Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay

Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).     

End-of-life expectations and experiences among nursing home patients and their relatives—A synthesis of qualitative studies

Objective

Synthesize research about patients’ and relatives’ expectations and experiences on how doctors can improve end-of-life care in nursing homes.

Methods

We systematically searched qualitative studies in English in seven databases (Medline, Embase, PsycINFO, CINAHL, Ageline, Cochrane Systematic Reviews and Cochrane Trials). We included 14 publications in the analysis with meta-ethnography.

Results

Patients and families emphasized the importance of health personnel anticipating illness trajectories and recognizing the information and palliation needed. Family members who became proxy decision-makers reported uncertainty and distress when guidance from health personnel was lacking. They worried about staff shortage and emphasized doctor availability. Relatives and health personnel seldom recognized patients’ ability to consent, and patients’ preferences were not always recognized.

Conclusion

Nursing home patients and their relatives wanted doctors more involved in end-of-life care. They expected doctors to acknowledge their preferences and provide guidance and symptom relief.

Practice implications

High-quality end-of-life care in nursing homes relies on organization, funding and skilled staff, including available doctors who are able to recognize illness trajectories and perform individualized Advance Care Planning.     

Genetic and epigenetic alterations of 9p21 gene products in benign and malignant tumors of the head and neck

The multistep process of tumorigenesis has not been decoded to date, although numerous investigations into probable molecular changes have meanwhile been conducted. However, not only DNA changes or loss of alleles cause deregulation of gene function, but also epigenetic alterations (e.g. methylation) result in functional loss. The INK4a-ARF (CDKN2A) locus, located on chromosome 9p21, encodes two functionally distinct tumor suppressor genes, p14ARF and p16INK4a, which play active roles in the p53 and Rb tumor suppressive pathways. We therefore examined not only p16 and p14 proteins, but also alterations of the INK4a-ARF locus, including methylation and loss of heterozygosity in benign and malignant tumors of the head and neck (squamous cell carcinomas and pleomorphic adenomas). In benign pleomorphic adenomas, methylation of p14ARF was found in 1 out of 42 (2%) cases, whereas alterations of p16INK4a occurred in 12/42 (29%) pleomorphic adenomas. In HNSCC, methylation of p16INK4a occurred in 16 out of 50 (32%) carcinomas. P14ARF was found to be methylated in 8 out of 50 cases (16%). Our results demonstrate that alterations of the INK4a-ARF locus are frequent and important events not only in the carcinogenesis of malignant, but also in benign tumors.     

Semi-conducting properties of titanium dioxide surfaces on titanium implants

The properties of the TiO₂ layer on titanium implant surfaces are decisive for good contact with the surrounding tissue. The oxide properties can be deliberately changed by for example chemical etching, ion incorporation or anodisation. In the present study impedance spectroscopy was used to study the semi-conducting properties of the naturally formed oxide for different pre-treatment of the surface. A turned surface was used as a reference and both physical (blasting) and chemical (hydrofluoric acid etching) treatments were investigated. Blasting of a titanium sample introduces defects in the metal surface and the study clearly shows that also the oxide layer contains defects leading to a higher number of charge carriers (increased conductivity) compared with the oxide on the turned surface. The hydrofluoric acid etching of the blasted surface results in an oxide film with even higher conductivity. Indication of the defect oxide structure for fluoride treated samples was also seen when analysing the TiO⁺/Ti⁺ ratio from ToF-SIMS data. The lowest value of this ratio was obtained for the HF etched sample, indicating a less stoichiometric oxide compared to the other surfaces. This is a result of incorporation of fluoride ions in the oxide, as proven by adsorption studies on a TiO₂ suspension. The results were treated in the context of surface complexation and two surface complexes were identified. Our results are discussed in relation to pull-out data on rabbit. The pull-out forces depend primarily on surface roughness but the contribution from the hydrofluoric acid etching might be explained by fluoride ion incorporation and the resulting increase in oxide conductivity.     

Four and one-half-year follow-up of the effectiveness of diphtheria-tetanus toxoids-acellular pertussis/Haemophilus influenzae type b and diphtheria-tetanus toxoids-acellular pertussis-inactivated poliovirus/H. influenzae type b combination vaccines in Germany

BACKGROUND: Recently an increase in the number of invasive Haemophilus influenzae type b (Hib) cases was observed in the United Kingdom, which coincided with a temporary change from diphtheria-tetanus toxoids-wild-type pertussis to diphtheria-tetanus toxoids-acellular pertussis (DTaP) Hib vaccines. A study in Germany based on approximately 2 years of follow-up, estimated vaccine effectiveness (VE) of DTaP/Hib and DTaP-inactivated poliovirus/Hib combination vaccines against invasive Hib disease to be high. OBJECTIVES: To assess VE of DTaP-containing Hib vaccines against Hib in Germany with the use of extended follow-up of case surveillance and vaccine uptake. SUBJECTS AND METHODS: Cases with confirmed systemic Hib infections in children born between June 1, 1996 and December 31, 1998 were ascertained by a nationwide active surveillance system from January 1998 through June 2002. A representative subcohort of 667 children born in the same time frame was randomly sampled in a nationwide vaccine coverage survey. VE was determined with a case-cohort approach of Cox regression with time-dependent covariates. RESULTS: Thirty-six cases of Hib disease were reported. Of these, 10 were vaccinated with DTaP-containing Hib vaccines only and 20 were not vaccinated. Of the 10 vaccinated cases, 4 had received an incomplete primary series (1-2 doses), and 6 had received the full primary series (3 doses), 3 of whom also received the booster dose. VE of combination vaccines against invasive Hib infection was 89.6% [95% confidence interval (CI), 67.0-96.7] for an incomplete primary series, 96.7% (95% CI 87.7-99.1) for a full primary series and 98.5% (95% CI 94.5-99.6) for a booster dose (irrespective of priming). CONCLUSION: Hib combination vaccines containing acellular pertussis antigens continue to be highly effective in Germany.     

Post-cesarean surgical site infections according to CDC standards: rates and risk factors. A prospective cohort study

OBJECTIVES: The aim of this study was to document the true incidence of post-cesarean surgical site infections (SSI), according to the definition of the US Centers for Disease Control and Prevention (CDC), and to identify independent risk factors for infection. DESIGN: Prospective population-based cohort study in Norway. Setting. Sykehuset Asker og Baerum HF, a secondary community hospital, associated with the University of Oslo (UiO), Norway, accounting for 2,000 deliveries per year. Participants. All cesarean deliveries during a 12-month period from September 2003. Main outcome measures. Rate and risk factors for SSI. RESULTS: The total rate of SSI was 8.9%, with an observation period of 30 days post-operatively, compared to 1.8% registered at hospital discharge. The total response rate was 100%. There was no significant difference in SSI rate in elective or emergency cesarean section (CS), respectively. All SSI were superficial. We found 2 significant independent risk factors: operating time > or =38 min and body mass index (BMI) >30. CONCLUSION: The rate of SSI is underestimated if the observation time is limited to the hospital stay. Operating time exceeding 38 min substantially increases the risk of SSI. The finding of no significant difference in SSI rate between elective and emergency CS should lead to a different approach concerning the use of antibiotics: subgroup at risk (operating time > or =38 min and BMI >30) may benefit from antibiotics in relation to the operation, whether the CS is an emergency or elective operation.     

Large measles outbreak at a German public school, 2006

BACKGROUND: In 2006, a large measles outbreak (n = 614) occurred in Duisburg city, Germany, with 54% of cases aged >9 years. An investigation was launched to determine reasons for the resurgence of measles, assess vaccination coverage and vaccine effectiveness (VE). METHODS: A retrospective cohort-study was undertaken at a Duisburg public school affected early in the outbreak. We distributed questionnaires to all 1250 students aged 10-21 years and abstracted vaccination records. Cases were identified according to a standard clinical case definition. RESULTS: Questionnaires were returned by 1098 (88%) students. Vaccination records were abstracted from 859 students, of whom 820 (95.4%) had received at least one, 605 (70.4%) 2, and 39 (4.5%) no dose(s) of measles-containing vaccine (MCV). Coverage with 2 doses was higher in younger students. We identified 53 cases (attack rate = 5%). Measles-virus sequencing revealed genotype D6. After excluding students vaccinated in 2006 and those with a history of measles, the attack rate was 53% in unvaccinated students, 1.0% in students with one, and 0.4% in those with 2 MCV-doses. VE was 98.1% (95% CI: 92-100%) in students with one and 99.4% (95% CI: 97-100%) with 2 MCV-doses. Based on observed attack rates in vaccinated and unvaccinated students with vaccination records and in students without vaccination records, one-dose-coverage among all participating students was estimated at 91%. CONCLUSIONS: VE was high. Vaccination coverage was, however, insufficient to prevent the outbreak. Immunization gaps were found especially in older students. To prevent further outbreaks and to achieve the goal of measles elimination in Germany, vaccination coverage must be increased.     

Detection of large deletions in the LDL receptor gene with quantitative PCR methods

Background  

Familial Hypercholesterolemia (FH) is a common genetic disease and at the molecular level most often due to mutations in the LDL receptor gene. In genetically heterogeneous populations, major structural rearrangements account for about 5% of patients with LDL receptor gene mutations.     

The prospects of single-cell analysis in autoimmunity

In the last decade, there has been a tremendous development of technologies focused on analysing various molecular attributes in single cells, with an ever-increasing number of parameters becoming available at the DNA, RNA and protein levels. Much of this progress has involved cells in suspension, but also in situ analysis of tissues has taken great leaps. Paralleling the development in the laboratory, and because of increasing complexity, the analysis of single-cell data is also constantly being updated with new algorithms and analysis platforms. Our immune system shares this complexity, and immunologists have therefore been in the forefront of this technological development. These technologies clearly open new avenues for immunology research, maybe particularly within autoimmunity where the interaction between the faulty immune system and the thymus or the target organ is important. However, the technologies currently available can seem overwhelming and daunting. The aim of this review is to remedy this by giving a balanced overview of the prospects of using single-cell analysis in basal and clinical autoimmunity research, with an emphasis on endocrine autoimmunity.