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HEPATITIS C VIRUS VIRAL LOAD

Categoría: Infectious diseases.
Días hábiles de entrega: 10
 

Venous blood obtained in tube with EDTA lid, Serum or Plasma.

Store and ship samples at temperatures between approximately 4-8 ° C; refrigerant gels or refrigerator ice can be used for it. The sample must reach the laboratory within 24 hours of being taken.

RNA extraction from the sample by commercial kits. Transcription of the RNA into cDNA with the Reverse transcription System kit (Promega, Madison, EU) using a random primer and a 5'NC HCV antisense primer. Real-time PCR using primers and SYBR® Green I No ROX Intercalators for the quantification of HCV RNA, which were designed from the sequence of the non-coding 5'NCR region of hepatitis C virus, according to Hong Yang J, et al. For the quantification, we construct a standard curve starting from the RNA extracted from the quantified serum of a positive HCV patient (positive control). Amplification of the cDNA was detected as an increase in fluorescence. A Step One thermal cycler from Applied Biosystems is used for the realization of the technique. Positive control: serum from HCV positive RNA patients with known viral load.

10 working days.

Negative: No amplification observed.

Positive: Amplification of HCV RNA x IU / ml LOG: x.

Limit of detection of the test of 100 IU / ml.

It is recommended that absolute values of viral load obtained by genomic amplification procedures be interpreted as any value within a range of 0.5 log (± 300 times) of the value obtained.

Genomik Laboratory has validated the test with positive and negative commercial controls; however, a negative result does not exclude the presence of HCV RNA below the sensitivity limit of the test, nor the possible presence of inhibitors of the PCR reaction. The reliability of the test is guaranteed by the use of systems for the prevention of contamination, in addition to the incorporation of an internal control of each test to avoid the appearance of false negatives.

The viral load test is useful in several areas:


Diagnosis: The test can detect the presence of HCV a few days after infection with HCV, before the antibody test is positive.


Confirmation of Chronic Infection: As noted above, up to 15% of people infected with HCV eradicate the infection. This process could take up to 6 months. However, antibody testing on these people will still give a positive result. A viral load test is used to determine if they have chronic infection.


To predict treatment success: People with a viral load less than 400,000 generally get better results.


Therapy management: The test determines if the treatment is controlling the virus. There are several viral load measurements for treatment response. These are described below. HCV viral load cannot be used in the same way as HIV viral load (see fact sheet 125). It is not a good indicator of the severity of HCV, or how fast the virus will progress. It is also more complicated to use it to evaluate the response to treatment. However, lower viral loads are associated with a better response to HCV therapy. In addition, higher viral loads are linked to an increased risk of HCV transmission, at least from pregnant women to their newborns.

Yang, J.Hg., Lai, J.P., Douglas, S., Metzger, D., Zhu, X.H., Ho, W.H. Real-Time R-PCR for quantitation of Hepatitis C Virus RNA. Journal of Virological Methods. 102 (2002) 119-128.



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