Implementation of modern - PowerPoint Presentation

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Implementation of modern molecular techniques in detecting viruses in the laboratory

What should we do in molecular laboratory for best result?

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ROLE OF MOLECULAR TESTING BY CNS SYNDROME

Dr Maryam SotoudehChildren Hospital Center

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Genetic disorder

Neoplasm

Infectious disease

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Infectious disease

Molecular diagnostic tests

and

Nucleic

acid amplification tests

(NATs)

used

synonymously

that detect

DNA

or

RNA

specific to infectious organisms (eg, bacteria, viruses) as a means of diagnosis. Such tests have dramatically impacted both accurate identification of a pathogen and prompt initiation of antimicrobial therapy are potentially lifesaving.

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S

pinal and brain tissue are normally

sterile body

sites

.

Any

evidence of a microorganism is

likely

to represent

infection.

 

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 The laboratory diagnosis of viruses has benefited probably more than the diagnosis of any other organism from molecular-based methods

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Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition Lela Buckingham

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Nucleic acid

amplification

techniques (NAAT) as the methods of

reference

are used to

diagnosis

of viral diseases and

management

of antiviral therapy 

.

This technology has reduced the use of viral culture based methods and serological assays in the clinical virology laboratory. 

CLSI-MM6

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Detection of PCR results using:

Real

time PCRHybridization

gel

/ capillary

electrofores

is

ELISA method

 

Direct sequencing

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Summary of quality assurance issues

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Molecular Diagnostics of Infectious Diseases Edited by Harald H. Kessler,2010

Pre-analytical

errors make up to 85% of all laboratory errors

,

95%

of them occurring

outside

the laboratory

.

M

olecular assays are sensitive to suboptimal pre-analytic conditions lead to: 10

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The quantity of

CSF for molecular

Dx

at least

2

ml (30–40 drops) in

children

at least 5 ml (100 drops) in adults

.

If Mycobacterium tuberculosis is suspected,

more fluid

is required.

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CSFTransportation : for

DNA studies (HSV, VZV ): at 2 to 8 oC

for RNA studies (Enterovirus) must be chilled immediately on wet ice and the RNA extracted within

one to four

hours.

If

specimens cannot be processed immediately, it should be placed

at -20

or -70 °C or lower.

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CSFStorage: DNA is stable at 22 to 25 °C (room temperature) for up to 24

hours at 2 to 6 °C for 72 hours or more

at -20 °C at least 1 year at -70 °C more than 1 year

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Hemolysis/BloodyHemoglobin inhibits enzyme activity. Specimens received in the laboratory should therefore be inspected for visual

signs of hemolysis. Hemoglobin and anticoagulants are removed effectively in most DNA and RNA isolation procedures

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Molecular Diagnostics

Fundamentals, Methods and Clinical Applications

Second Edition Lela Buckingham

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Bilirubin, bile salts, and hemin, which are derivatives of

hemoglobin, were also found to be PCR-inhibiting

Heme blockes

the active site

of

DNA polymerase activity

.

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CSF lacks common inhibitors of PCR such as heme, endonucleases, and exonucleases

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Potential causes for

false-negative

test results

●Sampling error

●Inhibition

of amplification

● Failure of the assay to amplify or detect the microorganism

●Insufficient

analytical sensitivity

●

Insufficient

clinical

sensitivity

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Potential causes for

false-negative

test results

●Sampling error (occurs at

low nucleic acid

concentration

due

toinherently

small test volumes used in molecular testing)

●

"

Inhibition" of amplification (due to a "bloody tap

,")●Failure of the assay to amplify or detect the microorganism (rare, usually due to unrecognized species genetic variation or high rate of mutation, as with RNA viruses)●Failure to test for the causative organism (eg, appropriate pathogen-specific test not performed, as most molecular tests detect only a single targeted pathogen)18

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Potential causes for false-negative test results●Insufficient analytical sensitivity

for the specific microorganism (due to low concentration of its nucleic acid

below the functional LOD)●Insufficient

clinical sensitivity

( low concentration

of the microorganism is

due

to pathophysiology of the disease [

eg

, West Nile virus],

prolonged treatment

, and/or immune clearance

)

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Potential causes for

false-Positive test results

●

Amplification of a

contaminating

organism

●Amplification of a

noncausative

"bystander"

●

Amplification of nucleic acid from a latent (not active) infection

●Lack of specificity of assay primer or probe sequences

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Potential causes for

false-negative test results

●Amplification of a contaminating organism at

the time of specimen collection

(

eg

, nonsterile technique, blood contamination of CSF) or post-collection (

eg

, in the laboratory

)

●Amplification of a

noncausative

"bystander" (

eg, microorganism present in circulating leukocytes that cross the blood-brain barrier during acute inflammation)●Amplification of nucleic acid from a latent (not active) infection●Lack of specificity of assay primer or probe sequences21

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Importance of clinical correlation

Test results should be analyzed in the context of the probability of infection

.

In

the

absence of an elevated leukocyte count or elevated protein level

in the CSF, the likelihood of a true positive molecular

is

significantly reduced, while the number of 

false-positive and uninterpretable

results increase.

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a positive NAT result for a specific pathogen may not always indicate CNS disease and

should be interpreted with

caution, Due to the

extreme sensitivity

of these

assays

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Limiting utilization of CSF molecular tests to

patients

with a

moderate to high pretest probability of having CNS disease

based on

clinical

presentation,

disease

prevalence,

CSF

parameters

increases

the predictive value of test results and reduces cost.24

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Multiplex or panel-based NATs

combine multiple individual NATs into a single test, at the same time.

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Multiplex or panel-based NATs

In 2015, the first commercial multiplex NAT (FilmArray

)for community-acquired meningitis and encephalitis was cleared by

FDA

This

multiplex NAT detects

14 bacterial, viral, and fungal

pathogens in just over

one hour

, including 

:

S.pneumoniae

,

N.meningitidis, H. influenzae, S.agalactiae (ie, groupB Streptococcus), Escherichia coli (K1 serotype only), Listeria monocytogenes, enterovirus, HSV-1, HSV-2, VZV, CMV, HHV-6, human parechovirus, Cryptococcus neoformans/gattii.26

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nearly 1 in 6 positive results (15.6 %) were false positives Thus, it is important

to perform standard microbiologic tests such as CSF bacterial culture

when infections are suspected even when the multiplex NAT is used and not use

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Nucleic acid detection methods are often

more sensitive

than conventional culture

-based or

antigen detection

and may detect organisms that are

nonviable or uncultivable

.

Except for

HSV

and

JC virus

,

the true clinical sensitivity of most molecular tests for CNS infections is not known because there are few studies utilizing a reference standard (eg, brain biopsy) for comparison.

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Next-generation sequencing

provide

a direct unbiased

analysis

E

xample

,

NGS of the CSF in diagnose

neuroleptospirosis

in a 14-year-old boy with

SCID ,

unexplained fever and progressive neurologic deterioration

Subsequent

testing (targeted PCR for Leptospira, Sanger sequencing, and IgM antibody by latex agglutination ELISA) confirmed the diagnosis. 29

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Commercial versus noncommercial assays Most commercially available molecular tests for CSF or CNS specimens have not been approved or cleared by the FDA

As a result, the majority of assays are not standardized between laboratories, and interlaboratory variability is common.

Analytical sensitivity may vary significantly, and quantitative values (eg, viral load, copy number) are not comparable between laboratories

.

For both commercial and noncommercial assays, testing specificity for a given organism depends on the uniqueness of the target being amplified.

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ROLE OF MOLECULAR TESTING :Meningitis

Bacteria — Gram stain and culture remain the methods of choice for diagnosis of bacterial meningitis. FDA-cleared multiplex

NAT, which detects most common causes of community-acquired (including neonatal) bacterial meningitis, may be useful as an adjunct to culture, especially in patients who have already received antibiotic treatment .

NAT is useful

in select cases when uncultivable or fastidious organisms are suspected (

eg

, 

Mycoplasma

 

spp

, 

Brucella

 

spp, or Tropheryma whipplei), although these assays are not widely available.Mycobacteria — Direct detection of M. tuberculosis from CSF (eg, tuberculous meningitis) is routinely available from reference laboratories and recommended as an adjunct to traditional mycobacterial culture .Mycobacterial culture should also always be ordered when a diagnosis of tuberculous meningitis is suspected given the poor sensitivity of molecular testing from CSF.31

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ROLE OF MOLECULAR TESTING BY CNS SYNDROME :Meningitis

Viruses — Use of molecular methods for the detection of enteroviruses and Herpesviridae (eg

, HSV, VZV], CMV], [

EBV]) is standard of care

Targeted

single-pathogen NATs

FDA-cleared

multiplex NAT, which detects enterovirus, HSV-1 and -2, VZV, CMV, human herpesvirus 6 (HHV-6), and

parechovirus

 

simultaneously, is also available

Fungi

and parasites

 — Fungal and parasite stains, culture, histology, and serology are usually preferred when suspected clinically, although Cryptococcus neoformans/gattii is included in an FDA-cleared multiplex NAT as well . 32

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Molecular tests for meningitis

Etiology

Availability

*

When inclusion of NAT is indicated

Preferred testing method(s)

¶

Bacteria

Common pathogens (eg, Streptococcus pneumoniae, Haemophilus influenzae)

Yes

Δ

Gram stain positive, culture negative; prior antibacterial therapy

Gram stain and 

culture(the methods of choice )Fastidious organisms (Mycoplasma spp, Tropheryma spp, Brucella spp)YesWhen clinically suspectedSerology, NAT +/– cultureMycobacterium tuberculosis◊YesWhen clinically suspected (poor sensitivity of molecular testing from CSF)AFB stain, culture, and NAT

Prior antibiotic therapy

§

Yes

Δ

Rarely

Culture prior to antibacterial therapy

Fungal

Cryptococcus

 spp

Yes

Δ

No

Antigen detection, culture

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Molecular tests for meningitis

Etiology

Availability

*

When inclusion of NAT is indicated

Preferred testing method(s)

¶

Adenoviruses

Yes

Rarely

Testing of alternative sites (eg, respiratory) +/– CSF

Arboviruses

Limited

When clinically suspectedSerology and NATCytomegalovirus (CMV)YesΔWhen clinically suspectedNAT, preferably quantitativeEpstein Barr Virus (EBV)YesWhen clinically suspectedSerology and NAT, preferably quantitativeEnterovirusesWidespreadΔWhen clinically suspected

NAT

Human herpesvirus 6 (HHV-6)

Yes

Δ

When clinically suspected

NAT, preferably quantitative

Herpes simplex virus (HSV)

Widespread

Δ

When clinically suspected

NAT

Influenza

Yes

Rarely

Testing of alternative sites (

eg

, respiratory) +/– CSF

Mumps

Limited

Rarely

Viral culture, serology, and NAT

Parechoviruses

¥

Yes

Δ

Primarily in young children (<5 years old)

NAT

Varicella-zoster virus

Yes

Δ

When clinically suspected

NAT

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Case study

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A27 y/o female comes by the rapid onset of fever, headache, seizures, and

impaired consciousness from 12 hours ago.

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Immediate CNS invasion via the trigeminal nerve or olfactory tract following an episode of primary HSV-1 of the oropharynx; most patients with primary infection are younger than 18 years of age●CNS invasion after an episode of recurrent HSV-1 infection, which is believed to represent viral reactivation with subsequent spread

●CNS infection without primary or recurrent HSV-1 infection, which is felt to represent reactivation of latent HSV in situ within the CNS

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Laboratory tests

CSF : lymphocytic pleocytosisincreased number of erythrocytes

elevated protein PCR

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Imaging study

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Abnormal signals were seen in the right temporal lobe and insular

cortex in MRI.

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PCR for HSV1 or HSV2?

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undetectable

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Laboratory testsThe

gold standard for the diagnosis of herpes encephalitis is the detection of HSV-DNA in the CSF by

PCR.sensitivity (98 percent) and specificity (94 to 100 percent) and is positive early in the course of illness.

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Viral culture?Viral culture of

CSF is only positive in

about 4 to 5 percent of patients with brain biopsy-proven HSV encephalitis

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CSF antigen and antibody determinations

.

●The use of purified HSV glycoprotein B to detect CSF

antibodies

has a sensitivity of 97

%and

a specificity of 100

%.

However, viral antibody titers, which rise fourfold over the course of the illness, are first positive after 10 days to 2 weeks of illness and are thus only helpful retrospectively

.

●HSV antigen can also be detected in CSF, but sensitivity and specificity of this test is lower than PCR assays

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in patients with a high probability

of HSV encephalitis (suggestive neuroimaging findings, CSF pleocytosis, positive EEG findings, or seizures), a negative PCR result reduces the disease likelihood to approximately 5

%. clinicians must consider alternative reasons for a false-negative CSF PCR result

, including

:

early

testing after the onset of symptoms

,

antiviral therapy,

presence

of PCR inhibitors (

eg

, hemoglobin)44

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While awaiting results of PCR testing, treatment for HSV encephalitis should be initiated. When

present, HSV DNA is detectable via PCR analysis of the CSF for at least two weeks and up to one month after the onset of clinical disease

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While awaiting results of PCR testing, treatment for HSV encephalitis should be initiated.

When

present, HSV DNA is detectable via PCR analysis of the CSF for at least

two weeks and up to one month

after the onset of clinical disease

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Conclusion

In molecular testing

, sample collection, storage, and

handeling

are important

in

Standard

laboratory procedures aimed at performing good laboratory practice is mandatory

.

Standardization and quality improvement are ever more needed.

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