Pediatric diagnosis of human immunodeficiency virus type 1 infection: the problem of false negative DNA polymerase chain reaction results.

Abstract

To The Editors: We read with interest the report from Kline et al. 1 describing a false negative HIV DNA PCR reaction in an infant infected with a non-B subtype of HIV-1. We and others in the United Kingdom are aware of this problem among infected children and adults 2–4. As discussed by Kline et al., 1 most in house and commercial assays, such as Roche Amplicor Version 1.0, have been developed and optimized for use with the B subtype of HIV-1, and their ability to detect non B subtypes is variable. This is of particular concern to us as ∼30% of patients attending clinics in South London are infected with non-B subtypes of HIV-1, predominantly subtypes A and C 5. In line with current UK guidelines, 4 we use HIV DNA PCR (Roche Amplicor Version 1.0) for pediatric diagnosis, and our local policy also includes use of an HIV RNA assay (Bayer-Versant HIV-1 RNA 3.0 bDNA). We were initially alerted to the problem of false negative DNA PCR results in three African infants born to HIV-infected women. In all three infants a high HIV viral load (>100 000 HIV RNA copies/ml) was detected in the absence of detectable HIV DNA using the Roche Amplicor Version 1.0 assay. However, when samples were tested with an alternative assay and a different primer set, HIV proviral DNA was detected. Two of the three mothers were subsequently shown to be infected with a non-B subtype of HIV. These findings prompted us to amend our procedures for pediatric diagnosis to reduce the risk of false negative DNA results. A maternal sample, ideally collected at or before delivery, is now routinely tested by the Roche Amplicor Version 1.0 assay to confirm that the primers can detect the virus to which the infant has been exposed. In cases in which maternal HIV DNA is not detected, an alternative assay with primers that can amplify maternal virus is used to test the infant. Current UK guidelines now recommend testing a maternal sample as part of routine pediatric diagnosis.4 During the last few years, we have tested samples from 108 HIV-infected pregnant women using the Roche Amplicor Version 1.0 assay. The majority of these women were African and likely to be infected with non-B subtypes of HIV-1. Seventeen (15.7%) had undetectable proviral DNA, and 10 (9.2%) gave equivocal results. Thus in our setting this commercial assay gave rise to potentially false negative results in up to 25% of samples and if additional tests had not been conducted this could have resulted in misdiagnosis in the infant. Kline et al. 1 suggest that testing samples in parallel with an HIV RNA assay is one approach to dealing with the problem of false negative HIV DNA results. This is the strategy that we use because a combination of assays, together with confirmation of results in a second sample, provide a secure diagnosis. However, quantitative HIV-1 RNA assays may give rise to low level (<1000 HIV RNA copies/ml) false positive results.6, 7 Indeed we have seen two infants in whom a low viral load has been detected intermittently by the Bayer bDNA assay in the absence of detectable HIV DNA or other markers of infection. These results were confirmed as false positives by an alternative HIV RNA assay. Kline et al. 1 conclude by suggesting that a more long term solution to false negative DNA results is the use of DNA assays which have been optimized and validated for use with non-B subtypes of HIV-1, and we would fully agree with this. However, the Roche Amplicor Version 1.5, which they suggest may be appropriate, is a prototype assay not currently available commercially other than in South Africa. However, a supplemental primer set, designed to improve the performance of the Amplicor 1.0 assay with non-B subtypes of HIV-1, is available from Roche Diagnostics. Although in some cases in our experience this has resolved the problem of false negative results, there are a small proportion of samples that are still not amplified and require further testing in a reference laboratory. Early diagnosis of pediatric HIV-1 infection is important; however, to ensure that all non-B subtypes of the virus are detected, further development of commercial HIV-1 DNA PCR assays is urgently required. Siobhan O’Shea, Ph.D. Jane Mullen, M.Sc. C. Y. William Tong, M.D.