David Waters

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.

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.

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.

HPV 16 antibody prevalence in Jamaica and the United States reflects differences in cervical cancer rates

Human papillomavirus (HPV) is widely accepted as the primary etiologic agent in the development of cervical cancer. DNA of a particular HPV type, HPV 16, is found in about half of tumors tested. Inconsistent with this causal relationship, however, population‐based studies of HPV DNA prevalence have often failed to find high rates of anogenital HPV infection in countries with high cervical cancer rates. To examine this issue, we used serology to compare HPV 16 exposure in healthy volunteer blood donors in the United States (n = 278) and similar subjects from a country with 3‐fold higher cervical cancer rates, Jamaica (n = 257). Jamaican sexually transmitted disease (STD) patients (n = 831) were also studied to examine in detail the relation of HPV 16 antibodies with sexual history. Serology was conducted using an ELISA employing HPV 16 virus‐like particles (VLPs). Age‐adjusted seroprevalence rates were greatest among male (29%) and female (42%) STD patients, intermediate in male (19%) and female (24%) Jamaican blood donors and lowest among male (3%) and female (12%) U.S. blood donors. The higher seroprevalence in women was significant, and prevalence tended to increase with age. In multivariate logistic regression, controlling for age and gender, Jamaican blood donors were 4.2‐fold (95% CI 2.4–7.2) and STD patients 8.1‐fold (95% CI 5.0–13.2) more likely to have HPV 16 VLP antibodies than U.S. blood donors. Among STD patients, HPV 16 antibodies were associated with lifetime number of sex partners and years of sexual activity, as well as other factors. Our data suggest that HPV 16 VLP antibodies are strongly associated with sexual behavior. Moreover, exposure to HPV 16 appears to be much greater in Jamaica than in the United States, consistent with the high rate of cervical cancer in Jamaica. Int. J. Cancer 80:339–344, 1999. Published 1999 Wiley‐Liss, Inc.

Estimating the time of HTLV-I infection following mother-to-child transmission in a breast-feeding population in Jamaica

Mother‐to‐child transmission of human T‐cell lymphotropic virus type I (HTLV‐I) is primarily due to prolonged breast‐feeding (>6 months) in the postnatal period. Most infant infections are not identifiable until 12 to 18 months of age by available whole virus Western blot serologic tests because of their inability to distinguish passively transferred maternal antibody from infant antibody. We investigated two methods to assess more accurately the time of infant infection. In prospectively collected serial biospecimens, HTLV‐I–specific immunoglobulin (Ig) isotypes of IgM and IgA were determined by Western blot and HTLV‐I proviral DNA was detected by polymerase chain reaction (PCR). IgA and IgG reactivity was assessed in periodic serum samples from 16 HTLV‐I–seropositive children while IgM reactivity was assessed in 9 of the 16 children. Approximately three to five samples were tested for each child. IgG reactivity was observed in 100% of children at 24 months of age and 73% of children at 6–12 months of age; however, this could represent maternal and not infant antibody. Both IgA and IgM reactivity were insensitive indicators of infection, with only 50% of children showing reactivity at 24 months of age. PCR testing was performed in biospecimens obtained from 11 of these children. An estimated median time of infection of 11.9 months was determined by PCR, which was similar to the median time to infection determined by whole virus Western blot (12.4 months; P = 0.72). PCR tests support a median time to infection that is similar to that estimated by whole virus Western blot. J. Med. Virol. 59:541–546, 1999.