A quantification of human cells using an ERV-3 real time PCR assay.

A novel approach to quantifying human cells using a real time PCR assay was developed. The target sequence used in the assay is a 135 bp segment within the unique 1.7 kb Hind III / Pst I fragment of the ERV-3 envelope gene. ERV-3 is a full-length human endogenous retrovirus present in known copy number in all human cells. The detection range of ERV-3 by real time PCR is from 10(6) to 10(1). The precision described, sensitivity and specificity of the assay indicate that the ERV-3 sequence is an accurate cell quantitation marker. The quantitative ERV-3 assay enables simple, fast, and reproducible detection and quantitation of the cell number. The assay can be used to determine the sample DNA conditions and also it can be used to adjust the quantitative DNA measurements of other target gene assays relative to the number of cell equivalents.

Use of abnormal and health psychology as topics in a classroom format to reduce alcohol and other drug abuse among college students at risk.

This study was done to assess whether classes containing topics derived from two college courses, Abnormal Psychology and Health Psychology, could be used in a class room format to reduce alcohol and other drug abuse among at-risk college students. Topics covered included stress and stress management, alcohol and other drug use and abuse, chronic illnesses and psychological disorders that develop from an unhealthy lifestyle, and factors that play a role in good health and well-being. Students were enrolled in a semester-long course for college credit as an alternative to punitive sanctions for on-campus alcohol violations and other drug violations. The Midwest Institute on Drug Use Survey and the CORE Alcohol and Drug Survey were administered on the first and last days of class. Analysis indicated a significant self-reported reduction in drug use and associated negative symptoms and behavioral effects. Women were more likely to report reductions in drug use than men.

Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load.

We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.

Quantitative proviral DNA and antibody levels in the natural history of HTLV-I infection.

The pathogenesis of human T-cell lymphotropic virus type I (HTLV-I) in adult T-cell leukemia/lymphoma (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) is poorly understood. We prospectively followed up and evaluated the virologic correlates of infection in transfusion recipients after seroconversion, in asymptomatic carriers, and in ATL and HAM/TSP patients. Proviral DNA levels (copies/105 lymphocytes) were determined by real-time automated polymerase chain reaction and antibody titers by end-point dilution by use of an HTLV-I enzyme-linked immunoassay. In early infection, proviral load was initially elevated (median, 212 copies/105 lymphocytes at time 1) and later decreased (median, 99 copies at time 2, and 27 copies at time 3). Corresponding antibody titers were low at time 1 (1:2154), had significantly increased by time 2 (1:12312), and were stable by time 3 (1:4694). These viral markers were significantly lower in asymptomatic carriers than in HAM/TSP or ATL patients. Therefore, proviral load and antibody titers may be useful as predictive markers of disease among carriers.

Does umbilical cord blood polymerase chain reaction positivity indicate in utero (pre-labor) HIV infection?

OBJECTIVE: To compare risk factors for infants whose cord blood was positive for HIV DNA with those who were cord blood-negative but found to be HIV DNA-positive in early infancy. METHODS: In 1994, infants born to HIV-infected women were enrolled in a study in Blantyre, Malawi. Birth weight and transmission risk factors from cord blood-positive infants were compared with cord blood-negative/HIV-positive infants on their first postnatal visit (4-7 weeks of age). Testing for HIV DNA on cord and peripheral blood was performed by polymerase chain reaction. RESULTS: Of 249 HIV-infected infants (overall transmission rate, 26%), 83 (33%) were cord blood-positive and 166 were initially cord blood-negative. The mean birth weight was 2.1% (59 g) lighter in cord blood-positive infants than initially cord blood-negative infants; initially cord blood-negative infants were 2.8% (80 g) lighter than uninfected infants born to HIV-infected women. There were no significant differences in the risk factors for infection between HIV-infected cord blood-positive and -negative infants; when transmission was increased, both HIV-infected cord blood-positive and -negative infants contributed to the increase in a similar proportion. INTERPRETATION: It was concluded that umbilical cord blood positivity for HIV DNA did not identity a subset of in utero HIV-infected infants and suggested that HIV-infected cord blood-positive and -negative infants have similar timing and routes of HIV infection.

Blood collection on filter paper: a practical approach to sample collection for studies of perinatal HIV transmission.

The use of dried blood spots lends itself to widespread application in large field studies, especially in remote areas. We present experience gained during a perinatal HIV transmission study in southern Africa in which dried blood spot samples were used for polymerase chain reaction (PCR) tests. In this study, 15,810 filter paper cards with dried blood spots were collected. Infants were seen at age 6 and 12 weeks, and PCR was routinely done in duplicate on each sample. Of 186 negative controls (infants born to HIV-negative women), two (1.1%) had a single strongly reactive PCR result; the repeated duplicates were both negative. In contrast, all 24 known positive samples were strongly positive in both tests. Results were available from 1,976 duplicate tests on 1,235 infants born to HIV-infected women. Based on the PCR result on a later sample, the positive predictive value was 97.6% if both replicates were strongly positive (absorbance: 0.8 OD450 U), 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive (but none strongly positive). When both replicates were negative, the negative predictive value was > or = 96.2%. Thus, when a single HIV PCR test has a strongly positive result, the infant is very likely to be infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months. Suggestions for sample collection, storage, and PCR testing are provided.

PIP: 15,810 filter paper cards with dried blood spots were collected during a perinatal HIV transmission study in southern Africa to be subjected to polymerase chain reaction (PCR) testing. Infants were seen at ages 6 and 12 weeks, with PCR routinely done in duplicate on each sample. Of 186 infants born to HIV-negative mothers, two had a single strongly reactive PCR result, while the repeated duplicates were both negative. All 24 known positive samples were strongly positive in both tests. Results were available from 1976 duplicate tests on 1235 infants born to HIV-infected women. Based upon the PCR result of a later sample, the positive predictive value was 97.6% if both replicates were strongly positive, 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive. When both replicates were negative, the negative predictive value was greater than or equal to 96.2%. Therefore when a single HIV PCR test has a strongly positive result, the infant is very likely infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months.

Serum HIV-1 RNA levels and time to development of AIDS in the Multicenter Hemophilia Cohort Study.

OBJECTIVE: To determine if the long-term incidence of the acquired immunodeficiency syndrome (AIDS) is related to human immunodeficiency virus type 1 (HIV-1) RNA levels measured early in HIV-1 infection. DESIGN: Epidemiologic cohort study. SETTING: Five hemophilia treatment centers in the United States. SUBJECTS: A total of 165 subjects with hemophilia and HIV-1 infection (age at HIV-1 seroconversion, 1-66 years) followed from 1979 to 1995. METHODS: The HIV-1 RNA level was measured by polymerase chain reaction over a range of 200 to 1 million or more HIV-1 RNA copies/mL in archived serum specimens collected 12 to 36 months (median, 27 months) after the estimated date of HIV-1 seroconversion. Kaplan-Meier methods were used to examine the risk of AIDS and proportional hazards models were used to estimate relative hazards. RESULTS: The HIV-1 RNA values were similar in subjects younger than 17 years at seroconversion (median, 5214 copies/mL) and those 18 to 34 years old (median, 4693 copies/mL), but higher in those 35 years or older (median, 12069 copies/mL) (P = .02 compared with each younger group). At 10 years after seroconversion, the proportions of subjects with AIDS were 72% among subjects with 100,000 or more HIV-1 RNA copies/mL measured 12 to 36 months after HIV-1 seroconversion (n = 9), 52% among subjects with 10,000 to 99,999 copies/mL (n = 55), 22% among subjects with 1000 to 9,999 copies/mL (n = 82), and 0% among subjects with fewer than 1000 copies/mL (n = 19) (P < .001). The age-adjusted relative hazard for AIDS for subjects with 10,000 or more copies/mL was 14.3 (95% confidence interval, 1.9-105.6) compared with subjects with fewer than 1000 copies/mL. CONCLUSIONS: The HIV-1 RNA level during early chronic HIV-1 infection is a strong, age-independent predictor of clinical outcome; low levels define persons with a high probability of long-term AIDS-free survival.

Perinatal intervention trial in Africa: effect of a birth canal cleansing intervention to prevent HIV transmission.

BACKGROUND: Perinatal transmission of human immunodeficiency virus (HIV) type 1 contributes significantly to infant mortality. Exposure in the birth canal may account for some transmission. We examined the efficacy of a birth canal washing procedure in reducing perinatal transmission in Malawi. METHODS: The infection status of infants of 3327 control women (conventional delivery procedures) was compared with that of 3637 infants of intervention-delivered women. The infants' HIV status was determined by polymerase chain reaction on dried blood spots collected at 6 and 12 weeks of age. The intervention consisted of manual cleansing of the birth canal with a cotton pad soaked in 0.25% chlorhexidine, which was done on admission in labour and every 4 h until delivery. FINDINGS: No adverse reactions to the intervention procedure were seen. 2094 (30%) of the enrolled women were HIV-infected, and 59% of their infants were seen in follow-up. Among 982 vaginal vertex singleton deliveries to HIV-infected women, 269 (27%) infants were infected. The intervention had no significant impact on HIV transmission rates (27% in 505 intervention women compared with 28% in 477 control women), except when membranes were ruptured more than 4 h before delivery (transmission 25% in the intervention group vs 39% in the control group). INTERPRETATION: If birth canal exposure is an important risk factor, different or additional methods to reduce the risk of perinatal HIV transmission should be tested. Alternatively, perhaps birth canal exposure is not a major contributor to perinatal infection risk.

PIP: In light of evidence that birth canal exposure to HIV may be a major means of maternal-fetal transmission of the infection, a clinical trial was performed to determine the safety and efficacy of cleansing the birth canal with a cotton pad soaked in 0.25% chlorhexidine. This cleansing took place on admission in labor and every four hours until delivery. After a pilot study in 160 women ensured the safety of the procedure, the study was designed so that all women giving birth in June, July, and November 1994 were part of the control group and those giving birth from August to October were assigned to the intervention group. The mother's HIV status was established, and infants were seen at 6 and 12 weeks postpartum. After adjustment of the sample, data were analyzed on 3327 controls and 3637 cases. 2094 of the total enrolled were infected with HIV. Of these, 59% brought their infants to follow-up at least once as did 61.5% of the mothers who were not infected. The mother-to-infant transmission rate in this study was 27.4%. There was no evidence that the intervention prevented transmission. The only case in which washing reduced transmission rates was in babies born to women whose membranes were ruptured for more than four hours before delivery (in these, the transmission rate was significantly lower in intervention women [25%] than in control women [39.4%]). This result points to a role for ascending infection, but, if this were the case, washing before rupture of the membranes should have and did not reduce risk. Possible reasons for the failure of this intervention are that there may have been inadequate cleansing, the chlorhexidine solution may have been too weak, or the role of birth canal exposure in transmitting the infection may be less than previously believed. It is also difficult to quantify peripartum transmission in infants who are breast fed. The multiple modes by which infection may be transmitted will make it difficult to discover a simple, effective, and affordable way to reduce maternal-child transmission in developing countries.

Prenatal stimulation and postnatal testosterone affects infanticide in female rats.

Prenatal handling, prenatal stress, and early postnatal exogeneous testosterone were examined in female rats for their effects on rat pup-killing and pup retrieval. During each of the last 5 days of pregnancy. Long-Evans rats received either 3 minutes of handling, 45 minutes of restraint and intense illumination or remained untouched. Half of the offspring of each group received testosterone from Day 1 after birth to Day 30. In adulthood, animals that received handling prenatally and testosterone postnatally killed pups more rapidly than any other group and a larger proportion did so than in the control groups. Animals not manipulated at any time retrieved pups more rapidly and a larger proportion did so than the combined other groups. The study suggests that prenatal handling interacts with testosterone presented immediately postnatally to increase infanticide in female rats. A variety of perinatal manipulations seem to suppress pup retrieval.

Effects of combining pilocarpine, d-amphetamine, and either a free feeding schedule or cyclic food deprivation on mouse killing by rats.

The combination of a drug which suppresses mouse killing by rats (d-amphetamine) , a drug which activates mouse killing (pilocarpine), and either ad-lib food or a 24-hour cyclic food deprivation schedule were examined for their effects on the mouse-killing response by rats (N = 53). Results showed that the presence of d-amphetamine prevented the activating effects of pilocarpine in rats with a fairly high killing propensity regardless of whether they were on the ad-lib food or the deprivation schedule. The study suggests that a drug which affects both eating behavior and mouse killing is more effective in determining behavioral outcomes than a drug which affects only mouse killing.