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The polymerase chain reaction (PCR) based assay of human immunodeficiency virus type 1 (HIV-1) RNA in plasma is now commercially available and is used widely for the assessment of antiretroviral treatments. The kit is called the AmplicorTM HIV-1 monitor test kit version 1.0 from Roche Diagnostics (Tokyo, Japan). However, this system is not sensitive enough for the accurate measurement of genetic subtypes A and E, and it gives falsely low titres for these virus subtypes.1,2 To surmount this problem, additional gag primers (AG primers) have been provided by Roche for research use (Ver. 1.0 plus). Furthermore, a new improved version (Ver. 1.5) was developed recently, which is said to yield accurate results not only on subtype B but on subtypes A and E. With the Ver. 1.0 plus kit, adding the AG primer set from the Ver. 1.5 kit to the PCR master mixture containing the Ver. 1.0 primer set makes it possible to amplify even subtype A and E viruses. In the Ver. 1.5 kit, the downstream primer is 12 bases downstream from the Ver. 1.0 primer position, whereas the upstream primer position is unchanged but the primer has two base substitutions. In our laboratory, we have examined over 1500 samples (148 cases) using the Ver. 1.0 kit, and among them, 150 samples (65 cases) were also measured with the Ver. 1.0 plus kit. As expected, most cases with the HIV-1 subtypes A and E, which could not be measured with the Ver. 1.0 kit, could be measured with the Ver. 1.0 plus kit. However, we were particularly interested in a few cases that gave unexpected results. Clinically and epidemiologically, these patients are not different from our other patients infected with HIV-1 subtypes A or E. As shown in table 1, case 1 gave equivalent results with all of the kits but in cases 2 to 5 higher results were obtained with the Ver. 1.0 plus kit than with the Ver. 1.0 kit. We measured these specimens with the Ver. 1.5 kit (kindly provided by Roche Diagnostics, Japan). In these five cases, all but case 2 revealed equivalent values with both the Ver. 1.0 plus and Ver. 1.5 kits. Unexpectedly, in case 2, only the Ver. 1.0 plus kit yielded the accurate results.
On the other hand, in case 6, a higher result was obtained with the Ver. 1.0 kit but not with the Ver. 1.0 plus or Ver. 1.5 kits. An additional two cases showed a similar trend, although the degree of discrepancy was less severe (data not shown).
To clarify the cause of these discrepant results, sequence analysis of the amplified regions of these cases was performed. The nucleotide sequences of these cases were found to be homologous to subtype A or E virus when they were aligned with the consensus sequences of HIV-1 subtypes A, B, and E obtained from the HIV Sequence Database WWW home page (Sequences. (Online.) http://hiv-web.lbnl.gov. 30 October 1999, last date accessed.). Surprisingly, the sequences of the primer regions of all cases were completely identical (fig 1). In conclusion, cases 2 and 6 are measurable by one of these kits, Ver. 1.0 plus and Ver. 1.0, respectively; and case 1 is measurable by all kits despite having the same nucleotide sequence in the primer regions as the other cases. The results of sequence analysis of the primer regions suggests that the minor differences in sequence between the virus and the primers does not always affect amplification efficiency in these kits. Although such cases might form a minor population among HIV-1 infections, these results indicate that some cases could not be measured by a single kit. As far as we have experienced, even if one kit fails to measure virus, the other will yield the expected viral load, as judged by disease history, CD4 count, treatment, and so on, suggesting that these kits are mutually complementary. If other methods, such as nucleic acid sequence based amplification or branched DNA (bDNA) systems, are available, it would be useful to test with them. We have found some cases of subtype A or E that have shown a higher viral load with bDNA (Chiron QuantiplexTM HIV RNA 2.0 assay Chiron Corporation, Emeryville, California, USA) than the Amplicor HIV-1 monitor test kit (data not shown). In patients infected with subtype B virus, the correlation between the Amplicor HIV-1 monitor test kit and the bDNA method was excellent (r = 0.904; n = 21). In conclusion, because the major difference in the three versions of the Amplicor HIV-1 monitor test kit is the primer set, we emphasise that for accurate quantitative measurement using this kit various additional primer sets that can amplify similar regions are needed.