. Environ. Microbiol. 2011; doi:10.1128/aem.02796-10 18

Taiwan showing O. tsutsugamushi strains isolated in scrub typhus patient. 2010~14 人常在由立克次體蟲媒以及適當的囓齒類動物共同存在之流行小島 (typhus island) 遭致感染人的感染機會和職業以及於感染地區的活動有關, 易感受者如軍隊進入地方性流行區域時, 約有 20 ~ 50% 的人會在數週至數月內發病台灣的恙蟲病病例近年有增加的趨勢, 各縣市均有病例報告, 病例主要分布在金門縣澎湖縣台東縣花蓮縣南投縣 19 Schema shows the life cycle of a Leptotrombidium mite. 2007 by Radiological Society of North America Jeong Y J et al. Radiographics 2007;27:161 172 20

傳染窩感染立克次體的恙蟎, 會經由垂直傳染而代傳立克次體, 並於其每個發育期中, 如卵幼蟲若蟲成蟲各階段均保有立克次體, 成為永久性感染動物宿主為囓齒類哺乳類 ( 羊猪狗貓 ) 鳥類( 鳥雞 ) 等, 其中又以囓齒類為主 Jerome Goddard Infect Med 17(4):236-239, 2000. 21 恙蟲病 ( Scrub typhus) 感染過程傳染方式被具傳染性的恙螨叮咬, 經由其唾液使宿主感染立克次體潛伏期 1-2 週, 通常為 9-12 天可傳染期不會直接由人傳染給人感染性及抵抗力受感染後對同一型別的立克次體有長期的保護力, 但對不同型別此保護力僅短暫存在對於生活在流行地區的人, 有可能第二次甚至第三次受感染, 不過通常症狀較輕微 22

Clinical manifestations and complications of patients with scrub typhus Fatal case Non-fatal case Clinical manifestations (%) (N = 18) (N = 279) P Fever 13 (72.2) 254 (91.0) 0.025 Chills 11 (61.1) 240 (86.0) 0.011 Sore throat 5 (27.8) 41 (14.7) 0.171 Headache 8 (44.4) 178 (63.8) 0.131 Myalgia 7 (38.9) 177 (63.4) 0.046 Arthralgia 0 (0.0) 12 (4.3) 1 Cough 7 (38.9) 104 (37.3) 1 Dyspnea 13 (72.2) 73 (26.2) < 0.001 Nausea/vomiting 6 (33.3) 100 (35.8) 1 Abdominal pain 6 (33.3) 88 (31.5) 1 Skin rash 5 (27.8) 182 (65.2) 0.002 Eschar 2 (11.1) 196 (70.3) < 0.001 Lymphadenopathy 1 (5.6) 25 (9.0) 1 Conjunctivitis 1 (5.6) 31 (11.1) 0.704 23 Am. J. Trop. Med. Hyg., 81(3), 2009, pp. 484 488 Clinical manifestations and complications of patients with scrub typhus Fatal case Non-fatal case Complications (%) (N = 18) (N = 279) P Central nervous system 14 (77.8) 33 (11.8) < 0.001 Confusion 8 (44.5) 29 (10.4) < 0.001 Seizure 2 (11.1) 2 (0.7) 0.019 Coma 4 (22.2) 2 (0.7) < 0.001 Respiratory system 12 (66.7) 75 (26.9) 0.001 Pulmonary edema 1 (5.6) 19 (6.8) 1 Interstitial pneumonia 2 (11.1) 11 (3.9) 0.182 Pleural effusion 3 (16.7) 40 (14.3) 0.732 Ventilator use 6 (33.3) 5 (1.8) < 0.001 Cardiovascular system 3 (16.8) 15 (5.4) 0.086 Pericardial effusion 1 (5.6) 4 (1.4) 0.27 Atrial fibrillation (new onset) 1 (5.6) 11 (4.0) 0.535 IHD (new onset) 1 (5.6) 0 (0.0) 0.061 Gastrointestinal system 0 (0.0%) 21 (7.5%) 0.256 Gastric ulcer 0 (0.0%) 18 (7.5%) 0.612 Pancreatitis 0 (0.0%) 1 (7.5%) 1 Upper gastrointestinal bleeding 0 (0.0%) 2 (7.5%) 1 Acute renal failure 13 (72.2) 54 (19.4) < 0.001 24

恙蟲病 ( Scrub typhus) 臨床表徵發燒 : 猝發性, 持續性高燒伴隨頭痛背痛惡寒盜汗淋巴結腫大等症狀焦痂 (eschar): 約有 50-80 %患者可在叮咬處發現潰瘍性焦痂, 大都為無痛性出疹 : 發燒 4-5 天起皮膚出現紅色斑狀丘疹, 由軀幹上部擴至四肢不出現於臉手掌及腳掌, 第 9-10 病日消退 25 恙蟲病 : 叮咬部位 26

恙蟲病 ( Scrub typhus) 焦痂 27 Figure: Eschar from the bite of a Leptotrombidium mite. The basic pathology of scrub typhus is a vasculitis secondary to direct endothelial injury, in multiple organs, by Orientia tsutsugamushi Jeong Y J et al. Radiographics 2007;27:161-172 28

Scrub Typhus: Radiological and Clinical Findings Findings No. of Lung zone Patients (%) Bilateral Unilateral Upper Lower Even Negative 21(28) Pulmonary abnormalities 53(71) 44 9 2 39 12 Reticulonodular opacities 30(40) 28 2 1 14 15 Ground-glass opacity 19(25) 17 2 0 11 8 Patchy consolidation 14(19) 6 8 0 12 2 Confluent consolidation 5(7) 5 0 0 3 2 Septal lines 27(36) 15 12 0 27 0 Subsegmental collapse 14(19) 4 10 0 14 0 Coarse nodules 6(8) 4 2 0 6 0 Hilar enlargement 19(25) 3 16* Pleural effusion 9(12) 7 2 Cardiomegaly 10(13) 29 Clinical Radiology (2000) 55, 140 144 Figure: Scrub typhus in a 68-year-old man with previously diagnosed usual interstitial pneumonia. Jeong Y J et al. Radiographics 2007;27:161-172 2007 by Radiological Society of North America 30

Figure 10a. Acute respiratory distress syndrome in a 47-year-old woman with scrub typhus. Jeong Y J et al. Radiographics 2007;27:161-172 2007 by Radiological Society of North America 31 Pediatric Scrub Typhus in Eastern Taiwan Scrub typhus should be considered in children with fever and hepatic dysfunction, particularly in those with a history of environmental exposure in an endemic area for scrub typhus. Eschar offers an important diagnostic clue, but not for all cases. Children with scrub typhus may develop serious complications and may even die if appropriate treatment is not given. Doxycycline is an effective antibiotic for pediatric scrub typhus in Taiwan. 32 Pediatr Neonatol 2009;50(3):96 101

Complications of Scrub Typhus Complications No. of patients (%) Pneumonia 12(36) ARDS 5(15) ARF 3(9) Myocarditis 1(3) Septic shock 1(3) Data from Clinical Radiology (2000) 55, 140 144 33 Journal of the American Academy of Nurse Practitioners 24 (2012) 160 165 An 81-year-old woman with scrub typhus. Initially, bilateral extensive consolidation is visible mainly in the lower zones. Cardiomegaly, pulmonary oedema and pleural effusions are also present. 34 Clinical Radiology (2000) 55, 140 144

Follow-up chest radiograph taken 10 days later, after treatment with doxycycline, shows clearing of both lower lungs and pleural effusions and a decrease in cardiac size. 35 Clinical Radiology (2000) 55, 140 144 Scrub Typhus-Associated CNS Complications Meningoencephalitis 1,5 Acute Disseminated Encephalomyelitis 2 Scrub typhus complicated by intracranial hemorrhage - A Case report. 3 Acute transverse myelitis 4 1. Ann Indian Acad Neurol 2012;15:141-4. 2. Acta Neurol Taiwan 2006;15:251-254. 3. Tzu Chi Med J 2005;17:111-4. 4. Diagn Microbiol Infect Dis. 2008;60(2):237-9. 5. Arch Neurol 2000;57:1770-2 36

Differentiate Diagnosis Coinfection with Leptospirosis and Scrub Typhus in Taiwanese Patients 1 The clinical differences between dengue and scrub typhus with acute respiratory failure in southern Taiwan 2 Acute Q fever and scrub typhus, southern Taiwan 3 Clinical Manifestations and Complications of Rickettsiosis in Southern Taiwan 4 1. Acute Q fever 2. Scrub typhus 3. Murine typhus 1. Am J Trop Med Hyg. 2007; 77(3): 525 527. 2. Infection 2012, DOI: 10.1007/s15010-011-0239-9 3. Emerg Infect Dis. 2009; 15(10): 1659 1661. 4. J Formos Med Assoc. 2002;101:385 92. 37 Comparative initial symptoms/signs between adult dengue and scrub typhus patients with acute respiratory failure Initial symptoms/signs Dengue (n = 18) Scrub typhus (n = 8) P-value Fever 17 (94.4%) 8 (100%) 1.000 Eschar 0 6 (62.5%) 0.001 Petechiae 8 (44.4%) 3 (37.5%) 1.000 Arthralgia 8 (44.4%) 1 (12.5%) 0.190 Myalgia 8 (44.4%) 1 (12.5%) 0.190 Headache 8 (44.4%) 2 (25%) 0.420 Gum bleeding 3 (16.7%) 1 (12.5%) 1.000 Chest pain 5 (27.8%) 2 (25%) 1.000 Cough 10 (55.6%) 8 (100%) 0.031 Dyspnea 10 (55.6%) 7 (41.2%) 0.190 Hemoptysis 2 (11.1%) 1 (12.5%) 1.000 Abdomen pain 11 (61.1%) 3 (37.5%) 0.401 Vomiting 7 (38.9%) 1 (12.5%) 0.360 Tarry stool 6 (33.3%) 2 (25%) 1.000 38 INFECTION 2012, DOI: 10.1007/s15010-011-0239-9

Comparative initial laboratory data between adult dengue and scrub typhus patients with ARF Dengue (n = sample size) Scrub typhus (n = sample size) P-value WBC ( 10 3 /μl) 7.40 ± 5.74(n = 18) 11.84 ± 4.95 (n = 8) 0.026 Hematocrit (%) 33.19 ± 8.64 (n = 18) 33.13 ± 5.54 (n = 8) 0.935 Platelet ( 10 9 /L) 42.2 ± 33.9 (n = 18) 104.1 ± 93.3 (n = 8) 0.008 PT (s) 12.82 ± 1.36 (n = 18) 10.74 ± 0.98 (n = 5) 0.007 APTT (s) 50.81 ± 10.08 (n = 18) 37.44 ± 4.06 (n = 5) 0.002 AST (U/L) 709.2 ± 1,381.4 (n = 18) 153.9 ± 68.5 (n = 8) 0.129 ALT (U/L) 543.8 ± 1,029.6 (n = 18) 153.5 ± 78.1 (n = 8) 0.461 ALP 115.0 ± 29.4 (n = 9) 178.8 ± 100.9 (n = 8) 0.114 Total bilirubin (mg/dl) 2.68 ± 1.29 (n = 12) 3.72 ± 3.67 (n = 8) 0.473 BUN (mg/dl) 64.6 ± 43.2 (n = 14) 20.9 ± 9.1 (n = 8) <0.001 Creatinine (mg/dl) 3.77 ± 3.37 (n = 17) 1.05 ± 0.37 (n = 8) <0.001 Albumin (g/dl) 2.47 ± 0.53 (n = 18) 2.80 ± 0.92 (n = 8) 0.531 39 INFECTION 2012, DOI: 10.1007/s15010-011-0239-9 Symptoms and Signs in 51 Patients with Rickettsial Infections Acute Q fever (n = 28) Scrub typhus (n = 16) Murine typhus (n = 7) Symptoms Fever 28 16 6 Chills 22 13 6 Cough 12 7 5 Sore throat 3 1 5 Headache 14 10 5 Diarrhea 6 1 1 Abdominal discomfort/pain 13 8 2 Nausea/vomiting 5 6 1 Arthralgia/myalgia 8 10 4 Signs Maculopapular rash 0 4 2 Eschar 0 6 0 Regional lymphadenopathy 0 6 0 Relative bradycardia 20 (n = 21) 11 (n = 15) 2 (n = 6) Hepatomegaly* 3 0 0 Splenomegaly* 0 3 1 40 J Formos Med Assoc. 2002;101:385 92.

Laboratory and Image Findings In 51 Patients with Rickettsial Infections Acute Q fever (n = 28) Scrub typhus (n = 16) Murine typhus (n = 7) Elevated aminotransferases 28 16 7 Hyperbilirubinemia 5 (n = 25) 6 2 Elevated C-reactive protein 27 16 7 Microscopic hematuria 4 0 0 Leukocytosis 2 6 3 Thrombocytopenia 2 7 4 Abnormal chest roentgenogram 3 (n = 25) 10 2 (n = 5) Hepatomegaly* 8 (n = 27) 1 (n = 13) 1 Splenomegaly* 8 (n = 27) 8 (n = 13) 3 *Detected by ultrasonography or computerized tomography. J Formos Med Assoc. 2002;101:385 92. 41 Diagnosis of Scrub Typhus 檢驗條件符合下列檢驗結果之任一項者, 定義為檢驗結果陽性 : 1. ( 一 ) 臨床檢體 ( 血液或皮膚傷口 ( 焦痂 )) 分離並鑑定出恙蟲病立克次體 (Orientia tsutsugamushi) 2. ( 二 ) 臨床檢體分子生物學核酸檢測陽性以間接免疫螢光染色法 (Indirect Immunofluorescene Assay,IFA) 檢測急性期 ( 或初次採檢 ) 血清,IgM 抗體 1:80 且 IgG 抗體 1:320 3. ( 三 ) 以間接免疫螢光染色法, 檢測成對 ( 恢復期及急性期 ) 血清, 恙蟲病立克次體特異性 IgM 或 IgG 抗體 ( 二者任一 ) 有陽轉或 4 倍上升 42

Summary of antigenic strains described by the selected studies, listed by the country where the indirect immunofluorescence assay was used. Blacksell S D et al. Clin Infect Dis. 2007;44:391 401 2007 Infectious Diseases Society of America 43 檢體採檢送驗事項 44

Drugs Chloramphenicol VS tetracycline Doxycycline 200 mg SD VS tetracycline 100 mg QID 7 days Doxycycline 100 mg BID 3 days VS tetracycline 100 mg QID 7 days Azithromycin 500 mg SD VS doxycycline 200 mg Daily 7 days Azithromycin 500 mg 3 days VS doxycycline 200 mg 7 days Telithromycin 800 mg daily 5 days VS doxycycline 200 mg 5 days Rifampicin 600 mg/900 mg VS doxycycline 200 mg 7 days Levofloxacin VS tetracyclines Summary of key treatment studies in scrub typhus Author/ Year Sheehyet al. 1973 Brownet al. 1978 Songet al. 1995 Kim et al. 2004 Phimdaet al. 2007 Kim et al. 2007 Wattet al. 2000 Tsaiet al. 2010 CT RCT RCT RCT RCT Type of study Quasi-RCT RCT Retrospective analysis Key finding Tetracycline superior to chloramphenicol doxycycline as effective as tetracycline doxycycline as effective as tetracycline Azithromycin equally effective as doxycycline. Defervescence slightly longer with doxycycline. Less gastrointestinal adverse effects with azithromycin Azithromycin 3-day equally effective as doxycycline. Defervescence longer with azithromycin. Less gastrointestinal adverse effects with azithromycin Teilithromycin as effective as doxycycline Rifampicin superior to doxycycline faster defervescence, fewer relapses (trial was in a geographical locality where doxycycline resistance was known to occur) Levofloxacin as effective as tetracyclines. Longer time to defervescence with levofloxacin. Higher mortality with levofloxacin in severe disease 45 RCT:Randomized controlled trial Trop Doct January 2011 vol. 41 no. 11 4 Intravenous Minocycline Versus Oral Doxycycline for the Treatment of Noncomplicated Scrub Typhus Journal of Microbiology, Immunology and Infection. 2011; 44:33-38 46

Levofloxacin vs Tetracycline for the Treatment of Scrub Typhus Kaplan-Meier curve of the time to defervescence in patients initially treated with levofloxacin and tetracycline antibiotics ( p = 0.0002). International Journal of Infectious Diseases;2010:14, e62 e67. 47 Treatment of O. tsutsugamushi Infection Tetracycline, doxycycline, or chloramphenicol is used to treat scrub typhus. Minocin 200mg/ IV/ st., 100mg/ IV/ q12h Doxycycline 100mg/ P.O./ q12h, for 14 days Chloramphenicol-resistant & doxycycline-resistant strains of O tsutsugamushi occur in the areas of northern Thailand. # Oral azithromycin was administered in a 500-mg dose on the first day, followed by 250 mg daily on days 2 to 5.* in children and pregnant women #Lancet 1996 Jul 13;348(9020):86-9 *Antimicrob. 48 Agents Chemother. 1999, 43: 2817-2818

治療及預後治療方式抗生素療法 : 四環黴素 (tetracycline) 類如 doxycycline minocycline 等氯黴素 (chloramphenicol) 預後未經治療, 死亡率可達百分之六十經治療後死亡率小於百分之五 49 References, Web sites and Further readings 1. Center for Disease Control Taiwan, ROC, 台灣疾病管制局 http://www.cdc.gov.tw/index.asp 2. Centers for Disease Control and Prevention, U.S.A, http://www.cdc.gov/ 3. World Health Organization, http://www.who.int/en/ 4. Mandell, Gerald L. et al. Principles and Practice of Infectious Disease. Sixth Edition Churchill Livingstone. October 22, 2004. pp. 1874. 5. Timmreck, Thomas C. An Introduction to Epidemiology. 2nd ed. 1998 Jones & Bartlett Publishers, Inc. Boston, United States of America. 6. CDC, Principles of Epidemiology. 3nd ed. 2006. Atlanta, GA: Centers for Disease Control and Prevention. 7. Wolfgang, A & Iris, P. Handbook of Epidemiology. 1st ed. 2005. Springer-Verlag Berlin Heidelberg, Germany. 8. Fred, B.; Pauline, van den D. ; Jianhong, W. Mathematical Epidemiology. 2008. Springer-Verlag Berlin Heidelberg, Germany. 9. Richard D.T. Farmer, Ross Lawrenson. Lecture notes on epidemiology and public health medicine. 5th ed. 2004.Blackwell Publishing, Inc., Massachusetts, USA. 10. Stefan Ma. Mathematical Understanding of Infectious Disease Dynamics. 2008 World Scientific Publishing. 11. Bonita R., R. Beaglehole, T. Kjellstrom. Basic epidemiology. 2nd ed. World Health Organization, 2006. 50