The diagnosis of perinatally acquired HIV infection can be established reliably as early as 2 months of age, or sometimes earlier, by viral culture (1) or PCR for proviral DNA (DNA PCR) (2–4). In the past several years, there have been investigations into the utility of quantitative RNA techniques (QRNA) for making the diagnosis, generally reporting high sensitivity and slightly lower specificity compared with DNA PCR (5,6). In two studies of QRNA in which a large proportion of the infants had received zidovudine (ZDV), there was no difference in QRNA sensitivity between ZDV recipients and nonrecipients (6,7). In predominantly breast-fed populations in Brazil and Uganda, where primary infection was thought to be common during the testing period, QRNA was reported to have an advantage because of its ability to detect temporary high elevations of viral quantity (8,9). Nevertheless, before using QRNA as a primary diagnostic tool, it is vital to understand its validity, particularly in the context of widespread perinatal antiretroviral prophylaxis. We studied the use of QRNA for the diagnosis of HIV infection in a cohort of non-breast-fed infants in the multicenter prospective Perinatal AIDS Collaborative Transmission Study (PACTS).
The Perinatal AIDS Collaborative Transmission Study is a prospective study of perinatal transmission and disease progression in a cohort of HIV-infected pregnant women and their infants. The study has been conducted at sites in Atlanta, Baltimore, and Newark and at a consortium of hospitals in New York City. Enrollment began between 1986 and 1990 at the various sites. Details of the enrollment process have been reported previously (10).
For the current study, analysis was performed on infants born from July 1, 1994 through June 30, 1998 whose HIV infection status was known and who had QRNA testing at least once prior to beginning antiretroviral therapy (ART) (excepting prophylactic neonatal ZDV). Analyses were limited to QRNA tests in infants ≤180 days of age. All QRNA results for these infants were used, including results of NASBA (NASBA HIV-1 RNA QT Amplification System; Organon-Teknika) and RNA PCR (Roche Amplicor; Roche Diagnostics Systems). The NASBA and Amplicor assays were performed in accordance with manufacturer's recommendations. The NASBA assay had a lower limit of quantitation of 1000 copies/mL, and the Amplicor assay had a quantitation limit of 400 copies/mL.
An infant was defined as infected with HIV if 1) two or more DNA PCR results were positive or 2) HIV ELISA and Western blot results remained positive after 18 months. An infant was defined as uninfected if 1) PCR results were repeatedly negative (minimum of two, at least one of these performed after 42 days of age) or 2) HIV ELISA results were nonreactive beyond 18 months of age.
Paired analyses were performed using the McNemar χ2 test. The comparison of viral loads by group was performed using the Kruskal-Wallis nonparametric test and the Wilcoxon rank sum test. Samples and data were collected after receiving informed consent from parents or a guardian according to the institutions' Human Investigations Committees.
There were 246 QRNA results available on 156 infants (121 tests in 54 infected infants and 125 tests in 102 uninfected infants). Of the 246 QRNA results, 225 (91%) were by the NASBA assay, 16 (7%) were by the Amplicor assay, and 5 (2%) were unspecified.
Sensitivity and specificity of single QRNA results are shown in Table 1. Overall results (i.e., irrespective of ZDV use) are in the row labeled “combined,” with a sensitivity of 29% in the first week of life, 79% from 8 to 28 days, increasing to 91% between 29 and 60 days, and remaining high thereafter. Specificity was 100% in all the periods except the 29- to 60-day period, when it was 93%. Of the three false-positive results, two had values of 1200 and 3500, respectively (i.e., in the very low range); in both cases, ZDV had been used by mother and infant. In the third case, the value was 130,000, and no ZDV had been used. Each of the three infants had a negative DNA PCR on that same day. When specificity and sensitivity were analyzed with a cutoff of 5000 copies instead of 400, specificity increased in the 29- to 60-day period from 93% to 98%. Using 5000 as the cutoff lowered the sensitivity in the following periods: 8 to 28 days (74%), 29 to 60 days (88%), and 61 to 120 days (92%). Positive predictive value of a single QRNA result was 100% in all periods except 29 to 60 days. Negative predictive value was 41% in the first week and 73% from 8 to 28 days, increasing to 93% at 29 to 60 days, 98% at 61 to 120 days, and 93% at 120 to 180 days (data not shown).
When sensitivity and specificity were stratified according to infant ZDV treatment (see Table 1), there appeared to be a nonsignificant trend to lower sensitivity in ZDV recipients between 8 and 120 days of age. Because there were no uninfected infants tested under 28 days of age in the group without ZDV prophylaxis, specificity of QRNA could not be examined in that group.
Viral load results according to maternal and/or infant ZDV use are shown in Table 2. Among infants <42 days of age, there was a significant difference in the distribution of viral loads between the four maternal-infant ZDV use groups (p = .005, Kruskal-Wallis nonparametric test). By the Wilcoxon rank sum test using a significance cutoff of 0.0083 (adjusted for multiple comparisons, derived from 0.05/6), infant viral loads were significantly lower in the group in which the mother and infant were treated with ZDV and in the group in which the mother alone was treated compared with those pairs who received no treatment (p = .007 and p = .005, respectively). Among children 42 to 180 days of age, there was also a significant difference in the distribution of viral loads between the four maternal-infant ZDV use groups (p = .004 by Kruskal-Wallis nonparametric test). By the Wilcoxon rank sum test, infant viral load in this age group was significantly lower only in the group where both the mother and infant were treated with ZDV compared with the group where only the mother was treated.
In the comparison of younger infants (<42 days of age) with older infants (42–180 days of age), median viral loads were higher in each of the mother-infant ZDV use groups in the older infants except in the group that did not use ZDV. The only statistically significant difference between the two age groups was when mothers used ZDV but not the infant, where the older infants had a significantly higher viral load (p = .003 by Wilcoxon sum rank test).
Both DNA PCR and QRNA results were available in 208 specimens. Comparing these paired results, there was no significant difference in the sensitivity and specificity of the two tests overall or in any time interval (data not shown). Concordance of DNA PCR and QRNA was ≥88% in each of the study's time frames.
DNA PCR has become the standard approach for the diagnosis of HIV infection in infancy. Interest in the use of QRNA for infant HIV diagnosis has increased, because the test is now used more often to guide initiation and monitoring of ART. QRNA tests also have specific advantages in situations where primary infant HIV infection is likely to occur during the diagnostic period (i.e., in breast-fed infants) (8,9). Before becoming routine, especially in non-breast-fed populations, it is important to establish whether the usefulness of quantitative HIV tests is enhanced or reduced by ART given to prevent mother-to-child transmission of HIV. This aspect is particularly relevant, because perinatal HIV prophylaxis using ART has become standard practice in the United States and many other countries after 1994 (10). In the current study, DNA PCR and QRNA were essentially equivalent for infant HIV diagnosis, with little practical effect of maternal/infant ZDV use on QRNA, despite a predictable reduction in infant viral load. Nevertheless, we cannot rule out the possibility that more intensive combination therapies used during the prenatal and perinatal periods could adversely affect the sensitivity of QRNA for infant diagnosis. The results in this report, although including some from the Amplicor assay, are predominantly from the NASBA assay; results may differ in populations in which a different assay predominated.
The sensitivity of QRNA in this non–breast-fed population was comparable to, although slightly lower than, that reported previously (5–7) and was similar to that of DNA PCR (2–4). There were 10 negative results in the first week of life among infants who were later found to be infected with HIV. Both the sensitivity and specificity of QRNA were comparable to those reported for qualitative NASBA (11) and noncommercial research qualitative assays (6). The sensitivity of QRNA appeared slightly lower in infants who had received ZDV, although the difference was not statistically significant. False-positive QRNA results may have occurred because of laboratory errors or errors in collection or labeling or because of the fact that the assays are configured for quantitation as opposed to detection. Our ability to define test specificity is reduced by the low number of uninfected infants tested, particularly in the first month of life.
Viral loads in this study were comparable to ranges previously described (12,13). It is not surprising that viral loads were usually lower in infants who had received ZDV with or without maternal ZDV use. Among the infants who were exposed to ZDV perinatally, the viral load tended to be higher in those 42 to 180 days of age compared with younger infants (<42 days of age), with a statistically significant difference found in infants when the mother received ZDV but not the infant.
There have been reports of more rapid disease progression in infected infants who received ZDV during the prenatal and perinatal periods compared with those who did not (8,14,15). This finding underscores the need for accurate rapid diagnosis in young infants. In the first month of life, when viral loads rapidly increase (11,13), these considerations are crucial, because initiation of highly active antiretroviral therapy (HAART) during this primary infection can dramatically reduce the viral load and potentially affect long-term outcome (16). We note that in an earlier analysis of the PACTS cohort, no infected child treated early with multidrug therapy progressed to AIDS or death by 1 year, regardless of early ZDV exposure (14).
Overall, QRNA appears comparable to DNA PCR for the diagnosis of HIV-exposed infants, although the viral load level is affected somewhat by the use of neonatal ZDV. In addition, QRNA occasionally produced a false-positive result during the second month of life. QRNA values in the low range (e.g., <10,000), in particular, should be interpreted with caution. Although QRNA techniques have been more expensive than DNA PCR, a new generation of RNA assays is being developed commercially, and these newer assays may well be financially competitive with DNA PCR assays. Our results indicate that QRNA assays are accurate for infant HIV diagnosis, regardless of perinatal ZDV use. Given the possibility of occasional specimen mix-up or contamination, using two assays at separate time points to verify infection status for clinical purposes is crucial (7). In the United States, QRNA may be most useful as a test to confirm a previous positive DNA PCR result, and in that situation, it can obviate the need for a second DNA PCR (6). Importantly, QRNA as a second-stage test has the advantage of providing quantitative results and can serve as a baseline test for institution of HAART. Finally, as increasing numbers of infants are exposed to maternal combination ART during pregnancy, further evaluation of the sensitivity of QRNA for early infant diagnosis will be necessary.
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