Prof Ahmed S AbdelMoneim College of MedicineTaif University 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 transfusi ID: 929963
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Slide1
Laboratory Diagnosis of Infectious Viral Diseases
Prof. Ahmed S. Abdel-Moneim
College of Medicine-Taif University
Slide2Outcomes
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,
Slide3Sampling
1)Specimen Selection
Right Place
Right time
Pathogenesis
Clinical
Signs
As soon as possible after the appearance of the clinical signs
Based on
Slide4Sampling
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.
Slide5Sampling for serology
Blood without anticoagulants, serum is separated.
If virus isolation is intended, another sample with anticoagulant [heparin, EDTA,..
etc
.] is needed
Slide62)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
Slide7Viral transport medium (VTM)
VTM typically consists of a protein such as gelatin and anti-microbial agents in a buffered salt solution.
Slide8Specimen 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.
Slide9Cool box
Dry
Ice
Slide10Sample 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
Slide11Blood 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
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
Slide13Tissue
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.
Slide14Urine
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
Slide15CNS samples
NEVER refrigerate CSF.
CSF should be submitted for bacterial culture and molecular testing.
Slide16Minimizing 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.
Slide17Centrifugation
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.
Slide18Treating 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.
Slide19Standard 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.
Slide20Risk 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.
Slide21Risk 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
Slide22Slide23Slide24Diagnostic 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.
Slide25BSL-4
Slide26BSL-3
Slide27BSL-2
Slide28Virology 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
Slide29Nucleic 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
Slide30Slide31Types 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.
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.
Slide33Serology
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).
Slide34Techniques
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.
Slide35Techniques
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
Slide36Laboratory 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)
Slide37Interpretation 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
Slide38HBsAg
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
Slide39Occult 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
Slide40Diagnosis 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
Slide41Anti-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
Slide42Endpoints 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
Slide43HIV 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
Slide44Window Period and HIV Infection
44
Slide45HIV Diagnostic Testing Algorithm
45
Slide46Diagnosis 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.
Slide47Viral 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.
Slide48Assessment 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).