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Detection and Identification of Microorganisms Detection and Identification of Microorganisms

Detection and Identification of Microorganisms - PowerPoint Presentation

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Detection and Identification of Microorganisms - PPT Presentation

Chapter 12 Target Microorganisms for MolecularBased Testing Those that are difficult or timeconsuming to isolate eg Mycobacteria Hazardous organisms eg Histoplasma Coccidioides ID: 306120

hiv detection high hepatitis detection hiv hepatitis high infection molecular methods good antigen antibody sensitivity diagnosis amplification tuberculosis viral assays tests control

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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. Slide32
Slide33

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.Slide35
Slide36

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. Slide38
Slide39

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.Slide40
Slide41

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.Slide47
Slide48

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.