Detection and Identification
of Microorganisms
Chapter 12
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. trachomatis
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 microorganisms
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.
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.
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 present
PCR Quality Control: Internal Controls
• Homologous extrinsic
• Controls for amplification
• Heterologous extrinsic
• Controls for extraction and amplification
• Heterologous intrinsic
• Human gene control
PCR Quality Control: Internal Controls
• Homologous extrinsic
• Controls for amplification
• Heterologous extrinsic
• Controls for extraction and
amplification
• Heterologous intrinsic
• Human gene control
Target sequence
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).
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 important
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 levels
Quality Control: False Negatives
• Improper collection, specimen handling
• Extraction/amplification failure: check internal controls
• Technical difficulties with chemistry or instrumentation: check
method and calibrations
Detection of Bacteria
Respiratory Diseases
• Respiratory infections are responsible for significant numbers of
infections and deaths worldwide.
• These infections are easily spread by inhalation
Bordetella (Whooping Cough)
• Bordetella pertussis is a Gram-negative, aerobic coccobacillus
• B. pertussis is nonmotile. Its virulence factors include pertussis toxin,
filamentous hemagglutinin, and tracheal cytotoxin.
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
high
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.
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.
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.
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.
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 acidfast-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.
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.
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 specimens
Tuberculosis
• Urine is being increasingly evaluated.
• A new method for detecting Mycobacterium tuberculosis (MTB) uses
combined IMS/ATP assay.
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.
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.
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.
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.
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.
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.
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
analysis
“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.
HIV
• Molecular testing is important in HIV, not only for detection of the
disease, but also continued monitoring of disease treatments.
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.
Detection of HIV
• There 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.
• 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.
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.
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.
Treatment of HIV
• In an HIV‐infected individual, the concentration of virus in the bloodst
ream or viral load (VL) can be a valuable tool for the clinical manage
ment 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.
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 clinicia
n to assess the success of treatment and detect treatment failure prior
to the onset of clinical symptoms.
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 variations
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.
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.
Hepatitis B Ags and Abs
Hepatitis B surface antigen Protein that is present on
(HBsAG)
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)
Used to detect previous
exposure to HBV;
Antibody produced in
response to HBV surface
antigen; levels in the blood
rise during the recovery
phase.
Hepatitis B Ags and Abs
Anti-hepatitis B core (antiHBc), 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 (antiHBc), 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.
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, antiHBe 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.
• 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.
Antimicrobial Agents
• Inhibit growth (-static); e.g., bacteriostatic, fungistatic
• Kill organisms (-cidal); e.g., bacteriocidal, fungicidal, viricidal
• Antimicrobial agents are classified by
1. -static/-cidal
2. Mode of action
3. Chemical structure
Antimicrobial Agents
(Sites of action)
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 organisms
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
investigations
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
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.
Pulsed-Field Gel Electrophoresis (PFGE)
M
O 1
2
3 4 5 6
M
O 1
2
3 4 5 6
O = outbreak strain
1–6 = isolates
= changes from
outbreak strain
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 strain
Interspersed Repetitive Elements
REP sequence inverted repeat
….GTGAATCCCCAGGAGCTTACATAAGTAAGTGACTGGGGTGAGCG….
ERIC sequence inverted repeat
GCC G/T GATGNCG G/A CG C/T NNNNN G/A CG C/T CTTATC C/A GGCCTAC
PCR amplification priming outward from repetitive elements generates strainspecific products.
Isolate A
Isolate B
M
A
B
Is the unknown (U) strain A or B?
M
A
B
U
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 typing
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–high
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 primaryresistance growth defects.
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.