Transcript Molecular Diagnosis Of Infectious Diseases

 Asim Farooq
 Shizza Fatima
Contents
 Introduction
 Need, Advantages and Disadvantages
 SARS and its Diagnosis
 Pertussis and Viral Pneumonia and their
Diagnosis
Infectious Disease
 Clinically evident illness
 Infection and presence of pathogenic agent in
host organism
 Sometimes contagious
 Viruses, Bacteria, Fungi, Protozoa,
Multicellular Parasites and Prions
 Primary and opportunistic pathogens
Molecular Diagnosis
 A technique of identifying organisms on the
basis of their genetic makeup
 Molecules specific to a particular organism
 Molecular sizes, structure, mass
 DNA, RNA and Proteins
 Cellular and molecular interactions
Why Molecular Diagnosis
 Need an accurate and timely diagnosis
Important for initiating the proper treatment
Important for preventing the spread of a
contagious disease
Continued…
 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
Continued…
 In situ detection of infectious agents
 Helicobacter pylori
 Toxoplasma gondii
 Agents present in low numbers
 HIV at early stages
 CMV in transplanted organs
 Organisms present in small volume specimens
 Intra-ocular fluid
 Forensic samples
Continued…
Molecular epidemiology
To identify point sources for hospital and
community-based outbreaks
To predict virulence
Culture confirmation
Molecular Techniques
 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
Target Amplification
Target amplification requires that the DNA to
be tested for be amplified, i.e., the number of
copies of the DNA is increased.
Advantages
High sensitivity
Can theoretically detect the presence of a single
organism
High specificity
Can detect specific genotypes
Can determine drug resistance
Can predict virulence
Speed
Quicker than traditional culturing for certain
organisms
Continued…
Simplicity
Some assays are now automated
Disadvantages
Expensive
So specific that must have good clinical data to
support infection by that organism before
testing is initiated.
Will miss new organisms unless sequencing is
done as we will be doing in the lab for our
molecular unknowns
May be a problem with mixed cultures –
would have to assay for all organisms causing
the infection.
Severe Acute Respiratory Syndrome
 SARS coronavirus
 Outbreak in China and Hong Kong in 2002
 Flu, fever, myalgia, lethargy, cough, sore
throat, shortness of breath
 Positive-strand, enveloped RNA viruses
 13 known genes and 14 known proteins
 Large pleomorphic spherical particles with
bulbous surface projections that form a corona
Molecular Diagnosis
 Viral selection
Viral loads maximum in lower tract specimens
Also found in gastrointestinal tract and feces
SARS-CoV RNA has been detected in blood,
cerebrospinal fluid, urine, and tears
 Viruses obtained from different sources are then
subjected to RNA extraction and then
amplification through PCR
RNA Extraction
Testing multiple specimens
 Nucleocapsid transcripts
 nuc and pol genes
Interpretation of Results
 For a positive result, repeat it again or repeat it with
a different genomic locus
 False-positive specimens can occur with poorly
designed primers
 A negative result from an infected patient could be
due to the presence of PCR inhibitors that co-purify
with RNA, a poor quality specimen, or a specimen
lacking virus
 Negative PCR results for specimens from the upper
respiratory tract could trigger sampling from the
lower respiratory tract where the titers of virus are
higher
Pertussis
Molecular Diagnosis
A study was carried out on 5 patients for
molecular diagnosis of pertussis
Age of the patients ranged from 35 days to 3
months, and one patient was 13 years old.
The clinical histories of the five patients
varied, but all had a cough and other
respiratory symptoms.
Hematoxylin and eosin staining of lung tissues
from the patients showed bronchopneumonia
Silver staining (Steiner's method)
demonstrated coccobacilli in all patients, while
Gram's staining showed gram-negative bacilli
in only patients 2 and 4.
From the tissue specimen β-globin gene was
amplified
Utilizing PCR technique
Each PCR mixture consisted of
A 300 nM concentration of each primer
10 μl of DNA extract
 High-fidelity PCR master mix (containing 1.5
mM MgCl2 and a 0.2 mM concentration of
each deoxynucleoside triphosphate)
And an enzyme mixture of Taq and Tgo DNA
polymerases in a 50-μl volume.
 When DNAs extracted from clinical samples of lung
tissue infected with
 Bacillus anthracis
 Group A Streptococcus
 Group B Streptococcus
 Haemophilus influenzae
 Legionella pneumophila
 Staphylococcus aureus
 Streptococcus pneumoniae, or Yersinia pestis
 And from liver tissue infected with spotted-fevergroup Rickettsia
Sequencing the 181-bp amplicons of the
IS481 gene from the clinical isolates and the
five patients.
Results of sequencing
 At nucleotide 100 in all five clinical isolates of B.
holmesii, a mixture of nucleotide bases C and A
occurred, with C slightly more predominant than A.
 In contrast, an A was always present at the same
nucleotide position in all five patient and clinical
isolates of B. pertussis.
 An analysis of the sequence from the reverse strand
confirmed that a mixture of nucleotide bases G and T
occurred at the homologous position in all five B.
holmesii isolates, while only a T occurred in theB.
pertussis isolates and the five patient isolates.
The patient history and clinical information are
beneficial in differentiating between B.
holmesii and B. pertussis. Although B.
holmesii is known to cause septicemia and, in
some instances, respiratory illnesses in
adolescents and adults, the respiratory illness
caused by B. holmesii is mild compared with
that caused by B. pertussis, and no deaths have
been attributed to B. holmesii.
To quantify the amounts of DNA extracted
from human tissues, a real-time assay to detect
an 80-bp region of the human RNase P gene
was performed
Results of PCR
The specific real-time pertussis toxin assay,
which targets a single-copy gene,
demonstrated that all specimens from the five
patients were positive for B.
Results of diagnosis
Of the five patients diagnosed with B.
pertussis infection in this study, the
epidemiologic data support the PCR results for
four who were in contact with ill family
members
Patient 2 confirmed influenza case without an
epidemiologic link but was determined to be
infected with B. pertussis by PCR tests
Patient 4 presented as a presumed SIDSrelated fatality; however, conventional and
real-time PCR tests, an immunohistochemical
assay, and an epidemiologic link to a known B.
pertussis case established that the infant was
infected with B. pertussis.
Viral Influenza
Antiviral agents
Specific antiviral agents such as M2 channel
inhibitors (amantidine and rimantidine) or
 NA inhibitors (oseltamivir and zanamavir) can
be prescribed; however, these drugs are
effective only when given within the first 24 h
following infection.
Diagnostic approaches
Serological tests such as the HAI test have
been used to detect seroconversion of
influenza virus
Nasopharyngeal swabs and NPA are the
preferred specimens for influenza virus
detection
Isolation of influenza virus was historically
performed in embryonated hen eggs or tube
cultures of primary monkey kidney, MadinDarby canine kidney (MDCK), or A549 cells.
CPE consistent with influenza virus can be
visualized by light microscopy
Molecular diagnosis
Molecular tests for influenza virus detection
include
Reverse transcriptase PCR (RT-PCR)
 NASBA
 LAMP
RT-PCR
In the case of RT-PCR, nucleic acid is reverse
transcribed into cDNA using virus-specific
oligonucleotide primers
Several different gene targets have been used
for amplification including the matrix, HA,
and NS protein genes
NASBA
(Nucleic acid sequence based
amplification)
A primer-dependent technology that can be
used for the continuous amplification of
nucleic acids in a single mixture at one
temperature.
Working
 RNA template is given to the reaction mixture, the first
primer attaches to its complementary site at the 3' end of the
template
 Reverse transcriptase synthesizes the
opposite, complementary DNA strand
 RNAse H destroys the RNA template (RNAse H only
destroys RNA in RNA-DNA hybrids, but not singlestranded RNA)
 the second primer attaches to the 5' end of the DNA strand
 T7 RNA polymerase produces a complementary RNA
strand which can be used again in step 1, so this reaction is
cyclic.
LAMP
(Loop mediated isothermal
amplification)
LAMP is a novel approach to nucleic acid
amplification which uses a single temperature
incubation thereby obviating the need for
expensive thermal cyclers.
Uses