The impact of sleep deprivation and nighttime light exposure on clock gene expression in humans.

AIM: To examine the effect of acute sleep deprivation under light conditions on the expression of two key clock genes, hPer2 and hBmal1, in peripheral blood mononuclear cells (PBMC) and on plasma melatonin and cortisol levels. METHODS: Blood samples were drawn from 6 healthy individuals at 4-hour intervals for three consecutive nights, including a night of total sleep deprivation (second night). The study was conducted in April-June 2006 at the University Medical Centre Ljubljana. RESULTS: We found a significant diurnal variation in hPer2 and hBmal1 expression levels under baseline (P<0.001, F=19.7, df=30 for hPer2 and P<0.001, F=17.6, df=30 for hBmal1) and sleep-deprived conditions (P<0.001, F=9.2, df=30 for hPer2 and P<0.001, F=13.2, df=30 for hBmal1). Statistical analysis with the single cosinor method revealed circadian variation of hPer2 under baseline and of hBmal1 under baseline and sleep-deprived conditions. The peak expression of hPer2 was at 13:55 ± 1:15 hours under baseline conditions and of hBmal1 at 16:08 ± 1:18 hours under baseline and at 17:13 ± 1:35 hours under sleep-deprived conditions. Individual cosinor analysis of hPer2 revealed a loss of circadian rhythm in 3 participants and a phase shift in 2 participants under sleep-deprived conditions. The plasma melatonin and cortisol rhythms confirmed a conventional alignment of the central circadian pacemaker to the habitual sleep/wake schedule. CONCLUSION: Our results suggest that 40-hour acute sleep deprivation under light conditions may affect the expression of hPer2 in PBMCs..

Twenty-four mini-pool HCV RNA screening outside a blood transfusion setting: results of a 2-year prospective study.

The usefulness of 24 mini-pool hepatitis C virus (HCV) RNA screening was evaluated in a 2-year prospective study carried out on a total of 6432 consecutive anti-HCV negative specimens in a routine diagnostic laboratory setting. A total of 268 mini-pools were tested using an automated commercial PCR assay for qualitative detection of HCV RNA, with a lower limit of detection of 50 IU/ml. Eighteen (0.28%) anti-HCV negative/HCV RNA positive serum samples obtained from 12 patients (all intravenous drug users), were detected. Ten patients responded to an invitation for follow-up testing. Five, three and one patient seroconverted in the first, second and third follow-up sample, respectively. One patient had not seroconverted by the end of the study period. The interval between the first HCV RNA positive sample and the first anti-HCV positive samples was 24-192 days. The costs of detecting a single anti-HCV negative/HCV RNA positive sample and a single anti-HCV negative/HCV RNA positive patient using the 24 mini-pool HCV RNA screening strategy were estimated to be around euro 643 and 965, respectively. It was shown that screening for HCV infection using the 24 mini-pool HCV RNA screening strategy can also be both useful and cost effective outside a blood transfusion setting.

Resolving repeatedly borderline results of Hybrid Capture 2 HPV DNA Test using polymerase chain reaction and genotyping.

The Hybrid Capture 2 HPV DNA Test (hc2) (Digene Corporation, Gaithersburg, MD) is at present the only FDA approved assay for routine detection of human papillomavirus (HPV) infections. A significant analytical inaccuracy of the hc2 near to cut-off was reported recently. To address this problem, 240 samples with repeatedly borderline/equivocal/indeterminate hc2 results (samples with repeated RLU/CO values between 0.4 and 4.0) were tested using the PGMY09/PGMY11 consensus PCR and genotyping in order to resolve their high-risk HPV status. All PGMY09/PGMY11 PCR negative samples were tested in addition using CPI/IIg consensus PCR. A false negative rate of 11.3% and false positive rate of 19.1% were recorded in the samples with repeatedly borderline hc2 results. The corresponding hc2 false reactivity rates in 95 samples selected at random which were clearly hc2 negative (samples with RLU/CO values less than 0.4) and 124 samples selected at random which were clearly hc2 positive (samples with RLU/CO values more than 4.0) were 4.2% and 5.6%, respectively. The proportion of hc2 false reactivity increased with proximity to the hc2 cut-off value. According to the results of the present study, the introduction of an hc2 grey-zone and retesting of samples with repeatedly borderline hc2 results by an alternate HPV detection method, such as the PGMY09/PGMY11 consensus PCR and genotyping, is recommended.

False-positive result of a confirmatory human immunodeficiency virus line immuno assay in an apparently healthy individual--a case report.

A case of a false-positive result of human immunodeficiency virus (HIV) confirmatory immunoblot-based assay is described. Repeatedly borderline reactive anti-HIV screening enzyme immunoassay result obtained in a local hospital resulted in directing the sample to the Slovenian HIV/AIDS Reference Laboratory. In the Reference Laboratory, both anti-HIV screening assays and confirmatory Western blot were negative, while a confirmatory test INNO-LIA HIV I/II Score (Innogenetics, Ghent, Belgium) was anti-HIV-1 positive due to sgp120 and gp41 reactivity. The results of serological testing of the second sample obtained three weeks later were completely identical, while in the third sample obtained 5 months later, seroreversion was observed. Due to a negative dynamics in anti-HIV serological profile and repeatedly negative results of the molecular tests for HIV-1 and HIV-2, HIV infection was excluded and the results of test INNO-LIA HIV I/II Score were finally interpreted as false positive.

Distribution of human papillomavirus genotypes in plucked eyebrow hairs from Slovenian males with genital warts.

In the present study, the presence and distribution of human papillomaviruses (HPVs) in the plucked eyebrow hairs obtained from 49 Slovenian male patients with genital warts were investigated. Using polymerase chain reaction (PCR) with three sets of degenerate primers targeting all known HPV genotypes, HPV DNA was found in 31 (63.3%) of 49 eyebrow hair samples. Epidermodysplasia verruciformis (EV) associated HPV specific Ma/Ha nested PCR system detected HPVs in 27 (55.1%) and CPI/CPIIs primers that amplify the majority of cutaneous/EV HPV genotypes in 20 (40.8%) of 49 samples tested. The CPI/CPIIg PCR specific for E1 open reading frame of genital HPVs showed the presence of HPV DNA in 10 (20.4%) of 49 specimens. Direct sequencing of the Ha PCR products showed the presence of three putative new HPV genotypes, named SIBX1, SIBX2 and SIBX3. Similarly, three potential new HPV genotypes, SIBX4, SIBX5 and SIBX6, were detected by sequencing CPI/CPIIs PCR products. In total, at least 24 different HPV genotypes were detected in 31 HPV DNA positive samples of plucked eyebrow hairs. The results of our study showed that the use of a combined degenerate primer PCR approach considerably improves the HPV DNA detection over individual primer sets and greatly improves the detection of different HPV genotypes in the plucked eyebrow hairs.