Analysis of HIV-1 in the cervicovaginal secretions and blood of pregnant and nonpregnant women.

OBJECTIVES: To detect HIV-1 in cellular and acellular fractions of cervicovaginal secretions obtained by cervicovaginal lavage (CVL) and evaluate viral genotypes in the HIV-1-positive CVL samples. STUDY DESIGN/METHODS: This study consists of 37 HIV-1-seropositive pregnant and nonpregnant women from the United States. A total of 63 paired CVL and blood samples were collected. HIV-1 DNA from cervical cells (CC) and virion RNA from cervical supernatant (CS) was detected by gag polymerase chain reaction (PCR) assays. The HIV-1 genotypes were determined by analyzing the nested PCR-amplified V3 region sequences of the HIV-1 gp120 envelope gene. RESULTS: Within this cohort, 95% of the women were on single or combination antiretroviral therapy. Of the pregnant women, 63% of samples had HIV-1 viral DNA in the CC, and 29% of samples were positive for viral RNA in the CS. Among nonpregnant women, 71% of samples were positive for HIV-1 DNA in CC, and 46% of samples tested positive for virion RNA in CS. Plasma viral load ranged between 10,000 and 100,000 copies/mL and showed significant correlation with the detection of HIV-1 RNA in the CVL; this relation was less apparent with viral DNA in CC. The viral blood and CVL specimens were further analyzed by evaluating the genotypes of HIV-1 variants. In most patients, a high degree of similarity was observed between the viral sequences derived from blood and CVL samples. Two patients demonstrated closely related but somewhat distinct genotypic variants in CVL and blood. One subject showed clear compartmentalization in which distinct viral genotypes were observed in CVL and blood. Based on V3 loop analyses of gp120, with one exception, the cervicovaginal secretions harbored viral populations with a macrophage (CCR5)-tropic phenotype. CONCLUSIONS: This study demonstrates the unique characteris tics of HIV-1 strains in the genital secretions of a relatively large cohort of HIV-1-infected women in the United States. These results are important for further analysis of HIV-1 transmission and pathogenesis in vivo and for rational vaccine design.

Protective role of beta-chemokines associated with HIV-specific Th responses against perinatal HIV transmission.

To examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as beta-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with beta-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with beta-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.

Epstein-Barr virus DNA in the blood of infants, young children, and adults by age and HIV status.

Polymerase chain reaction (PCR) methodology was used to detect Epstein-Barr virus (EBV) DNA in peripheral blood mononuclear cells (PBMCs) from children and adults whose HIV status (i.e., infected or uninfected) is known. Initial EBV infections especially occurred in children between the ages of 7 and 24 months. EBV-positive children with vertically acquired HIV infection tended to have a detectable blood level of EBV DNA for a period of years, and their EBV DNA blood levels often exceeded 10,000 copies/0.1 ml of blood--hundreds of times higher than levels typically found in EBV-positive, HIV-uninfected children of the same age. EBV DNA was found in PBMCs in 26% of 49 HIV-infected mothers who were sampled during their pregnancy, but the median EBV DNA level in their EBV-positive samples was low--only 50 copies/0.1 ml blood. In limited tests with specimens from children infected with both HIV and EBV, high blood levels of EBV DNA unexpectedly appeared to be associated with decreased blood levels of HIV DNA (p = .063).

HIV DNA blood levels in vertically infected pediatric patients: variations with age, association with disease progression, and comparison with blood levels in infected mothers.

Blood levels of HIV DNA in our vertically infected pediatric patients typically followed a characteristic age-related pattern: continuously increasing with increasing age to a peak between ages 4 and 8 months, and thereafter rather steadily declining. Median HIV DNA levels peaked about 3 months earlier in children who by age 24 months developed more severe rather than less severe HIV disease. Children at particular risk of developing severe HIV disease by age 24 months commonly had > 800 HIV DNA copies per 0.1 ml of blood at age 3 weeks to 2 months, > 1,000 copies at 2 to 4 months, and > 2,500 copies at ages 4 to 6 months. Near the time of delivery mothers who transmitted HIV had significantly higher median blood levels of HIV DNA than mothers who did not transmit, but median HIV DNA levels in infected mothers as a group were low compared with those in pediatric patients > or = 1 month of age.

Viral studies of mothers with human immunodeficiency virus infection at delivery and their infants in the first 3 days of life.

OBJECTIVE: We studied 49 mother-infant pairs for human immunodeficiency virus (HIV) (a) to assess the virological and immunological status of HIV-infected mothers at delivery and their infants within the first 3 days of the infant's life, and (b) to correlate these findings with eventual infection outcome in the infant. METHOD: Maternal blood from women in labor and infant's blood within 3 days of life were tested for the titer of HIV immunoglobulin G (IgG) antibody, for presence of HIV by culture, for p24 antigen, for HIV DNA by polymerase chain reaction (PCR), and for absolute T-helper cell count (CD4). RESULTS: Eight infants were in the confirmed infected (CI) group, with a transmission rate of 21%. Thirty infants were in the confirmed uninfected (CU) group. In the mother, mean anti-HIV IgG titer was 1:2600 (CI group) and 1:3350 (CU group); in the infant, the mean titer was 1:3250 (CI group) and 1:2710 (CU group). Eighty-seven percent of the mothers were culture-positive in the CI group compared to 33% in the CU group (p = 0.005). Eighty-seven percent of CI infants were PCR-positive at birth; none was PCR-positive in the CU group (sensitivity = 87%; specificity = 100%). Sixty-two percent of CI infants were culture-positive at birth, whereas none was positive in the CU group (sensitivity = 62%; specificity = 100%). Of the uninfected infants, 23% were positive for p24 antigen at birth. CONCLUSIONS: HIV IgG antibody titers in mothers and their infants at birth were markedly elevated in both CI and CU groups but were not protective against infection. However, the high titers explain the long duration of this antibody in the blood of infants born to infected mothers. Culture positivity in the mother at delivery correlated highly with eventual infection in the infant (p = 0.005). HIV antigen, specifically p24 antigen, was detectable in uninfected infants when tested at birth.

Human immunodeficiency virus infection in infants during the first 2 months of life. Reliable detection and evidence of in utero transmission.

OBJECTIVE: To evaluate the clinical utility of a polymerase chain reaction (PCR) method for detecting human immunodeficiency virus (HIV) infection in infants 2 months of age or younger who were born to HIV-positive mothers. DESIGN: Prospective, longitudinal study lasting 3 years. The PCR tests were performed with coded peripheral blood mononuclear cell lysates, and results were compared with findings using Centers for Disease Control and Prevention (CDC) (Atlanta, Ga) criteria defining HIV infection in children. SETTING: Hospitals, particularly a pediatric hospital in Washington, DC. PATIENTS: Newborns, young infants, and HIV-infected mothers. OUTCOME MEASURE: Presence or absence of pediatric HIV infection using CDC criteria compared with a diagnosis based on the detected presence or absence of HIV proviral DNA using PCR testing. RESULTS: One or more blood samples obtained by 62 days of age from 30 (94%) of 32 HIV-infected infants were positive for HIV by routine PCR testing. Blood samples from 32 infants now confirmed to be uninfected tested negative for HIV. Human immunodeficiency virus DNA was detected in blood samples obtained within 48 hours of birth from eight of nine infected infants. In six of these newborns as well as most older infants, HIV DNA was present in such quantity that it was detectable in specimens equivalent to 0.01 mL or less of the original blood sample. CONCLUSIONS: Our PCR procedure can reliably detect the presence or absence of HIV infection during the first 2 months of life. The frequent presence and not uncommon high titer of HIV DNA within 48 hours of birth suggest that much of the transmission of HIV from mother to infant occurs well before birth.

Cellular immune factors associated with mother-to-infant transmission of HIV.

OBJECTIVE: To study a possible correlate of protection in mother-to-infant transmission of HIV infection. In particular, to determine whether lack of HIV-specific T-helper (TH) function as indicated by HIV and non-HIV antigen-stimulated interleukin (IL)-2 production of mother and/or newborn peripheral blood leukocytes (PBL) is associated with mother-to-infant transmission of HIV. METHODS: PBL from 21 HIV-seropositive pregnant women and 23 cord blood leukocytes (CBL) from their offspring were studied for in vitro TH function by IL-2 production in response to HIV and non-HIV antigens. Polymerase chain reaction (PCR) and viral culture assays were performed to determine HIV infection of the infants. RESULTS: PBL from 10 out of 21 (48%) mothers and from eight out of 23 (35%) CBL samples responded to two or more out of five synthetic gp 160 envelope (env) peptides. Three of the 23 (13%) offspring were shown to be HIV-infected by PCR and/or viral culture on follow-up. All three infected infants were from a subset whose CBL did not exhibit env-specific TH immunity. CONCLUSION: Our results demonstrate that fetal T cells can be primed to HIV env determinants in utero, suggest that HIV-specific TH immunity may be protective in newborns, and provide a possible means for identifying newborns who are at risk for HIV infection.

Laboratory methods for early detection of human immunodeficiency virus type 1 in newborns and infants.

Cumulative data on serological testing of newborns and infants have shown that (i) maternal and newborn anti-HIV-1 IgG titers are high at delivery, which may explain the persistence of antibody in the infants of seropositive mothers; (ii) in some situations, serial HIV-1 antibody testing may identify infected infants; and (iii) detection of anti-HIV-1 IgA or IgM is specific for infection but the sensitivity of this assay may be compromised in certain situations, such as when infected infants are hypogammaglobulinemic or when the rise and fall of HIV-1-specific IgM synthesis following acute infection has been completed before delivery of the infant. Cumulative data on PCR, viral culture, and tests for antigen in newborns and infants have shown that (i) among all age groups, viral culture is probably the most specific test available for detection of HIV-1, as PCR and the p24 antigen test may (though rarely) give false-positive results; (ii) the sensitivity of these tests increases in the order of antigen, culture, and PCR, with relatively insensitive results in the first 3 months of life for all of these tests; (iii) the sensitivity of all of these tests improves and approximates 90 to 100% when infants over 6 months of age are tested; and (iv) data regarding the sensitivity, specificity, and usefulness of these virological assays in infants under 3 months of age are very scant and inconclusive.

Maternal and fetal infections.

Acquired immunodeficiency virus infections had recently been the leading cause of death in minority women of reproductive age in several urban cities in the United States. In addition, more than 80% of infants with human immunodeficiency virus type 1 infection acquired the infection perinatally. These alarming data have spawned a prodigious body of information about perinatal human immunodeficiency virus type 1 infection. Some of the most recent advances are highlighted in this review. Revolutionary techniques, initially developed in molecular biology, such as in situ hybridization, the polymerase chain reaction, and gene sequencing, have also recently made a significant contribution to the diagnosis and better understanding of viral and bacterial infections. For example, organisms such as cytomegalovirus, herpes simplex, Chlamydia, parvovirus, Pneumocystis carinii, and toxoplasma are now detectable by polymerase chain reaction methods. The most recent information in these areas is also briefly reviewed.

Detection of human immunodeficiency virus type 1 infection in young pediatric patients by using polymerase chain reaction and biotinylated probes.

Polymerase chain reaction (PCR) testing using up to four primer pairs and biotinylated probes was 97.9% sensitive (188 of 192 specimens positive) and 100% specific (267 of 267 specimens negative) for detecting the presence or absence of human immunodeficiency virus (HIV) DNA in peripheral blood mononuclear cells from pediatric patients whose HIV status has been confirmed. SK38/39 and SK145/150 were the most sensitive primer pairs, respectively detecting HIV DNA in 95.6 and 95.9% of peripheral blood mononuclear cell specimens from HIV-infected children and collectively detecting all adequately tested PCR-positive specimens. Primer pairs SK29/30 and SK68/69 respectively detected HIV DNA in only 76.4 and 76.6% of HIV-positive specimens. Among infants born to HIV-seropositive mothers, 30 who subsequently were confirmed to be infected were sampled when they were less than or equal to 6 months of age; in all but one infant, HIV DNA was found in the first specimen collected. Among the nine youngest infected infants tested, all were PCR positive by 38 days of age. PCR methods thus have reliably detected vertically transmitted HIV infection early in life.