Viral diagnostics eac for finals

VIRAL DIAGNOSTICS

February 2012

Friday, March 2, 2012

REMINDER: REMAINING SCHEDULE

Modifications:

PH Viruses merged with Emerging and Re-emerging

Viruses (March 2)


PLENARY GROUPS: Advances in Laboratory Diagnostics (Then and Now)

• INFLUENZA (AH1N1 and SEASONAL FLU) by Advincula to Bundang Group

• INFLUENZA (AVIAN FLU) by Cailao to Emerciana Group

• DENGUE by Faderog to Jamias Group

• HIV by Lao to Manahan Group

• HEPATITIS A/B/C by Pedregosa to Santos, Dana Group

• SARS by Santos, Fatima to Velasco Group


LABORATORY DIAGNOSIS OF

VIRAL INFECTIONS


STORAGE AND COLLECTION OF BIOLOGICAL SPECIMENS FOR

VIRAL TESTING



VIRAL TESTING• What types of specimens are

collected to diagnose?





• Respiratory tract infections: Nasal and bronchial washings, throat and nasal swabs, sputum






• Eye infections: throat and conjunctival swab/scraping







• Gastrointestinal tract infections: stool and rectal swabs








• Vesicular rash: vesicle fluid, skin scrapings









• Maculopapular rash: throat, stool, and rectal swabs










• CNS (encephalitis and meningitis cases): stool, tissue, saliva, brain biopsy, cerebrospinal fluid











• Genital infections: vesicle fluid or swab












• Urinary tract infections: urine












• Urinary tract infections: urine

• Bloodborne infections: blood


General Categories

•Direct Examination

• Indirect Examination (Virus Isolation)

•Serology


DIRECT EXAMINATION


DIRECT: clinical specimen is examined directly for the presence of virus particles, virus antigen or viral nucleic acids

• Electron Microscopy morphology / immunoelectron microscopy

• Light microscopy histological appearance - e.g. inclusion bodies

• Antigen detection immunofluorescence, ELISA etc.

• Molecular techniques for the direct detection of viral genomes


ELECTRON MICROSCOPY• BASIS: morphology

• MAGNIFICATION: 50,000

• USE:

• diagnosis of viral gastroenteritis by detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses

• detection of viruses in vesicles and other skin lesions, such as herpesviruses and papillomaviruses

• NOTE: With the availability of reliable antigen detection and molecular methods for the detection of viruses associated with viral gastroenteritis, EM is becoming less and less widely used


ELECTRON MICROSCOPY• SENSITIVITY & SPECIFICITY ISSUES:

• sensitivity and specificity of EM may be enhanced by immune electron microscopy

• virus specific antibody is used to agglutinate virus particles together and thus making them easier to recognize, or to capture virus particles onto the EM grid

• DISADVANTAGE:

• expense involved in purchasing and maintaining the facility

• poor sensitivity (at least 105 to 106 virus particles per ml in the sample required for visualization)

• observer must be highly skilled


Electronmicrographs of viruses commonly found in stool specimens from patients suffering from gastroenteritis. From left to right: rotavirus, adenovirus, astroviruses, Norwalk-like viruses. (Courtesy of Linda M. Stannard, University of Cape Town, http://www.uct.ac.za/depts/mmi/stannard/emimages.html)

Influenza Virus

Ebola Virus


IMMUNOELECTRON MICROSCOPY

NEW CASTLE DISEASE (Veits J et al. PNAS 2006;103:8197-8202)

PARAMYXOVIRUSESInt. J. Morphol., 28(2):627-636, 2010

COXSACKIE B4 VIRUS (http://www.rightdiagnosis.com)


http://www.rightdiagnosis.com

http://www.rightdiagnosis.com

LIGHT MICROSCOPY

• ASSUMPTION: Replicating virus often produce histological changes in infected cells.

• Viral inclusion bodies: collections of replicating virus particles either in the nucleus or cytoplasm

• EXAMPLES: negri bodies (RABIES) and cytomegalic inclusion bodies (CYTOMEGALOVIRUS / CMV)

• Not sensitive or specific; BUT useful adjunct in the diagnosis of certain viral infections


LIGHT MICROSCOPY

RABIEShttp://infectionnet.org’; http://virology-online.com

CYTOMEGALOVIRUShttp://www.meddean.luc.edu

http://en.citizendium.org


http://infectionnet.org

http://infectionnet.org

http://virology-online.com

http://virology-online.com

http://www.meddean.luc.edu

http://www.meddean.luc.edu



ANTIGEN DETECTION Immunofluorescence (IF)

• specimen: nasopharyngeal aspirates for respiratory viruses (e.g.. RSV, flu A, flu B, and adenoviruses)

• specimen: stool (rotavirus)

• specimen: skin scrapings (HSV)

• specimen: serum (HepB)*

• Advantage: rapid to perform; result being available within a few hours

• Disadvantage: technique is often tedious and time consuming; result difficult to read and interpret; sensitivity and specificity poor

• NOTE: quality of the specimen obtained is of utmost importance in order for the test to work properly

• * also categorized as serological assay



CYTOMEGALOVIRUShttp://www.med.upenn.edu


http://www.med.upenn.edu

http://www.med.upenn.edu


AFRICAN SWINE FEVERJournal of General Virology March 2005; 86 (3)

VARICELLA ZOSTER VIRUSBMJ Case Reports 2009; doi:10.1136/bcr.07.2008.0461


ANTIGEN DETECTION Molecular Probes

• Dot-blot and Southern-blot: use of specific DNA/RNA probes for hybridization

• Specificity: depends on the conditions used for hybridization

• Allow for the quantification of DNA/RNA present in the specimen

• Sensitivity: not better than conventional viral diagnostic methods.


ANTIGEN DETECTION Molecular Probes

EPSTEIN-BARR VIRUSMol Path 2000;53:255-261 doi:10.1136/mp.53.5.255

CYTOMEGALOVIRUShttp://www.fgsc.net


http://www.fgsc.net

http://www.fgsc.net

ANTIGEN DETECTION Molecular Probes (PCR)

• extremely sensitive technique (1 DNA molecule in a clinical specimen)

• ISSUES: contamination (danger of false + result); + result may not necessarily indicate the presence of disease

INFLUENZAhttp://www.nanohelix.net


http://www.nanohelix.net

http://www.nanohelix.net

ANTIGEN DETECTION Molecular Probes (PCR)


INDIRECT EXAMINATION


INDIRECT: the specimen into cell

culture, eggs or animals in an attempt to grow the virus (virus isolation)

• Cell Culture - cytopathic effect, haemadsorption, confirmation by neutralization, interference, immunofluorescence etc.

• Eggs pocks on CAM - haemagglutination, inclusion bodies

• Animals disease or death confirmation by neutralization


RECALL: Koch’s Postulates



1. Organism present only in diseased individuals



1. Organism present only in diseased individuals

2. Organism cultivated in pure culture from diseased individual


3. Organism causes disease when injected into healthy individuals



3. Organism causes disease when injected into healthy individuals

4. Organism re-isolated from infected individual from point 3.



• Isolate virus from diseased hosts

• Cultivation of virus in host cells

• Proof of filterability

• Production of a comparable disease when the cultivated virus is used to infect experimental animals

• Re-isolation of the same virus from the infected experimental animal

• Detection of a specific immune response to the virus

MODIFICATION TO THE KOCH’S POSTULATE

(T.M. River, 1937)


TYPES OF CELL CULTURE

• Primary cells - e.g. Monkey Kidney

• essentially normal cells obtained from freshly killed adult animals; can only be passaged once or twice

• Semi-continuous cells - e.g. Human embryonic kidney and skin fibroblasts

• taken from embryonic tissue; may be passaged up to 50 times

• Continuous cells - e.g. HeLa, Vero, Hep2, LLC-MK2, BGM

• immortalized cells i.e. tumour cell lines; may be passaged indefinitely

NOTE: Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses BUT are very expensive and it is often difficult to obtain a reliable supply. Continuous cells are the most easy to handle BUT range of viruses supported is often limited


PRESENCE OF GROWING VIRUS IS USUALLY

DETECTED BY:• Cytopathic Effect (CPE) - may be specific or

non-specific e.g. HSV and CMV produces a specific CPE, whereas enteroviruses do not

• Haemadsorption - cells acquire the ability to stick to mammalian red blood cells

• mainly used for the detection of influenza and parainfluenzaviruses.

NOTE: Confirmation of the identity of the virus may be carried out using neutralization, haemadsorption-inhibition, immunofluorescence, or molecular tests


Tissue culture cells are grown on coverslips on the bottom of shell vials

Detection of Herpes Virus Simplex 1 using the shell vial technique and immunofluorescence


Quantitative Plaque Assays

Quantitative Assays


CYTOPATHIC EFFECTS


CYTOPATHIC EFFECTS

• Visible results of viral infection

• Cell death by

• Multiplying viruses

• Inhibition of DNA, RNA or protein synthesis

• Effects on permeability of membrane


CYTOPATHIC EFFECTS

• Cytopathic effects (CPEs) of infected cells can be observed with inverted light microscopes

• Rounding/detachment from plastic flask

• Syncytia/fusion (Fusion of cells)

• Shrinkage

• Increased refractility

• Aggregation

• Loss of adherence

• Cell lysis/death

Cytopathic effect of HSV, enterovirus 71, and RSV in cell culture. Note the ballooning of cells in the cases of HSV and enterovirus 71. Note syncytia formation in the case of RSV. (Linda Stannard. University of Cape Town, Virology Laboratory, Yale-New Haven Hospital)


Quantitative AssaysTissue Culture Infectious Dose: TCID50

Measure cytopathic effects other than lysisConcentration of virus it takes to produce

cytopathic effect (CPE) in 50% of the dishes of cells infected with virus


Quantitative Assays

TCID50 Assays


PROBLEMS WITH CELL CULTURE

• Long period (up to 4 weeks) required for a result to be available

• Sensitivity is often poor and depends on many factors, such as the condition of the specimen, and the condition of the cell sheet

• Very susceptible to bacterial contamination and toxic substances in the specimen

• Many viruses will not grow in cell culture at all e.g. Hepatitis B and C, Diarrhoeal viruses, parvovirus etc.


SEROLOGY


SEROLOGICAL TESTS• ASSUMPTION:

• Primary Exposure: first antibody to appear is IgM, which is followed by a much higher titre of IgG

• Secondary exposure or Re-infection: level of specific IgM either remain the same or rises slightly But IgG shoots up rapidly and far more earlier than in a primary infection.

• ASSAYS AVAILABLE:

• EIA and RIA, one can look specifically for IgM or IgG (most sensitive)

• CFT and HAI, one can only detect total antibody, which comprises mainly IgG (not so sensitive)

• The sensitivity and specificity of the assays depend greatly on the antigen used

• Assays that use recombinant protein or synthetic peptide antigens tend to be more specific than those using whole or disrupted virus particles


SEROLOGY: detection of rising titres

of antibody between acute and convalescent stages of infection


NOTE: CRITERIA FOR PRIMARY INFECTION

• A significant rise in titre of IgG/total antibody between acute and convalescent sera

• however, a significant rise is very difficult to define and depends greatly on the assay used

• CFT and HAI: normally taken as a four-fold or greater increase in titre

• The main problem is that diagnosis is usually retrospective because by the time the convalescent serum is taken, the patient had probably recovered



• Presence of IgM

• EIA, RIA, and IF may be used for the detection of IgM

• offers a rapid means of diagnosis

• PROBLEMS: interference by rheumatoid factor, re-infection by the virus, and unexplained persistence of IgM years after the primary infection



• Seroconversion

• changing from a previously antibody negative state to a positive state e.g. seroconversion against HIV following a needle-stick injury, or against rubella following contact with a known case

• A single high titre of IgG (or total antibody)

• very unreliable means of serological diagnosis since the cut-off is very difficult to define


SEROLOGY: detection of rising titres

of antibody between acute and convalescent stages of infection

• Classical Techniques

• Complement Fixation Test

• Hemagglutination Inhibition Test

• Immunofluorescence Technique (IF

• Neutralization Tests

• Single Radial Hemolysis

• Newer Techniques

• Radioimmunoassay (RIA)

• Enzyme Immunoassay (EIA)

• Particle Agglutination Tests

• Western Blot (WB)

• Recombinant Immunoblot assay (RIBA)


LIMITATIONS OF SEROLOGY

• For viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide with the development of antibodies (detection of IgM or rising titres of IgG in the serum of the patient would indicate active disease)

• Many viruses often produce clinical disease before the appearance of antibodies such as respiratory and diarrheal viruses (any serological diagnosis would be retrospective and therefore will not be that useful)

• There are also viruses which produce clinical disease months or years after seroconversion e.g. HIV and rabies (mere presence of antibody is sufficient to make a definitive diagnosis)


DISADVANTAGES OF SEROLOGY



• Long length of time required for diagnosis for paired acute and convalescent sera




• Mild local infections may not produce a detectable humoral immune response (e.g. HSV genitalis)





• Extensive antigenic cross-reactivity between related viruses may lead to false positive results (e.g. Japanese B encephalitis and Dengue)






• Immunocompromised patients often give a reduced or absent humoral immune response







• Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result







• Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result

• Patients given blood or blood products may give a false positive result due to the transfer of antibody


HEMAGGLUTINATION/HEMAGGLUTINATION-INHIBITION/

COMPLEMENT FIXATION TEST


• Some viruses agglutinate RBCs

• Mumps, measles, influenza

• Hemagglutination

• Clumps RBCs


HEMADSORPTION TEST

Hemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus (Courtesy of Linda Stannard, University of Cape Town).


ENZYME-LINKED IMMUNOSORBENT

ASSAY (ELISA)


HIV & ELISA


WESTERN BLOT TO CONFIRM HIV


Figure 5.21b: The structure of HIV-1.

Image courtesy of Bio-Rad Laboratories

Figure 5.21c: The typical results of a Western blot testing patient serum for HIV-1 antibodies.

(c)(b)


WESTERN BLOT TO CONFIRM HIV


SEROLOGY AND HEPATITIS


USUALLY DIAGNOSED BY SEROLOGY

• Hepatitis Viruses - hepatitis A, B and C infections are usually diagnosed by serology as these viruses cannot be routinely cultured

• including the test for HBsAg

• HIV - HIV infection is normally diagnosed by serology

• The only instance when serology cannot be relied on is in diagnosing HIV infection in the newborn


USUALLY DIAGNOSED BY SEROLOGY

• Rubella and parvovirus - rubella and parvovirus infections are usually diagnosed by serology

• difficult to isolate and parvovirus cannot be isolated by routine cell culture

• onset of clinical symptoms for these infections coincide with the appearance of antibodies and thus there is little need for other means of diagnosis

• EBV - although EBV serology is reliable, the heterophile antibody test is usually used for diagnosing cases of infectious mononucleosis


MAY BE DIAGNOSED BY SEROLOGY BUT NOT METHOD OF CHOICE

• HSV - although CFT and other serological tests are available for HSV, HSV infections are usually diagnosed by cell culture

• Electron microscopy, immunofluorescence and PCR are available as rapid diagnostic methods

• Serology is not that reliable in the case of HSV infections, in particular reactivations

• CMV - although serology is available for diagnosing CMV infections, it is not reliable as most cases of CMV infections are a result of reactivation/reinfection

• Cell culture (including the DEAFF test) and rapid methods such as the CMV antigenaemia test and PCR are preferred means of diagnosis



• Respiratory viruses - diagnosis of respiratory virus infections is more commonly made by cell culture or more rapidly by immunofluorescence of the clinical material

• CFT and HAI techniques are usually used for serology and any diagnosis is going to be retrospective

• Enteroviruses - enterovirus infections are usually diagnosed by cell culture

• Serology has a very limited role to play as available tests such as neutralization, are cumbersome to perform and in any case, the diagnosis would be retrospective



• Rabies - serology is used along with other direct detection methods in diagnosing rabies and it may be used to check for immunity after vaccination

• Arboviruses - arbovirus infections may be diagnosed by serology or virus isolation

• Arboviruses will not usually grow in routine cell cultures and may require mosquito cell lines or animal inoculation.


NOT NORMALLY DIAGNOSED BY SEROLOGY

• Diarrhoeal viruses - diagnosis is going to be retrospective

• normally diagnosed by electron microscopy and the detection of viral antigens by ELISA or particle agglutination

• Papovavirus - serology is of virtually no value in diagnosing papovavirus infections

• Poxviruses - serology is of little value in diagnosing poxvirus infections


SAFETY FIRST!

Figure 5.25b: A CDC researcher working on a BSL-4 infectious agent.

Figure 5.25c: A CDC scientist showers in a protective suit before leaving a BSL-4 laboratory.


QUIZ TIME!Friday, March 2, 2012

• Draw the general serologic course of a viral disease/infection and label properly (6 points)

• Give one direct examination to diagnose a given viral pathogen and cite one virus that can use this method for diagnosis (8 points)

• Give one indirect examination to diagnose a given viral pathogen cite one virus that can use this method for diagnosis (8 points)

• Give one serological examination to diagnose a given viral pathogen cite one virus that can use this method for diagnosis (8 points)


Viral diagnostics eac for finals

Health & Medicine

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