critique of the theory and practicalities of viral diagnostic methods

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Viral diseases are increasingly becoming complex in the present medical world. There are a handful of diagnostic methods that are available and helpful but only to certain extend. In case of U.K it was found that by the year an average of 3,500 HIV diagnoses are performed. From past twenty years many new viruses and a few of them with subtypes are identified. A few are HIV with subtypes 1&2, Human T-cell lymphotrophic virus 1 & 2 [10]. Some of the improvements in nucleic acid based detection techniques helped to get in depth with viruses life cycle, morphology, genotypes, sequence and its resistant patterns. In recent years there in an increase in the development of antivirals for viruses of commercial and non commercial interest. With increase in spread of viral infections there is a necessity of developing and moving towards new diagnostic approaches.

Key words: Cytopathic Effect (CPE), Cytomegalovirus (CMV), Transcription Mediated Amplification (TMA), PCR.

Detection Methods:

The viral disease diagnosis is divided into two methods. One is the direct method and the other is indirect method. These methods are employed in common to detect most of the viruses. But in case of certain specific viruses some specific diagnostic methods are employed. The major difference in between the direct and the indirect methods are; the direct method is an conventional method which uses cell culture to isolate the virus and some other some other techniques for the detection of viral antigens . While in case of the indirect method it depends upon the antibody responses to viral infections in serum and other body fluids [9].

Cell Culture:

This is one of the conventional methods that come under the category of direct detection method. Weller and Enders [1] are the first people who isolated virus from the cell culture. The major importance of this method is its ability to provide a viable isolate which can be used for further characterization. The virus present in the cell culture is identified based on the Cytopathic Effect (CPE) [1], which is the change of morphological forms in the cells. Different cell cultures are used for the isolation of different viruses. Enterovirus and respiratory viruses are isolated by using monkey kidney cell line.

Cytomegalovirus (CMV) which found mostly in urine samples is isolated using the human fibroblast cells [1]. Some changes are made for the common culture methods in order to decrease the time taken for the culture. Shell vial culture method is a new method that reduces the time period required for the viral growth and involves incubation of the centrifuged sample which is then detected by using fluorescent antibody staining [3]. Genetically engineered cell lines are a new breakthrough in which the viral receptors are inserted directly into the indicator cell lines (contains transfected genes) that leads to the expression of promoter. The reporter enzyme β-galactosidase is triggered by the activation of promoter identifies the viruses like HIV [11].

Electron Microscopy:

Many of the viruses like enteroviruses, reoviruses, adenoviruses, etc can be easily detected and diagnosed using the Electron Microscopy (EM) [6]. As the viruses are divided based up on their size, structure it was easy to identify a certain type of virus using EM. One of the major advantages of this method is its ability to detect viral particles in various types of clinical samples like biopsy samples, urine samples, crust, plasma, etc and the ease in preparation of specimens [7]. Immune electron microscopy is another branch of EM which helps to increase the sensitivity in detecting virus by using a specific antiserum that attaches to the required viral antigen.

Even this method has the ability to identify viral particles at higher accuracy it lacks usage due to its drawbacks as it consumes more time and lack of sensitivity when compared to other methods as it can recognise 105-106 particles per ml [8].

Nucleic Acid Detection:

Nucleic acid detection method is one of the ground breaking achievements in the field of diagnostic virology. Using these methods it is easy to identify a virus due to higher sensitivity of the techniques. Some of the techniques used for this method are bDNA (branched chain DNA), Transcription Mediated Amplification (TMA) and Polymerase Chain Reaction (PCR). Among these three techniques PCR is widely used [1]. In case of the bDNA technology it is easy to monitor therapeutic responses in case of viruses like HIV-I based up on the rise or fall of the copy number.

bDNA achieves higher sensitivity through signal amplification of the bDNA probe [12]. PCR is the most widely used technique as this can be applied to virus of any kind [4]. Another advantage of PCR is its sensitive detection ability of detecting specific nucleic acids [1]. PCR has the capability to detect more than one virus. Other type of PCR called RT-PCR (Reverse Transcriptase-PCR) which is run by the addition of an enzyme called reverse transcriptase it can also detect the viral RNA [10].

Real Time PCR is also a widely used due to its high sensitivity, speed of reaction, low risk of product contamination as it being a closed system and its ability for mutational analysis. Another Nucleic Acid detection technique called Transcription Mediated Amplification is used to detect the sexually transmitted diseases [13]. Some of the major drawbacks of PCR are quantification of variables mostly at the early stages of amplification process, expensive, rarely available and require considerable expertise.

Immunofluorescence & Antigen Detection:

Immunofluorescence is very useful technique in searching the location of virus specific antigens accumulated in the nucleus or cytoplasm of the infected cell [5]. This technique was first found by Coons et al. One of the major advantages of this technique is to diagnose the disease immediately in the patient's specimen. Some of the immunoflourescent methods are Fluorescent Antibody (FA) [4] staining and immunoperoxidases staining.

Gel diffusion and latex agglutination are the two techniques that are used previously for direct detection of viral antigens but currently very rare in use [4]. The FA labelling technique was used majorly for the detection of respiratory viruses and the method is improved drastically by specimen preparation through cytocentrifugation and continuous staining with different antibodies of different fluorescent labels [16]. Some of the viruses that are detected are influenza, RSV, para influenza, HSV and adenovirus [1]. The major advantage of this method is its rapidity.

Enzyme Immuno Assays (EIA):

EIA is a technique which is mostly employed on humans (herpetic infections, etc) for serodiagnosis [15]. This technique is highly sensitive (detects antiviral antibodies), specific and can be easily performed [3]. In application with viral detection, the drawback for this method is, as it has a limited clinical utility. The viruses that are specific for the IgM antibodies to the viral capsid antigen are CMV, hepatitis A virus, EBV and for IgM antibodies to the hepatitis B core antigen are hepatitis B, parvovirus, Mumps, Measles and Rubella [1].

Diagnosis of Specific viral Diseases:

The diagnostic methods of specific viral diseases vary depending upon the nature of infection. In Mucocutaneous infections method employed for these infections in FA staining and had a specificity of 80% [1].

While in case of Respiratory Infections the methods employed for most of these infections are FA and EIA and had a specificity of 80%-100%. CNS infections (acute meningitis, acute encephalitis and opportunistic infections) the major bases of diagnosis are Nucleic Acid Amplification Tests (NAAT). For HIV and human retroviruses the common diagnostic method is EIA and had a specificity of 99.9%` [1].

Problems and Possible Solutions:

Some of the problems that are majorly associated with in the molecular diagnostics are quantitative techniques which can be solved using real time amplification techniques [2]. Most of the molecular diagnostic methods are time consuming so an automated system is always recommended which is always helpful for disease management. More work has to be done on major damage causing viruses like HIV, Hepatitis B, C viruses, CMV and Human papilloma virus [2].

Apart from these some of the common problems which may leads to poor results or damage to the process are going through the process of preparing specimen to obtaining end results and handling errors. These errors can be overcomed by maintaining strict hygiene conditions, usage of coloured reagents, rechecking the labelling and usage of information systems (barcodes, RFID Tags, etc) to obtain 100% precision starting.