Chapter 12 Target Microorganisms for MolecularBased Testing Those that are difficult or timeconsuming to isolate eg Mycobacteria Hazardous organisms eg Histoplasma Coccidioides ID: 306120
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Slide1
Detection and Identification of Microorganisms
Chapter 12Slide2
Target Microorganisms for Molecular-Based Testing
Those that are difficult or time-consuming to isolate
e.g.,
Mycobacteria
Hazardous organisms
e.g.,
Histoplasma
,
Coccidioides
Those without reliable testing methods
e.g.,
HIV, HCV
High-volume tests
e.g.,
S.
pyogenes
,
N.
gonorrhoeae
, C. trachomatisSlide3
Applications of Molecular-Based Testing in Clinical Microbiology
Rapid or high-throughput identification of microorganisms
Detection and analysis of resistance genes
Genotyping
Classification
Discovery of new microorganismsSlide4
Specimen Collection
Preserve viability/nucleic acid integrity of target microorganisms.
Avoid contamination.
Maintain appropriate time and site of collection (blood, urine, other).
Use proper equipment (coagulant, wood, or plastic swab shafts).
Commercial collection kits are available.
The Clinical and Laboratory Standards Institute (CLSI) has guidelines for proper specimen handling.Slide5
Sample Preparation
Consider the specimen type (stool, plasma, CSF).
Consider the number and type of organisms in the sample.
Inactivate inhibitors (acidic polysaccharides in sputum or polymerase inhibitors in CSF).
Inactivate
RNases
.Slide6
PCR Detection of Microorganisms: Quality Control
PCR and other amplification methods are extremely sensitive and very specific. For accurate test interpretation, use proper controls.
Positive control
: positive template
Negative control
: negative template
Amplification control
: omnipresent template unrelated to target
Reagent blank
: no template presentSlide7
PCR Quality Control: Internal Controls
Homologous extrinsic
Controls for amplification
Heterologous extrinsic
Controls for extraction and amplification
Heterologous intrinsic
Human gene controlSlide8
Target sequence
PCR Quality Control: Internal Controls
Homologous extrinsic
Controls for amplification
Heterologous extrinsic
Controls for extraction and amplification
Heterologous intrinsic
Human gene controlSlide9
Sensitivity vs Specificity
Sensitivity
and
specificity
are statistical measures of the performance of a
test.
Sensitivity
(also called the
true positive
rate
)
measures the proportion of actual positives which are correctly identified as such (e.g. the percentage of sick people who are correctly identified as having the condition).
Specificity
measures the proportion of negatives which are correctly identified as such (e.g. the percentage of healthy people who are correctly identified as not having the condition, sometimes called the true negative rate).Slide10
Sensitivity vs Specificity
When screening for a disease, sensitivity is more important (you can confirm with a more specific test later)
When confirming a disease, specificity is more importantSlide11
Quality Control: False Positives
Contamination: check reagent blank
Dead or dying organisms: retest 3
–
6 weeks after antimicrobial therapy
Detection of less than clinically significant levelsSlide12
Quality Control: False Negatives
Improper collection, specimen handling
Extraction/amplification failure: check internal controls
Technical difficulties with chemistry or instrumentation: check method and calibrationsSlide13
Detection of Bacteria
Respiratory Diseases
Respiratory infections are responsible for significant numbers of infections and deaths worldwide.
These infections are easily spread by inhalation
Slide14
Bordetella (
W
hooping Cough)
Bordetella
pertussis
is a Gram-negative, aerobic
coccobacillus
B.
pertussis
is
nonmotile
. Its virulence factors include pertussis toxin, filamentous hemagglutinin, and tracheal cytotoxin.Slide15
Bordetella
In the US, it killed 5,000 to 10,000 people per year before a vaccine was available. Worldwide in 2000, according to the WHO, around 39 million people were infected annually and about 297,000 died.
Because of concerns regarding the vaccine, numbers continue to be highSlide16
Bordetella
There are three species that cause most of the
pertussis
seen in humans:
B.
pertussis
causes the most severe whooping cough in children.
B.
parapertussis
and
B.
holmesii
cause a less severe whopping cough.Slide17
Bordetella
In view of its enormous sensitivity and specificity
rapid PCR
based detection of
B.
pertussis
has attracted
much attention
in recent years. The chromosomal regions
that have
been used as targets for
B.
pertussis
specific PCR include :the adenylate cyclase toxin (ACT) gene a region upstream of the porin gene , Pertussis toxin (PT) promotor region, and repeat insertion sequences.
Among these, repeat insertion sequence IS481 region being present in multiple copies (80–100) in B. pertussis, is a target of choice for amplification and detection with greater sensitivity.Slide18
Bordetella
However,
B
.
holmesii
also contains
regions homologous to IS481
.
Thus
PCR targeting
IS481
will generate a positive DNA product
for both
B. pertussis and B. holmesii as observed. As a result it although will provide high sensitivity, it will lack specificity. However, since there are two single nucleotide changes (A/C and C/T variation) in alleles of B. holmesii
genome; a DNA probe can be diagnostic.Slide19
Bordetella
However, there is another insertion sequence, (IS 1001), that is present in
B.
holmesii
but not in
B.
pertussis
.
Thus, testing for both insertion points will give a differential diagnosis.Slide20
Tuberculosis
The presence of acid-fast-bacilli (AFB) on a
sputum smear
or other specimen often indicates TB disease. Acid-fast microscopy is easy and quick, but it does not confirm a diagnosis of TB because some acid-fast-bacilli are not
Mycobacterium tuberculosis
.
Therefore, a
culture
is done on all initial samples to confirm the diagnosis. (However, a positive culture is not always necessary to begin or continue treatment for TB.) A positive culture for
M. tuberculosis
confirms the diagnosis of TB disease. Slide21
Tuberculosis
Although sputum smears are the gold standard for diagnosis of tuberculosis, sensitivity in HIV/TB
coinfection
cases is low, indicating a need for alternative methods.
Also culture can take as long as 3 weeks.
Urine is being increasingly evaluated.
A new method for detecting
Mycobacterium tuberculosis
(MTB) uses combined IMS/ATP assay.Slide22
Tuberculosis
If the smear is positive, PCR or gene probe tests can distinguish
M. tuberculosis
from other
mycobacteria
.
Target probe is the 16S
rRNA
sequence. Turnaround is reduced to about 2-3 hours.
But sensitivity is not good enough for clinical specimensSlide23
Tuberculosis
Urine is being increasingly evaluated.
A new method for detecting
Mycobacterium tuberculosis
(MTB) uses combined IMS/ATP assay.Slide24
Tuberculosis
Immunomagnetic
separation (IMS) is used to concentrate and recover pathogenic
mycobacteria
, including MTB . IMS also enables specific target capture and decreases particulate interference in detection assays. Slide25
Tuberculosis
ATP bioluminescence assays have demonstrated utility in
bacteriuria
(
bacteria in urine) screening , quality control of BCG vaccines, and MTB antibiotic susceptibility testing.
The determination of ATP using bioluminescence uses the ATP dependency of the light emitting luciferase catalyzed oxidation of
luciferin
for the measurement of extremely low concentrations of ATP. Luminescence is measured using a
luminometer
.Slide26
Tuberculosis
Combining
immunocapture
with an ATP-based cell viability assay can provide rapid, specific,
semiquantitative
detection of live cells.
The method can provide rapid, specific detection of MTB in urine. The method is easy to perform and could be used in settings where the rate of HIV/TB co-infection is high.Slide27
Detection of bacterial STDs
Historically, the diagnosis of sexually transmitted diseases (STDs) has been difficult. The introduction of molecular biology techniques in microbiological diagnosis and their application to non-invasive samples has produced significant advances in the diagnosis of these diseases.
Overall
, detection of
Neisseria
gonorrhoeae
by molecular biology techniques provides a presumptive diagnosis and requires confirmation by culture in areas with a low prevalence.
For
Chlamydia trachomatis
infections, these techniques are considered to be the most sensitive and specific procedures for mass screening studies, as well as for the diagnosis of symptomatic patients. Slide28
Detection of bacterial STDs
Diagnosis of
Mycoplasma
genitalium
infection by culture is very slow and consequently molecular techniques are the only procedures that can provide relevant diagnostic information
.
For
Treponema
pallidum
, molecular techniques can provide direct benefits in the diagnosis of infection
.
Molecular methods are advisable in
Haemophilus
ducreyi, because of the difficulties of culture and its low sensitivity.Slide29
Detection of Viruses
Because of the difficulty in growing and identifying viruses, it is this field that have benefitted tremendously from the introduction of molecular based methods.Slide30
Viruses
“Classical methods” of detection include antibody detection, antigen detection, and culture.
Molecular methods of detection include target, probe, and signal amplification.
Tests are designed for identification of viruses, determination of
viral load
(number of viruses per
mL
of fluid), and genotyping by sequence analysisSlide31
“Classic” Antibody detection
IgM
and
IgG
Levels
IgM
is the first antibody produced by the body when it is exposed to a virus. The
IgM
test is used to screen for early detection of infection and is used to diagnose the disease during onset.
IgG
antibodies develop later and remain present for many years, usually for life, and protect against further infection by the same virus. Slide32Slide33
HIV
Molecular testing is important in HIV, not only for detection of the disease, but also continued monitoring of disease treatments.Slide34
Detection of HIV
The first marker that becomes detectable after infection is the HIV RNA, indicated by the green line. This is detectable by current molecular methods at about 11 days from the time of infection or exposure to HIV
.
The second marker that becomes detectable in the laboratory is the HIV p24 antigen, indicated in the purple line. This is detectable by day 16 from exposure
.
And finally, the HIV antibodies that are detectable by current commercial assays occur at about day 22 from the date of infection. So the most widely used serologic tests, which are HIV antibody screening tests, are actually the least sensitive in picking up HIV infection compared to the other 2 markers.Slide35Slide36
Detection of HIV
T
here
are basically 2 windows of HIV infection for detection.
The
first is the
seroconversion
window, which starts from time of infection, indicated by the first arrow on the left-hand side of the timeline, to the time point where antibody becomes detectable. So, this
seroconversion
window period actually includes the eclipse period and the acute infection period.
The
eclipse period is the period at which time that only molecular tests can detect the presence of HIV RNA. Slide37
The acute infection is the period between viral infection detectable by molecular tests and a serologic response, which is detectable by serologic assays.
Now the incidence window is the period from the time of antibody detection first from the infected individual until a specific time point where the serologic assay can determine recent infection. So that particular window period is also known as the recent infection.
And
, after this assay-specific detection point for recent infection, we see long-standing HIV infection. Slide38Slide39
Detection of HIV
The
most widely used
methods for HIV detection are
the ELISA assays, the enzyme-linked
immunosorbant
assays, which can come in the form of enzyme immunoassay or
chemoluminescent
immunoassay. They can detect either HIV-1 antibodies, or HIV-2 antibodies, or HIV-1 p24 antigen, or a combination of HIV-1 and -2; and then lastly the fourth generation serologic test is a combination of antibody and p24 antigen.
And
, basically, there are 2 methods, the
immunochromatography
method, as well as the membrane
immunoconcentration
method. These rapid test devices are available to detect either HIV-1 antibodies alone, HIV-2 antibodies alone, or a combination of HIV-1 and -2 antibodies.The final group of serologic tests are the so-called supplemental tests, also known as confirmatory tests for HIV-1 and -2 antibodies. And there are essentially 2 methods that are commercially available: one is the Western blot and the other is the Immunoblot.Slide40Slide41
Detection of HIV
Early
in the epidemic of HIV infection, the first tests that were available for diagnosis or detection of HIV are viral cultures using CD4 cells; these are the human helper T cells that are infected by HIV viruses. And, using these viral cultures, one is able to detect production of viral p24 antigens in the supernatant of cell cultures from CD4 cell lines.
PCR
assays for qualitative and quantitative detection of HIV-1 and -
2 are also available.
For qualitative PCR assays, one could detect HIV-1
proviral
DNA. This is the DNA that is incorporated into the CD4 whole cells, DNA that belong to HIV-1 viral genome. One could also detect a combination of HIV
proviral
DNA and RNA and also laboratory-developed assays, particularly in certain research investigator laboratories, one could also design a qualitative detection of HIV-2 RNA. For quantitative assays, there are commercially available and FDA- approved assays for quantifying HIV-1 RNA, and then there are laboratory-developed assays for quantifying HIV-2 RNA.
Two other commercial laboratory tests available utilize transcription-mediated amplification for qualitative detection of HIV-1
RNA,
and the branched DNA
method, which utilizes signal amplification for quantitation of HIV-1 RNA. Slide42
Treatment of HIV
In an HIV‐infected individual, the concentration of virus in the bloodstream or viral load (VL) can be a valuable tool for the clinical management of the infection.
Broadly
, there are three clinical uses for quantifying HIV in plasma:
diagnosing
acute HIV
infection
determining
prognosis and disease
progression
therapeutic
monitoring. Slide43
Treatment of HIV
Unlike antibody detection, which is confounded by the trans‐placental transfer of maternal
IgG
antibodies, VL can also be useful in diagnosing babies born to HIV‐positive
mothers.
However
monitoring VL is most
relevant
as a biomarker to
monitor the
therapeutic
efficacy.
Quantifying viral load in plasma enables a clinician to assess the success of treatment and detect treatment failure prior to the onset of clinical symptoms. Slide44
Test Performance Features for Viral Load Measurement
Characteristic
Description
Sensitivity
Lowest level detected at least 95% of the time
Accuracy
Ability to determine true value
Precision
Reproducibility of independently determined test results
Specificity
Positive results are true positives
Linearity
A serial dilution of standard curve closely approximates a straight line
Flexibility
Accuracy of measurement of virus regardless of sequence variationsSlide45
Hepatitis
Viral hepatitis, including hepatitis A, hepatitis B,
hepatitis
C,
hepatitis D and hepatitis E are
distinct diseases that affect the liver and have different hepatitis symptoms and treatments
.
Each virus is different and the only commonality between them is that they all cause an inflammation of the liver.
There is more concern over hepatitis B and C because they are
bloodborne
pathogens.Slide46
Hepatitis
Old methods for detecting hepatitis revolved around antibody and antigen detection. For example the next couple of pages of this
powerpoint
describe the markers looked for in diagnosing Hepatitis B.Slide47Slide48
Hepatitis B Ags
and Abs
Hepatitis B surface antigen (
HBsAG
)
Protein that is present on the surface of the virus; will be present in the blood with acute and chronic HBV infections
Often used to screen for and detect HBV infections; earliest indicator of acute hepatitis B and frequently identifies infected people before symptoms appear; undetectable in the blood during the recovery period; it is the primary way of identifying those with chronic infections.
Hepatitis B surface antibody (anti-HBs)
Antibody produced in response to HBV surface antigen; levels in the blood rise during the recovery phase.
Used to detect previous exposure to HBV;Slide49
Hepatitis B Ags
and Abs
Anti-hepatitis B core (anti-
HBc
),
IgM
IgM
antibody to the hepatitis B core antigen (The hepatitis B core antigen is present only in infected liver cells; it cannot be detected in the blood.)
First antibody produced after infection with HBV; used to detect acute infection
Anti-hepatitis B core (anti-
HBc
), Total
Both
IgM
and IgG antibodies to hepatitis B core antigen
Can be used to help detect acute and chronic HBV infections; it is produced in response to the core antigen and usually persists for life.Slide50
Hepatitis B Ags
and Abs
Hepatitis B e-antigen (
HBeAG
)
Protein produced and released into the blood by actively replicating hepatitis B virus
Unlike the surface antigen, the e-antigen is found in the blood only when the HBV virus is actively replicating.
HBeAg
is often used as a marker of ability to spread the virus to other people (infectivity).
Anti-hepatitis Be antibody (Anti-
HBe
)
Antibody produced in response to the hepatitis Be antigen
In those who have recovered from acute hepatitis B infection, anti-
HBe
will be present along with anti-HBc and anti-HBs. In those with chronic hepatitis B, anti-HBe can be used to monitor the infection and treatment.Slide51
New methods of hepatitis detection are now available for all of the various types of hepatitis. Using nucleic acid detection has allowed much earlier detection of the viruses and has decreased the window for detection down from 90 days to less than one week after infection.Slide52
Antimicrobial Agents
Inhibit growth (-
static
); e.g., bacteriostatic,
fungistatic
Kill organisms (-
cidal
); e.g.,
bacteriocidal
, fungicidal,
viricidal
Antimicrobial agents are classified by
-static/-
cidal
Mode of actionChemical structureSlide53
Antimicrobial Agents
(Sites of action)Slide54
Mechanisms for Development of Resistance to Antimicrobial Agents
Enzymatic
inactivation of agent
Altered
target
Altered
transport
of agent in or out
Acquisition of
genetic factors
from other resistant organismsSlide55
Advantages of Molecular Detection of Resistance to Antimicrobial Agents
Mutated genes are strong evidence of resistance.
Rapid detection without culturing
Direct comparison of multiple isolates in epidemiological investigationsSlide56
Molecular Epidemiology
Epidemic
: rapidly spreading outbreak of an infectious disease
Pandemic
: a disease that sweeps across wide geographical areas
Epidemiology
: collection and analysis of environmental, microbiological, and clinical data Slide57
Molecular Epidemiology
Phenotypic analysis measures biological characteristics of organisms.
Molecular epidemiology is a genotypic
analysis targeting genomic or plasmid DNA.
Species-, strain-, or type-specific DNA sequences are the sources of genotype information.Slide58
Pulsed-Field Gel Electrophoresis (PFGE)
O = outbreak strain
1–6 = isolates
= changes from outbreak strain
M O 1 2 3 4 5 6
M O 1 2 3 4 5 6Slide59
Criteria for PFGE Pattern Interpretation: Rule of Three
Category
Genetic Differences*
Fragment Differences*
Epidemiological Interpretation
Indistinguishable
0
0
Test isolate is the same strain as the outbreak strain.
Closely related
1
2
–
3
Test isolate is closely related to the outbreak strain.
Possibly related
2
4
–
6
Test isolate is possibly related to the outbreak strain.
Different
>
3
>
6
Test isolate unrelated to the outbreak.
*Compared to the outbreak strainSlide60
Interspersed Repetitive Elements
REP sequence inverted repeat
ERIC sequence inverted repeat
PCR amplification priming outward from repetitive elements generates strain-specific products.
Is the unknown (U) strain A or B?
Isolate B
Isolate A
M A B U
M A BSlide61
Other Genotypic Methods Used to Type Organisms
Plasmid fingerprinting with restriction enzymes
RFLP analysis
Amplified fragment length polymorphism (AFLP)
Interspersed repetitive elements
Ribotyping
spa
typing
Multilocus
sequence typingSlide62
Comparison of Molecular Epidemiology Methods
Method
Typing
Capacity
Discriminatory
Power
Reproducibility
Ease of
Use
Ease of
Interpretation
Plasmid analysis
good
good
good
high
good
PFGE
high
high
high
moderate
good
moderate
Genomic RFLP
high
good
good
high
moderate
–
poor
Ribotyping
high
high
high
good
high
PCR-RFLP
good
moderate
good
high
high
RAPD
high
high
poor
high
good
–
high
AFLP
high
high
good
moderate
high
Repetitive
elements
good
good
high
high
high
Sequencing
high
high
high
moderate
good
–h
ighSlide63
Viral Genotyping
Viral genes mutate to overcome antiviral agents.
Gene mutations are detected by sequencing.
Primary resistance mutations
affect drug sensitivity but may slow viral growth.
Secondary-resistance mutations
compensate for the primary-resistance growth defects.Slide64
Summary
Molecular-based methods offer sensitive and direct detection of microorganisms.
Due to high sensitivity and specificity, proper quality control is critical for molecular testing.
Several molecular methods are used to type bacterial strains in epidemiological investigations.
Target, probe, or signal amplification procedures are also used to determine viral load.