Laboratory Diagnosis of Infectious Viral Diseases - PowerPoint Presentation

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Laboratory Diagnosis of Infectious Viral Diseases

Prof. Ahmed S. Abdel-Moneim

College of Medicine-Taif University

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Outcomes

Knowledge

Determine the method of sample collection, transport processing and storage.

Enlist the laboratory investigations conducted for routine screening of different virological investigations including blood transfusion.

Enlist the incriminated viruses in different common affections and their preferred samples.  

Determine the laboratory diagnosis of common viral diseases and their significance in diagnosis.

Skills

Interpret the laboratory findings in different laboratory techniques used for clinical virology including serological markers and

nucleic acid amplification test

(NAAT).

Regards,

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Sampling

1)Specimen Selection

Right Place

Right time

Pathogenesis

Clinical

Signs

As soon as possible after the appearance of the clinical signs

Based on

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Sampling

Alive patient

Secretions and excretions from the affected part and blood from febrile patients should be collected .

Dead patient

Affected tissue and drainage lymph nodes.

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Sampling for serology

Blood without anticoagulants, serum is separated.

If virus isolation is intended, another sample with anticoagulant [heparin, EDTA,..

etc

.] is needed

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2)Specimen Transport and Storage

Labeling of each specimen

Adding Virus transport media

Protein

Source

Antibiotic mix

Fluid part

Stabilize virus

Prevent microbial

contamination

Prevent desiccation

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Viral transport medium (VTM)

VTM typically consists of a protein such as gelatin and anti-microbial agents in a buffered salt solution.

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Specimen transport

Enveloped viruses such as RSV and CMV are extremely labile at room-temperature and freeze-thaw cycles, whereas non-enveloped viruses such as enteroviruses tolerate these conditions well.

Large or solid samples should be placed in wide mouth sterile container.

Sample is stored at -20

⸰

C or most suitably at -70

⸰ C.Short ShipmentSample is transported in an insulated container containing crushed ice.Long Shipment

Plastic bags containing dry ice (-70 ⸰ C)…………dry iceGlycerol buffer saline (50%) can be used for transportation at room temp since glycerol prevent any microbial growth.

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Cool box

Dry

Ice

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Sample label

Barcode should contain the following:

Demographic data [age, sex, name, file number, city, occupation]

Specimen

type.

Tests

requested.

Date and time at which the specimen was taken.Clinical

informationSymptoms

Date

of onset

Suspected pathogens

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Blood sample for virus isolation

kept refrigerated until processing.

Blood sample for serology Serum should be separated as soon as possible and divided into small aliquots and kept at 4 C for short storage or at -20 C for long storage

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Specimen Processing

For virus detection 50% suspension

For virus isolation 10% suspension

Stool

Pea size aliquot of faeces is

placed in VTM or sterile PBS

The clear supernatant is used

after centrifugation

Swab

(rectal, vaginal,pharyngeal..etc)

The tip of the swab is dipped in

VTM and forced against the wall

of the tube 6-12 times.The fluid is centrifuged and the supernatant is used for

virus isoaltion

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Tissue

Homogenize the tissue in sterile

mortar using sterile sand

.

After centrifugation, the clear

supernatant is used.

Vesicular fluid

Clear used as it is

Turbid used after

centrifugation

Skin scrape

Homogenize in sterile

PBS or VTM

.

After centrifugation, the clear

supernatant is used.

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Urine

CSF

Synovial fluid

Blood is taken without anticoagulant.

Serum is separated as soon as

possible and kept in small aliquots

at -20C till used

B

L

OOD

Blood is taken on anticoagulant.

Mononuclear cells are separated by Ficoll solution, and kept at -70C till used in virus isolation

Clear

Turbid used after centrifugation

Virus isolation

Serology

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CNS samples

NEVER refrigerate CSF.

CSF should be submitted for bacterial culture and molecular testing.

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Minimizing equipment and technique-related hazards

Pipettes

Mouth pipetting is banned at all times.

Pipetting devices should be inspected routinely for leakage.

Hypodermic needles and syringes

These are the most hazardous pieces of equipment in common use.

Many needle stick accidents occur when the needle is being recapped. It is important that used needles are promptly disposed of properly into harden "sharps containers“ which are then autoclaved before disposal.

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Centrifugation

Sealed centrifuges should be used when centrifuging infectious material.

Care should be taken to ensure that the centrifuge tubes are not cracked or flawed.

Tubes should not be more than three-quarters full.

Tubes should be capped and they and the buckets should be balanced carefully to avoid vibration, which may lead to breakage.

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Treating contaminated laboratory waste

Sterilization by moist heat (autoclaving) or dry heat (hot air oven)

Chemical disinfection

The most commonly used disinfectants:

Sodium hypochlorite, phenolics, and alcohols and aldehydes.

Disposable gloves and safety spectacles, goggles, or a visor should be worn by anyone handling strong disinfectants.

Full-face respirators should be worn when rooms are being fumigated with formaldehyde.

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Standard operating procedure(SOP)

SOP also provides information on specimen collection, laboratory safety instructions, purpose and limitations of the procedure, and interpretation of results.

SOP provides complete details of how exactly a test or a procedure is carried out in a laboratory.

The procedure manual should be in detail so that an inexperienced technologist can perform the procedure without additional information.

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Risk Group 1

(no or low individual and community risk)

·

A microorganism that is unlikely to cause human or animal disease.

Risk Group 2

(moderate individual risk, low community risk)

·

A pathogen that can cause human disease but is unlikely to be a serious hazard to laboratory workers, the community or the environment.·

Examples include herpesviruses.

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Risk Group 3

(high individual risk, low community risk)

·

A pathogen that usually causes serious human but does not ordinarily spread from one infected individual to another.

·

Human immunodeficiency virus (HIV), hepatitis B virus (HBV), hantaviruses, rabies, and yellow fever virus.

Risk Group 4

(high individual and community risk)· A pathogen that usually causes serious human disease and can be readily transmitted from one individual to another.

· Examples include Ebola, smallpox, avian influenza viruses, Nipah virus, SARS-CoV, SARS-CoV-2

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Diagnostic virology laboratories must be designed for Biosafety level2(BSL2)or above.

At least one national laboratory that is equipped with Biosafety level 3(BSL3) facility is required.

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BSL-4

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BSL-3

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BSL-2

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Virology techniques used in diagnosis

Techniques used in diagnostic virology.

Cell culture [

Not used for routine diagnosis

]

Antigen detection

Fluorescent antibody stainingImmunoperoxidase antibody stainingEnzyme immunoassayLatex agglutination test

Rapid chromatographic streps

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Nucleic acid detection [

nucleic

acid

amplification

techniques

(NAATs)]Polymerase chain reaction/ real time PCR [GOLD STANDARD TEST]Minipool [MP] of 10-12 samples were used then if positive, individual test

was conducted.Other nucleic acid amplification methodsElectron microscopy [Not used for routine diagnosis]CytologyHistologyImmunohistochemistryIn situ hybridizationSerology

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Types of PCR

Nested PCR

- use to synthesize more reliable product - PCR using an outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.

RT-PCR (reverse transcriptase): RNA is converted into ssDNA by reverse transcriptase then PCR is conducted.

Multiplex PCR:

Detection of two or more target in the same reaction

Real time PCR

: Products are detected automatically by using fluorescent signals.

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Real Time PCR

It is based on detection of a fluorescent signal produced proportionally during amplification of a PCR product.

A fluorescent reporter, increases in direct proportion to the amount of PCR product in a reaction.

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Serology

Acute [recent] infection

The presence of virus specific IgM (IgG may or may not be present)

Rise of the IgG titers in two subsequent samples.

4 fold or more increase in

titre

of IgG or total

Chronic [old] infection

IgG

alone

(and

absence of

IgM).

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Techniques

and

the

viruses

they detect

Techniques and the viruses they detect

Specimen typeClinical infectionAg detection

Molecular techniques

Throat swab, Nasopharyngealaspirate or Bronchoalveolar Lavage

Respiratory infection

Influenza A/B, parainfluenzaviruses, RSVRespiratory viruses

plus metapneumovirus,bocavirus, respiratory

coronaviruses.

Cerebro

-spinal fluid

Meningitis,

encephalitis

 

HSV 1 and 2 viruses, enteroviruses,

mumps virus.

 

Faeces

(Stool)

 

Viral gastroenteritis

 

Rotavirus,

norovirus, adenovirus

Clinically suspected.

Available in only limited

centres

–norovirus, enteric adenoviruses

40/41, rotavirus.

Blood (serum)

Hepatitis B, C, parvovirus B19

 

HBV, HCV and parvovirus B19.

Blood (EDTA)

HIV, HTLV 1, CMV,

EBV, BK virus

 

HIV,

HTLV

1,

CMV,

EBV,

BK

virus.

Urine

CMV, BK and JC

virus, measles,

mumps.

 

CMV, BK and JC virus, measles

v

irus, mumps virus.

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Techniques

and

the

viruses they

detectTechniques and the viruses

they detectSpecimen type

Clinical infectionAg detectionMolecular techniques

Vesicle fluid/lesion swab

Herpes, chickenpox, shingles

Herpes simplex virus 1 and 2, varicella-zoster virus

Genital swabs Cervicitis, vaginal or urethral discharge or lesions

Herpes simplex virus 1 and 2,

Herpes simplex virus 1 and 2,

Eye infections – conjunctival/ corneal swab as indicated

Conjunctivitis or keratitis

Possible but not preferred

Enteroviruses, adenoviruses, herpes simplex virus, varicella- zoster virus

Tissue biopsies

Aa indicated

Possible but not preferred

Depending upon suspected virus

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Laboratory diagnosis of miscellaneous viral infections.

Measles; acute infection Measles IgM antibody

Rubella

Acute infection Rubella IgM antibody, culture

Congenital infection, in utero Culture or reverse transcriptase PCR analysis of amniotic fluid

Congenital infection, postpartum Rubella IgM antibody, culture

Mumps;

acute infection Mumps IgM antibody, culture

ParvovirusAcute infection Parvovirus B19 IgMChronic infection PCR analysis of serumAplastic crisis PCR analysis of serum

Congenital infection, in utero PCR analysis of amniotic fluidCongenital infection, postpartum Parvovirus IgM antibodyHHV-6; roseola PCR analysis of leukocytes in the absence of HHV 6 IgG antibodyBK; hemorrhagic cystitis,nephropathy PCR analysis of urine (hemorrhagiccystitis) or plasma (nephropathy)

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Interpretation of Hepatitis B Serologic Test Results

Tests

Results

Interpretation

HBsAg

anti-HBc

anti-HBs

negative

negative

negativeSusceptible

HBsAganti-HBcanti-HBs

negativepositivepositive

Immune due to natural infection

HBsAganti-HBcanti-HBs negative

negative

positive

Immune due to hepatitis B vaccination

HBsAg

anti-

HBc

IgM

anti-

HBc

anti-HBs

positive

positive

positive

negative

Acutely infected

Prof. Ahmed Sayed Abdel-Moneim

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HBsAg

anti-

HBc

IgM

anti-

HBc

anti-HBs

positive

positivenegativenegative

Chronically infectedHBsAganti-HBcanti-HBs negative

positivenegative

Interpretation unclear; four possibilities:  Resolved infection (most common) False-positive anti-

HBc, thus susceptible "Low level" chronic infection

Resolving acute infection Prof. Ahmed Sayed Abdel-Moneim

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Occult or latent hepatitis B virus (HBV)

Occult or latent hepatitis B virus (HBV) infection is defined as infection with detectable HBV DNA and undetectable surface antigen (HBsAg) and/or Ab in patients' blood.

It is due to suppression of HBV replication and inhibition of HBV proteins synthesis.

Prof. Ahmed Sayed Abdel-Moneim

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Diagnosis of acute and chronic HCV infection

EASL CPG HCV. J Hepatol 2018;

69:461–511.

Recommendations

All patients with suspected HCV infection should be tested for anti-HCV

Ab

in serum or plasma as first-line diagnostic test

In cases of suspected acute hepatitis C, in immunocompromised patients and patients on haemodialysis, serum or plasma HCV RNA testing should be part of the initial evaluation

If anti-HCV Ab detected, HCV RNA should be determined by a sensitive molecular method (LLOD: ≤15 IU/mL)-

Lower Limit of Detection

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Anti-HCV Ab+, HCV RNA



individuals should be retested for HCV RNA 12 and 24 weeks later to confirm definitive clearance

Serum or plasma HCV core antigen (a marker of HCV replication) can be used instead of HCV RNA to diagnose acute or chronic HCV infection when HCV RNA assays are not available and/or not affordable

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Endpoints of therapy

EASL CPG HCV. J Hepatol 2018;

69:461–511.

Recommendations

Main endpoint

Undetectable serum or plasma HCV RNA by sensitive assay

(LLOD

≤15

IU/mL) 12 weeks (SVR12) [sustained virological response] or 24 weeks (SVR24)

after EOT [End Of Treatment response]

Alternative

endpoint

Undetectable HCV core

antigen in serum or plasma

by EIA 24 weeks (SVR24) after EOT

In patients with detectable HCV core antigen prior to therapy, if HCV RNA assays are not available and/or not affordable

Grade of evidence

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HIV diagnosis

Test generation

Antigens

Detects

p24 antigen

IgM

IgG

Time to first positive (weeks)

Examples of test kits

1st

viral lysatenono

yes6-12-2nd

recombinant/syntheticnono

yes4-6OraSure OraQuick3rdrecombinant/synthetic

Use labelled antigen as conjugateno

yes

yes

3-4

Trinity Uni-Gold,

Vitros

HIV1/2

Very high sensitivity and able to detect IgM antibody; reduced the window period considerably

4th

recombinant/synthetic- includes p24 antibody to detect p24 in serum

yes

yes

yes

2-4

Genetic Systems Aptima, Abbott HIV Combo test

http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/HIVtesting-newmethods.htm

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Window Period and HIV Infection

44

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HIV Diagnostic Testing Algorithm

45

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Diagnosis of newly borne and infants.

These maternal antibodies may persist in the infant for as long as 18 months.

A definitive diagnosis of HIV-1 infection can only be made on the basis of two positive HIV-1 DNA or RNA assay results at six weeks after birth (sensitivity >98%).

For infants born to HIV-1–infected mothers, it has been recommended that diagnostic testing with HIV-1 DNA or RNA assays be performed within the first 14 days of life, at one to two months of age, and again at three to six months of age.

If any of these test results are positive, repeat testing is recommended to confirm the diagnosis of HIV-1 infection.

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Viral load testing

HIV-1viral load measurement is useful for monitoring ART treatment.

It is predicted that with successful therapy a fall of 1.5 to 2 log in plasma viral load occurs within 4-6 weeks.

With successful ART, it should become undetectable in four to six months of therapy.

The Roche

Amplicor

HIV-1 monitor test, version 1.5, is used widely. The assay uses gag specific primers for the highly conserved region. The lower limit of detection with the standard assay kit is 400 RNA copies/ml and the upper limit is 750 000 RNA copies/ml.

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Assessment of Quality System

A quality system can be assessed either through an onsite inspection (audit) or by sending known but undisclosed material to the laboratories for testing (quality assessment scheme).

The latter can be done within an institute by internal staff(internal quality assessment scheme–IQAS) or through an external agency (

externalquality

assessment scheme–EQAS).