Question

1. Describe each of the techniques listed below in the following areas: 1) sample analysis, 2) viral detection, and 3) diagnosis.

- Direct Examination of Specimen

                        a. Electron and Light Microscopy

                        b. Antigen detection immunofluorescence, ELISA, etc.

c. Molecular techniques for the direct detection of viral genomes      

  

Answer 1

1. Direct Examination of Specimen

1.            Electron Microscopy morphology / immune electron microscopy

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

3.            Antigen detection immunofluorescence, ELISA etc.

4.            Molecular techniques for the direct detection of viral genomes   

2. Indirect Examination

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

2.            Eggs pocks on CAM - haemagglutination, inclusion bodies

3.            Animals disease or death confirmation by neutralization   

3. Serology

Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.

Classical Techniques       Newer Techniques

1. Complement fixation tests (CFT)          1. Radioimmunoassay (RIA)

2. Haemagglutination inhibition tests      2. Enzyme linked immunosorbent assay (EIA)

3. Immunofluorescence techniques (IF) 3. Particle agglutination

4. Neutralization tests    4. Western Blot (WB)

5. Single Radial Haemolysis          5. Recombinant immunoblot assay (RIBA), line immunoassay (Liatek) etc.

               

1. Direct Examination

Direct examination methods are often also called rapid diagnostic methods because they can usually give a result either within the same or the next day. This is extremely useful in cases when the clinical management of the patient depends greatly on the rapid availability of laboratory results e.g. diagnosis of RSV infection in neonates, or severe CMV infections in immunocompromised patients. However, it is important to realize that not all direct examination methods are rapid, and conversely, virus isolation and serological methods may sometimes give a rapid result. With the advent of effective antiviral chemotherapy, rapid diagnostic methods are expected to play an increasingly important role in the diagnosis of viral infections.

1.1. Antigen Detection

Examples of antigen detection include immunofluorescence testing of nasopharyngeal aspirates for respiratory viruses e.g.. RSV, flu A, flu B, and adenoviruses, detection of rotavirus antigen in faeces, the pp65 CMV antigenaemia test, the detection of HSV and VZV in skin scrappings, and the detection of HBsAg in serum. (However, the latter is usually considered as a serological test). The main advantage of these assays is that they are rapid to perform with the result being available within a few hours. However, the technique is often tedious and time consuming, the result difficult to read and interpret, and the sensitivity and specificity poor. The quality of the specimen obtained is of utmost importance in order for the test to work properly.

1.2. Electron Microscopy (EM)

Virus particles are detected and identified on the basis of morphology. A magnification of around 50,000 is normally used. EM is now mainly used for the diagnosis of viral gastroenteritis by detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses. Occasionally it may be used for the detection of viruses in vesicles and other skin lesions, such as herpesviruses and papillomaviruses. The sensitivity and specificity of EM may be enhanced by immune electron microscopy, whereby 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. The main problem with EM is the expense involved in purchasing and maintaining the facility. In addition, the sensitivity of EM is often poor, with at least 105 to 106 virus particles per ml in the sample required for visualisation. Therefore the observer must be highly skilled. 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.

1.3. Light Microscopy

Replicating virus often produce histological changes in infected cells. These changes may be characteristic or non-specific. Viral inclusion bodies are basically collections of replicating virus particles either in the nucleus or cytoplasm. Examples of inclusion bodies include the negri bodies and cytomegalic inclusion bodies found in rabies and CMV infections respectively. Although not sensitive or specific, histology nevertheless serves as a useful adjunct in the diagnosis of certain viral infections.

ELISA was developed in 1970 and became rapidly accepted. A wide variety of assay principles can be used in ELISA techniques. Currently the most important ones are;-

1.            Competitive methods

2.            Sandwich methods

3.            Antibody capture methods

Competitive methods

One component of the immune reaction is insolubilized and the other one labeled with an enzyme. The analyte can then be quantified by its ability to prevent the formation of the complex between the insolublized and the labelled reagent. Advantages of this approach are that only one incubation step is necessary and that the "prozone effect" at high analyte concentrations cannot occur. Disadvantages are that the concentration range in which the analyte can be quantified without sample dilution is rather narrow and that the antigen or antibody (in cases where either may be present in a sample e.g. hepatitis B) produce the same response, and can therefore cannot be distinguished in a one step assay.

Sandwich (Indirect) methods

1.            The method in which the same component of the immune reaction (e.g. the antibody) is used in the insolubilized and the enzyme labelled form. The other component, the analyte (i.e. the antigen in the sample forms a bridge between the two reagents.)

2.            The method in which one component (usually the antigen) is used in an insolubilized form to bind the analyte from the sample (the antibody),which is subsequently determined by addition of labelled second antibody against the same class of antibody as the analyte antibody or protein A.

In principle, quantification can be achieved over an extremely wide analyte concentration range in such sandwich methods. The "prozone effect" can be avoided in the following ways;- (i) using sequential incubation steps for sample and label, or (ii) by using monoclonal antibodies. Modification of the test in (2) so that antibodies of a specific class such as IgM, can give spurious results if antibodies from other immunoglobulin classes are also present in the sample. Also RF ( rheumatoid factor ) is known to be a potentially interfering factor.

Sandwich inhibition methods

The sample containing the analyte (usually antibody) is pre-incubated with a fixed amount of its binding partner (ie. the antigen ) after which the remaining amount of antigen is determined in a sandwich assay. These methods usually are complicated and have a limited measuring range. The method allows for simultaneous detection of antibody or antigen, if either of these 2 analytes is present in the sample.

Antibody capture methods

These methods used to detect antibodies of specific immunoglobulin subclasses, by first reacting the sample with e.g. insolubilized anti-IgM,and subsequently with either enzyme labelled antigen followed by enzyme linked antibody. Neither antibodies from other immunoglobulin subclasses nor rheumatoid factor interfere significantly in such assays. They are widely used for the diagnosis of acute infections by IgM detection. These assays may be used for detecting IgG and IgA. Considering trends towards simplification of assays and quantification of analytes over a wide concentration range. It must be expected that competitive and sandwich inhibition methods will decrease in importance, and the sandwich and antibody capture methods will be the main assay principles in the future.

Assay Characteristics

The use of monoclonal antibodies has lead to many improvements in ELISA systems.

1.            Higher sensitivity ;- either by selection of antibodies with a extremely high affinity, or by reduction of the height and variability of the background reaction, which makes very low concentrations of analyte more readily detectable.

2.            Higher specificity ;- by avoiding the presence of any antibody in the assay system with specific reactivity against non-analyte epitopes, and by selecting combinations of monoclonal antibodies which may further increase specificity.

3.            Higher practicality ;- e.g. by introducing simultaneous incubation of label, solid phase and sample without risk of "prozone effect".

The enzyme label ;- Most of the assays employ horse-radish peroxidase, alkaline phosphatase, or B-D-galactosidase. The most interesting recent developments has been in new methods to detect these enzymes rather than the use of new enzymes. Fluorimeters were introduced in 1984 for the detection of alkaline phosphatase and B-D-galactosidase. Methods are available to detect horse radish peroxidase by means of chemilumininescence. Fluorimetric and luminometric methods offer higher sensitivity and a wider measuring range than conventional spectrometry. TMB is gradually replacing mutagenic substrates such as OPD, leading to increased sensitivity and safety.

Immunofluorescence (IF) is widely used for the rapid diagnosis of virus infections by the detection of virus antigen in clinical specimens, as well as the detection of virus-specific IgG or IgA or IgM antibody. The technique makes use of a fluorescein- labelled antibody to stain specimens containing specific virus antigens, so that the stained cells fluoresces under UV illumination. In the case of direct IF, the specimen is probed directly with a specific labelled antibody against a particular virus antigen. In the case of indirect IF, the specimen is first probed with a non-labelled specific antibody, followed by a labelled antibody against the first antibody. Direct or indirect IF can be used for the detection of virus antigen, whereas indirect IF is virtually always used for the detection of antibody. Indirect IF possess the advantage of an extra amplification step for the signal, however, it requires an extra step in comparison to direct IF.

Detection of viral antigens

IF is most commonly used for the detection of respiratory viruses in respiratory specimens. Nasopharyngeal aspirates are the best specimens to use and is usually collected from babies less than 12 months old. However, there are no reasons why nasopharyngeal aspirates cannot be collected from older children and adults. A number of respiratory viruses can be detected by direct or indirect IF, including RSV, influenza A and B, adenoviruses and parainfluenza viruses. However, the sensitivities vary greatly between different viruses. The method is most useful in the case of RSV where antiviral treatment is available for severely ill babies. IF is also widely used for the detection of HSV infections, from vesicle lesions and brain lesions, for VZV and CMV infections. However in the case of CMV infection, the sensitivity of IF on clinical specimens directly is low, with the possible exception of the CMV antigenaemia test.

A typical indirect IF procedure for the detection of viral antigens is as follows;- cells from the clinical specimen are immobilized onto individual wells on a slide. Specific polyclonal or monoclonal sera is then added to each well and the slide is incubated at 37oC for 30 to 60 minutes. The slide is then washed 3 times for 5 minutes each with PBS and fluorescein labelled antibody against the first antibody is added. The slide is further incubated at 37oC for 30 to 60 minutes and washed again. The slide is then prepared for microscopy. Specific monoclonal or polyclonal sera raised against the viral antigen can be used. Monoclonal sera offer the advantage of increased sensitivity and specificity. However, one must be certain that it can detect all the different strains of the virus.

IF is highly dependent on the quality of the specimen. In many instances it has proved to be more sensitive than equivalent EIAs. This is because a firm diagnosis can be made on the identification of a few cells only that contain fluorescence of the right colour and with the correct antigen distribution. One of the criticisms of IF is that it is labor intensive and requires highly skilled staff for the reading the specimen.

Molecular biology techniques for the direct detection of viral genomes in the specimen will play an increasingly important role in the clinical virology laboratory in the 21st century. Molecular techniques can be divided into two categories: those that do not involve amplification i.e. hybridization with nucleic acid probes, and those that involve amplification e.g. PCR, LCR, NASBA etc.

Nucleic Acid Probes

Nucleic acid probes are segments of DNA or RNA that have been labeled with enzymes, antigenic substrates, chemiluminescent moeities, or radioisotopes. They can bind with high specificity to complementary sequences of nucleic acid. Probes can be directed to either DNA or RNA targets and can be from 20 to thousands of bases long. The presence and the quantity of hybrids after hybridization is determined by the detection of the label. Probes are usually synthesized by one of the following three methods.

1. Oligonucleotide probes - these are usually less than 50 bases long and are synthesized chemically

2. PCR - will produce probes from 50 to several hundred bases long

3. Cloning - will produce probes several thousand bases long e.g. probe for complete HBV genome

Altering the stringency of the reaction will alter the sensitivity and specificity of the hybridization. Stringency relates to the number of mismatched base pairs that can be tolerated when two nucleic acid molecules come together to form a double stranded molecule. Stringency is affected by several variables, including the temperature, salt concentration, and the pH of the hybridization reaction. High stringency is achieved by using buffers of low salt concentration or by conducting the hybridization reaction and stringency washes at higher temperatures. The higher the stringency of reaction, the less likely it is for mismatched base pairs to stay together.

The hybridization reaction may be carried out completely in solution phase whereby both the target nucleic acid and the probe are free to interact in the reaction mixture. Solution hybridization has the advantages of being rapid to the carry and carry a higher sensitivity than solid phase hybridization. This is the approach taken by Abbott with their quantitative HVB-DNA assay. Nucleic acid bound to a solid surface are still available to participate in hybridization reactions. However, the sensitivity tends to be lower than that of liquid hybridization. However, this technique greatly facilitates the handling of multiple samples. The dot-blot and sandwich hybridization assays are commonly used in this respect. In situ hybridization assays, in which whole cells or tissue sections are put through the hybridization process has become an important research tool.

Despite having been around for many years, hybridization assays are still not in common use in the clinical virology laboratory. The main reason is that its sensitivity is not usually higher than far simpler conventional virological techniques such as cell culture and viral antigen detection.

Polymerase Chain Reaction

PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique. It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest. These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94oC), annealing of primers to their complementary sequences (50oC), and primer extension (70oC) result in the exponential production of the specific target fragment. Further sensitivity and specificity may be obtained by the nested PCR technique, whereby the DNA is amplified in two steps. In the first step, an initial pair of primers is used to generate a long sequence that contain the target DNA sequence. A small amount of this product is used in a second round of amplification, which employs primers to the final target DNA.

Detection of DNA sequence product of the PCR assay may be performed in several ways. The least sensitive and specific method is to size fractionate the reaction product on an agarose or acrylamide gel and stain the DNA with ethidium bromide. A more sensitive technique involves the attachment of DNA to a membrane through dot or slot-blot techniques followed by hybridization with a labelled homologous oligonucleotide probe. Alternatively,   the   PCR product may be probed directly by   liquid   oligomeric hybridization. However, these techniques provides no information on size of the amplified product and thus could not exclude the possibility that the product originated from a region of the human genome which exhibits homology with the target CMV sequence. The most sensitive and specific detection methods result from combining the size information of gel electrophoresis with the improved sensitivity and specificity of hybridization techniques. This may be achieved by gel electrophoresis followed by Southern transfer and hybridization, or through liquid oligomeric hybridization followed by gel electrophoresis.

Advantages of PCR:

1.            Extremely high sensitivity, may detect down to one viral genome per sample volume

2.            Easy to set up

3.            Fast turnaround time

Disadvantages of PCR

1.            Extremely liable to contamination

2.            High degree of operator skill required

3.            Not easy to quantitate results

4.            A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

5.            Other Amplification Techniques

6.            Following the heels of PCR, a number of alternative in-vitro amplification techniques have been developed, of which some are now available commercially. Examples of these alternative techniques include ligase chain reaction (LCR), nucleic acid sequence based amplification/isothermal amplification (NASBA), strand displacement amplification, Q? Replicase method, and branched DNA probes. Of these techniques, LCR, NASBA and branched DNA are now available commercially in an automated or semi-automated format. A NASBA assay is available for the quantification of HIV-RNA (Organon), and an LCR assay is available for the detection of chlamydia (Abbott). Branched DNA assays are available for the detection of quantification of HIV-RNA, HBV-DNA, and HCV-RNA (Chiron).

7.            With the exception of the branched DNA probe, all these techniques involve exponential amplification of either the target nuclei acid or the probe. Therefore, they are all as susceptible to contamination as PCR. The branched DNA system is really an intermediate between classical hybridization techniques and the newer in-vitro amplification techniques. It is not as sensitive as those techniques which involve exponential amplification but is considerably more sensitive than the classical hybridization techniques. Below is a brief summary of the features of the different amplification methods available.