Serodiagnosis of viral infection

Presentation on theme: "Serodiagnosis of viral infection"— Presentation transcript:

1 Serodiagnosis of viral infection

2 How to identify an unknown virus infection?
Virus isolation and identification (sometime danger and not easy) PCR or RT-PCR Antibody screening: “Blind” immunoscreening of cDNA expression libraries

3 Discovry of Hepatitis C Virus (HCV)

4 Serological expression cloning (SEREX) approach to defining tumor or virus antigens

5 Significance of antibody against virus
Protective, such as anti-HBsAb Infected, such as anti-HCV, anti-HIV Why? Home work: please search medline, find out the original papers and describe how HCV and HIV were identified to be associated with post transfusion hepatitis and AIDS, respectively.

6 Outline Epstein-Barr Virus (EBV) Hepatitis virus (HAV, HBV, HCV)
Human Immunodeficiency Virus (HIV)

7 Epstein-Barr Virus (EBV)
1932年Paul跟Bunnell發現infectious mononucleosis (IM)患者的heterophil antibody效價顯著增高 1964年Epstein由Burkitt’s Lymphoma中發現病毒,便取名為Epstein-Barr Virus(EBV)

8 Epidemiology 世界各地分布極廣,經常感染兒童,尤其是落後地區兒童血清EBV抗體陽性反應高達80%
兒童感染EBV通常無臨床症狀,僅出現fever或輕微上呼吸道症狀 青少年或成人感染則易出現infectious mononucleosis (IM)為一種急性或亞急性.良性.具傳染性之淋巴球增生疾病,好發於年輕人 主要傳播途徑-saliva 口腔上皮細胞有EBV receptor--CD21(又稱為CR2)

9 EBV病毒特性 屬於herpes virus 病毒大小150-200nm Double strand DNA
有capsid也有envelope 病毒複製時最早出現抗原為EBNA、EMA，之後出現兩種EA 最後出現VCA、LMA

10 EBV

11 Specific antibody responses in IM
Anti-VCA IgM用來診斷IM具有良好特異性 Anti-EBNA則可視為曾經感染EBV的指標

12 EBV-Associated Tumors
Burkitt’s lymphoma(BL) ：散佈世界各地，但以非洲瘧疾流行區域最多 好發於3-15歲兒童，6-7歲發生率最高 100％具有對EBV的抗體，其中以anti-VCA比一般對照血清的平均值高8-10倍

13 鼻咽癌NPC 為一種惡性鱗狀上皮細胞腫瘤 anti-VCA IgA 比一般對照血清平均值高10倍 anti-EA/D也會升高EA/R則否
anti-VCA和anti-EA/D兩者與腫瘤體積有關，能作為預後的指標

14 Laboratory diagnosis 發病2-3週間肝臟酵素會上升；30％患者膽紅素會上升3-8mg/dL 血清檢查分兩大類：
1.specific antibody測定 2.heterophil antibody測定 小於4歲兒童有70-80 ％嗜異型抗體呈陰性反應，故幼童檢查必須測定專一性抗體 IM患者有85％會呈現嗜異型抗體positive，若為negative則可能為CMV或其他如rubella、toxoplasma、adenovirus所引起

15 Hematologic examination
IM患者有3％為coomb’s test positive 淋巴球總數比常人多(≧50％) ，主要為atypical lymphocyte增加(≧10％) EBV只感染B淋巴球 ，但是B、T淋巴球皆會因EBV感染而變成atypical lymphocyte (T淋巴球是因為病毒抗原或B淋巴球抗原而產生的免疫反應)

16 Serologic test of EBV-specific antibodies
檢anti-VCA或anti-EA可採用間接免疫螢光分析法，測定anti-EBNA則市採用抗補體間接免疫螢光法 P3HR-1可用來測定anti-VCA IgG、IgA、IgM Raji細胞株用於EBNA的測定 EA測定必須將病毒細胞株以sodium butyrate、halogenated pyrimidines或腫瘤促進因子處理，使EBV表現EA抗原

17 EBV感染之血清反應 － ＋ ±或－ 判讀 抗體種類 Anti-VCA IgM Anti-VCA IgG Anti-EA Anti-EBNA
PB 無感染 － 急性感染 ＋ ±或－ 最近曾感染 過去曾感染 過去曾感染，可能再度活化

18 heterophile antibody IM的heterophile antibody又稱為Paul-Bunnell antibody
其他抗體 (1)serum sickness antibodies：注射過馬血 清會出現的heterophil antibody (2)forssman antibody：天竺鼠腎臟物質免疫動物所引出的抗體

19 heterophile antibody被不同細胞抗原所吸附的情形
抗體來源 綿羊RBC 馬RBC 牛RBC 天築鼠腎臟 IM ＋ － Serum sickness Forssman-indused

20 各種 heterophile Ab TEST Paul-Bunnell test Davidsohn-differetial test
Monospot test

21 Paul-Bunnell presumptive test

22 Absorbed Heterophile Test (Davidsohn Differential)

23 EB capsid Ab (IgM) 目的 原理 方法 應用 測定血清中EBV VCA抗原之IgM抗體，為EBV正在感染的指標
IFA- 抗原: 分泌EBV的Burkitt‘s lymphima之HR1 cell Ab 1: serum中任何EBV VCA之IgM Ab 2: FITC conjugated anti-human IgM 方法 1. Serum以RF吸收劑做1:10稀釋，讓RF吸收劑作用15mins 2. 滴片，37 ℃放置90mins 3. PBS wash，泡在PBS震盪5mins，將slide吹乾 4. 加conjugate， 37 ℃反應30mins 5. 同步驟3. 6. 封片，螢光顯微鏡觀察 應用 EBV VCA IgM代表急性感染的抗體，不須依賴傳染性單核球增 多症狀（IM），即可判斷EBV感染。因幼兒感染EBV通常不會 出現明顯的症狀及嗜異性抗體。 23

24 EB capsid Ab (IgG) 目的 原理 方法 應用 測定血清中EBV VCA抗原之IgG抗體，為EBV感染的指標
IFA- 抗原: 分泌EBV的Burkitt‘s lymphima之HR1 cell Ab 1: serum中任何EBV VCA之IgG Ab 2: FITC conjugated anti-human IgG 方法 1. Serum作序列稀釋 2. 滴片，室溫放置30mins 3. PBS wash，泡在PBS震盪5mins，將slide吹乾 4. 加conjugate， 室溫反應30mins 5. 同步驟3. 6. 封片，螢光顯微鏡觀察 應用 當傳染性單核球增多症發病後，VCA IgG效價會保持在高效價達 數月之久，而後逐漸下降，並持續終生存在低效價。EBV高效 價抗體常見於布氏淋巴瘤（Burkitt’s lymphoma）、鼻咽癌及免 疫控制的病患。 24

25 EB capsid Ab (IgA) 目的 原理 方法 應用 測定血清中EBV VCA抗原之IgM抗體，為EBV正在感染的指標
IFA- 抗原: 分泌EBV的Burkitt‘s lymphima之HR1 cell Ab 1: serum中任何EBV VCA之IgA Ab 2: FITC conjugated anti-human IgA 方法 1. Serum與RF吸收劑作用15mins，再作序列稀釋 2. 滴片，37 ℃放置90mins 3. PBS wash，泡在PBS震盪5mins，將slide吹乾 4. 加conjugate， 37 ℃反應30mins 5. 同步驟3. 6. 封片，螢光顯微鏡觀察 應用 研究報告指出：大多數的NPC患者腫瘤增大時 EBV VCA IgA抗 體也會增加，此抗體可當作預後的指標。 25

26 Hepatitis

27 Outline The causative agent of hepatitis
Clinical manifestation/Pathology Immunology of hepatitis infections Laboratory diagnosis Immunization

28 Hepatitis A Virus Piconavirus Enterovirus 72
Known as infectious/epidemic hepatitis Naked icosahedral capsid, positive-sense, single-stranded RNA

29 Pathology Fecal-oral route
Viral is shed in large quantity into the stool Can’t initiate chronic infection Not associated with hepatic cancer Antibody protection against reinfection is lifelong

30 Clinical manifestation
Flu-like: fatigue, nausea, fever Jaundice (icterus):Yellow eyes and skin abdominal pain, loss of appetite, diarrhea average incubation period : 28 days (range: 15－50 days)

31

32 Laboratory diagnosis ALT and AST :20- to 50-fold elevations ;ALT/AST (De Ritis) ratio>1 Clinical syndrome Serology :ELISA or RIA or Competitive immunoassay anti-HAV IgM ＝＞recent infection anti-HAV IgG ＝＞previous infection

33

34 Immunization Inactivated HAV vaccine : Havrix & Vaqta ,only one serotype, infects only humans＝＞help ensure the success of immunization program Prophylaxis with serum immunoglobulin :80 to 90% effective in preventing clinical syndrome

35 Recommended dosages and schedules of hepatitis A vaccines
Age group Dose Volume # Doses Schedule Havrix (Glaxo-SmithKline) 2–18 years 720 El.U.* 0.5 ml 2 0, 6–12 mos. 19 years and older 1440 El.U.* 1.0 ml Vaqta (Merck & Co.) 25 U** 0, 6–18 mos. 50 U** *El.U. = Elisa Units **U = Units

36 Hepatitis B Virus Hepadnavirus Known as serum hepatitis
Enveloped DNA virus, circular, partly double-stranded

37 Pathology Transmission Acute or chronic ,symptomatic or asymptomatic
depend on the person’s immune response Replicate in the hepatocytes with minimal cytopathic effect Cell-mediated immunity and inflammation are responsible for causing the symptoms and resolution of the HBV infection by eliminating the infected hepaocytes

38

39 Clinical manifestation
Flu-like: fatigue, nausea, fever Jaundice (icterus):Yellow eyes and skin abdominal pain, loss of appetite, diarrhea average incubation period : (45－160 days)

40

41

42 Laboratory diagnosis A battery of serological tests are used for the diagnosis of acute and chronic hepatitis B infection. HBsAg - used as a general marker of infection. HBsAb - used to document recovery and/or immunity to HBV infection. anti-HBc IgM - marker of acute infection.

43 anti-HBc IgG - past or chronic infection.
HBeAg - indicates active replication of virus and therefore infectiveness. Anti-HBe - virus no longer replicating. However, the patient can still be positive for HBsAg which is made by integrated HBV. HBV-DNA - indicates active replication of virus, more accurate than HBeAg especially in cases of escape mutants. Used mainly for monitoring response to therapy.

44

45

46 Tests Results  Interpretation Recommendation HBsAg negative Not Immune - has not been infected, but is still at risk for possible future infection. Needs protection. Get the vaccine. HBsAb (anti-HBs) HBcAb (anti-HBc) Immune - surface antibodies present. You may have been already vaccinated. Or you have recovered from a prior hepatitis B infection. You cannot infect others. The vaccine is not needed. positive negative or positive

47 Tests Results  Interpretation Recommendation HBsAg positive New infection or a Chronic Carrier - positive surface antigen, which means hepatitis B virus is present. You can spread the virus to others. Find a doctor who is knowledgeable about hepatitis B for further evaluation. HBsAb (anti-HBs) negative HBcAb (anti-HBc) negative or positive Unclear. Several different interpretations are possible. You may need to have these tests repeated. *See below. The vaccine may or may not be needed. Find a doctor who is knowledgeable about hepatitis B for further evaluation. HBsAb  (anti-HBs) HBcAb (anti-HBc)*

48

49 Immunization HBV vaccine :
subunit vaccine genetically engineered and is produced by insertion of a plasmid containing S gene for HBsAg into yeast, Saccbaromyces cerevisiae three dose with secondary and third given 1 and 6 months after the first 95% will develop protective antibody

50 Hepacare (formerly, Hepagene)
NAME TYPE VACCINE COMPANY STATUS Hepatitis B Vaccines - Recommended for those at risk and patients with chronic HCV Engerix B Recombinant HBV GlaxoSmithKline, Philadelphia,  PA Market, USA Recombivax HB Merck, West Point, PA Gen Hevac B Aventis Pasteur, Lyons, France Market, Europe Hepacare (formerly, Hepagene) HBV preS1, preS2 PowderJect, Oxford, U.K. Bio-Hep B HBV S, preS1, PreS2 Biotech. Gen. Corp., Iselin, N.J. Market, Israel Hepavax Gene Berna Biotech, Switzerland

51 Interferon-α :chronic HBV infection
Hepatitis B immunoglobulin : particular efficacious within 48 hours of the incident Limousine/Lamivudine : a nucleoside analogue reverse transcriptase inhibitor

52 Hepatitis C Virus Flavivirus
Known as Non-A, non-B-post-transfusion hepatitis Enveloped, positive, single-stranded RNA

53 Pathology Transmission :blood ,semen ,vaginal secretion
Liver damage :caused by cell-mediated immunity Anti-HCV isn’t protective immunity to HCV may not be lifelong

54

55 Clinical manifestation
80% of persons have no signs or symptoms. Flu-like: fatigue, nausea, fever Jaundice (icterus):Yellow eyes and skin abdominal pain, loss of appetite, diarrhea average incubation period : 14－180 days

56

57

58 Laboratory diagnosis Screening test
Anti-HCV antibodies rise after two months and are detectable for several years if the patient is chronically infected.

59 EIA (Enzyme Immunoassay)
Detect anti-HCV Sensitivity is highly dependent on the clinical status acutely infected nonimmunocompromised persons :50-70% chronically infected nonimmunocompromised persons :97-99%

60 Laboratory diagnosis Confirmatory test
NAT (nucleic acid testing ) a. RT-PCR and Branched-chain DNA b. detect the virus as early as 1~2 weeks after exposure c. single positive :confirm HCV infection d. single negative :not confirm that the patient is not viremic or has recovered from HCV

61 RIBA (Recombinant Immunoblot Assay )
a. 4 recombinant viral proteins on nitrocellulose strips b. positive :≧ 2 positive bands c. Indeterminate :1 positive band anti-HCV result can’t be determined Repeat RIBA testing at least 1 month later d. negative :EIA false positive

62

63 Immunization No vaccine No post-exposure prophylaxis
Recombinant interferon-α alone or with ribavirin

64 Other hepatitis virus Hepatitis D (HDV):defective RNA virus need HBV provide envelop, coinfection with HBV or superinfection with HBV. Hepatitis E (HEV): similar to HAV in transmission and clinical course of the disease. In pregnant women about 10% result in fulminant hepatitis, detect by PCR or ELISA. Hepatitis G (HGV): flavivirus-like RNA virus in hepatitis patients, detected by PCR.

65

66 HIV - HIV structure - HIV-1 & HIV-2 - Disease - HIV transmission
- The immunology of HIV infection - Vaccines - Laboratory diagnosis

67 HIV (human immunodeficiency virus)
Sphere-shaped virus Lipid envelope Icosahedral capside Two copies of linear + ssRNA Viral protease, reverse transcriptase, integrase Proviral DNA (provirus) -ssRNA所反轉錄出的病毒DNA gag , pol , env Budding from host cell

68 HIV structure Envelope protein: gp120, gp41 Matrix protein: p17
(p7.p9) gp120 p17 p24 Envelope protein: gp120, gp41 Matrix protein: p17 Nucleocapsid structural protein: p24 Bound on RNA: p7, p9

69 HIV genome gag: p15(p7,p9) , p17 , p24 pol: p31 env:gp120 , gp41

70 Acquired immunodeficiency syndrome (AIDS)
後天免疫缺乏症候群主要由HIV-1感染引起 。 HIV-2也會造成AIDS , 但比例相當低 , 3000萬個case中約600個病人是感染HIV-2 , 主要在西非。 HIV virus 會攻擊CD4 cell , 進而引起免疫低落 , 而併發其它伺機性感染 , i.e. Kaposis sarcoma

71 Transmission of HIV 傳染方式: - Sexual contact (同性.異性)
- Blood or blood product transfusion - Needle sharing by drug users - Transplantation of tissues or organs - Breast feeding (7%) 感染部位: - M cells: rectal mucosa - Dendritic cells (i.e. Langerhans’ cells): female vagina , foreskin epithelia

72 HIV infect host cells 宿主細胞種類:
HIV主要攻擊具有表面有CD4分子的細胞 - CD4+ T cells - Dendritic cells - Macrophages - Monocytes 部分HIV會攻擊不具有CD4分子的細胞 - Astrocytes (星狀細胞) - some epithelial cells - spermatozoa (經由alternative receptor for gp120)

73 M-tropic and T-tropic HIV

74 Course of HIV infection
1. 有明顯的 viremic stage , 且伴隨有類似感冒的症狀 , 約在感染後數週內 , 此時可偵測到病毒顆粒和病毒RNA。 p24 Ag + , virus culture + , PCR + 2. 空窗期(window period) , 此時病毒Ag titer降低至幾乎消失 , 只測得到p24 , 且anti-HIV Ab 也還未出現 。 3. Seroconversion , 此時anti-HIV Ab 出現 , 通常感染後2週~6個月抗體會出現 , Ag下降至測不出 , 難virus culture。 (95%病人會在3~6個月內出現抗體;5%病人在6~18個月時才出現抗體) 4. 臨床潛隱期(outwardly latent phase) , 這個時期平均約10年, CD4+ T cell 數量大減 , 可偵測到virus RNA(100%) , anti-HIV Ab 高 , 血漿中HIV量低 , 淋巴器官中的HIV高。

75 Course of HIV infection
5. 伺機性感染和惡性腫瘤期 由於此時的CD4+ T cell數量已下降到極低 , 全身免疫系統已經被嚴重破壞 , 會造成許多微生物的感染 , ex: Candida , CMV , HSV , Mycobacterium tuberculosis 等的感染 。 惡性腫瘤如: Lymphoma , Kaposi’s sarcoma 為最常見的。 此時周邊血液的HIV病毒量上升 , CD4+ T cell 中的proviral DNA 上升 , anti-HIV Ab 下降。 這一時期可維持數月至數年 , 最後病人死亡。

76

77 HIV infection

78 Vaccines:目前仍在研究階段 Recombinant protein vaccines
-gp120 recombinant protein -AIDSVAX -Phase III clinical tirals -引起anti-HIV Ab反應 -Safe Attenuated viral vaccines -Cellular & humoral immunity -High risk DNA vaccines -Using plasmide -活化特定的B cell , CD4+ T cell , CD8+ T cell - 反應的量不足以確定後來的保護作用有沒有效

79 Laboratory diagnosis:
Serological test 先用免疫反應ELISA(or EIA)測定Ab , 再用western blot test做確定 HIV nucleic acid amplification

80 Serological Test 大部分方法是檢測 Antibody 檢體收集方法
USA Food and Drug Administration(FDA): -Urine Based Antibody Tests -HIV-1 Oral Specimen Collection Device

81 Urine Based Antibody Tests
Calypte HIV-1 Urine EIA Tests Enzyme immunoassay (ELISA) gp160 envelop protein 如果urine檢體結果為positive, 要再使用serum做第二次的免疫分析 優點:尿液中的HIV不大具有感染性 , 對實驗 室人員來說是一種保護。 目前FDA通過可使用尿液做檢體去測HIV-1的western blot kit

82 HIV-1 Oral Specimen Collection Device
採取檢體後(Ab) , 利用 Oral Fluid Vironostika HIV-1 Microelisa System 或 OraSure HIV-1 Western Blot kit 檢測

83 Saliva versus Oral Mucosal Transudate as Clinical Specimens
是早期使用來避免危害性的檢體收集的方法 將口水吐到杯子裡 IgA & 少量的IgG 收集效果沒有OraSure device好(因為主要是檢測IgG)

84 Serological Test Anti-HIV antibody detection Western blot
(1)Indirect enzyme immunoassay for HIV (2)Double antigen sandwich assay to detect anti-HIV antibody (3)Antibody capture assay for HIV Western blot Antigen detection - Amplification assay

85 (1) Indirect enzyme immunoassay for HIV----ELISA

86 (2) Double antigen sandwich assay to detect anti-HIV antibody ----EIA

87 (3)Antibody capture assay for HIV ---ACON

88 Western Blot P WP N I I gp160 gp120 當serological screening test 為positive時 , 使用western blot來確定IgG Ab 是否對HIV Ag具有專一性 +: (1)p31 or p24 (2)gp160 or gp120 p66 p55 p51 gp41 p31 p24 p17 p15

89

90 Antigen detection: Amplification assay
測Ag,所以在無抗體時期有很高的sensitive 可測定: 突發性感染 , 被針頭刺到的病人 , 先天性 HIV感染(因為母親的Ab只會存在幾個月) Antibody tests可以無法提供病人作為anti-viral therapy有效與否的測定 , 因為即使Ag load減低了 , Ab 的 titer 仍會一直維持很高。 Antigen detection tests 可用來監測drug therapy , 但臨床上不夠sensitive , 較常使用 病毒的核酸片段放大。

91 Antigen detection: Viral Load Assay
測定serum or plasma中的HIV particles數量 Standard method to monitor anti-viral therapy 可作為突發性感染或是針刺感染的測定 , 以及新生兒HIV的測定。 (1)the Amplicor HIV-1 Monitor by Roche Diagnostics (2)HIV-1 QT by Organon Teknika of Merieux SA (3)HIV-1 RNA 3.0 (Bayer)

92 HBsAg HBsAb HCV Ab HIV Ab
目的 檢測serum中HBsAg、 HBsAb 、HCV Ab 、HIV Ab的量 原理 Microparticle enzyme immunoassay 方法 1. Analytes bind to microparticles 2. Immune complex binds to glass fiber matrix 3. Conjugate completes the immune complex 4. MUP converts to MU

93 應用 HBsAg HBsAb HCV Ab HIV Ab
HBsAg表面抗原為感染Ｂ型肝炎病毒最早出現的血清病毒標記；急性感染痊癒者，約6個月後產生保護性的表面抗原抗體(HBsAb)。Ｂ型肝炎帶原者未產生HBsAb，其HBsAg會持續存在。 HBsAb HBsAb表面抗原抗體具保護力，表示病人對Ｂ型肝炎病毒已具免疫力，可作為施打Ｂ型肝炎疫苗成效之評估。 HCV Ab HCV抗體陽性表示已感染了C型肝炎病毒，此抗體不具保護力。診斷治療需一併參考臨床表現， C型肝炎為主要之輸血後肝炎，患者易演變為慢性肝炎及肝癌。 HIV Ab 僅為HIV 抗體篩檢，在感染HIV病毒 4-12週後出現，陽性反應須再以western blot確認。注意採檢時機，空窗期無法測得。

94 CMV HSV 1&2 Measles Rubella
目的 檢測CMV、 HSV 1&2 、 Measles、Rubella之IgG或IgM抗體 原理 酵素免疫法(ELISA) Ag Human fibroblast Monkey kidney cells Simian kidney cells 方法 1. 將serum做稀釋(測IgM要與RF吸收劑作用15mins) 2. Well加入serum，37 ℃放置60mins 3. Wash 4次，加conjugate 4. Wash 4次，加呈色受質溶液 5. 室溫放置30mins，加入終止溶液 6. 1小時內判讀

95 應用 CMV HSV 1&2 1. CMV感染後會終身存在體內，其IgG抗體也終身存在 。篩檢此抗體可減低血清檢驗陰性的受贈者在輸血或
移殖後，感染來自抗體陽性的捐贈者所轉移的病毒。 2. IgM抗體證明最近曾有CMV感染(初次感染或病毒再活 化)，可持續12-16週，或依病情而延續表現長達八個月 以上，但並非所有CMV IgM陽性的病毒感染者都有臨 床上的症狀。 HSV 1&2 1. 不是診斷的重要依據，HSV IgG主要是偵測病人體內 HSV的免疫狀況。 2. 近期感染人類單純皰疹病毒(HSV)時，會出現此IgM抗 體，提供診斷治療可減低HSV感染導致腦炎的致死 率。

96 應用 Measles Rubella 1. 麻疹感染後其抗體會終身存在，IgG抗體主要是偵測 病人體內的免疫狀況，因抗體具有保護性，也可作為
施打疫苗成效之評估。 2. 一般出疹後IgM 1-2週才會出現，30-60天後消失。此 IgM抗體陽性表示最近曾感染麻疹病毒。 Rubella 1. 感染出疹後4天，其 IgM抗體會出現，4-6週後消失， IgM抗體陽性表示最近感染德國麻疹病毒。 2. Rubella IgG主要是偵測病人體內德國麻疹的免疫狀況 ，德國麻疹的IgG抗體具有保護性。