Association of Serum 25-Hydroxyvitamin D With Prevalence, Incidence, and Clearance of Vaginal HPV Infection in Young Women.

BACKGROUND: We assessed the association between serum 25-hydroxyvitamin D levels and genital human papillomavirus (HPV) prevalence, incidence, and clearance among female participants in the HPV Infection and Transmission among Couples through Heterosexual activity (HITCH) Cohort Study. METHODS: We genotyped HPV DNA in vaginal samples and quantified baseline serum 25-hydroxyvitamin D levels using Roche's Linear Array and Total vitamin D assay, respectively. We used logistic and Cox proportional hazards models, respectively, to estimate adjusted odds ratios (ORs) and hazard ratios (HRs) with 95% confidence intervals (CIs). RESULTS: There was no association between vitamin D levels (every 10-ng/mL increase) at baseline and HPV prevalence (OR, 0.88; 95% CI, .73-1.03) or incidence (HR, 0.88; 95% CI, .73-1.06), but we observed a modest negative association with HPV clearance (HR, 0.76; 95% CI, .60-.96). Vitamin D levels <30 ng/mL, compared with those ≥30 ng/mL, were not associated with HPV prevalence (OR, 0.98; 95% CI, .57-1.69) or incidence (HR, .87; 95% CI, .50-1.43), but they were associated with a marginally significant increased clearance (OR, 2.14; 95% CI, .99-4.64). We observed consistent results with restricted cubic spline modeling of vitamin D levels and clinically defined categories. HPV type-specific analyses accounting for multiple HPV infections per participant showed no association between vitamin D levels and all study outcomes. CONCLUSIONS: This study provided no evidence of an association between low vitamin D levels and increased HPV prevalence, acquisition, or clearance.

An LC-MRM assay for the quantification of metanephrines from dried blood spots for the diagnosis of pheochromocytomas and paragangliomas.

The quantitation of metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) - referred to as metanephrines -- by LC-MS/MS is the gold-standard for screening for pheochromocytoma and paragangliomas (PPGLs), tumours of the adrenal gland and the peripheral nervous system. An assay for metanephrines from dried blood spots (DBSs) would be of high clinical utility as it simplifies sample collection, enables remote sampling, and could increase compliance with the clinical recommendation for supine sampling. Moreover, DBS sampling facilitates the measurement of blood-derived metanephrines in pediatric patients - where DBSs are well-established - in order to diagnose neuroblastomas. Here, we adapted an established derivatization-based LC-MRM-MS assay for plasma catecholamines, and optimized the sample extraction, LC, and MS parameters to produce a fast, sensitive, and robust method for the measurement of metanephrines from DBSs, including 3-methoxytyramine. The DBS samples were excised, derivatized with phenyl isothiocyanate (PITC) on-spot, extracted, and measured by LC-MRM-MS. To validate assay suitability and performance, we assessed the linearity, precision, accuracy, recovery, and matrix effects of the method, and determined the stability of metanephrines in DBSs under different storage conditions. Assay performance for NMN, MN, and 3-MT was sufficient for quantitation from a single DBS within a linear range from 40 to 2000 pg/mL. MN and NMN were stable in DBSs for 2 weeks, whereas 3-MT was stable for one week regardless of storage temperature. Altogether, this work represents the first quantitative LC-MS/MS method for metanephrines from DBSs and provides a novel opportunity for the diagnosis of PPGLs and neuroblastomas in the future.

Maximizing confidence in a negative result: Quantitative sample adequacy control.

Quantitative PCR (qPCR) is a leading screening tool, permitting rapid detection of pathogens and the maintenance of effective infection control programs. Unfortunately, qPCR assays frequently do not incorporate Sample Adequacy Control (SAC). A SAC controls for the quantity, quality and adequacy of the specimen. Without SAC, the confidence in a negative result remains questionable and the efficacy of screening is compromised. Ultimately, the exclusion of SAC from qPCR may result in false negative results. One should consider SAC to be an integral critical type of laboratory control; addressing diverse analytical problems, such as sample adequacy, sample processing and assay inhibition. Following distribution of cycle threshold values (Cq) of Influenza A positive results and Cq values of SAC, obtained from nasopharyngeal swabs, we showed that the confidence in a negative result cannot be guaranteed in the presence of a weak positive SAC signal (late Cq values). Herein, we explain why widespread inclusion of sample adequacy control in routine screening is blocked. A protocol and methods for SAC threshold establishment are offered.

Laboratory-developed test for detection of acute Clostridium difficile infections with the capacity for quantitative sample normalization.

We describe a laboratory-developed test intended for the detection of acute Clostridium difficile infections (CDI) with the capacity for quantitative sample normalization. The test is based on the detection of the tcdB gene. However, this biomarker is also present among people without symptoms, implying that individuals with diarrhea, not caused by C. difficile may nonetheless test positive. Therefore, clinical diagnosis based on this format of testing can be challenging. In order to improve diagnostic assays capability, tcdB-based quantification methods were suggested as a potential solution, however they did not increase clinical specificity. We report methodology for a dual biomarker monitoring (total bacterial load and tcdB assay), allowing for the calculation of the relative presence of tcdB in the total bacterial population in the tested samples. We believe that this approach is clinically relevant to current assays and can improve CDI testing algorithms.

Rectal swab screening assays of public health importance in molecular diagnostics: Sample adequacy control.

Rectal swabs are routinely used by public health authorities to screen for multi-drug resistant enteric bacteria including vancomycin-resistant enterococci (VRE) and carbapenem-resistant enterobacteriaceae (CRE). Screening sensitivity can be influenced by the quality of the swabbing, whether performed by the patient (self-swabbing) or a healthcare practitioner. One common exclusion criterion for rectal swabs is absence of "visible soiling" from fecal matter. In our institution, this criterion excludes almost 10% of rectal swabs received in the microbiology laboratory. Furthermore, over 30% of patients in whom rectal swabs are cancelled will not be re-screened within the next 48h, resulting in delays in removing infection prevention measures. We describe two quantitative polymerase chain reaction (qPCR)-based assays, human RNAse P and eubacterial 16S rDNA, which might serve as suitable controls for sampling adequacy. However, lower amounts of amplifiable human DNA make the 16s rDNA assay a better candidate for sample adequacy control.

Appropriate vitamin D loading regimen for patients with advanced lung cancer.

BACKGROUND: Most patients attending cancer clinics have hypovitaminosis D. Correcting or preventing this abnormal condition could mitigate the emotional and physical complications of their disease, but clinical trials of vitamin D therapy in this setting are hindered by the unavailability of safe, effective and practical loading dose regimens. METHODS: In this single arm open-label pharmacokinetic trial, outpatients with advanced lung cancer consumed 20,000 IU vitamin D daily with the largest meal of the day for 14 days followed by 10,000 IU per day for a further 7 days. Plasma concentrations of 25-hydroxyvitamin D [25(OH)D], parathyroid hormone, calcium, vitamin C and C-reactive protein were measured on protocol days 0, 14 and 21, and serum vitamin D binding protein (VDBP) concentrations on days 0 and 21. As a secondary objective, preliminary information was obtained regarding clinical effects of rapid vitamin D loading on mood and symptoms by administering appropriate questionnaires two times at baseline and after 14 and 21 days of vitamin D therapy. RESULTS: Of the 91 patients enrolled in the study, 85 % had hypovitaminosis D and 41 % had hypovitaminosis C. Plasma VDBP concentrations were in the normal range. The vitamin D load increased the average plasma 25(OH)D concentration to 116 ± 34 nmol/L (mean ± SD); the median concentration was 122 nmol/L (interquartile range 103-134); VDBP concentrations did not change. Final plasma 25(OH)D concentrations were subnormal (<75 nmol/L) for 13 % of the patients and sub-target (<120 nmol/L) for 44 % of them. In most cases, subnormal and sub-target 25(OH)D concentrations were attributable to obesity and/or a low baseline 25(OH)D concentration. Mood and symptom scores did not change significantly throughout the 3-week protocol. CONCLUSION: Hypovitaminosis D and C are very common in outpatients with advanced lung cancer. A vitamin D load of 20,000 IU per day for 14 days failed to achieve the target concentration in 44 % of the participants in this trial. These results suggest that a loading dose of 30,000 IU per day for 14 days would be safe and effective for patients who are obese or at risk of severe hypovitaminosis D. The preliminary nature of the study design, and the failure to achieve target 25(OH)D concentrations for a large proportion of the patients, do not allow any firm conclusion about the clinical effects of correcting hypovitaminosis D in this patient population. Nevertheless, no evidence was obtained that partial correction of hypovitaminosis D greatly improved mood, reduced distress or relieved cancer-related symptoms. This trial was registered at clinicaltrials.gov as NCT01631526.

Metabolic origin of hypovitaminosis C in acutely hospitalized patients.

OBJECTIVE: Recent studies have indicated a high prevalence of hypovitaminosis C in acutely hospitalized patients. It is unclear whether hypovitaminosis C in this setting represents deficiency or tissue redistribution of the vitamin as part of the acute-phase response. METHODS: We administered vitamin C for 1 wk to acutely hospitalized, but not critically ill patients with hypovitaminosis C, on the assumption that a large increase in plasma and mononuclear leukocyte vitamin C concentrations, a decrease in metabolic markers of oxidative stress, or an improvement in psychologic mood state would implicate the initial condition as nutritional deficiency rather than tissue redistribution. RESULTS: Vitamin C administration increased plasma and mononuclear leukocyte vitamin C concentrations from subnormal (16.3 ± 12.4 µmol/L and 6.5 ± 5.5 mmol/L, respectively) to normal (71.0 ± 30.9 µmol/L, P < 0.0001, and 8.2 ± 6.8 mmol/L, P < 0.015); the mood disturbance score improved by 33% (P < 0.008). There was no increase in plasma glutathione concentrations or a reduction in plasma or mononuclear leukocyte malondialdehyde concentrations. An inverse relation was observed between plasma C-reactive protein and plasma vitamin C concentrations (P = 0.006). CONCLUSION: Although associated with systemic inflammation, the metabolic features of hypovitaminosis C in acutely hospitalized, non-critically ill patients are more consistent with deficiency than with tissue redistribution.