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1.  Optimal methods for collecting and storing vaginal specimens for prostate-specific antigen testing in research studies☆ 
Contraception  2012;87(6):830-835.
Prostate-specific antigen (PSA) detected in vaginal fluid can be used in studies of HIV/sexually transmitted infection (STI) and pregnancy prevention as an alternative to relying on participant reports of exposure to semen. Optimal methods for collecting and storing specimens for this testing have not been determined.
Study Design
We conducted a controlled, in vitro experiment of 550 specimens spiked with semen to determine the effects of swab type (five types), storage conditions of the swabs (room temperature with or without desiccant or at −80°C without desiccant) and time from collection to testing (seven intervals over the course of 12 months) on the identification of PSA. We performed factorial analysis of variance to identify factors influencing PSA detection.
Concentrations of PSA detected in the swabs declined with time of storage over the 1-year experiment (p<.01). The 1-mL, rayon-tipped swab stored immediately at −80°C following collection performed best.
If immediate testing or freezer storage is not feasible, investigators should use a swab with 1-mL capacity with processing and testing as soon as possible after specimen collection.
PMCID: PMC4452941  PMID: 23121826
Biomarker; In vitro experiment; Prostate-specific antigen
2.  Trends and Correlates of Good Perinatal Outcomes in Assisted Reproductive Technology 
Obstetrics and gynecology  2012;120(4):843-851.
To estimate trends in good perinatal outcomes (singleton live births at term with birthweight more than 2,500 g) among live births after assisted reproductive technology in the United States from 2000 to 2008, and associated factors among singletons in 2008.
Using retrospective cohort data from the National Assisted Reproductive Technology Surveillance System from 2000 to 2008, we calculated relative change and χ2 tests for trend in the proportion of good perinatal outcomes among assisted reproductive technology live births (n=444,909) and liveborn singletons (n=222,500). We conducted univariable analyses followed by multiple logistic regression to estimate the effects of various characteristics on the outcome among singletons born in 2008 after fresh, nondonor assisted reproductive technology cycles (n=20,780).
The proportion of good perinatal outcomes among all liveborn neonates increased from 38.6% in 2000 to 42.5% in 2008, whereas it declined marginally among singletons from 83.6% to 83.4%. One previous birth, transfer of fewer than three embryos, and the presence of fewer than three fetal hearts on 6-week ultrasound examination were associated with good perinatal outcome among singletons. Non-Hispanic black race, tubal factor infertility, uterine factor infertility, ovulatory disorder, and 5-day embryo culture were associated with reduced odds for a good outcome. The strongest association was the presence of one fetal heart compared with more than two (adjusted odds ratio 2.43, 95% confidence interval 1.73–3.42).
From 2000 to 2008, good perinatal outcomes increased among assisted reproductive technology live births. Among singleton live births, odds for good outcome were greatest with the presence of a single fetal heart and lowest in women of non-Hispanic black race.
PMCID: PMC4454287  PMID: 22996102
3.  A Lower Degree of PBMC L1 Methylation in Women with Lower Folate Status May Explain the MTHFR C677T Polymorphism Associated Higher Risk of CIN in the US Post Folic Acid Fortification Era 
PLoS ONE  2014;9(10):e110093.
Studies in populations unexposed to folic acid (FA) fortification have demonstrated that MTHFR C677T polymorphism is associated with increased risk of higher grades of cervical intraepithelial neoplasia (CIN 2+). However, it is unknown whether exposure to higher folate as a result of the FA fortification program has altered the association between MTHFR C677T and risk of CIN, or the mechanisms involved with such alterations. The current study investigated the following in a FA fortified population: 1) The association between MTHFR C677T polymorphism and risk of CIN 2+; 2) The modifying effects of plasma folate concentrations on this association; and 3) The modifying effects of plasma folate on the association between the polymorphism and degree of methylation of long interspersed nucleotide elements (L1s), in peripheral blood mononuclear cell (PBMC) DNA, a documented biomarker of CIN risk.
The study included 457 US women diagnosed with either CIN 2+ (cases) or ≤ CIN 1 (non-cases). Unconditional logistic regression models were used to test the associations after adjusting for relevant risk factors for CIN.
The 677CT/TT MTHFR genotypes were not associated with the risk of CIN 2+. Women with CT/TT genotype with lower folate, however, were more likely to be diagnosed with CIN 2+ compared to women with CT/TT genotype with higher folate (OR = 2.41, P = 0.030). Women with CT/TT genotype with lower folate were less likely to have a higher degree of PBMC L1 methylation compared to women with CT/TT genotype with higher folate (OR = 0.28, P = 0.017).
This study provides the first evidence that the MTHFR 677CT/TT genotype-associated lower degree of PBMC L1 methylation increases the risk of CIN 2+ in women in the US post-FA fortification era. Thus, even in the post-FA fortification era, not all women have adequate folate status to overcome MTHFR 677CT/TT genotype-associated lower degree of L1 methylation.
PMCID: PMC4193871  PMID: 25302494
4.  Biological Markers of Sexual Activity: Tools for Improving Measurement in HIV/Sexually Transmitted Infection Prevention Research 
Sexually transmitted diseases  2013;40(6):10.1097/OLQ.0b013e31828b2f77.
Research on interventions to prevent HIV and other sexually transmitted infections (STIs) is heavily influenced by participant reporting of sexual behavior, despite uncertainty about its validity. Exclusive reliance on participant self-report often is based, overtly or by implication, on 4 assumptions: (1) no feasible alternatives exist; (2) misreporting can be minimized to levels that can be disregarded; (3) misreporting tends to underreport sensitive behaviors; and (4) misreporting tends to be nondifferential with respect to the groups being compared. The objective of this review are to evaluate these assumptions, including a review of studies using semen biomarkers to evaluate the validity of self-reported data, and to make recommendations for applying biological markers of semen exposure detectable in women to further strengthen research on HIV/STI prevention. Increasing evidence shows that semen biomarkers provide an important means of assessing and augmenting the validity of studies on HIV/STI prevention. Additional biomarkers are needed to assess male exposure to vaginal sex and both male and female exposure to anal sex. Methods and study designs that incorporate biomarkers into studies collecting self-reported behavioral data should be considered where possible.
PMCID: PMC3812692  PMID: 23677018
5.  Perspective: Strategies for Developing Biostatistics Resources in an Academic Health Center 
Biostatistics—the application of statistics to understanding health and biology—provides powerful tools for developing research questions, designing studies, refining measurements, analyzing data, and interpreting findings. Biostatistics plays an important role in health-related research, yet biostatistics resources are often fragmented, ad hoc, or oversubscribed within academic health centers (AHCs). Given the increasing complexity and quantity of health-related data, the emphasis on accelerating clinical and translational science, and the importance of conducting reproducible research, the need for the thoughtful development of biostatistics resources within AHCs is growing.
In this article, the authors identify strategies for developing biostatistics resources in three areas: (1) recruiting and retaining biostatisticians; (2) efficiently using biostatistics resources; and (3) improving biostatistical contributions to science. AHCs should consider these three domains in building strong biostatistics resources, which they can leverage to support a broad spectrum of research. For each of the three domains, the authors describe the advantages and disadvantages of AHCs creating centralized biostatistics units rather than dispersing such resources across clinical departments or other research units. They also address the challenges biostatisticians face in contributing to research without sacrificing their individual professional growth or the trajectory of their research team. The authors ultimately recommend that AHCs create centralized biostatistics units, as this approach offers distinct advantages both to investigators who collaborate with biostatisticians as well as to the biostatisticians themselves, and it is better suited to accomplish the research and education missions of AHCs.
PMCID: PMC3610776  PMID: 23425984
6.  Randomized Controlled Trial on the Effectiveness of Counseling Messages for Avoiding Unprotected Sexual Intercourse During Sexually Transmitted Infection and Reproductive Tract Infection Treatment Among Female Sexually Transmitted Infection Clinic Patients 
Sexually transmitted diseases  2013;40(2):10.1097/OLQ.0b013e31827938a1.
The effectiveness of counseling messages to avoid unprotected sex during short-term treatment for curable sexually transmitted infections is unknown.
We randomized 300 female STI clinic patients 18 years or older with cervicitis and/or vaginal discharge in Kingston, Jamaica, in 2010 to 2011, to 1 of 2 counseling messages for their course of syndromic treatment: abstinence only or abstinence backed up by condom use. At a follow-up visit 6 days afterward, we collected vaginal swabs to test for prostate-specific antigen (PSA), a biological marker of recent semen exposure, and administered a questionnaire assessing sexual behavior.
No differences were found in the proportions of women testing positive for PSA at follow-up in the abstinence-plus-condom group (11.9%) and abstinence-only group (8.4%) (risk difference, 3.5; 95% confidence interval, −3.5 to 10.5). There also was no significant difference in reporting of unprotected sex between groups. Reporting a history of condom use before enrollment significantly modified the effect of counseling arm on PSA positivity (P = 0.03). Among those reporting recent condom use, 10.3% in the abstinence-only arm and 4.8% in the abstinence-plus-condom arm tested positive for PSA. Conversely, among those not reporting recent condom use, 6.5% in the abstinence-only arm and 17.3% in the abstinence-plus-condom arm had PSA detected.
We found no evidence to support the superiority of either counseling message. Post hoc analyses suggest that women with recent condom experience may benefit significantly more from abstinence-plus-condom messages, whereas women without such experience may benefit significantly more from abstinence-only messages. Providers should weigh individual condom use history when determining the most appropriate counseling message.
PMCID: PMC3811001  PMID: 23321990
7.  Predictors of Unprotected Sex Among Female Sex Workers in Madagascar: Comparing Semen Biomarkers and Self-Reported Data 
AIDS and behavior  2010;14(6):10.1007/s10461-010-9742-8.
Research on the determinants of condom use and condom non-use generally has relied on self-reported data with questionable validity. We identified predictors of recent, unprotected sex among 331 female sex workers in Madagascar using two outcome measures: self-reports of unprotected sex within the past 48 h and detection of prostate-specific antigen (PSA), a biological marker of recent semen exposure. Multivariable logistic regression revealed that self-reported unprotected sex was associated with three factors: younger age, having a sipa (emotional partner) in the prior seven days, and no current use of hormonal contraception. The sole factor related to having PSA detected was prevalent chlamydial infection (adjusted odds ratio, 4.5; 95% confidence interval, 2.0–10.1). Differences in predictors identified suggest that determinants of unprotected sex, based on self-reported behaviors, might not correlate well with risk of semen exposure. Caution must be taken when interpreting self-reported sexual behavior measures or when adjusting for them in analyses evaluating interventions for the prevention of HIV/STIs.
PMCID: PMC3826447  PMID: 20625928
Female sex workers; Condoms; Biological markers; Prostate-specific antigen; Africa
8.  Good Performance of Rapid Prostate-Specific Antigen Test for Detection of Semen Exposure in Women: Implications for Qualitative Research 
Sexually transmitted diseases  2009;36(8):501-506.
Prostate-specific antigen (PSA) is a valid biomarker of semen exposure in women and has been used to assess reliability of self-reported sexual behavior as well as serve as a proxy measure for condom efficacy. Quantitative PSA tests are expensive and require specialized equipment. A simple, rapid, and inexpensive test for PSA would facilitate semen biomarker evaluation in a variety of research settings. This study evaluated the performance of a rapid PSA test compared with a quantitative assay to identify semen in vaginal swab specimens.
We tested 581 vaginal swabs collected from 492 women participating in 2 separate research studies in Bangladesh and Zimbabwe. PSA in vaginal secretions was detected using the quantitative IMx (Abbott Laboratories) assay and the ABAcard p30 (Abacus Diagnostics) rapid immunochromatographic strip test.
The ABAcard test was 100% sensitive (95% confidence interval [CI], 98%–100%) and 96% specific (95% CI, 93%–97%) compared with the quantitative test in detecting >1.0 ng PSA/mL vaginal swab eluate. Rapid PSA results were semiquantitative and correlated well with PSA concentrations (κ = 0.88; 95% CI, 0.85–0.90).
Rapid PSA detection requires no instrumentation and can be performed easily and economically. Having rapid PSA results available immediately following interview provides opportunities to explore discrepancies between the objective marker of recent semen exposure and self-reported behaviors.
PMCID: PMC3790320  PMID: 19455082
9.  A higher degree of LINE-1 methylation in peripheral blood mononuclear cells, a one-carbon nutrient related epigenetic alteration, is associated with a lower risk of developing cervical intraepithelial neoplasia 
The objective of the study was to evaluate LINE-1 methylation as an intermediate biomarker for the effect of folate and vitamin B12 on the occurrence of higher grades of cervical intraepithelial neoplasia (CIN 2+).
Study included 376 women who tested positive for HR-HPVs and were diagnosed with CIN 2+ (cases) or ≤ CIN 1 (non-cases). CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of LINE-1 methylation of peripheral blood mononuclear cells (PBMCs) and of exfoliated cervical cells (CCs) as the independent predictors of primary interest. In analyses restricted to non-cases, PBMC LINE-1 methylation (≥70% vs. <70%) and CC LINE-1 methylation (≥54% vs. <54%) were the dependent variables in logistic regression models that specified the circulating concentrations of folate and vitamin B12 as the primary independent predictors.
Women in the highest tertile of PBMC LINE-1 methylation had 56% lower odds of being diagnosed with CIN 2+ (OR = 0.44; 95% CI, 0.24-0.83; P = 0.011) while there was no significant association between degree of CC LINE-1 methylation and CIN 2+ (OR = 0.86; 95% CI, 0.51-1.46; P = 0.578). Among non-cases, women with supra-physiologic concentrations of folate (>19.8 ng/mL) and sufficient concentrations of plasma vitamin B12 (≥ 200.6 ng/mL) were significantly more likely to have highly methylated PBMCs compared to women with lower folate and lower vitamin B12 (OR = 3.92; 95% CI, 1.06-14.52; P = 0.041). None of the variables including folate and vitamin B12 were significantly associated with CC LINE-1 methylation.
These results suggest that a higher degree of LINE-1 methylation in peripheral blood mononuclear cells, a one-carbon nutrient related epigenetic alteration, is associated with a lower risk of developing cervical intraepithelial neoplasia.
PMCID: PMC3070926  PMID: 21463750
methylation; cervical; neoplasia
10.  A higher degree of methylation of the HPV 16 E6 gene is associated with a lower likelihood of being diagnosed with cervical intraepithelial neoplasia 
Cancer  2010;117(5):957-963.
Even though HPV 16 is the most common HPV genotype associated with cancerous lesions of the cervix, only a fraction of HPV 16 infected women are diagnosed with pre-cancerous lesions of the cervix. Therefore, molecular changes in HPV 16 rather than infections per se may serve as better screening or diagnostic biomarkers. The purpose of the study was to evaluate whether methylation status of specific regions of the HPV E6 gene promoter and enhancer is independently associated with the likelihood of being diagnosed with higher grades of cervical intraepithelial neoplasia (CIN 2+).
The study included 75 HPV 16 positive women diagnosed with CIN 2+ or ≤ CIN 1. Pyrosequencing technology was applied to quantify methylation at 6 cytosine guanine dinucleotide (CpG) sites of the HPV 16 E6 promoter and enhancer. CIN 2+ (yes/no) was the dependent variable in logistic regression models that specified the degree of methylation of the CpG sites of the HPV 16 E6 gene as the primary independent predictors. All models were adjusted for demographic, lifestyle, known risk factors for cervical cancer and circulating concentrations of “cancer-protective” micronutrients.
The odds of being diagnosed with CIN 2+ was 79% lower when the degree of methylation of the HPV 16 enhancer and promoter sites were ≥9.5% (OR= 0.21; 95% CI, 0.06–0.79; P=0.02).
Results suggested that CpG methylation is independently involved in the biology of HPV-16 as well as in the development of higher grades of CIN.
PMCID: PMC3023831  PMID: 20945322
HPV 16; methylation; cervical; neoplasia
11.  A retrospective follow up study on maternal age and infant mortality in two Sicilian districts 
BMC Public Health  2011;11:817.
Infant mortality rate (IMR) is a key public health indicator. Maternal age is a well-known determinant of pregnancy and delivery complications and of infant morbidity and mortality. In Italy the Infant Mortality Rate was 3.7/1000 during 2005, lower than the average IMR for the European Union (4.94/1000). Sicily is the Italian region with the highest IMR, 5/1000, and neonatal mortality rate (NMR), 3.8/1000, with substantial variation among its nine districts.
The present study compared a high IMR/NMR district (Messina) with a low IMR/NMR district (Palermo) during the period 2004-2006 to evaluate potential determinants of the IMRs' differences between the two districts and specifically the impact of maternal age.
The Death Causes Registers identified all deaths during the first year of life recorded among infants born to residents of the two districts in 2004-2006. For every case, available hospital charts records were abstracted using a standardized form designed to capture information on potential determinants of infant death. For each district and for each year, IMRs and NMRs were computed. Chi-squared statistics tested the significance of differences between district-specific IMRs. A Poisson regression model was used to analyze the relationship between maternal age, district of residence and IMR.
The 246 death registry-confirmed cases included 143 (58.1%) males and 103 (41.2%) females, with mean age at death of 33.3 days (SD: 64.5, median: 5.5). The average IMR for 2004-2006 was significantly higher for the Messina district than for the Palermo district (p = 0.0001). The IMR ratio was 1.6 (95%CI: 1.2 - 2.1). The IMRs declined from 2004 to 2006. A significant interaction (p = 0.04) between maternal age and district of residence was documented.
The association between advanced maternal age and infant deaths in the Messina district was due in part to the excess of newborns from advanced age mothers, but also to increased risk of death among such newborns. The significant interaction between district of residence and maternal age indicated that the IMR excess in the Messina district cannot be explained by disproportionately high live birth rates among older mothers and suggested the hypothesis that health care facilities in the Messina district could be less well prepared to provide assistance to the excess of high risk pregnancies and deliveries, as compared to Palermo district.
PMCID: PMC3206483  PMID: 22011347
12.  Hunting in the Rainforest and Mayaro Virus Infection: An emerging Alphavirus in Ecuador 
The objectives of this report were to document the potential presence of Mayaro virus infection in Ecuador and to examine potential risk factors for Mayaro virus infection among the personnel of a military garrison in the Amazonian rainforest.
Materials and Methods:
The study population consisted of the personnel of a garrison located in the Ecuadorian Amazonian rainforest. The cross-sectional study employed interviews and seroepidemiological methods. Humoral immune response to Mayaro virus infection was assessed by evaluating IgM- and IgG-specific antibodies using ELISA.
Of 338 subjects studied, 174 were from the Coastal zone of Ecuador, 73 from Andean zone, and 91 were native to the Amazonian rainforest. Seroprevalence of Mayaro virus infection was more than 20 times higher among Amazonian natives (46%) than among subjects born in other areas (2%).
Age and hunting in the rainforest were significant predictors of Mayaro virus infection overall and among Amazonian natives. The results provide the first demonstration of the potential presence of Mayaro virus infection in Ecuador and a systematic evaluation of risk factors for the transmission of this alphavirus. The large difference in prevalence rates between Amazonian natives and other groups and between older and younger natives suggest that Mayaro virus is endemic and enzootic in the rainforest, with sporadic outbreaks that determine differences in risk between birth cohorts of natives. Deep forest hunting may selectively expose native men, descendants of the Shuar and Huaronai ethnic groups, to the arthropod vectors of Mayaro virus in areas close to primate reservoirs.
PMCID: PMC3249982  PMID: 22223990
Alphavirus; Amazon; Ecuador; Mayaro virus
13.  Vaginal swab specimen processing methods influence performance of rapid semen detection tests: A cautionary tale 
Contraception  2010;82(3):291-295.
Detection of semen biomarkers in vaginal fluid can be used to assess women’s recent exposure to semen. Quantitative tests for detection of prostate-specific antigen (PSA) perform well, but are expensive and require specialized equipment. We assessed two rapid immunochromatographic strip tests for identification of semen in vaginal swabs.
Study Design
We tested 581 vaginal swabs collected from 492 women. Vaginal secretions were eluted into saline, and PSA was measured using the quantitative IMx (Abbott Laboratories) assay. Specimens were also tested using the ABAcard p30 test (Abacus Diagnostics) for detection of PSA and RSID-Semen test (Independent Forensics) for detection of semenogelin (Sg).
Vaginal swab extraction using saline was compatible with direct assessment of vaginal swab eluates using ABAcard for PSA detection, but not for Sg detection using RSID. The rapid PSA test detected 91% of specimens containing semen compared to 74% by the rapid Sg test.
Investigators are urged to optimize vaginal swab specimen preparation methods for performance of RSID or other tests to detect semen components other than PSA. Previously described methods for PSA testing are not uniformly applicable to other tests.
PMCID: PMC2921540  PMID: 20705160
semen; prostate-specific antigen; human semenogelin protein; vaginal swabs; rapid test
14.  Biomarker Validation of Reports of Recent Sexual Activity: Results of a Randomized Controlled Study in Zimbabwe 
American Journal of Epidemiology  2009;170(7):918-924.
Challenges in the accurate measurement of sexual behavior in human immunodeficiency virus (HIV) prevention research are well documented and have prompted discussion about whether valid assessments are possible. Audio computer-assisted self-interviewing (ACASI) may increase the validity of self-reported behavioral data. In 2006–2007, Zimbabwean women participated in a randomized, cross-sectional study that compared self-reports of recent vaginal sex and condom use collected through ACASI or face-to-face interviewing (FTFI) with a validated objective biomarker of recent semen exposure (prostate-specific antigen (PSA) levels). Of 910 study participants, 196 (21.5%) tested positive for PSA, an indication of semen exposure during the previous 2 days. Of these 196 participants, 23 (11.7%) reported no sex in the previous 2 days, with no difference in reported sexual activity between interview modes (12.5% ACASI vs. 10.9% FTFI; Fisher's exact test: P = 0.72). In addition, 71 PSA-positive participants (36.2%) reported condom-protected vaginal sex only; their reports also indicated no difference between interview modes (33.7% ACASI vs. 39.1% FTFI; P = 0.26). Only 52% of PSA-positive participants reported unprotected sex during the previous 2 days. Self-report was a poor predictor of recent sexual activity and condom use in this study, regardless of interview mode, providing evidence that such data should be interpreted cautiously.
PMCID: PMC3888136  PMID: 19741042
biological markers; condoms; data collection; epidemiologic measurements; HIV; prostate-specific antigen; sexual behavior
15.  The Protective Effect of Family Strengths in Childhood against Adolescent Pregnancy and Its Long-Term Psychosocial Consequences 
The Permanente Journal  2010;14(3):18-27.
Background: Few reports have addressed associations between family strengths during childhood and adolescent pregnancy and its consequences. We examined relationships among a number of childhood family strengths and adolescent pregnancy, risk behavior, and psychosocial consequences after adolescent pregnancy.
Methods: Our retrospective cohort of 4648 women older than 18 years (mean age, 56 years) received primary care in San Diego, CA. Outcomes included adolescent pregnancy and psychosocial consequences compared with number of the following childhood family strengths: family closeness, support, loyalty, protection, love, importance, and responsiveness to health needs.
Results: Of the cohort, 3082 participants (66%) reported 6 or 7 categories of childhood family strengths. Teen pregnancy occurred in 39%, 33%, 30%, 25%, 24%, 21%, and 19% of those with 0 or 1, 2, 3, 4, 5, 6, and 7 childhood family strengths, respectively (p for trend < 0.00001). When childhood abuse and household dysfunction were present, adjusted odds ratios (ORs) for adolescent pregnancy demonstrated an increasingly protective effect as numbers of childhood family strengths increased from 0 or 1 to 2 or 3, 4 or 5, and 6 or 7 (1.0 to 0.80), (1.0 to 0.80, 0.60, and 0.54, respectively). These findings were partly explained by progressive delays in initiation of sexual activity as the number of childhood family strengths increased. Adjusted ORs for psychosocial problem occurring decades later decreased as the number of childhood family strengths increased from 0 or 1 to 2 or 3, 4 or 5, and 6 or 7 (job problems, 1.0, 0.8, 0.6, 0.4; family problems, 1.0, 1.1, 0.7, 0.6; financial problems, 1.0, 0.9, 0.9, 0.6; high stress, 1.0, 1.1, 0.9, 0.8; uncontrollable anger, 1.0, 0.7, 0.7, 0.4).
Conclusions: Childhood family strengths are strongly protective against adolescent pregnancy, early initiation of sexual activity, and long-term psychosocial consequences.
PMCID: PMC2937841  PMID: 20844701
16.  Mandatory fortification with folic acid in the United States appears to have adverse effects on histone methylation in women with pre-cancer but not in women free of pre-cancer 
To evaluate whether mandatory fortification of grain products with folic acid in the US is associated with changes in histone methylation in cells involved in cervical carcinogenesis.
Cervical specimens obtained before (1990 to 1992) and after mandatory folic acid fortification (2000 to 2002) were used to examine the degree of histone methylation (H3 Lys-9) by immunohistochemistry. 91 women (51 before and 40 after fortification) were diagnosed with cervical intraepithelial neoplasia (CIN) grade 3 or carcinoma in situ (CIS) and sections utilized in the study also contained normal, reactive or metaplastic cervical epithelium, CIN 1 or CIN 2. 64 women (34 before and 30 after fortification) were free of CIN and these sections contained only normal or reactive cervical epithelium. Immunohistochemical staining for H3 Lys-9, its assessment in different cell or lesion types and data entry were blinded for fortification status. For each cell type or lesion category we used PROC MIXED in SAS with the specimen identifier as a random effect and the robust variance estimator to estimate age- and race-adjusted intensity score for H3 Lys-9 in the pre- and post-fortification periods.
Degree of H3 Lys-9 methylation was significantly higher (P < 0.0001) in ≥CIN 2 lesions (CIN 2, CIN 3 and CIS) than in ≤CIN 1 lesions (CIN 1, normal, reactive and metaplastic), in both pre- and post-fortification CIN 3/CIS specimens. Age- and race-adjusted mean H3 Lys-9 score was significantly higher in all cell or lesion types in CIN 3/CIS specimens obtained in the post-fortification period compared to pre-fortification period (P < 0.05, all comparisons). In contrast, in specimens obtained from women free of CIN, Lys-9 methylation in normal/reactive cervical epithelium was significantly lower in post-fortification specimens than in pre-fortification specimens (P = 0.03).
Higher levels of Lys-9 methylation in ≥CIN 2 compared to ≤CIN 1 lesions suggest that higher Lys-9 methylation is associated with progression of lower grade CIN to higher grade CIN. Higher Lys-9 methylation in cervical tissues of women diagnosed with CIN 3 in the post-fortification period than in pre-fortification period suggest that fortification may adversely affect histone methylation in already initiated cells. Lower Lys-9 methylation in normal/reactive cervical cells of women free of CIN in the post-fortification period than pre-fortification on the other hand suggests that fortification is likely to protect against initiation of carcinogenic process in the cervix. These results suggest that mandatory fortification with folic acid in the US seems to have different effects on cancer depending on the stage of carcinogenesis. Because this is the first study to report folic acid fortification-associated differences in histone methylation and because of the limitations inherent to the approach we have taken to demonstrate these differences, validation of the results in other study populations or with other techniques for assessing histone methylation is necessary.
PMCID: PMC2971712  PMID: 21072283
folic acid; fortification; histone methylation; cervix
17.  The Effect of Uncertainty in Exposure Estimation on the Exposure-Response Relation between 1,3-Butadiene and Leukemia 
In a follow-up study of mortality among North American synthetic rubber industry workers, cumulative exposure to 1,3-butadiene was positively associated with leukemia. Problems with historical exposure estimation, however, may have distorted the association. To evaluate the impact of potential inaccuracies in exposure estimation, we conducted uncertainty analyses of the relation between cumulative exposure to butadiene and leukemia. We created the 1,000 sets of butadiene estimates using job-exposure matrices consisting of exposure values that corresponded to randomly selected percentiles of the approximate probability distribution of plant-, work area/job group-, and year specific butadiene ppm. We then analyzed the relation between cumulative exposure to butadiene and leukemia for each of the 1,000 sets of butadiene estimates. In the uncertainty analysis, the point estimate of the RR for the first non zero exposure category (>0–<37.5 ppm-years) was most likely to be about 1.5. The rate ratio for the second exposure category (37.5–<184.7 ppm-years) was most likely to range from 1.5 to 1.8. The RR for category 3 of exposure (184.7–<425.0 ppm-years) was most likely between 2.1 and 3.0. The RR for the highest exposure category (425.0+ ppm-years) was likely to be between 2.9 and 3.7. This range off RR point estimates can best be interpreted as a probability distribution that describes our uncertainty in RR point estimates due to uncertainty in exposure estimation. After considering the complete probability distributions of butadiene exposure estimates, the exposure-response association of butadiene and leukemia was maintained. This exercise was a unique example of how uncertainty analyses can be used to investigate and support an observed measure of effect when occupational exposure estimates are employed in the absence of direct exposure measurements.
PMCID: PMC2760421  PMID: 19826555
1,3-butadiene; epidemiology; methods; leukemia; workplace exposures; uncertainty analysis
18.  Anamnestic Immune Response to Dengue and Decreased Severity of Yellow Fever 
A protective immunity against yellow fever, from cross-reactive dengue antibodies, has been hypothesized as an explanation for the absence of yellow fever in Southern Asia where dengue immunity is almost universal. This study evaluates the association between protective immunity from cross-reactive dengue antibodies with yellow fever infection and severity of the disease. The study population consisted of military personnel of a jungle garrison and its detachments located in the Ecuadorian Amazonian rainforest. The cross-sectional study employed interviews as well as seroepidemiological methods. Humoral immune response to yellow fever, Mayaro, Venezuelan equine encephalitis, Oropouche, and dengue 2 infections was assessed by evaluating IgM and IgG specific antibodies. Log-linear regression analysis was used to evaluate age and presence of antibodies, against dengue type 2 virus, as predictors of yellow fever infection or severe disease. During the seroepidemiological survey, presence of dengue antibodies among yellow fever cases were observed in 77.3% cases from the coastal region, where dengue is endemic, 14.3% cases from the Amazon and 16.7 % cases from the Andean region. Dengue cross-reactive antibodies were not significantly associated with yellow fever infection but significantly associated with severity of the disease. The findings of this study suggest that previous exposure to dengue infection may have induced an anamnestic immune response that did not prevent yellow fever infection but greatly reduced the severity of the disease.
PMCID: PMC2840959  PMID: 20300401
Antibody; Cross-protection; Dengue; Immunology; Yellow fever
19.  Mandatory Fortification with Folic Acid in the United States is associated with Increased Expression of DNA Methyltransferase 1 in the Cervix 
The objective of this study was to evaluate whether mandatory fortification of grain products with folic acid in the USA is associated with changes in DNA methyltransferase (Dnmt) 1 expression in cells involved in cervical carcinogenesis.
Archived specimens of cervical intraepithelial neoplasia (CIN) diagnosed before (1990-92) and after mandatory folic acid fortification (2000-02) were used to examine the expression of Dnmt 1 in specific lesions involved in cervical carcinogenesis by immunohistochemistry. The total number of lesions examined was 101 in the pre-fortification period and 96 in the post-fortification period. Immunohistochemical staining for Dnmt 1, its assessment and data entry were blinded with regard to the fortification status.
Age- and race-adjusted mean percentage of cells positive for Dnmt 1 or the Dnmt 1 score was significantly higher in all lesion types (i.e., normal cervical epithelium, reactive cervical epithelium, metaplastic cervical epithelium, CIN or carcinoma in situ) detected in the post-fortification period compared to pre-fortification period (P < 0.05, all comparisons). The degree of Dnmt 1 was significantly higher (P < 0.0001) in ≥ CIN 2 lesions compared to ≤ CIN 1 lesions, regardless of the fortification group.
These results suggest that mandatory fortification with folic acid in the United States seem to have resulted in a change in the degree of expression of Dnmt 1 in cells involved in cervical carcinogenesis. Because the approach we have taken to demonstrate these differences have limitations inherent to a study of this nature and this is the first study to report a folate fortification associated change in Dnmt 1, validating these results in other study populations and or with other techniques of assessing Dnmt 1 will increase the scientific credibility of these findings.
PMCID: PMC2215322  PMID: 18065205
Folic acid; Fortification; DNA Methyltransferase; Cervix
20.  Assessing Yellow Fever Risk in the Ecuadorian Amazon 
This study reports results of a cross-sectional study based on interviews and seroepidemiological methods to identify risk factors for yellow fever infection among personnel of a military garrison in the Amazonian rainforest. Clinical symptoms and signs observed among yellow fever cases are also described. Humoral immune response to yellow fever, Mayaro, Venezuelan equine encephalitis, Oropouche, and dengue 2 infection was assessed by evaluating IgM and IgG specific antibodies. A yellow fever attack rate of 13% (44/341, with 3 fatal cases) was observed among military personnel. Signs of digestive track bleeding (14.6%) and hematuria (4.9%) were observed among the yellow fever cases. In 32.2% of the cases, we measured high levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase with maximum levels of 6,830 and 3,500, respectively. Signs of bleeding or jaundice were observed in some cases, and high levels of transaminases were seen. The epidemiological and laboratory investigations demonstrated that the military personnel were affected by a yellow fever outbreak. The association between clearing the rainforest and also being at the detachments with yellow fever infection confirms that clearing is the main factor in the jungle model of transmission, which takes place deep in the Amazonian rainforest.
PMCID: PMC2840941  PMID: 20300380
Disease outbreak; Ecuador; Military personnel; Risk factors; Yellow fever
21.  The Protective Association of MTHFR Polymorphism on Cervical Intraepithelial Neoplasia is Modified by Riboflavin Status 
We previously reported that women polymorphic for the methylenetetrahydrofolate reductase (MTHFR) gene were less likely to have cervical intraepithelial neoplasia (CIN) 2 or 3 (odds ratio [OR] = 0.40, 95 % confidence interval [CI]: 0.21, 0.78, p=0.007). In the current study, we tested whether this protective association is modified by the circulating riboflavin status in the same study population.
Riboflavin status was assessed by the erythrocyte glutathione reductase (EGR) assay and expressed in terms of an EGR activity coefficient (EGRAC). The status of MTHFR polymorphism, riboflavin, and circulating concentrations of folate, vitamins B-12, A, E, C and total carotene were ascertained in 170 White and 265 African-American women positive for the cervical presence of high-risk human papilloma virus (HR-HPV). Presence/absence of CIN 2 or 3 was determined histologically, and associations with risk factors were examined using multiple logistic regression. Eighty women with CIN 2 or 3 lesions were compared to 355 women without cervical lesions. Based on the median EGRAC of 1.4, women were grouped into low (> 1.4) and high (≤1.4) riboflavin status.
Women with MTHFR polymorphism and low riboflavin status were significantly less likely to have CIN 2 or 3 than the referent group of women without the polymorphism and high riboflavin status (OR = 0.35, 95% CI: 0.13, 0.92, p= 0.034). MTHFR polymorphism was not associated with CIN 2 or 3 in women with high riboflavin status (OR = 0.51, 95% CI: 0.22, 1.19, p = 0.119), nor were any of the associations influenced by folate levels.
A further inactivation of polymorphic MTHFR by low riboflavin status and a resulting shift in the folate metabolic pathway toward DNA synthesis may explain these observations. The practical implications of this complex gene-nutrient-disease interaction will require further investigation.
PMCID: PMC2025704  PMID: 17303386
MTHFR; riboflavin; cervical; neoplasia
22.  Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men 
Journal of Clinical Microbiology  2001;39(11):3927-3937.
Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities.
PMCID: PMC88466  PMID: 11682509

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