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Polymerase chain reaction (PCR) AKA "viral load" does not prove viral presence or activity
Hveragerthi Views: 6,326
Published: 9 years ago
This is a reply to # 1,548,978

Polymerase chain reaction (PCR) AKA "viral load" does not prove viral presence or activity


 From one of my old posts in response to a poster named Gerha:


>Diagnosis of hepatitis C 

Hepatitis c is a distinct entity of hepatitis which can be identified with reasonable accuracy by antibody testing and confirmation by detection of Hepatitis C viral RNA in in blood. What are molecular tests for HCV? As previously described, HCV is an RNA virus. The code of the genetic material, HCV RNA, is unique to this virus. Several types of tests (assays) are available to measure the HCV RNA in a person's blood. These tests are referred to as molecular tests because they examine the virus at the molecular level. The two most common systems for measuring HCV RNA are the reverse transcription polymerase chain reaction (RT-PCR) assay and the branched chain DNA (bDNA) assay. Recently, a third type of assay, called transcription-mediated amplification (TMA), has been released.-Gerha


I especially loved the part "reasonable accuracy by antibody testing". They say pretty much the same thing about the HIV tests, but they claim an almost 100% accuracy. Of course that is far from the truth. There are over 65 known causes for false positives with HIV antibody tests. This mostly due to serological cross reactivity. Then there are also false positives due to exogenous antibodies, and exposure without chronic infection. As far as PCR (viral load) goes that has been proven to have its own set of problems. The sailor they claimed died of HIV in the 50s was "confirmed" by PCR. Though 2 years after the fact they admitted that the samples were contaminated in the lab. And part of the claim that HIV came from monkeys was based again on tissue samples taken from wild caught monkeys that again were later proven to have been contaminated in the lab. Other factors also affect the accuracy, such as annealing time and reagents. We also have to keep in mind that PCR does not detect the presence of viruses, but rather the presence of DNA fragments, fragments that can have shared coding with other viruses. As I pointed out before I posted an earlier study about the DNA testing, in which about 40% of the time the doctors could not agree on what virus was being detected. Also keep in mind that this is the least sensitive of the PCR tests. So they claim that it is the least accurate. Though since it is less subject to cross contamination it could actually be considered more accurate. Then there is the new TMA. First of all the blood sample must be spun down and the serum frozen for shipment to the lab within 4 hours, which is not always going to happen. Though the man concern is sensitivity. They claim PCR is near 100% accurate, but then read this:

"A study presented at this year's 51st American Association for the Study of Liver Disease (AASLD) meeting reported on results using a new hepatitis C virus (HCV) test which utilizes a molecular diagnostics technology called "transcription mediated amplification" (TMA). The results of this study showed that more than one-third of the patient samples that tested negative for HCV using the conventional PCR test were actually positive when tested by the new TMA test." So why is this important? Well for one thing it proves that PCR is not near 100% accurate as has been claimed for years. Secondly, one of the main reasons PCR is not accurate is due to its high sensitivity, which makes it highly prone to contamination. For instance if the tech coughs or sneezes the samples will be contaminated with their DNA that will be amplified. And it has been shown that contaminated gloves can cross contaminate test wells. If you have ever worked in a lab you know that techs do not change their gloves between samples. Therefore cross contamination is a real concern with PCR, and increases with increased sensitivity. And TMA is exactly that, more sensitive than PCR. Here is some more for you on the problem of contamination:

"Technician proficiency can vary and increases in direct relation to experience. Intra-assay contamination can occur, including aerosolization, splashing, and carry-over.




This link talks about all of the precautions that must be taken to help reduce, not eliminate, contamination (read G and H). Now how many of these are followed precisely in a busy lab environment? As far as your statement of "As previously described, HCV is an RNA virus. The code of the genetic material, HCV RNA, is unique to this virus." read the following links:

Proclamations of the National Academy of Sciences, USA (1990) 87:2057

Hepatitis C Virus Shares Amino Acid Sequence Similarity with Pestiviruses and Flaviviruses as well as Members of Two Plant Virus Supergroups

'We report here that HCV shares an even greater degree of protein sequence similarity with members of the pestivirus group (i.e., bovine viral diarrhea virus and hog cholera virus), which are thought to be distantly related to the flaviviruses. In addition, we find that HCV shares significant protein sequence similarity with the polyproteins encoded by members of the picornavirus-like plant virus supergroups. These data suggest that HCV may be evolutionarily related to both plant and animal viruses.'



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