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    DR Sonnie P. Talavera

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    Yna del Castillo
    Viral culture and identification methods are important for diagnosing viral infections and guiding patient management. Common techniques include growing viruses in cell cultures, using shell vial cultures for rapid identification, and molecular methods like nucleic acid testing. Accurate diagnosis allows for proper treatment and infection control. A variety of cell types can support growth of different viruses. Primary cells and cell lines are used, and hybrid cell cultures have been developed to detect more respiratory and enteric viruses.

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    DR Sonnie P. Talavera

    Uploaded by

    Yna del Castillo
    137 views36 pages
    Viral culture and identification methods are important for diagnosing viral infections and guiding patient management. Common techniques include growing viruses in cell cultures, using shell vial cultures for rapid identification, and molecular methods like nucleic acid testing. Accurate diagnosis allows for proper treatment and infection control. A variety of cell types can support growth of different viruses. Primary cells and cell lines are used, and hybrid cell cultures have been developed to detect more respiratory and enteric viruses.

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    INTROVIROLOGYmedtech

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    Viral culture and identification methods are important for diagnosing viral infections and guiding patient management. Common techniques include growing viruses in cell cultures, using shell vial cultures for rapid identification, and molecular methods like nucleic acid testing. Accurate diagnosis allows for proper treatment and infection control. A variety of cell types can support growth of different viruses. Primary cells and cell lines are used, and hybrid cell cultures have been developed to detect more respiratory and enteric viruses.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    137 views36 pages

    DR Sonnie P. Talavera

    Uploaded by

    Yna del Castillo
    Viral culture and identification methods are important for diagnosing viral infections and guiding patient management. Common techniques include growing viruses in cell cultures, using shell vial cultures for rapid identification, and molecular methods like nucleic acid testing. Accurate diagnosis allows for proper treatment and infection control. A variety of cell types can support growth of different viruses. Primary cells and cell lines are used, and hybrid cell cultures have been developed to detect more respiratory and enteric viruses.

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    of 36

    m  m

    r r r 

    DR SONNIE P. TALAVERA
    r   
    ¬ Viruses are the most frequent cause of
    human infectious diseases
    ± range from trivial colds to fatal
    immunoimpairment and opportunistic infections.
    ¬ Virus isolation in cell culture and viral antigen
    identification
    ± community hospital laboratories to diagnose
    infection
    ± molecular methods.
    ¬ Accurate viral diagnosis is fundamental for
    optimal patient management
    ± reasonable use of antiviral drugs,
    ± reduction of unnecessary lab testing
    ± inappropriate antimicrobial therapy,
    ± application of infection control procedures and
    for public health control of community outbreaks.
    ¬ any common viruses grow in vitro in cell
    culture
    ± primary, diploid or heteroploid cell lines;
    ¬ cytopathic changes that develop in the cell culture
    monolayer
    ± detection of viral antigen in the cells
    ¬ fluorescence-labeled specific antibodies.
    ± propagate in newborn mice or embryonated egg
    tissue,
    ¬ .
    ¬ Shell vial culture technique uses the cell
    monolayer
    ± short incubation (1-3 days), the monolayer is
    stained with fluorescein-labeled specific viral
    antibody.
    ± ID of herpes simplex virus (HSV),
    cytomegalovirus (C V), varicella zoster virus
    (VZV), respiratory viruses, and enteroviruses.
    ¬ Hybrid culture systems employ two cell lines
    ± recovery of the common respiratory viruses
    (influenza, parainfluenza, respiratory syncytial
    virus (RSV), and adenovirus), the enteroviruses,
    and HSV/VZV.
    ¬ Nucleic acid hybridization and nucleic acid
    amplification tests (NAAT)
    ± DNA or RNA viral genome.
    ± human papillomavirus (HPV), C V, HSV, and
    parvovirus B19 in tissue;
    ¬ NAATs such as polymerase chain reaction,
    strand displacement amplification, and
    nucleic acid sequence-based amplification
    ± molecular diagnosis beyond culture or antigen detection
    methods.
    ¬ Direct visualization of viral particles by
    electron microscopy
    ± ID morphologic features of agents of
    gastroenteritis
    ¬ rotavirus, enteric adenovirus, norovirus, astrovirus and
    calicivirus;
    ± none of these viruses replicates in standard cell
    culture lines.
    ¬ Viral serology has two major applications ±
    diagnosis of recent infection and
    determination of immunity.
    ± Ig -specific antibody during acute illness or 4-fold rise
    in IgG antibody between acute and convalescent sera
    designates   .
    ± Persistent specific IgG is marker of  
    
    for
    several viruses such as measles, mumps, VZV, hepatitis
    A and B.
    ¬   

      
    
    
    
     
    
    r r
    ¬ community hospital with adult and pediatric
    primary care and high risk obstetrics,
    neonatology, hematology±oncology, and HIV
    services.
    ¬ Average detection time for most viruses is
    often 2 days or less
    ¬ reflects the use of rapid antigen assays,
    ¬ shell vial cultures with short incubation
    schedules,
    ¬ nucleic acid amplified tests.
    ¬ Overall recovery rates for viruses are high±
    16-23%± several times greater than for
    routine bacterial blood or stool cultures,
    ¬ any common viruses exhibit seasonal
    variations
    ¬ Influenza, respiratory syncytial virus, and
    parainfluenza virus 1 and 2 circulate every
    winter.
    ¬ Adenovirus, parainfluenza virus 3,
    cytomegalovirus, and herpes simplex
    infections occur year-round.
    ¬ Enterovirus disease clusters in late summer
    and early autumn.
    Y  

    ¬ clinically significant viruses replicate in vitro,


    ¬ virus recovery correlates well with acute
    disease.
    ¬ culture
    ± labor-intensive,
    ± requires technical proficiency,
    ± means at least one-day turnaround for positive
    results.
    Y  
    ¬ Replication in newborn mice or embryonated eggs
    ± essential for some fastidious viruses
    ¬ Tissue culture cells are divided into three
    categories:
    ± primary cell cultures,
    ± diploid cell lines,
    ± heteroploid cell lines.
    Y  

    ¬ Primary cell cultures


    ± prepared directly from the parent organ (e.g.,
    monkey or rabbit kidney)
    ± by mincing and trypsinization
    ± mixture is transferred to a tube or shell vial
    where the cells attach
    ± form a confluent monolayer on the glass surface.
    Y  

    ¬ Primary cell cultures


    ± Overlay medium with buffered isotonic inorganic
    salts, glucose, amino acids, vitamins, and
    cofactors at a physiologic pH
    ± Eagle minimum essential medium in Earl or
    Hanks balanced salt solution ±
    ¬ to maintain a healthy monolayer but not promote cell
    proliferation.
    Y  

    ¬ Diploid cell lines


    ± usually human diploid fibroblasts (HDF) derived
    from lung or newborn foreskin,
    ± majority of cells have a normal diploid
    chromosome number.
    ± propagated in serum-rich growth medium
    ¬ examples include RC-5, WI-38, and foreskin
    fibroblasts.
    ± serially subcultured 20-50 times;
    Y  

    ¬ Heteroploid cell lines


    ± derived from malignant tumors and can be
    passaged indefinitely;
    ± they are aneuploid and divide rapidly.
    ± Heteroploid lines include HEp-2 (laryngeal
    carcinoma), HeLa (cervical carcinoma), and
    A549 (laryngeal carcinoma).
    ¬ Primary cultures must be checked for
    contamination with endogenous virus.
    Y  

    ¬ Diploid and heteroploid cell lines should be


    tested periodically
    ± for m  contamination
    ± continued sensitivity to viral infection.
    ¬ Hybrid¶ cells are a new concept; the
    monolayer is a mixture of two cell types that
    together support growth of a wide range of
    viruses.
    Y  

    ¬ R- ix contains A549 and mink lung cells ±


    ± respiratory agents±influenzas, parainfluenzas,
    respiratory syncytial virus (RSV), and
    adenovirus.
    ¬ E- ix contains Buffalo green monkey kidney
    and A549 cells -- enteroviruses.
    Y  

    ¬ Tube monolayers are versatile:


    ± Viral replication in tube monolayers -- inverted
    phase-contrast microscope for cytopathic
    changes
    ± a single cell line may support growth of several
    viruses,
    ± each producing distinctive cytopathic effect
    (CPE)
    Y  

    ¬ Tube monolayers are versatile:


    ± other identifying features (hemagglutination,
    hemadsorption, interference).
    ± cytopathic effect (CPE)
    ¬ 1-3 days (herpes simplex virus [HSV])
    ¬ may not be recognized for 5-20 days (RSV, varicella
    zoster virus [VZV], C V);
    ¬ extended incubation for 2 weeks or more.
    Y  

    ¬ shell vial technique


    ± cell sheet rests on a round coverslip, monolayer
    side up, in a shell vial.
    ± specimen is inoculated into the vial, which is
    then centrifuged to enhance viral attachment to
    the cell surface.
    ± 1-3 days' incubation, the monolayer is tested for
    specific viral antigen, usually with a 
    
     
    
    ± ideal for identification of a single virus (HSV) or
    limited number of viruses (RSV and influenza),
    Y  

    ¬ shell vial technique


    ± faster detection of slow-growing agents such as
    C V and VZV.
    ¬ HSV vesicular genital lesions are excellent candidates
    for shell vial culture; sensitivity is almost 100%
    turnaround for negative specimens is decreased from
    1 week to 1 day.
    ¬ comparable or superior to tube methods for recovery
    of C V from urine,
    ± backup tube culture is recommended to maximize C V
    recovery from peripheral blood, body fluids and tissue
    biopsies.

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