Prospective Study of Trichomonas vaginalis Infection and Prostate Cancer Incidence and Mortality: Physicians' Health Study

Jennifer R. Stark; Gregory Judson; John F. Alderete; Vasanthakrishna Mundodi; Ashwini S. Kucknoor; Edward L. Giovannucci; Elizabeth A. Platz; Siobhan Sutcliffe; Katja Fall; Tobias Kurth; Jing Ma; Meir J. Stampfer; Lorelei A. Mucci

Disclosures

J Natl Cancer Inst. 2009;101(20):1406-11. 

In This Article

Study Subjects and Methods

Study Population

The Physicians' Health Study[13,14] was initiated in 1982 as a randomized, double-blind, placebo-controlled trial of aspirin and β-carotene for the primary prevention of cardiovascular disease and cancer. The study included 22 071 healthy US male physicians aged 40–84 years at baseline. Before being randomly assigned to a treatment group, 14 916 (68%) of the 22 071 men provided a blood sample.[15] These participants constitute the study base for the nested case–control study.

We included 673 case subjects who were diagnosed with prostate cancer up to 18 years after blood collection (1982–2000) and who had available plasma samples. We selected 673 control subjects from the population at risk at the time of the case subject's diagnosis (ie, those who had provided blood, had not had a prostatectomy, and had not reported a diagnosis of prostate cancer at the time the case subject was diagnosed with prostate cancer). For statistical efficiency, control subjects were individually matched to case subjects by age (within 1 year), smoking status (never, former, or current), and follow-up time.

Laboratory Assessment

Plasma from prospectively collected blood samples from each case subject and his matched control subject (stored at –80°C) was thawed and assayed for antibodies against T vaginalis with an assay that detects IgG antibodies against purified, recombinant α-actinin protein from T vaginalis. Enzyme-linked immunosorbent assays were optimized with known negative and positive pooled plasma of uninfected individuals and patients with trichomonosis, respectively, that gave reproducible readings after incubation with microtiter wells containing immobilized α-actinin. In this study, paired plasma samples from case and control subjects were diluted at 1:10 (vol/vol) in phosphate-buffered saline–Tween-20 containing 5% skim milk, and 100 µL of the diluted plasma was added to each well of a 96-well plate (Nunc, Rochester, NY). After incubation for 3 hours at 37°C, the plates were washed three times with phosphate-buffered saline–Tween-20 followed by the addition of 100 µL of secondary goat anti–human IgG (Fc-specific) conjugated to horseradish peroxidase at a 1:1500 dilution in phosphate-buffered saline–Tween-20 containing 5% skim milk to each well. Plates were incubated again for 1 hour at 37°C and then washed three times with phosphate-buffered saline–Tween-20. Color was allowed to develop by adding 100 µL of substrate solution per well (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); phosphate–citrate buffer with 0.03% sodium perborate, Sigma Chemical Co, St. Louis, MO) according to the manufacturer's recommendations, and plates were incubated at room temperature for 10 minutes. Absorbance values at a wavelength of 405 nm were then obtained by examining the supernatants spectrophotometrically with an enzyme-linked immunosorbent assay plate reader (Bio-Tek instruments, Inc, Winooski, VT).

Case–control sample pairs were assayed in adjoining wells, with blinding of laboratory personnel as to the case–control status of the samples. All samples were tested in duplicate and inferences were based on the mean of duplicate values. To create absorbance scores, we used a control plasma panel consisting of pooled plasma from known seronegative patients and four plasma samples with increasing seropositivity. We divided the mean duplicate absorbance value for each seropositive sample in the control panel by the mean duplicate absorbance value of the seronegative control plasma to obtain a minimum positive to negative (P/N) ratio for each absorbance score (0 = 1 to < 1.81; 1 = 1.81 to < 2.78; 2 = 2.78 to < 3.31; 3 = 3.31 to < 4.07; or 4 = ≥4.07). The positive to negative ratio was computed for all case subjects with prostate cancer and all control subjects, and the resulting values were then compared with the specified cut points determined from the control panel to assign an absorbance score (ie, 0, 1, 2, 3, or 4). Samples from the control panel were included with each plate to monitor reproducibility; values for these samples always fell within the previously determined range. Samples with absorbance scores of 3 or 4 were considered positive for history of trichomonosis. We also included 29 quality-control duplicate or triplicate samples that were randomly distributed across plates. Concordance in serostatus was achieved for 26 of 29 (90%) of the quality-control samples; 17 of 26 of the concordant replicate samples were seropositive.

Statistical Analysis

We used conditional logistic regression to analyze prostate cancer risk according to serostatus adjusting for matching factors. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by comparing men who were T vaginalis seropositive at baseline with men who were T vaginalis seronegative. We additionally controlled for randomization to aspirin assignment and body mass index (continuous) and evaluated risk within subgroups of stage and grade at diagnosis. All P values were from two-sided statistical tests, with α of .05 considered to be statistically significant.

Analyses were undertaken with the SAS Statistical Analysis version 9.1.3 (SAS Institute, Cary, NC). The research protocol was approved by the institutional review board at Partners Healthcare. Questionnaire data were collected with implied consent, and biomarker data were collected with written authorization.

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