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Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC–MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011–2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5 nmol/l; 100 %), UMFA (1·21 nmol/l; 99·9 %), MeFox (1·53 nmol/l; 98·8 %), and THF (1·01 nmol/l; 85·2 %) were mostly detectable. 5-FormylTHF (3·6 %) and 5,10-methenylTHF (4·4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7 %); UMFA (4·0 %), non-methyl folate (4·7 %) and MeFox (4·5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's r< 0·4) but significant (P< 0·05) correlations with physiological and lifestyle variables. Fasting, kidney function, smoking and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiological and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.

The Quansys multiplex (Q-Plex) measures ferritin (Fer), soluble transferrin receptor (sTfR), C-reactive protein (CRP), α-1-acid glycoprotein (AGP), and retinol-binding protein (RBP). We compared Q-Plex results with reference-type assays and evaluated Q-Plex performance. Pearson correlation and Lin's concordance coefficients between the Q-Plex and reference assay were: Fer 0.98 and 0.91, sTfR 0.88 and 0.35, CRP 0.98 and 0.98, AGP 0.82 and 0.81, and RBP 0.68 and 0.31, respectively. The median relative difference between the Q-Plex and reference assay were: Fer -2.4%, sTfR 107%, CRP 0.03%, AGP -1.3%, and RBP 51%. The Q-Plex intra-assay CVs were <5%; the inter-assay CVs were higher: Fer 11%, sTfR 14%, CRP 9.3%, AGP 7.5%, and RBP 19%. EDTA plasma produced 74% higher Q-Plex sTfR concentrations compared to serum. Analyte stability was good for ≤5 freeze-thaw cycles. After adjusting Q-Plex data to the reference assays, sensitivity and specificity were >85% for Fer and CRP; specificity was >85% for sTfR, AGP, and RBP. Using performance criteria derived from biologic variation, Fer, CRP, and AGP met the minimum allowable imprecision (<10.7%, <31.7%, and <8.5%, respectively) and difference from the reference assay (<±7.7%, <±32.7%, and <±10.3%, respectively), while sTfR and RBP exceeded these thresholds (<8.5% and <7.8% for imprecision and <±7.7% and <±12% for difference, respectively). The Q-Plex measures multiple biomarkers simultaneously, is easy to perform, and uses small sample volumes. With some improvements in accuracy and precision (i.e., sTfR and RBP), this assay could be a useful tool for low-resource laboratories conducting micronutrient surveys for epidemiologic screening applications. These findings need to be verified using other populations, particularly those with inadequate micronutrient status.

Aflatoxin exposure is endemic in developing countries with warm, humid climates that promote toxigenic mold growth on crops and foodstuffs. Estimating human aflatoxin exposure is key to identifying and abating contamination sources. Serum aflatoxin B1 bound to albumin lysine (AFB1-lys) is a preferred exposure biomarker, but field sample collection, processing, transportation, and storage logistics are challenging. We validated an improved LC-MS/MS method for serum AFB1-lys and applied it to three field sampling challenges: transportation/storage (elevated temperature); collection/processing (hemolysis); and sample type substitution (heparinized plasma). Our new LC-MS/MS method had a LOD of 0.03 ng/mL, accuracy (mean spike recovery) of 112%, total imprecision (replicate pool measurements) ≤5% at ≥0.2 ng/mL, and results that were 95.1% similar (mean percentage similarity) to an established method. AFB1-lys in human serum spiked with serum from aflatoxin-dosed rats was stable for 14 days at both ambient (22.5 °C) and elevated (38 °C) temperatures. Simulated hemolysis (adding 0.25–3 mg hemoglobin) did not affect AFB1-lys accuracy at ≥0.5 ng/mL but caused 10–25% signal suppression. Heparinized plasma AFB1-lys was 99.0% similar to serum but interfered with albumin measurements (bromocresol green) causing spurious low bias. Further investigation is warranted, but our findings suggest that AFB1-lys is pre-analytically robust.

Objectives: To study the prevalence of circumcision in the United States and to examine the association between circumcision and herpes simplex virus Type 2 (HSV-2) infection. Methods: As part of National Health and Nutrition Examination Surveys from 1999 to 2004, 6174 men were interviewed about circumcision status and sexual behaviors, and were tested for HSV-2 antibodies. Medical artwork was used to aid the reporting of circumcision status. Results: The overall prevalence of circumcision was 79% and varied by race/ethnicity (88% in non-Hispanic whites, 73% in nonHispanic blacks, 42% in Mexican Americans, and 50% in others). For men born in the United States from 1940 through 1979, the prevalence of circumcision increased, with larger increases in non-Hispanic blacks and Mexican Americans than in non-Hispanic whites; the prevalence of circumcision decreased significantly in those born in the 1980s (84%) compared to those born in 1970s (91%) (P <0.001). Circumcision status was not associated with sexual behaviors we assessed. In multivariate analyses, circumcision was not associated with HSV-2 infection (P = 0.47). Conclusions: The prevalence of circumcision apparently peaked in those born in the 1970s and declined in those born in the 1980s. Circumcision was not associated with HSV-2 infection.

Seroprevalence of and coinfection with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in the United States were analyzed by use of data from a nationally representative survey (National Health and Nutrition Examination Survey III, 1988–1994). Evidence was explored for possible protection by prior HSV-1 infection against infection and clinical disease with HSV-2. Overall, 27.1% of persons aged ≥12 years were seronegative for HSV-1 and HSV-2; 51.0% were seropositive for HSV-1 only, 5.3% for HSV-2 only, and 16.6% for both HSV-1 and HSV-2. The seroprevalence of HSV-2 was higher in persons with HSV-1 antibody. Approximately 76% of persons who had HSV-2 antibody also had HSV-1 antibody. Persons seropositive for HSV-2 only reported a history of genital herpes more frequently (16.2 %) than persons seropositive for both HSV-1 and HSV-2 (5.9 %). The seroprevalence of HSV-1 and age at infection may influence the epidemiology of clinical genital herpes, even if prior HSV-1 infection does not prevent HSV-2 infection.

Infection with human papillomavirus (HPV) type 16 accounts for about half of cervical cancers worldwide. This study investigated the seroepidemiology of HPV-16 infection in the United States by using a population-based survey. Serum samples and questionnaire data were collected from 1991 to 1994 for the National Health and Nutrition Examination Surveys. HPV-16–specific IgG antibody was detected by use of an HPV-16 virus-like particle ELISA. HPV-16 seropositivity in the US population aged 12–59 years was 13.0% (95% confidence interval, 11.5%–14.7%). Seroprevalence was higher in women (17.9%) than in men (7.9%). Age, race/ethnicity, and number of lifetime sex partners were associated with HPV seropositivity in women. Race/ethnicity, age at first intercourse, urban/nonurban residence, years of sexual activity, and having had sex with a man were associated with HPV seropositivity in men. Information on HPV-16 seroepidemiology will be important for designing prevention efforts including vaccine programs

Objectives: To describe demographic and behavioral characteristics and the prevalence of HIV and herpes simplex virus type 2 (HSV-2) infections in men who had sex with men identified through a nationally representative, population-based survey. Methods: As part of National Health and Nutrition Examination Surveys in 2001-2006, men 18 to 59 years of age were interviewed about sexual behavior using audio computer assisted self-interview and were tested for antibodies to HIV and HSV-2. Results: Of the 4319 men interviewed, 5.2% reported having ever had sex with men (MSM). MSM were more likely than non-MSM (those reporting female partners only) to have first sex at <15 years (31.9% vs. 17.3%), have >10 lifetime sex partners (73.6% vs. 40.8%), and have ever used cocaine (46.1% vs. 26.6%) (all < 0.004). Among MSM, the prevalence of HIV and HSV-2 was 9.1% and 18.4%, respectively. Only 44.5% of MSM reported their sexual orientation as homosexual or gay. Comparing with bisexual and heterosexual MSM, homosexual MSM reported the highest number of lifetime male partners and had the highest HIV prevalence (16.5%). Conclusions: In this population-based sample of men in the United States, self-reported same-sex behavior and homosexual orientation are strong markers for high risk of HIV infection.

The Quansys multiplex (Q-Plex) measures ferritin (Fer), soluble transferrin receptor (sTfR), C-reactive protein (CRP), [alpha]-1-acid glycoprotein (AGP), and retinol-binding protein (RBP). We compared Q-Plex results with reference-type assays and evaluated Q-Plex performance. Pearson correlation and Lin's concordance coefficients between the Q-Plex and reference assay were: Fer 0.98 and 0.91, sTfR 0.88 and 0.35, CRP 0.98 and 0.98, AGP 0.82 and 0.81, and RBP 0.68 and 0.31, respectively. The median relative difference between the Q-Plex and reference assay were: Fer -2.4%, sTfR 107%, CRP 0.03%, AGP -1.3%, and RBP 51%. The Q-Plex intra-assay CVs were 85% for Fer and CRP; specificity was >85% for sTfR, AGP, and RBP. Using performance criteria derived from biologic variation, Fer, CRP, and AGP met the minimum allowable imprecision (<10.7%, <31.7%, and <8.5%, respectively) and difference from the reference assay (<±7.7%, <±32.7%, and <±10.3%, respectively), while sTfR and RBP exceeded these thresholds (<8.5% and <7.8% for imprecision and <±7.7% and <±12% for difference, respectively). The Q-Plex measures multiple biomarkers simultaneously, is easy to perform, and uses small sample volumes. With some improvements in accuracy and precision (i.e., sTfR and RBP), this assay could be a useful tool for low-resource laboratories conducting micronutrient surveys for epidemiologic screening applications. These findings need to be verified using other populations, particularly those with inadequate micronutrient status.

Objectives: To estimate the prevalence of same-sex sexual behavior in women in the United States; to describe demographic and behavioral characteristics and the prevalence of herpes simplex virus type 2 (HSV-2) infection. Methods: As part of the National Health and Nutrition Examination Surveys during 2001-2006, women aged 18 to 59 years were interviewed about sexual behaviors using audio computer assisted self-interview. Persons aged 14 to 49 years were tested for antibodies to HSV-2. Results: Among sexually experienced women aged 18 to 59 years, 7.1% (95% confidence interval, 6.1-8.2) reported ever having had sex with a woman (WSW-ever) and 2.7% in the past year. The prevalence of WSW-ever correlated negatively with age, highest (9.4%) in 18 to 29-year-olds and lowest (5.5%) in 50 to 59-year-olds. Among WSW-ever, 52.6% self-identified as heterosexual/straight, 28.3% as bisexual, and 19.1% as homosexual/lesbian. Among WSW-ever, demographic characteristics were similar but sexual behaviors were different by sexual orientation: 31.3% of heterosexuals, 38.9% of bisexuals, and 12.9% of homosexuals reported first sex at age 14 or younger (P = 0.005); the median number of lifetime male partners was 10.8, 17.6, and 2.9, respectively (P < 0.0001). Among WSW-ever, the prevalence of HSV-2 was 45.6% in heterosexuals, 35.9% in bisexuals, and 8.2% in homosexuals (P = 0.001). In comparison, among women who reported no same-sex partners, the prevalence of HSV-2 was 23.8%. Conclusions: In this population-based sample of women, selfreported same-sex behaviors were increasingly more prevalent in younger women. Compared with homosexual WSW-ever and women who reported never having sex with other women, heterosexual or bisexual WSW-ever had higher HSV-2 seroprevalence.

We evaluated the prevalence of antibodies to human papillomavirus (HPV) type 16 in a representative sample of children 6–11 years of age in the United States. Serum samples and questionnaire data were collected between 1991 and 1994, for the National Health and Nutrition Examination Survey III. HPV-16–specific immunoglobulin G antibodies were detected by an HPV-16 L1 virus-like particle–based enzyme-linked immunosorbant assay. Overall, 2.4% of 1316 children 6–11 years of age were seropositive. Seroprevalence was higher in boys than in girls (3.5% vs. 1.2%; P=.08) and in children >7 years of age than in children ⩽7 years of age (3.3% vs. 0.4%; P<.05). None of the variables tested for, including race/ethnicity, socioeconomic status, and urban or rural residence, were significantly associated with HPV-16 seropositivity. To explain HPV-16 seropositivity in this population, further study is required