Monday, June 20, 2011

General diagnosis of viruses

Specimens collection and transport:
        Collection and transport of an appropriate sample to the laboratory is a vital factor in successful diagnosis (Smith and Yassin, 2000).
Specimens collection
Collection of samples that contain the highest titer of virus is most desirable. Preservation of the viral titer and viral infectivity until cell cultures can be inoculated is essential. Body sites and collection methods vary according to the type of infection and viral etiology e.g. clinical samples collected from body sites such as skin and the genital tract, which is usually contaminated with microbial flora, are collected with
dacron or polyester swab and placed in viral transport medium (VTM) but CSF which is expected to be free of contamination, is collected in sterile containers (Leland and Ginocchio, 2007).
Table (1): shows Specimens useful for viral studies (Specter and Bendinelli, 2005).
Specimen type
Disease category (example)
Nasopharyngeal swab or aspirate 
Respiratory tract infection, certain exanthemas, CNS infection (especially Enteroviruses).
Throat wash or swab
Respiratory tract infection.
Respiratory infection (HSV, CMV).
Bronchoalveolar lavage
Lower respiratory infection (CMV, Influenza virus, PIV).
Rectal swab or stool
Gastroenteritis and Enteroviruses.
Meningitis, encephalitis.
Brain biopsy
Meningitis, encephalitis (HSV, Rabis virus).
Vesicle fluid or lesion scraping
Endocervical swab
Genital infections (HSV).
Eye swab
Ocular infections (Adenovirus, HSV).
CMV, BK virus, Mumps virus.
Antibody studies (most viruses).

            Several VTM are commercially available. Most VTM consist of buffered isotonic solution with some types of protein as albumin, gelatin or serum to protect less stable viruses. Antibacterial and antifungal agents are added to prevent overgrowth of bacteria and fungi (Nauschuetz and Learmonth, 2007).
Ideally, all specimens collected for viral detection should be placed in ice and transported to the laboratory at once. If a delay is unavoidable, the specimen should be refrigerated not frozen until processing occurs. Every attempt should be made to process the specimen within 12 to 24 hours of collection. Under unusual circumstances, specimens may need to be held for days before processing. For storage up to 5 days, hold specimen at 4°C. Storage for 6 or more days should be at -70°C. Specimens for freezing should first be diluted in VTM. Significant loss of viral infectivity may occur during prolonged storage, especially for the more labile enveloped viruses (Forbes et al, 2007).                   
Methods of laboratory diagnosis:
1. Direct Microscopy :
         Electron microscope (EM) with negative staining methods can be used to directly examine specimens for the presence of viral particles. EM is particularly helpful to detect non cultivatable or fastidious viruses. The major limitation are low sensitivity and specificity, the expertise needed, successful detection requires the presence of 105  or greater particles/ml. EM is effective for identification of virus morphologically by family, which may be sufficient clinically in many cases, and can be enhanced by use of antibodies (immune EM), which permits identification of specific viruses (Petric and Szymanski, 2000).
2. Virus isolation:
              It is still the gold standard method for virus detection. Traditionally, three methods are used for virus isolation; cell culture, animal inoculation and embryonated eggs. Animal inoculation is extremely costly and used only in research laboratories e.g. certain Coxsakie A viruses are isolated in suckling mice and embryonated eggs are rarely used e.g. Influenza viruses. Cell culture is the most commonly used by clinical virology laboratories (Nauschuetz and Learmonth, 2007).

Traditional cell culture
            There are three basic types of cell cultures. Primary cultures are obtained from tissue removed from an animal. The tissue is finely minced and then treated with trypsin to disperse cells then the cells seeded            onto a surface to form monolayer, as flask or test tube. Primary cell lines can only be passaged a few times e.g. primary monkey kidney cell (Nauschuetz and Learmonth, 2007).
            Diploid cell lines are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages); with increasing passage diploid cells become more insensitive to viral infection e.g. human neonatal lung culture. Continous cell lines have variable number of chromosomes (haploid) and are capable of more prolonged  perhaps indefinite growth that have been derived from diploid cell lines or from malignant tissues e.g. HEP2(derived from human laryngeal carcinoma). The type of cell culture used for viral cultivation depends on the sensitivity of the cells to a particular virus (Knipe, 2001).

Table (2): shows Different cell lines sensitive to common viruses (Specter and Bendinelli, 2005).
Cell line
A549 cells
Adenovirus, HSV.
HeLa cells
HEP-2 cells
Adenovirus, HSV, RSV.
Human embryonic kidney
Adenovirus, BK virus, HSV.
Human diploid fibroblasts
CMV, HSV, VZV, Enteroviruses, Rhinoviruses.
Madin-Darby canine kidney (MDCK)
Influenza viruses.
NCI-H 929
Adenovirus, Enteroviruses, Mumpes,
Primary monkey kidney (PMK)  cells
Measles virus, PIV, RSV.
Primary rabbit kidney cells
Enteroviruses, Influenza viruses, PIV.

Centrifugation-Enhanced shell vial culture
       This technique is simple method that more rapidly identifies the virus than the traditional viral culture. Cells are grown on a round coverslip in a shell vial. The shell vial is inoculated with the clinical sample and then centrifugated to promote viral absorption. The shell vial is incubated for 24-48 hours, after which the cells are scraped from the coverslip, and the DFA technique is performed using a varity of Abs (Nauschuetz, 2000).
Identification of viruses detected in cell culture:
            Detection of virus in cell culture depend on cytopathic effects (CPE) which are morphological changes noted as a result of viral replication. CPE of cells may be clumping, destruction, granulation, rounding or vaculation, giant cell or syncytia formation, or retractile cells. In some circumstances, viruses will not cause a visible CPE and it is necessary to resort to other methods to detect their presence. Mumps, Influenza and Parainfluenza viruses will not normally cause CPE or   little CPE but addition of guinea pig red blood cells (RBCs) to infected primary monkey kidney cells will result in adherence of the RBC to the cells (hemadsorption) and Rubella virus may be detected by interference with echovirus 11 to replicate and cause cell destruction of African green monkey kidney cells (Smith and Yassin, 2000).                                   
3. Antigen detection:
          Immunological methods are highly effective for detection of viral antigens (Ag). They offer high degree of sensitivity and specificity, are rapid, and the costs are reasonable. Virtually all methods use antibodies that are tagged with a fluorochrome, enzyme, or radiolabel (Schutzbank and McGuire, 2000).
A) Immunofluorescence (IF):
           The direct staining of clinical specimens using monoclonal or polyclonal antibodies (Abs) bound to fluorescent dye. This may be direct  or indirect techniques. In direct IF, a single fluorochrome-labeled Ab is used. Indirect IF uses two Abs, one specific for the Ag and a second fluorochrome-labeled Ab to the immunoglobulin (Madeley and Peiris, 2002).
B) Enzyme Immunoassay (EIA):
Enzyme Immunoassay can perform to detect antigens in clinical specimens. This versatility has resulted in wide spread use of EIA in diagnostic virology. Most commonly these assays are performed using a solid phase format, as ELISA, with the particular target reagent bound to plastics in a microtiter well. EIA are generally high specific and sensitive, and rapid, within minutes to hours (Leland, 2000).
C) Radioimmunoassays (RIA):
Radioimmunoassays have fallen out of use for most viral assays due to exposure to radioisotope and the high cost of disposal of radioactivity.  RIA rivals EIA in specificity and sensitivity (Mushahwar and Brawner, 2000).

4. Serology:
            This approach is currently the most widely used. Typically during acute primary infection, virus specific IgM Abs appear in serum between 5-10 days after initiation of infection, reach peak concentration at 2-4 weeks, and then decline to undetectable level at 2-4 months, although they may last considerably longer in some infections. In          the course of persistent infections, IgM Ab is usually negative, although they can reappear during episodes of reactivation of virus. Thus, detecting IgM to a given virus, with or without the corresponding IgG, even in a single serum sample obtained during the acute phase of illness, denotes current or very recent primary infection or reactivation of persistent infection by that virus. By contrast, the presence of virus specific IgG but not IgM signifies past infection (Specter and Bendinelli, 2005).
5. Molecular methods (nucleic acid detection):
        A wide variably of commercial assays are available to detect viral nucleic acid directly or after amplification. These procedures are rapidly becoming the standards for diagnostic virology and it is more sensitive than antigen detection and cultures include polymerase chain reaction (PCR), Reverse -Transcriptase PCR and others. These procedures allow detection of viruses (e.g. Enteroviruses) as well as quantitation of the viruses (e.g. CMV, HIV) which guide antiviral therapy.  The principle is the hyperdization of nucleic acid probe (single strand DNA or RNA) to specific nucleic acid sequence in the specimen followed by detection of the paired hybrid. The probe is labeled with enzyme, chemiluminescent molecule, or radioisotopes to facilitate detection of the hyperdization product .
If you need more information read Lectures on applied clinical microbiology 

No comments: