Diagnostic Medical
Microbiology
29 Safar 1430
25 Febuary 2009
Objectives and Learning Outcomes
►
To acquire basic skills in the identification and isolation of
microbial pathogens.
►
Demonstrate understanding of the principles of diagnostic
medical microbiology and the clinical correlations.
Make observations, understand the fundamental elements of
experimental design, generate and analyse data on
pathogenesis and control of diseases.
Demonstrate proficiency in collection, interpretation, and
presentation of scientific data in medical microbiology when
conducting a research.
Communicate about case studies using appropriate oral and
written means.
►
►
►
Lecture Outline:
► Normal
Bacterial & Fungal flora
► Laboratory Aids in the selection of
Antimicrobial Therapy
► Diagnosis of Chlamydial infections
► Diagnosis of Viral Infections
► Diagnosis of HIV and AIDS
Normal Bacterial & Fungal Flora
Some organisms are considered pathogens whenever
they are found in patients. Eg.
► Many infections are caused by organisms that are
permanent members of normal flora.
► Gram-negative rods are strongly suspect as the cause of
pneumonia eg. Klebsiella pneumoniae
► In some cases, identification of normal flora is more
warranted, eg. in abdominal abscesses.
► Yeast in small numbers are common flora, not others.
► Viruses are usually not part of the normal flora, but
some latent viruses eg. _____________ or live vaccine
viruses _______________ occasionally appear in viral
cultures.
►
Laboratory Aids in the Selection of
Antimicrobial Therapy
►
►
►
►
Drug testing is essential in those bacteria commonly showing
resistance, primarily Staphylococcus sp., Neisseria gonorrhoea
etc. Testing on fungal and protozoan infections is difficult and
often unnecessary. Identification of infectious agents should
be attempted as soon as possible, secured before the
antimicrobic drug is given.
Choice of drug is based on “informed best guess”.
Disk diffusion susceptibility (or Kirby-Bauer) test – common
test, that measures the ability of drugs to inhibit the growth
of bacteria. The size of zones of inhibition vary with each
different drug.
Tube dilution tests give more sensitive and quantitative
results.
Laboratory Aids in the Selection of
Antimicrobial Therapy
Minimum inhibitory concentration (MIC) = measures the
concentration of an antibiotic necessary to inhibit growth of a
standardised inoculum under defined conditions. The end point,
or minimum inhibitory concentration is the last lowest
concentration of drug that gives clear broth, ie. free from
microbial growth. This is very useful in gauging the dosage
regimen necessary for patients.
► In addition, bactericidal effects can be estimated by subculturing
the clear broth onto antibiotic-free solid media. The result, which
is a reduction of colony-forming units by 99.9% below that of
the control, is called the minimal bactericidal concentration
(MBC).
► The results of antimicrobic sensitivity tests guide the physician’s
choice of a suitable drug. Continuous observation of patient’s
clinical response is imperative once antimicrobial therapy has
begun.
►
Laboratory Aids in the Selection of
Antimicrobial Therapy
► Drug
toxicity – choose one with high/low selective
toxicity for the infectious agent and high/low
human toxicity.
► Therapeutic index (TI) = the ratio of the dose of
the drug that is toxic to humans as compared to
its minimum effective (therapeutic) dose:
►
TI = toxic dose (µg/ml)
effective dose (µg/ml)
Hence, TI=10 is ______________than TI=1.2.
Laboratory Aids in the Selection of
Antimicrobial Therapy
► When
antimicrobic treatment fails, this may be
due to:
► i) the inability of the drug to diffuse into body
compartment (brain, joints)
► ii) a few resistant cells that did not appear in the
sensitivity test in vitro
► iii) infection caused by more than one pathogen
► Other considerations before administrating drugs:
allergy, pregnancy, underlying liver or kidney
diseases, age, any genetic or metabolic
abnormalities, drugs incompatibility, cost.
Diagnosis of Chlamydia
► Chlamydia
: Structure
► Outer cell wall resembles Gram-negative; with
relatively high lipid content.
► Cell wall is rigid but no peptidoglycan, hence no
effect on lysozyme. Perhaps it contains
tetrapeptide-linked matrix.
► Penicillin-binding proteins occur in chlamydiae.
► DNA and RNA are present in elementary and
reticulate bodies.
Diagnosis of Chlamydial Infections
Although Chlamydia sp. are bacteria, they are
obligate intracellular parasites. Hence,
procedures are much likely resembled those of
diagnosing viruses.
► Specimens are collective from respective sites of
infections, and carefully placed in transport medium:
►
Species
Infected sites
C. trachomatis
Oculogenital epithelial, conjunctival
scraping, endometrium scraping
Nasopharyngeal swab
C. pneumoniae
C. psittaci
Sputum, blood, biopsy material.
Chlamydiae
► Staining
properties:
► Giemsa stains: Elementary bodies –
purple; larger, noninfective reticulate bodies
stain – blue and host cytoplasm - blue.
► Fully formed, mature intracellular inclusions
of C trachomatis are compact masses near
nucleus (dark purple) in Giemsa stain.
► Gram staining – negative
Chlamydia
► Microscopy:
► Micro-organisms
can
sometimes be found in the
cytoplasm of squamous cells
from conjunctival scrapings
due to Chlamydia psittaci
infection. This organism
forms compact, oval-shaped
grape-like cluster in the cells’
cytoplasm (yellow arrow).
Sometimes dispersed
elementary bodies can be
seen.
Diagnosis of Chlamydia
Cell culture techniques for isolation of chlamydia species
involves inoculation of the C trachomatis and C psittaci
onto cycloheximide-treated McCoy cells (antimetabolites
that inhibit host cell replication but allows chlamydiae to
use available cell nutrient for growth), and C pneumoniae
requires pretreated HEp-2 cells.
► Immunoassay –EIAs are used to detect chlamydial
antigens from genital swab.
► Nucleic acid Hybridization – commercial kits with
nonradioisotopic probes are available for C trachomatis 16S
RNA sequences. PCR and ligase chain reaction (LCR) are
much more sensitive than culture.
► Serology: Complement fixation test is widely used to
diagnose psittacosis. The microimmunofluorescence
method is more sensitive than CF for measuring
antichlamydial antibodies. Detection of IgM against C
trachomatis is helpful in infant pneumonitis.
►
Diagnosis of Viral Infections
► Isolation
of a virus may not establish the cause of
a given disease. Some viruses persist in human
hosts for long periods.
► Care for specimens: refrigerated for up to 24hr
(except RSV) or freeze -60°C or colder.
► Virus is present in pharyngeal secretions; fluid and
scrapings of vesicular rashes; conjunctival swabs
and tears; brain tissue; and CSF; faeces.
► Direct fluorescent antibody is the tests of choice
for diagnosis because they provide answers within
a few hours after collection cf. virus culture.
Detection of Viral Growth
Lytic or cytopathic viruses replicate in cells and produce
alterations in cellular morphology (or cell death) and the
effect can be seen directly by light microscopy.
► For example, enteroviruses often produce cell rounding,
pleomorphism and eventual cell death; and measles and
RSV cause fusion of cells to produce ____________.
► Other viruses are detectable by their production of
haemagglutinins;
and by a method called interference whereby the virus
which produces no cytopathic effect in susceptible cell
culture, but can be detected by “challenging” the cell
culture with a different virus that normally produces a
characteristic cytopathic cell. The second virus fails to
infect the cell culture because of the interference by the
first virus, which is thus detected. Eg. Rubella virus.
►
A. Normal monkey kidney cell culture monolayer. B.Enterovirus
cytopathic effect in a monkey kidney cell monolayer. Note cell lysis
and monolayer destruction. (X 40)
C. Normal human diploid fibroblast cell monolayer. D. Cytomegalovirus
cytopathic effect in human diploid cell monolayer. Note rounded, swollen
cells in a focal area. (Χ 40)
In vivo Isolation Methods
► Embryonated
hen’s egg is still used for the initial
isolation and propagation of influenza A virus. The
egg is incubated to permit viral replication and
recognition.
► Animal host, commonly the mouse; suckling mice
in the first 48h of life are especially susceptible to
many viruses. Viral replication is based on
development of illness, with signs such as
paralysis, convulsions, poor feeding or death.
► Further test to elucidate the nature of infecting
virus – by histologic and immunofluorescent
examinations of tissues or by antibody detection.
Virus Identification
► On
isolation, virus can usually be identified by its
cultural characteristics. Further identification may
require adequate quantities for testing.
► Several diagnostic methods for viral identification
are:
► 1. Antigen detection
► 2. nucleic acid amplification and detection
► 3. nucleic acid hybridisation
► 4. measuring immune response to virus infection
► 5. immune electron microscopy.
Diagnostic Virology
► 1.
Antigen detection: this neutralisation method
works by neutralise virus infectivity by mixing it
with specific antibody.
► Immunofluorescence, enzyme immunoassay (EIA)
and latex agglutination are common methods.
► Commercial kits are available to detect many
viruses inc HSV1 and HSV2, influenza A and B,
RSV, adenovirus, parainfluenza virus, rotavirus,
cytomegalovirus.
► Advantage: allow detection of viruses that do not
readily grow in the cell culture (rotavirus, Hep A),
or that grow very slowly (CMV)
Diagnostic Virology
►
►
►
►
2. Nucleic Acid Amplification & Detection: commercial
assays are available which inc PCR, reverse-transcriptase
PCR.
Detects enteroviruses; and quantifies HIV-1, CMV, EBV.
3. Nucleic Acid Hybridisation: is a highly sensitive and
specific method.
4. Measuring immune response: cellular immunity may be
assessed by dermal hypersensitivity, lymphocyte
transformation, cytotoxicity tests. Humoral immune
responses- IgM appear initially and are followed by IgG.
IgM disappear in several weeks whereas IgG persist for
many years. The methods used are neutralisation test,
complement fixation, haemagglutination inhibition test, and
the immunofluorescence test.
Diagnostic Virology
► 5.
Immune Electron Microscopy (EIA): for viruses
not detectable by conventional techniques.
► Antigen-antibody complexes or aggregates formed
between virus particles in suspension are caused
by the presence of antibodies in added antiserum
and are detected more readily and with greater
assurance than individual virus particles.
► IEM detects viruses that cause enteritis and
diarrhoea.
HIV and AIDS
► HIV-1
occurs worldwide, while HIV-2 occurs
primarily in West Africa and other geographic
areas.
► Blood banks use very sensitive tests to detect HIV1 in donated blood.
► Characteristics of HIV:
► Family
,
RNA,
► Possess viral enzyme reverse transcriptase.
► Commonly assayed viral protein is p24. antibody
responses to several HIV gene product: env,
gp160, gp120, gp41, gag and pol.
Assays for Anti-HIV Antibodies
►
►
►
►
►
►
►
►
A. ELISA Enzyme-Linked Immunosorbent Assay:
Primary screening test for HIV-1 infection.
HIV-1 antigens immobilised on a solid surface (plastic
wells, or beads). Add patient’s serum. HIV-1 antibodies
bound to immobilised antigens are then detected with an
enzyme labeled anti-human IgG and a colorimetric
reaction.
Results interpretation: the amount of colour is
proportionately higher with higher HIV-1 antibodies.
ELISA for HIV-1 is extremely sensitive and specific.
What about infants born with HIV-infected mothers?
ELISA test at 2 months old:
ELISA test at 2 years age:
Principle of Enzyme Immunoassays
First Ab
Second antibody
for the Ag,
labelled with
enzyme
Add antigen
Assays for HIV-1 Antibodies
► B.
Western Blot:
► To measure specific HIV-1 antibodies to
confirm a positive ELISA result.
Western Blot
1. HIV-1 proteins are
separated by
electrophoresis.
► 2. HIV-1 proteins are
transferred onto
nitrocellulose strip.
► 3. the strip is incubated with
patient’s serum. The specific
HIV-1 antibodies are
subsequently detected using
an enzyme-linked antihuman
IgG.
► 4. positive colorimetric
reaction forms bands on the
nitrocellulose paper
corresponding to p24, gp41
and pg120/160.
►
B. Western Blot: Results
► i)
Positive test : any 2 bands corresponding to the
proteins.
► ii) no bands : negative result
► iii) bands that do not meet the criterion for a
positive test is an indeterminate results.
► False-positive and false-negative results are
relatively uncommon.
► Repeat positive ELISAs and indeterminate Western
blots.
► Infant born to an HIV-1-infected mother?
Assays to Detect HIV Infection
►
►
►
►
►
►
A. Detection of p24 Antigen:
ELISA to detect p24 antigen. p24 antigen is detectable during
the acute viraemic stage and in the late stages of AIDS.
A very small proportion of asymptomatic HIV-1 infected
persons are p24 antigen +.
B. Detection of HIV-1 RNA: commercial assays inc PCR.
NASBA, bDNA.
C. HIV-1 Proviral DNA: DNA extracted from mononuclear cells
obtained from anticoagulated peripheral blood. Perform PCR.
Useful for infants.
D. HIV-1 Culture: peripheral blood mononuclear cells from a
potentially infected patient are cocultured with uninfected
cells stimulated with phyto haemagglutinin and interleukin-2.
Cultures are looked for: multinucleated giant cells, HIV-1 rev
transcrip, p24 Ag. Time-consuming and expensive.
Objectives and Learning Outcomes
►
To acquire basic skills in the identification and isolation of
microbial pathogens.
►
Demonstrate understanding of the principles of diagnostic
medical microbiology and the clinical correlations.
Make observations, understand the fundamental elements of
experimental design, generate and analyse data on
pathogenesis and control of diseases.
Demonstrate proficiency in collection, interpretation, and
presentation of scientific data in medical microbiology when
conducting a research.
Communicate about case studies using appropriate oral and
written means.
►
►
►