Transcript Infectious diseases

Facilitator: Dr Rania M. H. Baleela
Email address: [email protected]
Diploma in Molecular Diagnosis of Infectious Disease by Rania M. H.
Baleela is licensed under a Creative Commons Attribution 3.0 Unported
License.
This course theory part
Is concerned with molecular diagnosis of
infectious diseases
2 lectures, 4 hours each
Will concentrate as much as possible on
infectious diseases from Sudan and the best
way they could be diagnosed
Contents (headlines)
Introduction to Infectious disease,
Introduction to genetics,
Introduction to molecular biology,
Introduction to molecular taxonomy,
Molecular Diagnosis of infectious disease
Sudan statistics, WHO
Sudan statistics, WHO
Sudan statistics, WHO
Malaria
It was raining here…..
El Fashir, Darfur
Aroma, Kassala
And every where
and the result will definitelly be…..
and …..
Infectious diseases
Are caused by transmission of specific pathogenic
agents to susceptible hosts
May be transmitted to humans:
1. Directly => from other infected humans or animals
2. Indirectly => through vectors (e.g. insects), airborne
particles or vehicles (e.g. contaminated: cloth, water,
milk, food, blood, plasma, etc)
Contagious (= by touch) diseases: are those
transmitted between humans without vectors or
vehicles
1. Malaria = communicable but not contagious
2. Syphilis = communicable & contagious
Epidemics
= the occurrence of cases in excess of what is normally
expected in a community or region
When describing an epidemic, describe:
1. The time period
2. Geographical region
3. Particulars of the population in which the cases
occur
An epidemic dynamics are determined by:
1. Characteristics of its agent
2. Its pattern of transmission
3. Susceptibility of its human hosts
Do you know John Snow (1813-1858)?
A British physician who is
considered one of the
founders of epidemiology for
his work identifying the
source of a cholera outbreak
in 1854.
http://www.culture24.org.uk/science-and-nature/medicine/art421823
A brief history
1823: 1st cholera pandemic dies down in the Caucuses before
reaching Europe.
1826-37: 2nd cholera pandemic breaks out starting in Russia,
then moving to Poland and subsequently the rest of Europe,
North Africa and the eastern seaboard of North America.
1831-32: 1st outbreaks of cholera in London: 6,536 die.
1838: John Snow qualifies as a doctor.
1842: Edwin Chadwick publishes a report: The Sanitary
Conditions of the Labouring Population ,that makes a clear
link between disease and living conditions.
1848-49: 2nd outbreak of cholera in London: 14,137 die.
A brief history cont.
1849: Albion Terrace Outbreak. The cholera outbreak
that leads John Snow to publish his theory that cholera
is water-borne in: On the Mode of the Communication
of Cholera.
1849: William Budd claims that cholera is caused by a
living organism or parasite that reproduces itself in
large numbers when it reaches the gut. He further
claims that water is the primary channel that the
parasite enters the body.
1883: Robert Koch discovered that a comma shaped
bacterium called the Vibrio cholerae was responsible
for cholera in human beings.
John Snow and Disease Mapping in Soho, London
map showing deaths from cholera in Broad Street, Golden Square and the surrounding area
between August and September 1854, taken from John Snow's publication, On the Mode of
Communication of Cholera
© London School of Hygiene and Tropical Medicine Archive
http://www.culture24.org.uk/science-and-nature/medicine/art421823
Traditional diagnosis
Microscopy was the gold standard for malaria
detection
Traditional diagnosis
Isolation and identification of Vibrio cholerae
serogroup O1 or O139 by culture of a stool
specimen remains the gold standard for the
laboratory diagnosis of cholera.
Vibrio cholerae diagnosis
Cary Blair media is ideal for transport, and the
selective thiosulfate–citrate–bile salts agar
(TCBS) is ideal for isolation and identification.
The Crystal VC® dipstick (an
immunochromatographic rapid test) rapid test
can provide an early warning to public health
officials that an outbreak of cholera is occurring.
The sensitivity and specificity of this test are
not optimal.
A molecular approach to diagnose cholera
Molecular quantitation and characterization of
Vibrio cholerae from different seafood obtained
from wetmarket and supermarket
Read online at:
http://khartoumspace.uofk.edu:8080/jspui/bits
tream/123456789/618/1/Molecular%20quantit
ation%20and%20characterization%20of%20v.%
20cholerae.pdf
Why use a molecular test to diagnose an infectious disease?
Need an accurate and timely diagnosis
Important for initiating the proper treatment
Important for preventing the spread of a
contagious disease
Leading uses for nucleic acid based tests
Nonculturable agents
Human papilloma virus
Hepatitis B virus
Fastidious, slow-growing agents
Mycobacterium tuberculosis
Legionella pneumophilia
Highly infectious agents that are dangerous to culture
Francisella tularensis
Brucella species
Coccidioidis immitis
In situ detection of infectious agents
Helicobacter pylori
Toxoplasma gondii
Agents present in low numbers
HIV in antibody negative patients
Cytomegalovirus (CMV) in transplanted organs
Organisms present in small volume specimens
Intra-ocular fluid
Forensic samples
Differentiation of similar agents (e.g. virus
genotypes associated with human cancers
Papilloma viruses)
Antiviral drug susceptibility testing
Non-viable organisms
Organisms tied up in immune complexes
Molecular epidemiology
To identify point sources for hospital and
community-based outbreaks
To predict virulence
Culture confirmation
What are the different types of nucleic acid molecular techniques
that are used?
Direct probe testing – better for identification than for
detection because it is not as sensitive as amplification
methods
Amplification methods – used to improve the
sensitivity of the nucleic acid testing technique
Target amplification
Probe amplification
Signal amplification
Combinations of the above
DNA Structure
In double stranded linear DNA,
1 end of each strand has a free
5’ carbon and phosphate and
1 end has a free 3’ OH group.
The two strands are in the
opposite orientation with
respect to each other
(antiparallel).
Adenines always basepair with
thymines (2 hydrogen bonds)
and guanines always basepair
with cytosines (3 hydrogen
bonds)
Direct probe testing
Hybridization – to come together through
complementary base-pairing.
Can be used in identification.
In colony hybridization the colony is treated to
release the nucleic acid which is then
denatured to single strands.
Labeled single-stranded DNA (a probe)
unique to the organism you are testing for is
added and hybridization is allowed to occur.
Unbound probe is washed away and the
presence of bound probe is determined by
the presence of the label.
Target amplification
Target amplification = the number of copies of
the DNA increase.
The enzyme DNA polymerase is used
Polymerase template and primer requirements
DNA polymerase cannot initiate synthesis on its
own.
It needs a primer to prime or start the reaction.
The primer is a single stranded piece of DNA that is
complementary to a unique region of the
sequence to be amplified.
Synthesis can occur only in the 5’ to 3’ direction
DNA replication is semiconservative
Some theoretical background
2. The central dogma of molecular biology
Some theoretical background
Proteins can be organized in 4
structural levels:
Primary (1°): The amino acid
sequence, containing members of a
(usually) twenty-unit alphabet
Secondary (2°): Local folding of the
amino acid sequence into α helices
and β sheets
Tertiary (3°): 3D conformation of the
entire amino acid sequence
Quaternary (4°): Interaction between
multiple small peptides or protein
subunits to create a large unit
Folding determines function
Misfolded proteins can mean the protein will
have a lack of functionality
•Even worse can be damaging or dangerous to
other proteins
•Too much of a misfolded protein can be worse
then too little of a normal folded one
•Can poison the cells around it
What is Protein Folding
Primary Structure
– 3-D conformation of a protein depends only on
its linear amino acid sequence
– In theory can be computed explicitly with only
this information
– One of the driving forces that is thought to cause
protein folding is called the hydrophobic
effect=> burial of the hydrophobic residues in
the core of the protein
Hydrogen Bonds
In both secondary structures
–Alpha-helix
–Beta-Sheets
Responsible for stabilization
Greatly effect the final fold of the protein
Why Fold Proteins
•Many genetic diseases are caused by
dysfunctional proteins
–By learning the structures we can learn the
functions of each protein
–Build better cures
–Understand mutation
–Assign structures functions to every protein
•Thus understand the human genome
•Decode the Human DNA
Why bother with structures when we have sequences?
In evolutionary related proteins structure is
much better preserved than sequence.
Structural motifs may predict similar biological
function .
Getting insight into protein folding. Recovering
the limited (?) number of protein folds.
Applications
Classification of protein databases by structure.
Search of partial and disconnected structural
patterns in large databases.
Extracting Structure information is difficult, we
want to extract “new” folds.
Speed up of drug discovery.
Detection of structural pharmacophores in an
ensemble of drugs (similar substructures in drugs
acting on a given receptor – pharmacophore).
Comparison and detection of drug receptor active
sites (structurally similar receptor cavities could
bind similar drugs).
The differential expression of
genes in healthy & diseased
tissue is usually highly
revealing
PCR in parasite diagnosis
Amplifies target sequences & increases sensitivity.
1.Ribosomal DNA/RNA.
Highly sensitive.
No good for closely related species.
2.Specific sequences of genomic DNA.
Highly specific for single species - not
sensitive.
3.Random primer amplification (RADP) PCR.
Very highly sensitive - not specific.
Nucleic acid based molecular diagnosis
Advantages:
 Genomic DNA constant -parasite & hosts unique
DNA sequences .
 Very sensitive - small biopsy.
 Probes can be designed with flexibility:
 Specific - detect single parasite species.
 Less specific - detect group of parasites.
Nucleic acid based molecular diagnosis
Disadvantages:
 Sometimes expensive.
 Radioactivity needed: newer nonradioactive probes.
 PCR can fail: - Contamination & false
positives.
 DNA probes do not distinguish between
dead & living parasites
Methods for molecular diagnosis e.g.
bDNA technology for HIV viral load
Gene©Xpert MTB/RIF assay: Rapid molecular
detection of tuberculosis and Rifampin
resistance
Loop-Mediated Isothermal DNA Amplification
(LAMP)
Leishmaniasis molecular diagnosis
bDNA
Sensitive, specific and reliable
Amplification of a target sequence is not required
cross-contamination between replicate samples due
to excessive amplicons or carryover is less likely in
bDNA assays
Can be used for:
viral load testing (HIV-1 and HPC)
Trypanosoma brucei detection
Antibiotic-sensitive and resistant Staphylococcus
detection
Papillomavirus detection
Hepatitis B virus detection