Medios de protección de las víctimas de este tipo delictivo en la legislación procesal

1.6.1 CELL CULTURE

Human astrovirus (HAstV-1 strain) was first cultured from faeces using a continuous human colon adenocarcinoma cell line (Caco-2 cells) and trypsin treatment of the sample (65). Other cell lines such as T-84 (adenocarcinoma cells) and PLC/PRF/5 (human liver hepatoma cells) have been used to isolate HAstV directly from faeces, however, Caco-2 cells remain the cell line of choice as they allow all HAstV strains to be propagated. In the past, cell culture coupled with RT-PCR (ICC/RT-PCR) was used to enhance virus detection (29, 66). Today, with the increased sensitivity of RT-PCR, cell culture followed by RT-PCR is normally only performed for detectionof HAstV in samples with low viral load, such as contaminated water samples (67).

1.6.2 ELECTRON MICROSCOPY (EM)

Astrovirus and norovirus were first identified through EM examination of faeces from children with diarrhoea. For detection of these viruses, 106 to 107particles per gram of stool are required (42). During viral infection, the numbers of virions excreted are usually less than this. Sensitivity of EM can be increased by using antisera to clump the virusand make

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detection easier. This method is known as immunoelectron microscopy (IEM) and was used to detect the first norovirus (4). Experienced technical personnel are required for EM, asonly a small percentage of virions show typical morphology. EM is not routinely used in

diagnostic laboratories due to high cost of equipment and lack of experienced personnel. It is also unsuitable for screening large numbers of specimens due to the time involved, high cost and low sensitivity.

1.6.3 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

The ELISA method is routinely used by most diagnostic laboratories for rapid detection of astrovirus and norovirus. It is rapid, has a low cost and allows screening of large numbers of specimens. Expensive equipment is not required, and the test is relatively easy to perform. Approximately 104 to 105 virions per gram of stool are required for detection (42).

Commercial kits are available for detection of both viruses. Second generation ELISA kits have improved sensitivity and specificity. They use a group-specific monoclonal antibody to capture the viral antigen, followed by a biotinylated monoclonal anti-virusantibody for detection. The sensitivities and specificities of the ELISA test varyaccording to the commercial kit used. There are two commercial kits available for astrovirus and norovirus antigen detection in Australia: RIDASCREEN by R-Biopharm and IDEIAby DAKO. According to the manufacturers, the sensitivities compared to RT-PCR range from 76.2- 98.3% and 83.0-98.0% for astrovirus and norovirus respectively. The specificities are claimed to be100% for both viruses.

1.6.4 MOLECULAR DETECTION

Since the first development of PCR for DNA detection (68) with addition of reverse transcription (RT) for RNA detection (69), nucleic acid detection has played an important role in diagnostic virology. PCR provides a very sensitive method to detect low levels of viral

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DNA/RNA in specimens. For RNA detection, RT needs to be performed to generate

recombinant DNA (cDNA) using the transcriptase before the PCR cycle. The PCR procedure in its simplest form involves two oligonucleotide primers that flank and define the target region to be amplified, and a reaction mix that contains the target nucleic acid, heat-stable DNA polymerase, salt solution and excess amounts of the four deoxynucleoside triphosphates (dNTPs). The mix is then placed in a thermal cycler for the 3 steps of the PCR cycle: (1) denaturation (2) annealing and (3) extension (Figure 1.8). This cycle is repeated 20-40 times and that results in a 105 – 106 increase in target nucleic acid concentration. RT-PCR is the most sensitive assay for astrovirus and norovirus detection. The sensitivity is higher than ELISA; 10 to 100 virus particles per gram of stool can be detected (42).

However, this sensitivity depends on various factors such as the primers used, RNA methods, reaction conditions and inhibitors. With astrovirus RT-PCR, primers from the highly conserved regions such as the 5’end of ORF2, the 3’ end of the genome, and the RdRp gene (13) are commonly selected as targets. However, primers targeting the hypervariable region of ORF2 have recently been used in the confirmative tests for genotype determination (15, 66). Norovirus, primer design is more challenging due to the high genetic diversity of the viral genotypes. Usually two seperate primer pairs are used to detect genogroups I and II. Different conserved regions of the genome have been targeted with varying success, including region A (RdRp gene, ORF1), region B (the 3’ end of ORF1), region C (5’ end of ORF2) and region D (3’end of ORF2). Recent studies favour the use of region C as the target for reliable

genotyping of GI and GII noroviruses as VP1 (ORF2) is the reference genomic region for establishing genotypes (70-72). Moreover, phylogenetic analysis of region C sequences

appears to give better strain identity and distinct clusters. The performance of RT-PCR assays can be effected by inhibitors present in clinical

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specimens (73). Stool specimens, for example, may contains inhibitors such as haemoglobin, anticoagulants, glycogen, fats, bacteria and drugs. Incomplete removal of these substances during nucleic acid extraction would lead to inhibition of the RT-PCR and hence false negative results. While nucleic acid extraction techniques, either manual or automated, usually are able to eliminate most inhibitors from specimens, laboratories using molecular assays must have methods to detect the presence of inhibitors. Many diagnostic laboratories are now using RT-PCR for detection. However, there are many laboratories that do not use RT-PCR for routine diagnostic testing due to the fact that

dedicated areas are needed for RT-PCR testing and equipment is expensive.

1.7 REVERSE TRANSCRIPTION-PCR