Viral diagnostics Flashcards
Methods most commonly used in viral diagnostic labs for virus cultivation
Traditional tube cultures
Shell vial cultures
Traditional tube cultures
The number and type of cell culture tubes inoculated with the clinical specimen depends on the specimen source and the viruses suspected to be present in the specimen.
Tube cultures are incubated for days to weeks depending on the specimen source and the suspected virus(es).
The cell cultures are examined by light microscopy for the development of cytopathic effects (CPE) such as cell vacuolization, rounding up of cells, cell fusion, and cell lysis
Hemadsorption test
Used for viruses that may make cytopathic effects slowly or not at all
Cell culture medium is removed from the culture tube and replaced with a solution of erythrocytes.
The culture tubes are incubated and examined by light microscopy.
If a hemadsorbing virus is present the erythrocytes will adhere in clumps to the cell monolayer.
Hemadsorption inhibition test:
We can use known Ab and if no hemagglutination once those are added, then we know that’s the virus
Shell vial cultures
Cell monolayers are grown on a round glass coverslip in a small shell vial. To inoculate the cells with the clinical specimen, the cell growth medium is removed and the processed clinical specimen is placed directly onto the cells.
The entire vial is then centrifuged at low speed for one hour to enhance virus attachment to cells and virus penetration of cells.
Fresh culture medium is added and the vials are incubated. At a designated time interval, virus infection is detected by staining cells on the coverslips with labeled virus-specific monoclonal antibodies and the stained coverslips are evaluated by fluorescent microscopy.
Advantage and disadvantages to viral culture techniques
Advantages:
- Capacity to isolate a wide variety of viruses
- If virus cultivation is successful, this provides a virus isolate that can be used for further studies such as antiviral susceptibility testing, serotyping, and epidemiology.
- Shell vials with centrifugation have shorter turnaround times and do not require the development of cytopathic effects (CPE).
Disadvantages:
- Long incubation times required for some viruses
- Inability of some viruses to proliferate in cell cultures
- Need for technical expertise to evaluate cell cultures for various forms of CPE.
Viral nucleic acid tests
Polymerase Chain Reaction (PCR)
PCR
Polymerase chain reaction
Most commonly used method for rapid virus diagnosis
Very sensitive!
Procedure:
- DNA or RNA is isolated from the patient’s clinical specimen or from cultured cells infected with the patient’s specimen.
- Reverse transcriptase (a retrovirus enzyme) is used to transcribe RNA to make a cDNA copy.
- The patient’s viral DNA or cDNA is then amplified by PCR using oligonucleotide primers specific for the suspected virus pathogen.
PCR assays can be either qualitative or quantitative. In qualitative PCR, the size of the amplified DNA (amplicon) is identified in agarose gels. Quantitative PCR, in which the amount of nucleic acid amplified is determined from a standard curve, is typically performed if viral loads are clinically relevant, that is, clinical decisions are dependent on the viral load.
In addition to determining the presence of virus nucleic acid, the nucleotide sequence can be determined from PCR amplicons.
Multiplex PCR assays for virus diagnosis
Designed to allow simultaneous detection of multiple different viruses that might be found in a particular anatomical site
Advantages and disadvantages of PCR detection
Advantages:
- High sensitivity and specificity
- short turnaround time
- Ability to detect evidence of virus infection for viruses that cannot be cultured in traditional cell cultures.
Disadvantages:
- Lack of standardized protocols or FDA-approved kits (most PCR assays for virus detection are developed within individual diagnostic laboratories)
- Technical expertise is required in-house for assay development
- Cost of equipment and reagents.
Rapid antigen detection tests
Rapid immunoassays
Western Blot (can also be serology test if testing for pt Ab to virus instead of viral antigens)
Direct immunofluorescence of infected cells from clinical specimens
Rapid immunoassays
A wide variety of FDA-approved commercial kits are available for the rapid detection of influenza A, influenza B, and respiratory syncytial virus (RSV) antigens in clinical specimens.
Very rapid (10-15 minutes to results), have a sensitivity range of 50-90%, a specificity range of 90-95%, and are used by many hospitals for rapid testing during influenza and RSV season.
Procedure (example of rapid flu):
- A nasal or throat swab is mixed with a buffer to extract influenza antigens from the swab.
- A test strip is placed into the buffer. The test strip is embedded with antibodies that recognize influenza A or B protein. These antibodies are labeled to permit a visual readout when concentrated. The test strip also contains unlabeled antibodies that recognize influenza A or B proteins. These unlabeled antibodies are immobilized along distinct lines further down the test strip.
- When the clinical specimen is applied to one end of the strip, it migrates by capillary action towards the opposite end. A virus protein that reacts with the labeled antibodies will also be bound by the immobilized antibodies as the complex migrates down the strip.
- The result is a visible colored line on the test strip indicating that protein is present in the sample. If no virus protein is bound to the antibody, it will move past the immobilized antibody and no color will develop. A color change at the control line (C) must occur for a valid assay.
Advantages and disadvantages of rapid antigen detection
Advantages:
- Rapid antigen tests have high specificity for RSV and influenza A and B viruses
- Results are very rapid (15 minutes or less)
- Minimal technical expertise is required to perform the tests.
Disadvantage:
- Comparatively poor sensitivity, making false negative results common.
- Some of these tests distinguish influenza A or B virus infection while others do not.
- These tests do not provide information on influenza A subtypes (e.g. H1N1 versus H3N2).
Western Blot
Uses antibodies to recognize viral proteins separated in an SDS polyacrylamide gel.
Samples containing viral proteins from a clinical specimen are subjected to electrophoresis through a polyacrylamide gel, which separates proteins based on their sizes.
Proteins are transferred onto nitrocellulose membranes, and virus specific antibodies recognize the bands containing antigenic viral proteins.
IMPORTNANT: Western blots can also be used to detect if patient sera contain specific antibodies against known virus proteins, as is done to identify patients infected with HIV. So, in some cases Western blots are used to detect viral antigens in clinical samples, and in other cases they are used to detect anti-viral antibodies in patient samples.
Direct immunofluorescence of infected cells from clinical specimens
Some clinical specimens, for example nasopharyngeal washes, contain virus-infected cells. These are centrifuged onto glass slides and monoclonal antibodies against each of the viruses that commonly infect this anatomic site are used to detect viral antigens in the cells. This is a fast and definitive assay, but quite labor intensive
Serology tests for viruses
ELISA and Western blot
Obtain a serum specimen from the patient on the first visit at the time of acute infection and a second serum specimen several weeks later during convalescence.
A positive test requires at least a four-fold increase in circulating antibody titer against the isolated virus between acute and convalescent serum specimens.
Hospital-associated diagnostic virology labs typically perform assays to detect anti-viral antibodies for a
limited number of viruses. Most samples for serology are sent to reference labs .
