Lesson 5

• Specimen Collection, Handling and Transport

  1. Obtain during the acute phase (maximal viral shedding-disease prodrome). Best time to collect is ASAP.
  2. Cotton/Dacron/rayon swabs are used. Calcium alginate swabs inactivates some viruses. (except Neisseria spp). Use aseptic technique.
  3. Aspirated secretions are preferable.
  4. Keep cool and moist, especially for tissue sample.
  5. Storage/transport temp: 4' C (<3/4 days); -70'C (>3/4 days)
  6. Viral Transport medium (VTM) for respiratory, swab and tissue specimens: most contains buffered isotonic solution with protein.
• Buffered isotonic solution with proteins

  • Albumin
  • Gelatin
  • Serum
  • Antibacterial agents
  • Antifungal agents: Leibovitz-Emory, Hank's BSS, Stuart's medium, Amies medium
• Swab Bud Fibers

  • Cotton - natural product that contains fatty acids: oleic acid. These can inhibit recovery of some bacteria if not used immediately. Sterilization by irradiation can make this inhibitive effect worse, especially transport device.
  • Viscose - a semi-synthetic material, where the starting component is wood pulp. Ideal for general purpose transport devices. Tips preserve the sample longer (no fatty acids)
• Swab Bud Fibers

  • Polyester - a fully synthetic material with high absorbency. Ideal for hygiene testing with most commercial luminometer techniques.
  • Alginate - natural organic fibre extracted in a process from seaweed. Swab tip dissolves fully in Calgon Ringers Solution.
• Normal Tip 


  5mm diameter
• Reduced Tip


  3mm diameter
• Mini Tip 


  2mm diameter
• Diagnostic Techniques

  1. Direct Detection: Microscopy: Brightfield, Electron, Fluorescent; EIAs: detection of antigens
  2. Nucleic Acid-Based Detection
  3. Viral Isolation: Cell Cultures
  4. Serologic Assays: Detection of Antibodies
• Brightfield Microscopy

  • best for detection poxviruses
  • detection of cytopathic effect (CPE)

  -Tzanck smear: Cowdry type A bodies (HSV - Herpes Simplex Virus and VZV - Varicella Zoster Virus)

  -Papanicolau (Pap) smear: HPV-associated koilocytes

  -Negri bodies (Rabies virus)
• Electron Microscopy

  • greater magnification: viral morphology
  • useful for detection of non-culturable viruses (Norwalk virus in stool filtrates)
  • uses negative stains (gold/silver/phosphotungstic)
  • rarely used in clinical laboratories: expensive, labor-intensive, and not very sensitive detection of viruses
• Fluorescence Microscopy/ Immunofluorescence/ Direct Fluorescent Antibody (DFA) test

  • detects various viral agents with increased sensitivity
  • respiratory specimens: adenovirus, influenza viruses A and B, measles virus, parainfluenza viruses (PIVs) 1 thru 4, and RSV
  • cutaneous lesions: HSV 1, HSV 2, VZV
  • blood: CMV
• Enzyme Immunoassays

  • most use multi-well microtiter plate assays

  -respiratory specimens: influenza virus A and RSV

  -serum/plasma: HBV and HIV-1

  -stool: enteric adenoviruses

  -cutaneous and conjunctival swabs: HSV

  • immunochromatography

  -respiratory samples: influenza virus A and B, RSV

  -stool and rectal swabs: enteric adenoviruses and rotaviruses

  • less sensitive than IF/ cell culture
  • most common viral testing
• Nucleic Acid-Based Detection

  • quantitative result may be obtained (not just +/-)
  • Adv: faster TAT, better sensitivity than culture and DFA, quantitation, detection of nonculturable viruses (Noroviruses and Hepa viruses), capability for simultaneous detection of multiple viruses (multiplex), potential for genetic characterization (genotyping)
  • Disadv: detection of active and inactivated virus, higher cost, need for specialized training and more complex facilities, lack of assays approved by US FDA
  • Examples: PCR, rtPCR, branched DNA assay, NASBA, Luminex system (multiplex)
• Viral Isolation

  • gold standard in clinical virology
  • methods:

  1. Cell culture - most common
  2. Animal inoculation - extremely costly
  3. Embryonated eggs - rarely used; enhances isolation of influenza viruses
• Cell Culture under Viral Isolation

  • culture of cells (not organized into a tissue) in vitro

  1. Primary cell lines
  2. Low passage/finite
  3. Continuous
• Primary cell lines

  • tissue removed from an animal; diploid
  • few passages only
  • ex: primary monkey kidney (PMK) cells
• Low passage / finite

  • diploid; limited passage of ~50 generation
  • ex: human neonatal lung, human diploid fibroblasts (HDF)
• Continuous

  • heteroploid; capable of infinite passages
  • ex: HEp2 (human epithelial laryngeal CA), A549 (human lung carcinoma), Vero (monkey kidney)
• Herpes Simplex Virus (HSV)


  PMK: -

  HDF: +++

  HEp2: +++

  Rabbit Kidney: ++++

  A549: +++

  CPE: large, rounded cells
• Cytomegalovirus (CMV)


  PMK: -

  HDF: +++

  HEp2: -

  Rabbit Kidney: -

  A549: -

  CPE: large, rounded cells
• Varicella Zoster Virus (VZV)


  PMK: -

  HDF: +++

  HEp2: -

  Rabbit Kidney: -

  A549: +/-

  CPE: foci or rounded cells; possible syncytium
• Enterovirus


  PMK: +

  HDF: +

  HEp2: ++

  Rabbit Kidney: -

  A549: +

  CPE: refractile, round cells in clusters
• Adenovirus


  PMK: +

  HDF: ++

  HEp2: +++

  Rabbit Kidney: -

  A549: ++

  CPE: large, rounded cells in clusters
• Respiratory Syncytial Virus (RSV)


  PMK: +/-

  HDF: +/-

  HEp2: +++

  Rabbit Kidney: -

  A549: ++

  CPE: syncytia
• Influenza, Parainfluenza


  PMK: +++

  HDF: +/-

  HEp2: -

  Rabbit Kidney: -

  A549: -

  CPE: variable - none to granular appearance
• Cytopathic Effects (CPE) on Cell Culture under Viral Isolation

  • Some very characteristic CPE can provide presumptive identification of viruses
  • ex: HSV - large, rounded cells (focal)
  • ex: CMV - HSV-like CPE, but grow more slowly and only in diploid fibroblasts
  • ex: VZV - also HSV-like
  • ex: Enterovirus - small, round cells (diffuse); Adenovirus - large, round cells (diffuse or focal, which may appear as cluster of grapes)
• Cytopathic Effects (CPE) on Cell Culture under Viral Isolation

  • ex: RSV - syncytia (giant, multinucleated cells)
  • many respiratory viruses does not produce characteristic CPE
  • Influenza CPE: granular appearance which is typically does not exhibited, so viral hemagglutinin protein is detected via hemadsorption and viral hemagglutination
• Hemadsorption

  • RBC suspension added to infected cell monolayer
  • (+) RBCs will adsorb/stick to the infected cells

  Viral Hemagglutination

  • Viral supernatant (from cell monolayer) + RBC suspension
  • (+) Agglutination
• Other Methods to Detect Virus on Cell Culture

  • Fluorescent antibody stains may also be used to screen cell cultures prior to release of negative results
  • IF, EIA, and nucleic-acid based tests may also be used
• Cell Culture Preparation

  • 35'C - temp used for dissociation of cells
  • after distribution of cells, tubes are incubated at 36-37'C until confluence of cells
  • 35'C - incubation for isolation of viruses (33'C for rhinoviruses and myxoviruses)
  • Round bottom tubes - read under conventional microscopes
  • Shell vial - contains a monolayer of cells; read under inverted microscopes
  • Roller drum - used for incubating tubes with gentle agitation
• Shell Vial Culture Technique

  • more rapid virus ID than traditional cell culture
  • shell vial: small, round, flat-bottomed tube, commonly screw-capped
  • Procedure: round coverslip in shell vial + cell monolayer + sample inoculated - centrifuged for viral absorption, incubated for 24-48 hrs, coverslip removed, IF technique is performed
  • Disadv: labor-intensive and increased TAT
• Serologic Assays

  • detection of antibodies after exposure
  • provides limited information and certain problems

  -measure host response, not detection of viruses

  -differing antibody-producing capabilities per host

  -antibody level does not always correlate with acuteness/ activity level of infection

  -cross-reactions may occur

  -difficult interpretation

  • diagnosis of recent recent infection: seroconversion
• Serologic Assays

  • indications for serologic testing

  -diagnosis of nonculturable viruses (hepa virus)

  -diagnosis of past or acute infection from various viral pathogens

  -determination of immune status (rubella, measles, VZV, HAV, HBV)

  -patient monitoring of immunosuppressed patient (transplant patients)

  -epidemiologic or prevalence studies