Viral Load: False Positives and Non-specific Primers

Douglas et al (Annals of Internal Medicine, 1996, Vol. 124, p803-815) analysed 96 studies evaluating the sensitivity and specificity of PCR for the diagnosis of HIV infection. These studies included 5739 "HIV-infected" persons and 8929 "uninfected" persons.

They found (page 808):

"Measured performance was extremely variable. When indeterminate PCR results were excluded, sensitivity ranged from 10% to 100% and specificity ranged from 40% to 100%"

They concluded (pages 809-810):

"the false positive and false negative rates of PCR that we determined are too high to warrant a broader role for PCR in either routine screening or in the confirmation of diagnosis of HIV infection. This conclusion is true even for the results reported from the more recent, high-quality studies that used commercially available, standardised PCR assays"

If what Noble says is true then false negative PCRs should be completely unheard of, they should NEVER occur.

Noble also says

"The primers used in HIV PCR are quite specific."

"The most common problem in PCR is carry-over contamination with amplified sequences from previous tests.

PCR is extremely sensitive. If amplified sequences form the previous tests are not completely removed then false positives will occur. Good laboratory practice can reduce the number of false positives but PCR is still not used for primary tests."

Keep these comments in mind as you read the following from Holguin et al 1998. AIDS, Vol. 12, p2076-2077:

"Since viral load tests have been approved for the quantification of viraemia in already known HIV-seropositive individuals, we were interested to know their specificity. For this purpose we selected 20 healthy volunteers, all of whom yielded negative results for HIV antibodies using different screening tests. Plasma from all of them were analysed by three different currently available HIV viral load tests: branched DNA (bDNA) signal amplification assay (Chiron), nucleic acid sequence based amplification (NASBA) Nuclisens, and Ultradirect reverse transcriptase (RT)-PCR Monitor. The detection limits of these assays are 500, 40 and 20 HIV RNA copies per ml respectively. Moreover, we used two different HIV-1 monitor kits (Roche, Madrid, Spain), one using primers exclusively designed for recognising HIV-1 subtype B and another with non-B primers."

"In summary, two samples yielded positive results by the bDNA assay, with values of 2020 and 10620 HIV RNA copies per ml. Another two specimens yielded false positive results by the NASBA Nuclisens, with values of 150 and 480 HIV RNA copies per ml. Finally, one of the 20 samples was interpreted as positive by the Ultradirect RT-PCR Monitor assay, with a value of 73 HIV RNA copies per ml. Moreover, using the monitor test with non-B primers, up to four of the 20 samples yielded positive values, ranging from 28 to 253 HIV RNA copies per ml. RESULTS WERE REPRODUCED IN MORE THAN HALF OF TESTED SPECIMENS FOR WHICH PLASMA VOLUMES WERE ENOUGH FOR REPEAT TESTING. The experiments were all performed by a single well-trained laboratory technician. Furthermore, CONTROLS RUN DURING THE STUDY EXCLUDED CONTAMINATION AS A SOURCE OF FALSE POSITIVE RESULTS."

It is obvious here, and the authors admit it, that the primers are engaging in non-specific hybridisation. In other words the authors admit the primers are non-specific.

It's worth emphasising that the researchers found 5 out of 20 (25%!) false positives with one set of primers and 4 out of 20 (20%) false positives with a different set of primers, moreover these results were REPEAT false positives in the cases where there was sufficient plasma left for retesting.