HIV DNA Polymerase Chain Reaction Research Articles

The use of dried blood spots on filter paper for the diagnosis of HIV-1 in infants born to HIV seropositive women

Polymerase chain reaction (PCR) is the most sensitive test to diagnose HIV-1 infection among infants born to HIV seropositive mothers. The purpose of this study was to evaluate the use of dried blood spot (DBS) specimens for PCR and to compare it with whole-blood stored in tubes for HIV-1 DNA PCR. Five hundred and seventy-seven whole-blood infant samples were tested using HIV-1 qualitative in-house nested DNA PCR. Three hundred and fifty-nine samples were from infants at 48 hours of birth and 218 samples at second month. All positive samples tested from whole-blood and every fifth negative sample were coated onto filter paper. DNA was extracted from the filter paper and was amplified using in-house nested PCR. Among the whole-blood samples tested using HIV-1 DNA PCR, 19 of 359 (5.29%) samples were HIV-1 positive and 340 (94.7%) were negative at 48 hours of birth. At second month, 19 (8.7%) of the 218 samples were positive and 199 (91.2%) were negative. Using dried filter paper, 18 samples (95%) tested positive from 19 positive samples (using whole-blood) and 1 tested negative at 48 hours of birth. The 68 negative samples tested using whole-blood were also negative in the DBS test (sensitivity 95% and specificity 100%). At second month, 19 were positive and 40 samples (every fifth sample of 199) were negative (sensitivity and specificity, 100%). PCR performed using DNA extracted from filter paper permits the diagnosis of HIV-1 infection among infants born to HIV-1 seropositive mothers. This assay is simple, rapid, sensitive and specific and can be used in resource limited settings.

THE USE OF DRIED BLOOD SPOTS ON FILTER PAPER FOR THE DIAGNOSIS OF HIV-1 IN INFANTS BORN TO HIV SEROPOSITIVE WOMEN

Polymerase chain reaction (PCR) is the most sensitive test to diagnose HIV-1 infection among infants born to HIV seropositive mothers. The purpose of this study was to evaluate the use of dried blood spot (DBS) specimens for PCR and to compare it with whole-blood stored in tubes for HIV-1 DNA PCR. Five hundred and seventy-seven whole-blood infant samples were tested using HIV-1 qualitative in-house nested DNA PCR. Three hundred and fifty-nine samples were from infants at 48 hours of birth and 218 samples at second month. All positive samples tested from whole-blood and every fifth negative sample were coated onto filter paper. DNA was extracted from the filter paper and was amplified using in-house nested PCR. Among the whole-blood samples tested using HIV-1 DNA PCR, 19 of 359 (5.29%) samples were HIV-1 positive and 340 (94.7%) were negative at 48 hours of birth. At second month, 19 (8.7%) of the 218 samples were positive and 199 (91.2%) were negative. Using dried filter paper, 18 samples (95%) tested positive from 19 positive samples (using whole-blood) and 1 tested negative at 48 hours of birth. The 68 negative samples tested using whole-blood were also negative in the DBS test (sensitivity 95% and specificity 100%). At second month, 19 were positive and 40 samples (every fifth sample of 199) were negative (sensitivity and specificity, 100%). PCR performed using DNA extracted from filter paper permits the diagnosis of HIV-1 infection among infants born to HIV-1 seropositive mothers. This assay is simple, rapid, sensitive and specific and can be used in resource limited settings.

Falso negativo en el diagnóstico de VIH-1

We report a case of a false negative diagnosis of HIV-1 infection in an African girl. Two HIV-1 DNA polymerase chain reaction (PCR) tests were negative at the second and fourth months of life. Because anti-HIV antibodies persisted when the patient was 18 months old, the HIV-1 RNA PCR test was performed with a positive result, confirming HIV-1 non-B subtype, recombinant A-G. The prevalence of non-B HIV-1 subtypes are increasing in Spain, which could be related to the phenomenon of immigration. Approximately one-third of HIV-infected foreigners have non-B subtypes and the percentage increases to 70 % of the African population in Spain. In non-B HIV-1 subtypes, false negative results and inconsistencies between viral load and CD4 count are more frequent. These subtypes also show a higher rate of resistance to protease inhibitors, which can have therapeutic implications.

Exposure to HIV and antiretroviral medication as a potential cause of necrotizing enterocolitis in term neonates

Desfrere et al. [1] recently reported an association between maternal HIV infection and necrotizing enterocolitis (NEC) in neonates, independent of the classic NEC risks such as prematurity, hypoxia/ischaemia, cardiac malformation, anaemia, and formula feeding. The authors hypothesized that altered IL-12 expression and zidovudine-induced mitochondropathy, observed in HIV-exposed infants, may contribute to the development of NEC. Their study was limited to preterm infants, and suggested that HIV exposure could increase the immature infant's risk of NEC. We observed NEC in a term neonate without identifiable risk factors other than than being born to an HIV-positive mother. A male infant was delivered by elective caesarean section at 37 + 4 weeks of gestation to an HIV-positive mother. Maternal CD4 cell counts were normal above 500 cells/μl, and the HIV viral load was 55 800 copies/ml before the initiation of therapy. At 31 + 2 weeks of gestation, antiretroviral treatment was started using nevirapine (400 mg/day), lamivudine (300 mg/day) and zidovudine (600 mg/day). At 35 weeks, the viral load was 64 copies/ml. The mother received 100 mg intravenous zidovudine intrapartum. The postnatal adaptation of the newborn was uneventful, birthweight was 3015 g, scores at 1, 5 and 10 min were 6, 10 and 10, respectively. Postnatal transmission prophylaxis in the neonate was initiated at 6 h according to the Berlin regimen [2] of intravenous zidovudine 1.3 mg/kg body weight four times a day; blood cell counts and IL-6 were within normal ranges (see Table 1). Oral feeding of formula milk was started. On the fifth day of life, the infant developed clinical signs of sepsis, with pale skin, floppyness, and blood-stained stools. Elevated IL-6 of 50 000 ng/nl and granulocytopenia of 1.3/nl confirmed the diagnosis. An abdominal X-ray revealed intestinal pneumatosis. Oral feeding and antiretroviral medication were discontinued. Intravenous fluids and antibiotic treatment, including ampicillin, gentamycin, and metronidazole, were initiated. The next day, an abdominal X-ray showed pneumoperitoneum, and the infant underwent surgical resection of perforated and necrotic bowel. Oral feeding was restarted on the tenth postoperative day. After 3 months, three consecutive HIV-DNA polymerase chain reaction tests showed negative results, therefore the vertical transmission of HIV-1 could be excluded.Table 1: Blood tests and intravenous zidovudine administration in the infant.The study of Desfrere et al. [1] has highlighted the fact that vertical HIV exposure may be associated with an elevated risk of NEC in preterm neonates. We can add to their observation by showing that HIV exposure and its treatment may also be associated with NEC in term newborns. Maternal HIV infection might contribute to the risk of NEC through an alteration of cytokine expression and decreased T-cell function [3–5]. Zidovudine used for transmission prophylaxis may further increase the risk of NEC through its recognized adverse effects, including anaemia, granulocytopenia, and gastrointestinal toxicity [6]. Little is known about the potential effects of other antiretroviral drugs used during pregnancy. In this case, hyperosmolarity or local zidovudine toxicity did not play a role, because zidovudine was administered parenterally, and anaemia or granulocytopenia were not present until the onset of NEC (Table 1). In our centre, 105 infants fullfilled Bell's criteria for NEC (grade IIa and more), 100 of them were born before 37 weeks of gestation. In three of the five term infants with NEC, cardiac malformations were identified. The remaining two were a hypotrophic infant and the HIV-exposed infant presented here. Among the factors predisposing to NEC in term neonates, congenital heart disease, severe intraunterine growth retardation, early feeding, gastroschisis, hypercoagulable states, sepsis, but not HIV exposure have been described [7]. We conclude that exposure to HIV and antiretroviral drugs do not only augment the preterm infant's genuine risk of NEC, but may be an independent risk factor for this disease, possibly by altering the cellular immunity of the neonate.

Polymerase Chain Reaction for Diagnosis of Human Immunodeficiency Virus Infection in Infancy in Low Resource Settings

Diagnosis of human immunodeficiency virus (HIV) is essential for accessing treatment. Current HIV diagnostic protocols for infants require adaptation and validation before they can be implemented in the developing world. The timing and type of HIV assays will be dictated by country-specific circumstances and experience from similar settings. The performance of an HIV-1 DNA polymerase chain reaction (PCR) test, and in particular a single test at 6 weeks of age, in diagnosing HIV subtype C infection acquired in utero or peripartum was assessed. A retrospective review of 1825 Amplicor HIV-1 DNA PCR version 1.5 tests performed between 2000 and 2004 in 2 laboratories in Johannesburg, South Africa on 769 effectively non-breast-fed infants from 3 clinically well characterized cohorts was undertaken. The HIV status of each infant was used as the standard against which the HIV PCR results were compared. The overall sensitivity and specificity of the HIV PCR test were 99.3 and 99.5% respectively. A single test was 98.8% sensitive and 99.4% specific in the 627 infants tested at 6 weeks of age (58 HIV-infected and 569 HIV-uninfected). Repeat testing of all positive HIV PCR tests minimized false positive results. In resource-poor settings where HIV PCR testing in an environment of good laboratory practice is feasible, a single 6-week HIV DNA PCR test can increase identification of HIV-infected children substantially from current levels. Further operational research on how best to implement and monitor such a diagnostic protocol in specific local settings, especially in breast-fed infants, is necessary.

Performance Characteristics of HIV-1 Culture and HIV-1 DNA and RNA Amplification Assays for Early Diagnosis of Perinatal HIV-1 Infection

Lambert JS, Harris R, Stiehm R, et al. J Acquir Immune Defic Syndr. 2003;34:512–519The diagnosis of HIV infection in a newborn exposed to HIV in utero is a challenge. In the early years of the epidemic, HIV clinicians monitored the decline of HIV antibody levels for up to 2 years after birth to confirm that a child was not HIV-infected. HIV infection in infants is now typically made by the detection of viral DNA sequences in peripheral blood mononuclear cells by means of a DNA polymerase chain reaction (PCR). Plasma HIV-RNA measurements with PCR may also be valuable but has the theoretic limitation of false-negative reactions resulting from early treatment of the mother and infant. The purpose of this study was to evaluate the performance of HIV DNA PCR, HIV RNA PCR, and HIV culture to identify infected infants exposed to the virus in utero.Infants participating in the Pediatric AIDS Clinical Trials Group protocol 185.Specimens from the infants (24 infected and 100 uninfected) obtained prospectively were studied with standard nucleic acid–amplification assays and peripheral blood mononuclear cell microcultures. The sensitivities, specificities, and positive and negative predictive values were calculated for each of the 3 assay systems.At birth the sensitivity of culture, DNA PCR, and RNA PCR were 21%, 11%, and 27%, respectively. By 6 weeks, the sensitivity had improved to 90%, 83%, and 95%. The specificity was 99% to 100% for all assays at all times.The authors concluded that the diagnostic performance of the RNA PCR assay matched or exceeded that of culture and DNA PCR. Because RNA assays require less blood volume and often can be performed more quickly at reference laboratories, it is suggested that RNA assays may be used for early diagnosis of HIV infection in infants.This study demonstrates that RNA PCR assays are effective for the diagnosis of HIV infection. However, it must be noted that cryopreserved specimens were used for these PCR assays and may have impacted the sensitivity of the DNA PCR. Additionally, we have had 3 false-positive RNA PCR assays in 2 newborns and 1 adolescent exposed to HIV. A negative RNA PCR at or after 6 weeks of age strongly indicates that an infant is not infected.

Perinatal transmission of multidrug-resistant HIV-1 despite viral suppression on an enfuvirtide-based treatment regimen

Perinatal transmission of multidrug-resistant HIV-1 despite viral suppression on an enfuvirtide-based treatment regimen

Dried Blood Spots Improve Access to HIV Diagnosis and Care for Infants in Low-Resource Settings

Effective health care delivery to the majority of perinatally exposed infants worldwide, including those enrolled in prevention of mother-to-child transmission programs, is hampered by lack of access to an HIV diagnosis in infancy. Dried blood spot collection from young infants for centralized HIV polymerase chain reaction (PCR) testing is attainable in low-resource settings, provided PCR methodology suitable for routine laboratory service is available. The accuracy of the Roche Amplicor HIV-1 DNA test version 1.5 (Branchburg, NJ) performed on dried blood spots collected prospectively on ordinary Whatman filter paper from a cohort of 300 6-week-old infants born to HIV-infected women in Johannesburg, South Africa, was assessed. Anonymous analysis of the blood spots using a unique DNA extraction procedure was performed in a routine diagnostic laboratory and the results compared with HIV DNA and RNA PCR liquid blood tests at age 6 weeks, and the HIV status of the infant. Dried blood spots were available for 288 infants (96%) of whom 25 (8.7%) were HIV infected. The Roche Amplicor assay yielded a sensitivity of 100% and a specificity of 99.6%. HIV DNA PCR tests on dried blood spots have the potential to improve health care delivery to HIV-affected children in low-resource settings right now.

Universal nevirapine upon presentation in labor to prevent mother-to-child HIV transmission in high prevalence settings.

To assess the uptake of and adherence to nevirapine to prevent mother-to-child HIV transmission among women of unknown HIV serostatus presenting in labor. We also assessed preliminary efficacy of the approach. Women of unknown HIV serostatus presenting in labor were offered single-dose nevirapine in a prospective cohort study. Two additional contemporaneous comparison populations were also studied. We measured uptake by counting the number of women that accepted enrollment when offered. We measured adherence with cord blood nevirapine assay. We measured preliminary efficacy with HIV DNA polymerase chain reaction of infant blood spots at 4-6 weeks of life. Of 1591 women approached in labor, 634 (40%) took up the intervention and received nevirapine, of whom 185 (29%) were HIV infected. Of 179 cord blood specimens from HIV-exposed infants that could be evaluated, 178 (99.4%) had nevirapine detected. This was higher than the 73 of 98 (74%) adherence rate observed in a comparison cohort in which women self-administered nevirapine before presenting to the labor ward (P < 0.001). Of 145 available infant specimens, 17 (11.7%) showed evidence of infection at 4-6 weeks, compared with 12 of 60 (20%) infants born immediately prior to study commencement whose HIV-infected mothers did not receive nevirapine (P < 0.05). Nevirapine without HIV testing upon presentation in labor was accepted by two-fifths of women. Because therapy is directly observed, adherence is nearly perfect. Labor ward dosing to enhance nevirapine coverage should be considered as an adjunct to antenatal nevirapine administration for prevention of mother-to-child transmission of HIV.

Maternal immune responses and risk of infant infection with HIV-1 after a short course Zidovudine in a cohort of HIV-1 infected pregnant women in rural Kenya.

To investigate the effects of short-course nucleoside reverse transcriptase inhibitor (Zidovudine, ZDW/AZT) on maternal immune responses and risk of infant infection with HIV-1 among rural-based mothers in western Kenya. A prospective cohort study involving HIV-1 seropositive pregnant mothers and their infants. One hundred and seven HIV-1 seropositive asymptomatic pregnant women and their infants. After informed consent, the women were enrolled at gestation age between 16-24 weeks. For cultural and economic reasons, all mothers were allowed to breast feed their infants. Short-course antepartum regime of AZT was administered to all mothers starting at 36 weeks gestation until start of labour. Maternal absolute CD4+ T cell subset assays were performed before 3rd trimester (about 36 weeks gestation) and after a 4-week therapy of AZT (at least one month post-nuptially). Infant HIV-1 status was determined by HIV-1 DNA polymerase chain reaction (PCR) on samples sequentially taken at 1, 2, 3, 4, 6 and 9 months and confirmed by serology at 18 months of age. Antepartum short-course orally administered AZT: 300mg twice-daily starting at 36 weeks gestation until start of labour, 300mg at labour onset and 300mg every three hours during labour until delivery. Maternal CD4+ T cell counts before and after AZT treatment. Determination of infant HIV-1 infection status. Among 107 women sampled, only 59 received full dose of AZT and thus qualified for present analysis. Of these, 12 infected their children with HIV, while 47 did not. Comparison of CD4+ T cells before and after AZT treatment scored a significant rise in all mothers (P = 0.01). This increase in CD4+ T cells was not significant among mothers who infected their infants with HIV-1 (P = 0.474). However, a significant rise in CD4+ T cells following AZT therapy was observed only in mothers who did not transmit HIV-1 to their infants (P=0.014). These data suggest that a rise in the CD4+ T cell counts following short AZT regimen, now widely in use in resource-weak countries, may be evidence of the active suppression of the replication of HIV. However, further studies to examine the multi-factorial effect of CD4+ lymphocytes and pregnancy on MTCT of HIV need to be carried out to help fully explain the effect of AZT on immune response and whether the CD4+T cell count can be used as a true test of immunological normalisation during antiretroviral therapy.

Myelomeningocele in a child with intrauterine exposure to efavirenz

The authors report a case of a full-term male neonate born to a 34-year-old woman with heterosexually acquired asymptomatic HIV infection, with a CD4 cell count of 390/mm3 (26%) and an HIV-RNA level of 7600, receiving zidovudine, stavudine and efavirenz therapy before pregnancy. She was counselled regarding the potential risks of this therapy and the necessity for adequate birth control. After a period of amenorrhoea she performed a pregnancy test, which was positive. For this reason antiretroviral therapy was switched to an alternative regimen with lamivudine, stavudine and nelfinavir at 24 weeks of pregnancy. Obstetric controls were performed regularly and no side-effects were reported. Before and during the whole pregnancy the patient was submitted to daily folic acid supplementation. Ultrasound surveillance performed at the second trimester of gestation showed regular fetal growth and the presence of a lumbar mass and a triventricular hydrocephalus. The baby was born at the 38th week of gestational age, weight 3450 g, length 49 cm and head circumference 34.5 cm, presenting with a lumbo-sacral mass compatible with a myelomeningocele of 13 cm diameter with full intact skin (Fig. 1). A cerebral ultrasound showed a triventricular hydrocephalus. Magnetic resonance imaging confirmed a large sacral myelomeningocele lesion (10 cm diameter). Laboratory evaluation showed hepatic, renal and metabolic assessment in the normal range. The serological test for HIV (enzyme-linked immunosorbent assay and Western blot) was positive. HIV-1 DNA polymerase chain reaction at birth on plasma and cerebrospinal fluid was negative. The newborn was submitted to surgical repair of the myelomeningocele lesion and ventriculo–peritoneal shunting. Prophylactic therapy with zidovudine 2 mg/kg a day trial was started.Fig. 1.: Myelomeningocele in efavirenz-exposed newborn.The aetiology of myelomeningocele and other neural-tube defects still remain to be completely understood, although genetics, race, socioeconomics and environmental factors are recognized [1]. The role of folic acid deficiency as the major cause of spina bifida has been recognized, but at least 30% of cases are not prevented by periconceptional multivitamins containing folic acid supplementation. Therefore other specific environmental causes have been involved; to date maternal diabetes and fetal exposure to some anti-epileptic drugs such as valproic acid have been identified [2]. Efavirenz is a potent non-nucleoside reverse transcriptase inhibitor that shows a durable suppression of HIV replication. In animal studies [3], efavirenz crosses the placenta and produces fetal blood concentrations similar to maternal blood concentrations. Teratogenic effects have been observed in three out of 20 fetuses from efavirenz-treated cynomolgus monkeys from the 20th to the 150th days of gestation in a developmental toxicity study. Drug-induced neural tube defects such as anencephaly and unilateral anophthalmia were observed in one fetus, microophthalmia was observed in another fetus, and cleft palate was observed in a third fetus. These effects were not noted on rats and rabbits probably because of different pharmacokinetics. The use of this drug is not allowed in pregnant women for two reasons: first, the teratogenic effects were observed in monkeys exposed at the same recommended dose as in humans; second, the drug follows the same metabolic pathway in humans and monkeys (the 8-OH conjugated glucuronide is the major metabolite in plasma and urine) [4]. To date, this case is the first report of neural tube defect in a human fetus accidentally exposed to efavirenz during the first month of pregnancy. This hypothesis is supported by strong evidence: (i) the extension of fetal exposure to efavirenz occurred within the first 28 days of gestation, exactly corresponding to the embryological period of neural tube closure [5]; (ii) the teratological effect observed in our neonate involved neural tube development, as did the anencephalia observed in fetuses from efavirenz-treated monkeys. In conclusion, this case suggests special considerations: the demonstration of a possible teratogenic effect in humans strengthens the recommendation that pregnancy should be avoided in women receiving efavirenz. Moreover, efavirenz-treated women should be carefully advised to shift to an alternative antiretroviral treatment before pregnancy. If our report is confirmed by further observations, we believe that the advisability of efavirenz proscription in women of reproductive age should be prudently considered. Carlo Fundarò Orazio Genovese Claudia Rendeli Enrica Tamburrini Elio Salvaggio

Broad Spectrum of Time of Detection, Primary Symptoms and Disease Progression in Infants with HIV-1 Infection

The relationship between time of HIV-1 detection, appearance of symptoms and disease progression was studied in all 24 HIV-1-infected infants from a cohort of 117 children who were born to HIV-1-infected mothers and monitored from birth. HIV isolation from plasma and mononuclear cells, HIV-1 DNA PCR (polymerase chain reaction) and, retrospectively, a quantitative assay for HIV-1 RNA were used for virus detection. Two infants possibly exhibited a symptomatic primary HIV infection. More children with than without symptoms during the first year of life progressed to immunological class 3 (P=0.013) and to AIDS or death (P=0.003) during follow-up. HIV-1 was detected within 4 days of age in 4 of 16 infants: 3 of them became symptomatic within 1 year, as did 6 of the remaining 12 infants (not statistically significant). All four infants in whom virus was detected within 4 days of age progressed to severe immunosuppression, compared to 6 of 14 in whom the virus detection test was initially negative prior to the first positive result (n.s.). Two children with previous repeatedly negative HIV detection tests were diagnosed with HIV-1 infection at 8 and 9 months, respectively. Repeated blood sampling is needed for the diagnosis of HIV-1 infection in perinatally exposed infants, and virus detection tests for exclusion of HIV-1 infection must be used with caution.

Thai Red Cross zidovudine donation program to prevent vertical transmission of HIV: the effect of the modified ACTG 076 regimen.

To evaluate the impact of the modified ACTG 076 zidovudine regimen on the risk for vertical HIV transmission. Observational retrospective evaluation of a prospective cohort. Thai Red Cross zidovudine donation program to reduce vertical HIV transmission. HIV-infected Thai women and their offspring. The modified regimen consisted of 500 mg zidovudine daily during pregnancy and 300 mg zidovudine every 3 h during labor, taken orally, and 2 mg/kg zidovudine syrup four times daily for 6 weeks to infants. Only infants with at least 1 HIV DNA polymerase chain reaction (PCR) result at age > or = 4 weeks were included. HIV infection was defined by having at least one positive PCR at age > or = 4 weeks. The transmission rate was calculated. Characteristics of women who did and did not transmit HIV to infants were compared. A total of 2891 women and their infants participated in the program and 726 infants of 719 women were included in the analysis. Forty-three infants were infected. The overall transmission rate was 6.0% (95% confidence interval, 4.4-8.0). There were no differences in maternal characteristics between transmitters and non-transmitters. The transmission rate in women who started zidovudine before 30 weeks' gestation was not significantly different from that in women who started zidovudine at or after 30 weeks' gestation: 5.7 versus 3.3%, respectively. This modified zidovudine regimen is effective in reducing vertical transmission in a country with predominant subtype E infection. A donation program for preventing vertical HIV transmission can be implemented in developing countries, as in Thailand.

Epidemiology of HIV infection in the newborn.

Vertical transmission of HIV infection can take place in utero, during delivery and postnatally through breastfeeding, with about three-quarters of infections occurring around the time of delivery in non-breastfeeding populations. In Europe, in the absence of specific interventions, the vertical transmission rate was 15-20%. High maternal load is the major risk factor for both intra-uterine and intra-partum mother-to-child transmission. Prematurity is the most common adverse neonatal outcome associated with maternal HIV infection. Earlier diagnosis of paediatric HIV infection than previously available is now possible with virological tests, particularly HIV DNA polymerase chain reaction. An estimated one fifth of infected children will have been diagnosed with AIDS or have died by 12 months of age, rising to a third by 6 years of age. Surgical and therapeutic interventions are effective in reducing vertical transmission risk, in addition to the avoidance of breastfeeding. Caesarean section delivery before labour and before rupture of membranes approximately halves the risk of transmission, while prophylactic zidovudine therapy according to the ACTG076 regimen reduces transmission by up to two-thirds, transmission is reduced even further with both interventions. Trials of short-course zidovudine regimens show their effectiveness in reducing vertical transmission, in breastfeeding and non-breastfeeding populations. Nevirapine has been shown to be significantly more effective than short course zidovudine regimens in breastfeeding populations, but is still under evaluation in non-breastfeeding populations additionally receiving routine anti-retroviral prophylaxis. Reports of a small number of serious adverse events in uninfected children exposed in utero or neonatally to antiretroviral therapy need further investigation. Trials of vitamin A supplementation to reduce vertical transmission have had negative results, while the effectiveness of vaginal lavage and passive immune therapy in reducing vertical transmission remains uncertain.

Prevention and Treatment of Pediatric HIV Infection (Contempo 1998: Updates Linking Evidence and Experience)

Research focusing on the timing and pathogenesis of vertical HIV-1 infection has led to the development of effective interventions to prevent vertical transmission of HIV-1. Prevention of vertical transmission of HIV-1 can best be achieved when women are identified as being HIV-1-infected as early as possible during pregnancy. Early treatment of pregnant women and their newborn children can substantially decrease the risk of transmission of the virus to the child. Since exposure to HIV in utero during childbirth and during breast-feeding can infect children women should undergo zidovudine treatment during pregnancy. In addition infants born to HIV-1-infected women should be tested at birth by HIV-1 DNA polymerase chain reaction and repeatedly during the first months of life to ensure the early identification of perinatally acquired HIV-1 infection and the prompt treatment of infected infants with a potent combination of antiretroviral drugs.

Evaluation of HIV1 infection status by HIV1 PCR and culture methodologies in a small cohort of intravenous drug users

A small cohort of high-risk intravenous drug users (IVDU) from the Baltimore, MD, area was evaluated for HIV1 infection status and viral load. Quantitative dilution endpoint HIV1 DNA polymerase chain reaction (PCR) results, from HIV proviral DNA from quantitated peripheral blood mononuclear cell (PBMC) lysates, were compared to the dilution endpoint results for HIV PBMC micrococulture. The quantitative dilution endpoint HIV1 PCR was more rapid, sensitive and reproducible. In addition, an HIV1 capture RT-PCR technique was used to qualitatively detect the presence or absence of intact HIV1 virus in IVDU plasma and was compared with plasma culture detection, for HIV1 viraemia. Using the results of the PCR techniques, a rapid molecular assessment of the HIV1 infection status can be attained, which is important, as the IVDU population can be difficult to study prospectively. The PCR techniques can also be used to assess HIV1 burden as well as the potential effectiveness of antiviral therapies. These molecular techniques can be used to monitor the progression of HIV in patients and to evaluate the clinical effects of concurrent substance abuse.

Human immunodeficiency virus infection of human bone marrow stromal myoid cells.

In order to investigate the potential susceptibility of bone marrow stromal myoid cells to human immunodeficiency virus (HIV), a myoid cell population devoid of all other cellular components of marrow environment was isolated from 3 normal adult bone marrow samples. The bone marrow myoid cells were infected with 3 different strains of HIV-1, 2 strains isolated from patients with acquired immune deficiency syndrome (AIDS) and HTLV-IIIB strain. To demonstrate successful infection and HIV production, culture supernatants, harvested weekly until 2 months post-infection, were tested for the presence of p24 antigen and infectious virus. Myoid cell monolayers obtained from the 3 different bone marrow samples were shown to be susceptible to infection. In particular, infection led to the presence of p24 antigen and of infectious virus in culture supernatants up to day 49 post-infection. After day 49, it was not possible to demonstrate the presence of infectious virus in culture supernatants and HIV-DNA polymerase chain reaction (PCR) failed to show viral genome in any of the cultures assayed. Our results demonstrate the susceptibility of myoid stromal cells to HIV infection and may provide an in vitro model for studying the effects of HIV infection in disregulation of the haemopoietic function of bone marrow environment.

The sensitivity of HIV-1 DNA polymerase chain reaction in the neonatal period and the relative contributions of intra-uterine and intra-partum transmission

To derive reliable estimates of the sensitivity of HIV-1 DNA polymerase chain reaction (PCR) in the neonatal period and to quantify the relative contributions of intra-uterine and intra-partum transmission. After reviewing studies on the early diagnosis of HIV-1 infection, investigators were asked to provide published and unpublished PCR test results on prospectively followed, non-breastfed, vertically infected children. Age-specific estimates of the sensitivity of PCR were derived using distribution-free methods for interval-censored data. Data on 271 infected children were combined for analysis. PCR detected HIV-1 DNA in an estimated 38% [90% confidence interval (CI), 29-46] of HIV-infected children tested on the day of, or day after, birth. Sensitivity was observed to rise rapidly in the second week of life, reaching 93% (90% CI, 76-97) by 14 days of age. The sensitivity of PCR in the neonatal period is higher than previously reported. This affects the clinical interpretation of an early negative test result and encourages the use of PCR as an endpoint for trials to evaluate interventions to reduce vertical transmission in non-breastfed populations. Approximately one-third of vertically acquired HIV-1 infection could be attributable to intra-uterine transmission.

Virologic, immunologic, and clinical evaluation of human immunodeficiency virus antibody status of symptom-free children born to infected mothers

To determine the prevalence of infection by the human immunodeficiency virus (HIV) in a population of symptom-free children who were born to HIV-infected mothers and who subsequently underwent seroreversion from an HIV antibody-positive to an HIV antibody-negative status. Cohort. Pediatric HIV program in a community setting. We used HIV DNA polymerase chain reaction (PCR) and coculture to detect the presence or absence of HIV in peripheral blood mononuclear cells of 134 children aged 6 to 53 months. All children had HIV antibody at birth and underwent a subsequent seroreversion to antibody-negative status. In 134 children with HIV antibody-negative status, 219 of 220 culture results and 242 of 247 HIV-1 DNA PCR assay results were negative. Six positive laboratory results were obtained for six different children, each of whom had negative results on multiple assays. For HIV-infected children, 56 of 62 cultures and 99 of 104 PCR evaluations showed positive results. There was no clinical or laboratory evidence of HIV infection in the group with HIV antibody-negative status. We were unable to find evidence of latent HIV type 1 infection in this cohort of symptom-free children who underwent seroreversion to HIV antibody-negative status. The loss of maternal HIV antibody in these children indicates the absence of HIV infection. False-positive PCR and culture results occurred sporadically, indicating that repeated analysis of HIV seropositivity in infants and children is necessary.

Development and validation of a polymerase chain reaction method for the precise quantitation of HIV-1 DNA in blood cells from subjects undergoing a 1-year immunotherapeutic treatment

To validate a quantitative polymerase chain reaction method developed to measure HIV-1 DNA in peripheral blood mononuclear cells. The assay was used to measure HIV-1 DNA in 15 consecutive blood samples taken from subjects enrolled in a multicenter, randomly allocated, double-blind, placebo-controlled trial using an HIV-1 immunogen. The assay was validated following the United States Pharmacopeia guidelines. The analytical parameters assessed were sensitivity, specificity, linearity and precision. The quantitative analysis was obtained by (1) co-amplifying HIV-1 DNA targets with an endogenous control (globin); (2) extrapolating the target values using HIV-1 and globin standard curves; and (3) normalizing the HIV-1 copy numbers to the globin copy numbers (genomic DNA load). With United States Pharmacopeia assay validation methodology, the HIV-1 DNA polymerase chain reaction assay proved to be sensitive, specific, linear and precise and the evaluation of the relative difference between two consecutive blood samples was reproducible. The intra-assay variability, which examines the reproducibility of replicates, was determined using a conservative assessment (tolerance intervals). We established that an increase of 60% or more in the number of DNA copies or a decrease of 38% or more was significantly greater than the variation due to random or experimental error and therefore attributed this variability to a significant change in the HIV-1 DNA copy number. We developed and validated a polymerase chain reaction method for the precise quantitation of HIV-1 DNA in peripheral blood mononuclear cells. This assay was able to detect changes in viral loads in HIV-1-infected asymptomatic subjects enrolled in a double-blind placebo-controlled trial using an HIV-1 immunogen.