Human immunodeficiency virus type 1 RNA polymerase chain reaction reasonably excludes infection in exposed infants

A nucleic acid sequence-based amplification (NASBA) assay that detects HIV RNA may be helpful in excluding perinatal HIV infection. We reviewed the records of 190 infants born to HIV-infected mothers. The sensitivity and specificity of the NASBA assay were 100% when measured at two time periods in the first 9 months of life. The HIV RNA NASBA assay should be considered a valid diagnostic test in this population. Most cases of HIV/AIDS cases in children <13 years of age in the US are vertically acquired. Currently 90% of HIV-infected mothers and/or their infants receive zidovudine during pregnancy and delivery and during the postpartum period. 1 This increase in zidovudine use has been accompanied by a decrease in the number of perinatally infected children. 2 Early determination of the HIV status of infants born to HIV-infected mothers is important for the medical management of these infants, because decisions regarding duration and type of antiretroviral treatment and prophylaxis for prevention of opportunistic infections are made based on this determination. Antibody testing in early infancy is impaired by the fact that maternal antibodies are passively transferred during pregnancy. Current recommendations from the Public Health Service (PHS) and CDC are to perform PCR with proviral DNA testing or plasma viral RNA. 3–6 Although either DNA-PCR or RNA-PCR is acceptable, most centers use DNA-based methods for early diagnosis. To date most studies involving RNA-PCR testing have assessed its use in establishing, rather than excluding, the diagnosis of perinatally acquired HIV infection. Thus there is little information about the use of RNA-PCR testing for exclusion of HIV infection in young infants. The purpose of this study was to determine the usefulness of the HIV RNA-PCR nucleic acid sequence based amplification (NASBA) assay to identify HIV-uninfected infants before seroreversion. Materials and methods. Study population. We reviewed the records of 190 infants followed in the Memphis Pediatric HIV/AIDS Program who were born to HIV-infected mothers between 1996 and 2000. Our policy is to follow all such infants at regular intervals from birth until HIV status is established in accordance with recommendations of the PHS and CDC. 3, 4 During the study period the HIV RNA-PCR NASBA assay was used as a routine virologic testing procedure. Inclusion criterion and definitions. We included in the study infants for whom at least two NASBA assays were done in the first 9 months of life (the first between 1 and 4 months of age and the second between 4 and 9 months of age) and two enzyme-linked immunosorbent assays (ELISA) in the first 12 months of life or at least one after 12 months of age. HIV status was defined according to criteria of the American Academy of Pediatrics. Infants were determined to be HIV-negative if they had at least two negative ELISA) assays if <12 months of age or a single negative ELISA if older than 12 months of age. HIV-infected status was defined as two positive virologic tests (culture, DNA-PCR, RNA-PCR) in infants <18 months of age or a single positive virologic test after 18 months of age. 7 Assay for plasma HIV RNA. Plasma HIV RNA was measured with a NASBA assay adapted for use with HIV (NASBA HIV-1 RNA QT kit; Organon Teknika, Durham, NC). The assay consists of four stages: nucleic acid release; nucleic acid isolation; nucleic acid amplification; and nucleic acid detection. The NASBA assay lower threshold of sensitivity is 400 HIV RNA copies/ml. The NASBA assay was performed following the manufacturer’s standard operating procedures. 8 Results. Of the 190 infants born to HIV-infected mothers followed in our clinic, 139 (73.2%) met the eligibility criterion. Of these infants 124 were HIV-negative and 15 were HIV-infected. Of the 124 infants who were HIV-negative, no infant had a positive HIV RNA-PCR at any time. Thus in our sample population the sensitivity and specificity of the HIV RNA-PCR NASBA assay in identifying HIV-uninfected infants were 100%. When there were two undetectable HIV RNA-PCR assessments (one performed after 1 month of age and the other performed after 4 months of age), HIV infection was considered reasonably excluded. Using seroreversion by HIV ELISA as the standard for HIV-negative status, the kappa coefficient of correlation was 1.0. The 15 HIV-infected infants were diagnosed between 3 and 106 days of age, with a median of 45 days of age. All were positive on their first NASBA assay and had viral loads between 11 000 and 1 500 000 RNA copies/ml (median, 425 000). Three of these infants had a NASBA assay done before 1 month of age with all three being positive. The earliest test was done at 3 days of age and detected a viral load of 11 000 RNA copies/ml. The other two infants were tested at 15 and 19 days of age and had 87 000 and 600 000 RNA copies/ml, respectively. These data confirm prior reports of the sensitivity of HIV RNA-PCR NASBA assay in identifying HIV-infected infants. 9, 10 Thirty-one of the infants in this study had concurrent HIV DNA-PCR studies. These were performed as part of the PACTG 316 study, and all results were returned as undetectable, confirming our findings with HIV RNA-PCR. A big concern was determining the HIV status of the 51 infants who did not meet the criterion for inclusion in this study. Thirty-five infants were excluded because the HIV RNA-PCR tests were not done during the specified periods for inclusion (between 1 and 4 months and between 4 and 9 months of age). All of these 35 infants eventually demonstrated evidence of HIV seroreversion by ELISA. An additional 8 infants were lost to follow-up and had only 1 negative HIV ELISA documented. However, all of them had enough virologic testing to be considered HIV-negative. There were 8 infants who had negative HIV ELISA tests before 4 months of age; their mothers had false positive tests by HIV ELISA during pregnancy. Discussion. In this study of infants born to HIV-infected mothers, the HIV RNA-PCR NASBA assay was highly sensitive and specific in determining the HIV status of the infants. Results were identical when the two testing periods (1 to 4 and 4 to 9 months of age) were evaluated individually and when the two assessments were combined to meet the CDC definition of “HIV-infection reasonably excluded.” These findings suggest that this assay can establish HIV status in this population. Despite the endorsement of the RNA-PCR test by the PHS and the CDC, to date there has been little information regarding utility of HIV RNA testing for determination of HIV status in infants born to HIV-infected mothers. In studies done in the United States, France and Brazil, the HIV RNA-PCR NASBA assay was more sensitive than a DNA PCR method in detecting HIV-infected infants. 9–12 Another study concluded that HIV-1 RNA reverse transcriptase PCR offered a slight advantage in sensitivity over DNA-PCR for diagnosis of HIV infection in young infants. 13 Because of the aggressive treatment of HIV-infected women during pregnancy, there are fewer HIV-infected infants born in the US. This has refocused the importance of testing on early exclusion of HIV infection and reassurance of the family regarding infection status. Although this is a retrospective study with its associated limitations, it is based on a real life scenario and a situation many physicians are likely to encounter. One of the limitations of the study is that 51 infants were not eligible because testing for them did not meet the defined eligibility criterion. However, the testing done to these infants is reassuring in that we know that none of these children had evidence of HIV infection. These results provide excellent reassurance about the sensitivity of the NASBA assay. Some potential advantages of the NASBA HIV RNA-PCR include its wide availability in commercial laboratories, offering the potential of earlier notification of results. The HIV RNA-PCR assay is also routinely used for management in adults and provides quantitative results (“viral load”), which are useful to follow response to antiretroviral therapy. Another advantage of the HIV RNA-PCR assay is that it uses plasma, rather than whole blood, which can be easily processed and frozen within a few minutes (∼10 min) and can be easily stored. Although we used the NASBA methodology in this study, it is reasonable to assume that similar results would be obtained using other RNA-PCR methodologies. Based on the evidence presented, the HIV RNA-PCR NASBA assay could be used to screen infants perinatally exposed to HIV at 1 and 4 months of age. Our rationale is based on the mode of transmission in the United States, where HIV-infected mothers are advised to avoid breast-feeding their infants. The avoidance of breast-feeding limits the risk of HIV transmission after the peripartum period and would allow the detection of all HIV-infected infants at an early age. Our results support the findings of previous studies demonstrating that the NASBA assay is sensitive early in life, particularly after 4 weeks of age. 9 In our series even the three HIV-positive infants tested before 1 month of age had detectable HIV RNA levels. Acknowledgments. This work was supported in part by Cancer Center Support CORE Grant P30 CA 21765 and the American Lebanese Syrian Associated Charities.