MICROBIOLOGY PPT LECTURE NOTES | Karya Tulis Ilmiah

1

2

Category of Sample

• Blood, Urine, Stool, nasal washing, nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab),

3

Laboratory Diagnosis

• Microscopy Identification

• Virus isolation and identification

• Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material

4

Microscopy Identification

• Light microscopy

• Fluorescent microscopy • Electron microscopy

5

Light microscopy

• Characteristic CPE • Inclusion Bodies

6 • Cell death

Cell rounding Degeneration Aggregation

Loss of attachments to substrate

• Characteristic histological changes:inclusion bodies in the nucleus or cytoplasm, margination of chromatin

• Syncytia: multinucleated giant cells caused by virus-induced cell-cell fusion

7

Fluorescent microscopy

8

Electron microscopy

• Direct detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus.

• Immune Electron microscopy: Human rotavirus; HAV;

9

Laboratory Diagnosis

• Microscopy Identification

• Virus isolation and identification

• Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material

10

Viral isolation and Identification

• Viral Growth and Cell culture • Viral Detection

• Viral Identification

11

Systems for the Propagation of

Viruses

• People

• Animals: cows, chickens, mice,rats, suckling mice • Embryonated eggs

• Organ and tissue culture Organ culture

Primary tissue culture Cell lines: diploid

12

Viral detection

• CPE

• Hemadsorption • Interfere

13

TCID50

(Tissue culture infective dose)

• TCID50 is defined as that dilution of virus

which will cause CPE in 50% of a given batch of cell culture

• TCID50= log10 of highest dilution giving 100%CPE +1/2 –

(total number of test units showing CPE)/ (number of test units per dilution)

14

Viral identification

• Complement fixation （

• Hemagglutination inhibition • Neutralization

• Immunofluorescence ( direct or indirect) • Latex agglutination

• In situ EIA • ELISA

15

Laboratory Diagnosis

• Microscopy Identification

• Virus isolation and identification

• Detection of viral proteins( antigens and enzymes)

• Detection of viral genetic material • Serologic procedures

16

Detection of viral proteins

( antigens and enzymes)

• Antigen detection ( ELISA, RIA, Western blot) • Hemagglutination and hemadsorption

• Enzyme activities( reverse transcriptase) • Protein patterns( electrophoresis )

17

Laboratory Diagnosis

• Microscopy Identification

• Virus isolation and identification

• Detection of viral proteins( antigens and enzymes)]

• Detection of viral genetic material

18

Detection of viral genetic material

• PCR ( Polymerase chain reaction)

• RT-PCR (Reverse transcriptase polymerase chain reaction)

• Southern （ DNA), Northern(RNA), and dot blots • DNA genome hybridization in situ(cytochemistry)

• Electrophoretic mobilities of RNA for segmented RNA viruses( Electrophoresis)

19

Laboratory Diagnosis

• Microscopy Identification

• Virus isolation and identification

• Detection of viral proteins( antigens and enzymes)] • Detection of viral genetic material

20

Serologic procedures

• If the antibody titer in the convalesent-phase serum sample is at least 4-fold higher than the

titer in the acute-phase serum sample, the patient is considered to be infected.

• In certain viral diseases, the presence of IgM antibody is used to diagnose current infection • Other nonspecific serologic tests are available

21

Serologic procedures

• Complement fixation （

• Hemagglutination inhibition • Neutralization

• Immunofluorescence ( direct or indirect) • Latex agglutination

• In situ EIA • ELISA

22

Viruses Diagnosed by Serology

• Epstein-Barr virus • Rubella virus

• Hepatitis A, B, C, D, and E viruses • HIV

• Human T-cell Leukemia virus

23

Prevention

Prevention

•

Successes of the Past

24

Active immunization

25

Overview of Active immunization

• Active immunization - administration of

antigen resulting in production of a specific immune response with immunologic

memory. Response may be cellular or humoral or both.

 _{Natural immunity - to diseases you have}

caught and successfully fought

26

Attributes of a good vaccine

• Ability to elicit the appropriate immune response for the particular pathogen

• Long term protection ideally life-long

• Safety vaccine itself should not cause disease • Stable retain immunogenicity, despite adverse

storage conditions prior to administration • In-expensive

27

LIVE VACCINES

•

_{Live attenuated organism}

•

_{Heterologous vaccines}

•

_{Live recombinant vaccines}

28

Live attenuated organism

• Organisms whose virulence has been

artificially reduced by in vitro Culture under adverse conditions, such as reduced

29

Heterologous vaccines

• Closely related organism of lesser virulence,

which shares many antigens with the virulent organism. The vaccine strain replication in

the host and induces an immune response that cross reacts with antigens of the virulent organism.

30

Live recombinant

• Vector

1. bovine vaccine 2. BCG

31

•Both cell mediated immunity and antibody response •Activates all phases of immune system. Can get humoral IgG and local IgA

•Raises immune response to all protective antigens. Inactivation may alter antigenicity.

•More durable immunity; more cross-reactive

•Immunity is long lived •Single dose

Advantages of Attenuated

Vaccines 2-1

32

Advantages of Attenuated

Vaccines 2-2

• Low cost

• Quick immunity in majority of vaccinees

• In case of polio and adeno vaccines, easy administration

• _{Easy transport in field}

• Can lead to elimination of wild type virus from

33

Disadvantages of Live Attenuated Vaccine

• Mutation; reversion to virulence (often frequent)

•_{Spread to contacts of vaccinee who have not}

consented to be vaccinated (could also be an

advantage in communities where vaccination is not 100%)

• Spread vaccine not standardized--may be back-mutated

• Poor "take" in tropics

• Problem in immunodeficiency disease (may spread to these patients)

34

Killed vaccines

• The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These vaccines are not

infectious and are therefore relatively safe. However, they are usually of lower

immunogenicity and multiple doses may be needed to induce immunity. In addition, they are usually expensive to prepare.

35

Killed vaccines

• Inactivated organism: rabies virus; epidmic type B encephalitis virus.

• Subunit Vaccines: Influenza virus( HA and NA)

36

Advantages of inactivated

vaccines

• Gives sufficient humoral immunity if boosters given

• No mutation or reversion

• Can be used with immuno-deficient patients • These vaccines tend to be able to

withstand more adverse storage

37

Disadvantages of inactivated

vaccines

• Many vaccinees do not raise immunity • poor, only antibody, no cell immediated

immune response

• response is short-lived and multiple doses are needed

• No local immunity (important)

• Inactivated, therefore can not replicate in the host and cause disease

• Failure in inactivation and immunization with virulent virus

38

New Methods

Selection of attenuated virus strain

• Varicella • Hepatitis A

Use monoclonal antibodies to select for virus with altered surface receptor

• Rabies • Reo

Use mutagen and grow virus at 32 degrees. Selects for

temperature-sensitive virus. Grows in upper respiratory tract but not lower

• ‘flu (new vaccine)

39

New Methods

Passage progressively at cold temperatures TS mutant in internal proteins

Can be re-assorted to so that coat is the strain that is this years flu strain

40 PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS Attenuated Donor Master Strain New Virulent Antigenic Variant Strain

X

Attenuated Vaccine Strain: Coat of Virulent strain with Virulence Characteristics of Attenuated Strain

41

New Methods

Deletion mutants

• Suppression unlikely (but caution in HIV)

• Viable but growth restrictions Problems

42

New Methods

• Recombinant DNA

•Single gene (subunit)

S-antigen mRNA cDNA

Express plasmid

S-antigen mRNA protein

Hepatitis B vaccine

43

Single gene (subunit) - problems

• Surface glycoprotein poorly soluble -

deletion?

• Poorly immunogenic

• _{Post-translational modifications} • Poor CTL response

44

Single gene (subunit) in

expression vector

Vaccinate with live virus

Canary Pox

• Infects human cells but does not replicate

• _{Better presentation} • CTL response

Vaccinia

Attenuated Polio

45

New Methods

Chemically synthesized peptide

•

_malaria

46

antibody

New methods

Anti-idiotype vaccine

epitope

Antibody

with epitope

binding site

47

antibody

Anti-idiotype vaccine

cont

Make antibody against antibody idiotype

Anti-idiotype antibody

Anti-idiotype

antibody mimics the epitope

48

Anti-anti-idiotype antibody

Anti-idiotype antibody

cont 2

Use anti-idiotype antibody as injectable vaccine

Antibody to anti-idiotype antibody Binds and neutralizes virus Anti-idiotype antibody Anti-anti-idiotype antibody Anti-anti-idiotype antibody

49

New Methods

New “Jennerian Vaccines”

• _{Live vaccines derived from animal strains of}

similar viruses

• Naturally attenuated for humans Rotavirus: Monkey Rota

80% effective in some human populations Ineffective in others

50

New Methods

New Jennerian Vaccines Bovine parainfluenza Type 3

Bovine virus is:

• _{Infectious to humans}

• Immunogenic (61% of children get good response)

• Poorly transmissable

51

New Methods

Second Generation Jennerian Vaccines

Rotavirus

11 segments of double strand RNA Two encode:

• VP4 (hemagglutinin) • VP7 (glycoprotein)

Co-infect tissue culture cells reassortment •10 segments from monkey rotavirus

• 1 segment outer capsid protein of each of four major rotavirus strains Efficacy >80%

Elicit neutralizing antibodies

52

Vaccines

•

_{1796 Jenner: wild type animal-adapted}

virus

•

_{1800’s Pasteur: Attenuated virus}

•

1996 DNA vaccines

53

DNA vaccines

• DNA vaccines are at present experimental , but hold promise for future therapy since they evoke both humoral and cell-mediated

immunity, without the dangers associated with live virus vaccines

54

DNA Vaccines

plasmid Muscle cell

Gene for antigen

Muscle cell expresses protein - antibody made CTL response

55

DNA Vaccines

• Plasmids are easily manufactured in large amounts • DNA is very stable

• DNA resists temperature extremes so storage and transport are straight forward

• DNA sequence can be changed easily in the laboratory. This means that we can respond to changes in the

infectious agent

• By using the plasmid in the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are

processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than

56

DNA Vaccines

• Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so that a broad spectrum vaccine could be produced

• The plasmid does not replicate and encodes only the proteins of interest

• No protein component so there will be no immune response against the vector itself

• Because of the way the antigen is presented, there is a CTL response that may be directed against any antigen in the

pathogen. A CTL response also offers protection against diseases caused by certain obligate intracellular pathogens (e.g. Mycobacterium tuberculosis)

57

DNA Vaccines

Possible Problems

• Potential integration of plasmid into host genome leading to insertional mutagenesis

• _{Induction of autoimmune responses (e.g.}

pathogenic anti-DNA antibodies)

• _{Induction of immunologic tolerance (e.g. where}

the expression of the antigen in the host may lead to specific non-responsiveness to that antigen)

58

DNA Vaccines

DNA vaccines produce a situation that reproduces a virally-infected cell

Gives:

• Broad based immune response • Long lasting CTL response

Advantage of new DNA vaccine for flu:

CTL response can be against internal protein

In mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins

59

Adjuvants

• Certain substances, when administered

simultaneously with a specific antigen, will enhance the immune response to that

60

Adjuvants in common use

• Aluminium salts

• Liposomes and immunostimulating complexes • Complet Freund’s adjuvant is an emulsion of

mycobacteria, oil and water • Incomplete Freund’s adjuvant • Muramyl di-peptide

61

Possible action modes of adjuvant

• By trapping antigen in the tissues, thus

allowing maximal exposure to dendritic cells and specific T and B lymphocytes

• By activating antigen-presenting cells to secrete cytokines that enhance the

recruitment of antigen-specific T and B cells to the site of inoculation

62

63

Smallpox

•

Variolation

•_{1% v. 25%}

mortality

•_{Life-long immunity} • No drift or shift

64

Smallpox

Vaccination

• Jenner 1796 : Cowpox/Swinepox

• 1800’s Compulsory childhood vaccination

• 1930’s Last natural UK case

• _{1940’s last natural US case} • 1958 WHO program

• _{October 1977: Last case}

65

Smallpox

_{• No animal reservoir} • Lifelong immunity

• Subclinical cases rare

• _{Infectivity does}

not precede overt symptoms

• One Variola serotype

• _{Effective vaccine} • Major commitment by governments

66

polio

• Killed virus vaccine(Salk, 1954)

• Live attenuated oral polio vaccine( Sabin, 1957)

• The inactivated Salk vaccines is

recommended for children who are immunosuppressed.

67

Small RNA virus Some drift…but not too far as non-viable

Sabin attenuated vaccine

~ 10 cases vaccine-associated disease per year • 50% vaccinees feces

• 50% contacts

• Vaccine-associated cases: revertants

• 1 in 4,000,000 vaccine infections paralytic polio • 1 in 100 of wt infections

Scandinavia: Salk dead vaccine

• No gut immunity

• Cannot wipe out wt virus

68 R ep o rt ed c as es p er 1 00 00 0 p o p u la ti o n 100 10 1 0.1 0.001 0.01

1950 1960 _{1970 1980 1990}

Inactivated (Salk) vaccine

Oral vaccine

Cases per 100,000 population United States

69 10000 1000 100 10 1 0 R ep o rt ed c as es

1950 1955 1960 1965 1970 1975

Killed (Salk) vaccine

Total cases

70 R ec ip ro ca l v ir us a nt ib o dy t ite r 512 128 32 8 2 1

Serum IgG Serum IgG

Serum IgM Serum IgM

Nasal and duodenal IgA Nasal IgA Serum IgA Serum IgA Duodenal IgA Days Vaccination _Vaccination

48 96 48 96

Killed (Salk) Vaccine Live (Sabin) Vaccine

71

Sabin Polio Vaccine

Attenuation by passage in foreign host

More suited to foreign environment and less suited to original host

Grows less well in original host Polio:

• Monkey kidney cells

• Grows in epithelial cells

• Does not grow in nerves

• No paralysis

•Local gut immunity (IgA) Pasteur rabies vaccine also attenuated

72

Salk Polio Vaccine

•

Formaldehyde-fixed

73

Polio Vaccine

Why use the Sabin vaccine?:

• Local immunity: Vaccine virus just like natural infection

• Stopping replication in G.I. Tract stops viral replication TOTALLY

• Dead Salk vaccine virus has no effect on gut replication

• No problem with selective inactivation

• Greater cross reaction as vaccine virus also has antigenic drift

74

Measles

• Live attenuated virus grown in chick embryo fibroblasts, first introduced in the 1960’s.

• Etiology: Measles virus • Incubation: 8 to 12 days

• Clinical Manifestations: cough, coryza, conjunctivitis , erythematous maculopapular rash

fever ,Koplik Spots ,complictions include Encephalitis, Pneumonia, and SSPE

75

Mumps

• Live attenuated virus developed in the 1960’s • MMR vaccine

• Etiology: Mumps Virus • Incubation: 16 to 18 days • Clinical Manifestations:

• swelling of the salivary glands

76

rubella

• Live attenuated virus • Etiology: Rubella Virus • Incubation: 14 to 21 days

• Clinical Manifestations: Congenital , cataracts • patent ductus arteriosus , deafness mental

retardation , Postnatal mild disease , erythematous maculopapular rash , postauricular

77

Hepatitis B

• Two vaccines are in current use: A serum derived vaccine

A recombinant vaccine • Etiology: Hepatitis B Virus

• Incubation: 120 days (average)

• Clinical Manifestations: jaundice ; anorexia • nausea and vomiting ; malaise

• complications include the development of a chronic carrier state with a high risk for Hepatocellular

78

Hepatitis A

• Formalin-inactivated , cell cultured-derived virus,

79

Yellow fever

• The 17D strain is a live attenuated vaccine developed in 1937.

• It is a highly effective vaccine which is

administered to residents in the tropics and travellers to endemic areas.

80

Rabies

No safe attenuated strain of rabies virus has yet been developed for human. Vaccines in current use include: a] The neurotissue vaccine

b] human diploid cell culture-derived vaccine, which is much safer.

There are two situation where vaccine is given: a] Post-exposure prophylaxis, followinf the bite of a rabid animal, Hyperimmune rabies globulin may also administered .

b] Pro-exposure prophylaxis is used for

protection of those occupation puts them at risk of infection with rabies.

81

Influenza

82

Varicella-Zoster virus

84

85

Modes of immunization

• Passive immunization - administration of

antibody-containing serum to provide immediate, but temporary protection. Doesn't activate a lasting specific immune response.

86

Natural

• Provides immunity for diphtheria, tetanus,

streptococcus, rubeola (red measles), rubella (German measles), mumps, polio, and

87

Artificial

• Often used as antitoxins for things such as black widow spider and snake bites,

botulism, and tetanus. Important for some infectious diseases such as rabies, since it provides immediate protection rather than waiting the 7-10 days for a protective

response to develop from active immunization.

88

Immunoglobulin

• “Normal”Immune globulin

89

“Normal”Immune globulin

Low titres of antibody to a wide range of human viruses

• Hepatitis A virus infection

• _{Parvovirus infection}

• _{Enterovirus infections (in neonates)} • _{HIV-infected babies}

90

Hyper-immune globulin

---

high titres of antibody to particular viruses

• Zoster immune globulin: prevention of varicella in immunocompromised children and neonates

• Human rabies immunoglobulin: post-exposure

prophylaxis in an individual who has been bitten by a rabid animal

• Hepatitis B immune globulin:non-immune individal who has been exposed to HBV

• RSV immune globulin: treatment of respiratory syncitial virus infections in the very young

91

92

Antiviral Therapy

• Antiviral chemotherapy • Interferon

• Gene therapy

93

Antiviral chemotherapeutic Agents

• Antiviral drugs are available to treat only a few viral diseases.

• The reason for this is the fact that viral

replication is so intimately associated with the host cell that any drug that interferes

significantly with viral replication, is likely to be toxic to the host

94

Targets for chemotherapeutic

agents

• Attachment to host cell • Uncoating –(amantadine)

• Synthesis of viral mRNA-(interferon) • Translation of mRNA-(interferon)

• Replication of viral RNA or DNA- (nucleoside anologues)

• Maturation of new virus proteins-(protease inhibitors)

96

Diseases for which effective

therapy is available

• AIDS:

Zidovudine （（（（ + Lamivudine （（（（ + protease inhibitors

• Influenza: Amantadine

• Herpes simplex virus: Acyclovir

• Varicella-Zoster virus: Acyclovir

• Cytomegalovirus : Gancyclovir （（（（ , Foscarnet （（ （

97

Nucleotide analogues

• Nucleotide analogues competes with

normal nucleotide for incorporation into viral DNA or RNA.

98

Interferon

• Direct antiviral effect ( prevents the infection of new cells) by a) degradation of viral

mRNA, and b) inhibition of protein synthesis • Enhancement of the specofic

immuneresponse by increasing the

expression of MHC class I molecules on the surface of infected cells, the interferons

increase the opportunity for specifif cytotoxic T cells to recognise and kill infected cells

99

Chinese Herbs

• _{（（（（（（（（（（（（（（} • _（（（

94

Targets for chemotherapeutic

agents

• Attachment to host cell • Uncoating –(amantadine)

• Synthesis of viral mRNA-(interferon) • Translation of mRNA-(interferon)

• Replication of viral RNA or DNA- (nucleoside anologues)

• Maturation of new virus proteins-(protease inhibitors)

96

Diseases for which effective

therapy is available

• AIDS:

Zidovudine （（（（ + Lamivudine （（（（ + protease inhibitors • Influenza: Amantadine

• Herpes simplex virus: Acyclovir

• Varicella-Zoster virus: Acyclovir

• Cytomegalovirus : Gancyclovir （（（（ , Foscarnet （（ （

97

Nucleotide analogues

• Nucleotide analogues competes with

normal nucleotide for incorporation into viral DNA or RNA.

98

Interferon

• Direct antiviral effect ( prevents the infection of new cells) by a) degradation of viral

mRNA, and b) inhibition of protein synthesis • Enhancement of the specofic

immuneresponse by increasing the

expression of MHC class I molecules on the surface of infected cells, the interferons

increase the opportunity for specifif cytotoxic T cells to recognise and kill infected cells

99

Chinese Herbs

• _{（（（（（（（（（（（（（（} • _（（（