Elevated cervical white blood cell infiltrate is associated with genital HIV detection in a longitudinal cohort of antiretroviral therapy-adherent women.

BACKGROUND: Identification of factors associated with the presence of human immunodeficiency virus (HIV) in female genital secretions is critical for intervention strategies targeting transmission and eliminating replication of genital virus. We sought to monitor the prevalence of genital HIV shedding in antiretroviral therapy-adherent women over time and to assess changes in the genital microenvironment. METHODS: Levels of cell-free HIV (HIV RNA) and HIV-infected cells (HIV DNA) were monitored in peripheral blood samples and cervical and vaginal fluid samples at monthly intervals in 11 women for 1 year. Genital tract infections and fluctuations in cervical and vaginal white blood cell counts were also evaluated at each study visit. RESULTS: Plasma HIV was undetectable at the majority of study visits; when detected, it was only at low levels. Throughout the study, genital HIV RNA and DNA were detected in each person. Combined genital HIV (RNA and DNA) was detected at 49.2% of study visits and was associated with an elevated concentration of cervical white blood cell infiltrate (odds ratio, 2.52 [95% confidence interval, 1.01-6.22]; P = .04). Infiltrate was not associated with a clinical disorder or patient-reported symptoms. CONCLUSIONS: Despite antiretroviral therapy adherence and clinically suppressed plasma viremia, HIV was intermittently detected in genital secretions and was associated with subclinical inflammation and cells trafficking to the cervical mucosa.

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C.

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at -20 degrees C. DBS were created by spotting 50-microl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at -20 degrees C or at 37 degrees C with approximately 85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at -20 degrees C, and 398 stored at 37 degrees C. A chi-square test showed fewer positive reactions for DBS stored at 37 degrees C (55%) than for those stored at -20 degrees C (78%) (P < 0.0001). Samples stored at -20 degrees C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37 degrees C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37 degrees C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.

Amniotic fluid has higher relative levels of lentivirus-specific antibodies than plasma and can contain neutralizing antibodies.

BACKGROUND: The in utero transmission rate of HIV-1 is estimated to be 10-15% in the absence of interventions and breastfeeding. Natural protective mechanisms involving lentivirus-specific antibodies may therefore exist to limit in utero transmission of lentiviruses. OBJECTIVES: HIV-1- and SIV-specific immunoglobulin G (IgG) levels in amniotic fluid samples from humans and rhesus macaques were assessed. STUDY DESIGN: HIV-1- and SIV-specific immunoglobulin G levels, relative to total IgG concentrations in amniotic fluid samples from humans and rhesus macaques, were determined using a quantitative Western blotting procedure. Amniotic fluid from rhesus macaques was tested for the ability to neutralize SIV infection of CEMX174 cells. RESULTS: The levels of HIV-1- and SIV-specific immunoglobulin G, relative to total IgG concentrations in amniotic fluid samples from humans and rhesus macaques, were approximately 3-10-fold higher than in plasma. The ability of antibodies in human amniotic fluid samples to neutralize viral infectivity could not be assessed, because zidovidine was present in the samples. Most amniotic fluid samples from rhesus macques not treated with antiretrovirals were able to neutralize SIV infectivity, except for a sample from a SIV positive rhesus whose infant was infected in utero. CONCLUSIONS: Active immunity to HIV-1 resulting in virus-specific antibodies in amniotic fluid exists, and may be a natural barrier to in utero infection. This may provide hope for stimulating neutralizing antibody via vaccine design.

Various viral compartments in HIV-1-infected mothers contribute to in utero transmission of HIV-1.

Perinatal HIV transmission occurs in utero or intrapartum. The mechanisms and timing of transmission are not clearly understood. To compare the genetic sequences of the V3 envelope region of infant's plasma HIV to that of the mother's plasma, peripheral blood mononuclear cells (PBMC) and vaginal secretions, and correlate with timing of transmission. All 3 infants had a positive HIV PCR in the first days of life, thus classified as in utero infections. In the first mother-infant pair, two different variants were present in the infant, one correlating with maternal PBMC virus and highly homologous to virus from vaginal secretions and the other identical to sequences in maternal plasma. In the second pair, the infant plasma virus was similar to that of maternal PBMC. In the third pair, the cord blood and infant plasma virus were highly similar to maternal vaginal virus. The presence of more than one HIV variant from the maternal blood and from the vaginal compartment in the cord blood of infants presumably infected in utero could point to more than one episode of transmission or, alternatively, to transmission of PBMC virus.

Efficient methodologies for sensitive HIV-1 RNA quantitation from plasma and vaginal secretions.

BACKGROUND: Quantifying HIV levels in mucosal secretions is essential to study compartmentalized expression of HIV and facilitate development of intervention strategies to prevent disease progression and transmission. OBJECTIVES: To develop a sensitive, reliable, and cost-effective technique to quantify HIV from blood and vaginal secretions that is compatible with efficient implementation in clinical research environments. STUDY DESIGN: A sensitive, reliable, internally-controlled real-time reverse transcriptase (RT) PCR assay, which uses the HIV-1 pol gene as a target (Hpol assay) was developed to quantify HIV levels in plasma and genital secretions, and compared to the widely used Roche Amplicor HIV-1 Monitor assay. In addition, a simplified method of sample collection and processing of genital secretions (self-collection and use of RNAlater with batch processing) was compared to provider collection of samples and immediate processing. RESULTS: The sensitivity and reliability of HIV levels detected by the assay described herein correlate well with measurements from Roche Amplicor HIV-1 Monitor assay for both plasma and vaginal secretions (R(2)=0.9179 and R(2)=0.942, respectively). The Hpol assay reproducibly quantifies a lower limit of 5 HIV-1 RNA copies per reaction, with low-levels of inter-assay and intra-assay variation. Additionally, vaginal viral levels and detection frequency did not differ significantly between the two the collection and processing methods. CONCLUSIONS: The methodologies developed here provide sensitive, reliable, and cost-effective quantification of HIV levels in plasma and mucosal secretions, and are compatible with efficient use in clinical research studies.

Genetic analysis of simian immunodeficiency virus expressed in milk and selectively transmitted through breastfeeding.

To develop effective intervention strategies that prevent breast milk transmission of human immunodeficiency virus (HIV), we must understand the specific viral properties and mechanisms responsible for infant infection. We have used lactating rhesus macaques infected with a pathogenic simian immunodeficiency virus (SIV) stock to analyze the viral genotypes expressed in plasma and milk throughout the disease course and to identify those variants ultimately transmitted to infants through breastfeeding. In these studies we observed mother-to-infant transmission of SIV/Delta(B670) by eight females during the chronic phase of disease, and we analyzed by heteroduplex tracking assays and sequence analysis the distribution and fluctuations in viral genotypes expressed. Each female expressed multiple V1 envelope genotypes in milk near the time of transmission, while a single genotype was found in each of the infants. Variants transmitted to infants were not expressed throughout the maternal disease course but were only detected near the time of transmission. The emergence of the transmitted genotype in the dam typically occurred in plasma before milk and was coincident with increased milk viral loads. Transmitted genotypes tended to be longer and more glycosylated and had a less negative charge over the V1 region compared to viral genotypes expressed in milk but not transmitted. These observations demonstrate that specific viral genotypes are selectively transmitted to infants through breastfeeding and support the hypothesis that transmission occurs as genotypes adapt for efficient expression in milk.

The antibody response to SIV in lactating rhesus macaques.

As a model of breast milk transmission of HIV, we characterized humoral immune responses in the milk and plasma of 14 female rhesus macaques with suckling infants. Total immunoglobulin levels in plasma and milk were similar in all females and could not be correlated with transmission to the infant. These females, however, had elevated milk IgG levels and decreased milk IgA levels as compared with levels in seronegative controls. SIV envelope-specific antibody responses developed similarly in all females over the first 14-28 days after inoculation; however, 2 females had significantly lower titers by 98 days after inoculation. These females, characterized as rapid disease progressors, were the only animals to transmit SIV through breast-feeding during the period of acute viremia (14-21 days after inoculation). The remaining 12 females developed similar levels of high-avidity SIV envelope-specific IgG in plasma and low, but detectable, levels of IgA in milk. Despite similar quantities of antibody in milk, transmission of SIV through breast-feeding occurred in 8 of 12 mother-baby pairs during the chronic phase of disease. These observations are comparable with those for HIV-infected women and indicate that the SIV-macaque model provides a unique resource for deciphering the functional role of antibodies in breast milk transmission of HIV.