Diagnostic techniques viruses
Since molecular techniques for detecting nucleic acids had been developed, the diagnosis of several viral diseases has been revolutionized in the clinical laboratories. In the majority of them, it has been introduced these methods for routine diagnosis, but some of those are being used only in reference settings. The main advantages of molecular techniques are its higher sensitivity and specificity compared with other diagnostic methods such as serological assays and culture methods, as well as its rapidity and possibility of automation.
From an epidemiological and clinical point of view, these features are very important for the diagnosis of some diseases such as CNS infections, in which the detection of microorganisms should be faster in order to treat rapidly the patients and isolate them to prevent viral transmission of disease. Among this fact, the automation also permits the performance of much more assays and its rapidity helping to improve the patient diagnosis.
Virology laboratories for clinical diagnosis should introduce some of these techniques in order to determine the main viruses implicated in human diseases, after to do an analysis of cost.
Laboratory director and technical coordinator should establish the workflow for these techniques enhancing the efficiency of the testing methods. This workflow should be done in an individualized way, taking into account the assays introduced and the special characteristics of the laboratory.
Moreover, the personnel should be trained according the best practices for this methodology at this time. National Center for Biotechnology Information , U.
Journal List Open Virol J v. Open Virol J. Published online Nov Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC. Abstract Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. Keywords: Automation methods, molecular diagnosis, molecular microbiology, nucleic acid techniques, PCR techniques, viral laboratory diagnosis.
Table 1. Open in a separate window. Signal Amplification Techniques In signal amplification assays, the signal is directly proportional to the amount of the target sequence present in the clinical specimen, reducing false-positive results due to cross contamination; also, the development of quantitative assays is more reliable.
Hybrid Capture Assays This system is a solution hybridization-antibody capture technique that uses a chemiluminescence detection system of the hybrid molecules. Target Amplification Techniques These techniques use enzyme-mediated processes, in which the enzymes synthesize several copies of target nucleic acid.
Multiplex PCR In the same reaction mixture, two or more primer sets designed for amplification of different targets are used [ 16 ]. Probe Amplification Techniques These methods differ from those that use target amplification in which the amplification products contain only a sequence present in the initial probes.
Ligase Chain Reaction Ligase chain reaction assay is based on the ligation of two adjacent synthetic oligonucleotide primers which hybridize to one strand of the target DNA. Cycling Probe Technology Cycling probe technology is a method for detection and quantification of low amounts of target DNA. Personnel Requirements Laboratory workers must be trained in both the pre-analytical specimen extraction and processing and the analytical procedures.
Facilities Requirements In order to minimize or decrease the risk of specimen contamination, it is necessary a physical separation of processes as well as to have reagents and equipment for use only in the molecular laboratory. Work Flow Design After selection and successful introduction of a molecular testing platform into the virology laboratory, work flow should be implemented at the same time that this technology is being introduced.
Table 2. Parvovirus B19 Infection with parvovirus B19 might cause asymptomatic infection or a wide spectrum of disease erythema infectiosum in children with arthropathy, severe anemia and systemic affectation as well as hydrops fetalis, congenital anemia and abortion if the infection is produced in pregnant women. Influenza and Parainfluenza Viruses Rapid laboratory diagnosis is critical for infection control, so several diagnostic tests have been developed and are available for the detection of influenza viruses.
Adenovirus PCR is a specific and sensitive assay for detecting adenovirus DNA from a wide variety of clinical specimens, but results must be always interpreted in the context of the clinical findings of adenovirus disease.
Enterovirus CNS Disease Enteroviruses such as echoviruses, parechoviruses echoviruses 22 and 23 and coxsackieviruses A and B could produce several diseases such as respiratory tract infections, aseptic meningitis, myocarditis and neonatal systemic enteroviral disease. Hepatitis Viruses Molecular techniques can be very useful for the diagnosis of viral hepatitis infections such as hepatitis A, B, C, D and E in cases in which serological assays are no conclusive.
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The ligation amplification reaction LAR - amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Identification of oseltamivir resistance among pandemic and seasonal influenza A H1N1 viruses by a His Tyr genotyping assay using the cycling probe method.
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Rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method. Methods for detecting directly either virus specific antigen 30 or nucleic acid 31 in clinical material have removed the need for virus cell culture.
An early concern that the high sensitivity of molecular amplification techniques would result in the detection of latent herpesvirus genome, so lowering the clinical specificity of virus nucleic acid detection, have been unfounded: the detection of viraemia has been found to correlate well with active CMV disease 32, 33 and CMV PCR is now often incorporated into routine post-transplant microbiological surveillance of susceptible patients.
The problem of drug resistant virus phenotypes is becoming more recognised with an increase in the number of immunosuppressed patients receiving long term antiviral treatment. The use of prophylactic antiviral treatment for reactive herpesvirus infection, and the use of combinations of drugs to suppress viral replication in HIV infection, has selected for mutations conferring drug resistance.
Under selective pressure, a drug resistant strain can quickly become the dominant phenotype, but molecular based assays have improved the clinical management of these patients by facilitating the detection of mutations conferring resistance. For the HIV genome, a commercial assay is available based on molecular amplification of the viral genome followed by hybridisation with mutation specific DNA probes. At present, these methods are used for the detection of resistance to the reverse transcriptase inhibitor class of antiviral compounds, but as a rapid and inexpensive approach it is likely to be used for other phenotypic determinants.
A similar technique has been used for genotyping HCV infection for epidemiological study and for predicting the response to treatment. Molecular assays have had an important role in the study of the natural history of HIV infection, and subsequently in the development of effective treatment strategies.
Quantitative molecular assays have shown that HIV-1 infection is characterised by high rates of virus production and clearance of both infected cells and cell free virions. The assessment of viraemia is now used as a prognostic indicator in HIV, 38 HCV, 39 and hepatitis B virus 40 infections, and is also used in monitoring the efficacy of antiviral treatment in systemic viral infections of immunosuppressed patients.
The introduction of molecular techniques to routine diagnostic virology laboratories may have been held back by the lack of assays developed commercially, the high initial costs, and the specialist training involved in establishing these techniques. Using the technology currently available, the areas described might represent their limits in clinical virology; however, it is probable that formats currently under development and further genome sequencing will spread molecular detection more widely throughout diagnostic microbiology.
Automation in virology laboratories for serology assays has been slow to transfer to molecular amplification techniques, but sequence detection and other downstream processes have now been automated successfully with the introduction of colorimetric based detection methods.
An important proposed development will be the ability to perform molecular assays on an automated platform currently widely used for serology assays. If these processes are automated successfully, it seems likely that for high throughput applications molecular assays will replace many traditional microbiological methods, which will become in contrast, both slow and expensive.
An exciting new development is the combination of thermal cycling for PCR nucleic acid amplification and fluorimetry in a single machine, such as the LightCycler instrument from Roche Molecular Biochemicals, able to support a number of fluorescent chemistries.
It is possible by measurement of the signal from DNA sequence specific fluorescent dyes to monitor amplification kinetics in real time, allowing for accurate, rapid, and cheaper viral load tests figs 1 and 2. These assays have the potential for accurate measurement over a wide range of viral concentrations.
This instrument drastically reduces the problem of contamination in PCR because the reaction is analysed in a sealed system without exposure to the environment. Viral genotyping and mutation analysis can also be performed within the system by characterisation of the PCR product. The technology facilitates assay automation because the amplification, data collection, and analysis can be performed by a single machine connected to a computer.
Measurement of hepatitis B virus HBV genome in serum using real time monitoring of polymerase chain reaction PCR amplification kinetics of a set of standards on the LightCycler instrument. Each line represents a PCR amplification. The concentration of HBV genome is proportionate to the PCR cycle at which the fluorescent signal increases above the background level represented by the horizontal line.
Additional information about the reaction detailed in fig 1 can be obtained from the melting curve analysis. Primer dimer formation occurs only at lowest input copy numbers. Genetic screening has encouraged a rapid development of this technology, 41, 42 and microbiological application has recently been described in screening for drug resistance in tuberculosis. This development, backed by advances in assay usefulness and reliability resulting from automation, might herald the end of the traditional stand alone clinical virology laboratory.
A DNA chip containing immobilised arrays of oligonucleotides for the study of gene expression. Specialised equipment is required for interpreting the patterns generated by hybridisation of DNA to the array but the technique is suited to automated, high throughput applications. You will be able to get a quick price and instant permission to reuse the content in many different ways. Skip to main content. Log in via OpenAthens. Log in using your username and password For personal accounts OR managers of institutional accounts.
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Log in via Institution. Email alerts. Article Text. Article menu. Molecular techniques for clinical diagnostic virology. Statistics from Altmetric. Commercially available influenza diagnostic tests do not specifically detect novel influenza A viruses and a positive result for influenza A virus cannot distinguish seasonal influenza A virus from avian or swine influenza A virus infections.
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