Lecture Week 14 Reference for Vaccines and Immunisations

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Lecture Week 14Reference for Vaccines and
Immunisations

• Schaechter chapters 45 and 55
• Schaechter chapter 32 for Polio vaccines. There
  is a table that compares live and killed
  vaccines.
• Brooks chapter 30 for viral vaccines (page
  407-410)
• Brooks. Selected chapters with subheading
  Prevention and control by vaccines chapters
  15, 16, 19,24,27,33,34,35,36, 37,39,40 and 42.

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Medical Diagnostic in Microbiology

• Medical Microbiology SBM 2044

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Diagnosing pathogens

• Why is this important?
• To guide the selection of appropriate therapy for
  the cause of an infection.
• Valuable information can be generated in four
  ways
• Microscopic examination of patient samples
• Cultivation and identification of microorganisms
  from patient samples
• Measurement of a pathogen-specific immune
  response in the patient.
• Detection of pathogen-specific macromolecules in
  patient samples.

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Assessing performance of laboratory tests

• No lab test is perfect
• Require skills to perform diagnostic techniques,
  as well as analysing and interpreting the results
• To describe the performance of value of all
  diagnostic tests
• Sensitivity the likelihood that it will be
  positive when the pathogen is present
• Specificity the likelihood that it will be
  negative if the pathogen is not present

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Diagnosing Infections by Microscopy

• Based on
• morphologic features
• staining properties
• movement
• Helminthic and protozoal infections are routinely
  diagnosed by microscopy.
• Fungal ? characteristic morphologic features
• e.g. Cryptococcus neoformans is an encapsulated
  yeast. Staining CSF with India ink, transparent
  capsule is visualised

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FIGURE 48-4 India ink preparation of
cerebrospinal fluid that shows many encapsulated
yeast cells, some of which are budding, that are
diagnostic of Cryptococcus neoformans meningitis.
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Diagnosing Infections by Microscopy

• Based on
• morphologic features
• staining properties
• movement
• Helminthic and protozoal infections are routinely
  diagnosed by microscopy.
• Fungal ? characteristic morphologic features
• e.g. Cryptococcus neoformans is an encapsulated
  yeast. Staining CSF with India ink, transparent
  capsule is visualised
• Viruses indirectly by the virus-induced changes
  in host cell morphology

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Laboratory findings of Cytomegalovirus A.
Nuclear inclusions and cytoplasmic inclusions
(arrows) in infected alveolar epithelium of an
infant with congenital CMV infection.
A
B
B. Immunohistochemical staining of infected cells
(arrow) in a renal biopsy specimen from a
transplanted kidney in a patient with CMV
infection.
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Stains

• When to stain culture for diagnosing?
• Provide info of presence of bacteria in a
  normally sterile body fluid
• Provide suggestive identification which allows
  for the empirical selection of antibiotics
• Confirmative diagnosis for certain clinical
  specimens
• G- diplococci inside the leukocytes of urethral
  pus ? (always) gonococci
• Acid-fast bacteria in coughed sputum ? M.
  tuberculosis
• Stains
• Gram stain, acid-fast, Giemsa, iodine and silver
  stains

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Giemsa-stained blood smears from Trypanosoma sp.
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Antibody-based identification FIGURE 58-2 The
cross-reactivity of three types of immunoglobulin
preparations. Cross-reactive Common
antibody-binding sites (epitopes) Specificity
is enhanced by monoclonal antibody against an
epitope on the antigen
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Diagnosing infections by Culture

• The most specific way identification of a
  particular pathogen in a patient sample
• However, is culturing practical for all microbes?
• Culturing media agar and broth, in aerobic or
  anaerobic conditions
• Assumption any organisms present in the
  specimen are causative and that the chemotherapy
  directed against them will be beneficial
• Open-ended vs. a specific pathogen
• Selective Thayer-Martin medium for gonococci, in
  non-sterile sites
• What factors might result in false positive
  cultures?

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Blood culture

• Blood sample is inoculated into a nutrient broth
  ? turbidity
• Then subcultured on agar plates for species
  identification
• Lysis-centrifugation method ? lyse blood cells
  and pellets are removed and inoculated directly
  onto agar-based medium

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More on culture techniques

• To determine phenotypic properties
• Motility, utilisation of various nutrient
  substrates, enzymes and toxin productions,
  byproducts of metabolism
• This would take days
• Antimicrobial sensitivity testing
• For therapeutic information

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Antibody-based Techniques

• More rapid method
• Serological tests measure the patients humoral
  immune response to an infection, those directed
  at specific microbial antigens
• Other serology of infectious diseases are
  agglutination, complement fixation, and
  neutralization
• Solid-phase assays, where either the pathogen or
  antigens from the pathogen fixed to a solid
  support, and then the patient serum is added to
  the system.

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Serology test
FIGURE 58-3 Direct ELISA serology for the
detection of specific antibodies in patient
serum.
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Western blot

• Most specific serologic methods available
• Antigenic molecules from a pathogen are first
  separated according to their size using
  electrophoresis
• Immunoblotting (Western blot) whereby defined
  antigens are placed on strips of nitrocellulose
  paper.
• Following incubation, the strip is treated with
  an enzyme-labeled Ab. Addition of the substrate
  for the enzyme allows detection of Ag-specific
  bound Ab by colorimetric reaction.
• Useful for Abs in HIV infection and Lyme disease.

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• Antigenic molecules from a pathogen are first
  separated according to their size using
  electrophoresis

Antigens are then blotted
The blot is then incubated with the patients
serum
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Interpreting Western Blot
Band pattern Interpretation Lane 1, HIV serum
(positive control) Lane 2, HIV- serum (negative
control) Lane A, Patient A Lane B, Patient B
Lane C, Patient C
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Diagnosing Infection by Detecting Microbial
Nucleic Acid

• To design a PCR, a specific sequence of microbial
  nuc acid must be known ? designing primers
• The PCR recipe
• two primers
• deoxyribonucleotides dNTPs
• heat-stable DNA polymerase
• Temperature shifts between heating (denaturation
  of DNA) and cooling (hybridization of the
  primers)
• The chain reaction results in a massive
  production of target DNA

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PCR
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Real-time PCR

• Amplicons (DNA amplified sequence) are quantified
  as they are generated using fluorescence label
  probe
• Intensity of fluorescence increases as the
  amplicons generated
• Useful in measuring viruses in human fluid
  samples
• e.g. HIV, CMV, EBV

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Microarrays

• Bacterial culture allows microbiologist to detect
  a variety of pathogenic organisms in a single
  method.
• That is also the beauty of microchip
• A microchip can be printed to contain hundreds of
  oligonucleotide probes
• Because bacteria have many conserved sequences,
  the design PCR primers will amplify a product
  (cDNA) from any bacterial species, but with
  different amplified sequences between the
  primers.
• Watch animation http//www.bio.davidson.edu/Cours
  es/genomics/chip/chip.html
• Play the Video game http//learn.genetics.utah.e
  du/units/biotech/microarray/

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Microarrays
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1
4
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1. PCR will amplify intervening sequences that
are different in each species 2. During the
amplification, the amplicons (cDNA) are labeled
with a fluorescent dye-conjugated primer 3.
Microarray has bound oligonucleotides (ssDNA)
representing the intervening sequences from
hundreds of bacterial species
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Reference

• Schaechter chapter 58
• or
• Brooks chapter 47
• QUIZ?