How to evaluate PCR assays for the detection of low‐level DNA

High sensitivity of PCR‐based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures . The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real‐time PCR‐based detection of low‐level DNA. The model was tested against experimental data of diluted cell‐free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell‐free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real‐time PCR‐based detection of low‐level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated.     

Satisfaction levels of paediatric surgeons in Canada

BACKGROUND:

Surgeons’ satisfaction levels may affect patient care and the stability of the surgical workforce.

METHODS:

A detailed Internet-based satisfaction survey was administered to paediatric surgeons in Canada in 2005 and 2012. Satisfaction was rated 1 (most) to 5 (least), in five areas: quality of life, financial compensation, work environment, academics and patient care.

RESULTS:

Responses were received from 21 surgeons in 2005 and 61 in 2012, representing 43% and 98% of practicing paediatric surgeons in Canada, respectively. Satisfaction levels were generally moderate to high in most areas during both years. In academics, surgeons were more satisfied in 2012 with the amount of teaching they provided (1.8 versus 2.2; P=0.02), and clinical research they performed (2.5 versus 3.0; P=0.04). In patient care, there was higher satisfaction with the ability to provide elective services without impediment (2.5 versus 3.0; P=0.02). Over the seven-year period, surgeons increasingly preferred the Canadian health care system over that of the United States (1.7 versus 2.2; P=0.02). In the 2012 survey, no differences in levels of satisfaction were found between male and female surgeons.

CONCLUSIONS:

During the recent seven-year period, satisfaction levels of paediatric surgeons in Canada have been stable with regard to quality of life, compensation and work environment, and improving in areas of academics and patient care. Male and female surgeons are equally satisfied. The Canadian health care system is preferred over that of the United States.     

Prof. Dr. med. Dr. med. dent. Gottfried P. F. Schmuth zum 65. Geburtstag

Ohne Zusammenfassung     

Hemorrhagic fever with renal syndrome in the Pomurje region of Slovenia – An 18-year survey

OBJECTIVE: To determine the etiology of hemorrhagic fever with renal syndrome (HFRS) in the north-eastern part of Slovenia (Pomurje region) together with demographic, epidemiological and clinical data on 25 patients from this region who were diagnosed and treated at the General Hospital in Murska Sobota between 1986 and 2003. METHODS: Medical records of patients with a discharge diagnosis of HFRS who were either hospitalized or referred to an infectiologist as outpatients were included in this retrospective study. Data on demographic characteristics, clinical manifestations and laboratory parameters were collected from the patients' records. In addition, all available follow-up records were examined and information on general health, blood pressure, basic blood and biochemical examination and urine analysis was collected. RESULTS: Infection with Puumala virus (PUUV) was indicated in 23 patients and Dobrava virus (DOBV) infection in two patients. The median age of patients was 39 years; 19 were male. The patients primarily had outdoor occupations. Most of the HFRS cases occurred between May and August. The most common findings were fever, vomiting, headache, myalgia, chills, cough, back and abdominal pain, and blurred vision. The most prominent laboratory abnormalities were elevated erythrocyte sedimentation rate and C-reactive protein concentration, thrombocytopenia, and leucocytosis with neutrophilia. The signs of renal dysfunction were observed in 24 of the 25 patients. Oliguric renal failure was seen in 13 of 23 (57%) PUUV-infected patients. Six of 23 (26%) patients infected with PUUV and one of two (50%) patients from the DOBV group had hypotension or developed signs of shock. Seven out of 15 (47%) patients had elevated protein concentration in cerebrospinal fluid (CSF). Sinus bradycardia was documented in 7 of 17 (41%) patients with PUUV infection. CONCLUSIONS: HFRS is endemic in the north-eastern part of Slovenia; PUUV and DOBV infections coexist, with PUUV being the main causative agent of HFRS. Demographic, clinical and laboratory findings in our patients with HFRS caused by PUUV were mostly in accordance with those published previously, but the ratio of patients with sinus bradycardia, oliguric renal failure and mildly elevated CSF protein concentration was rather high.     

Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA(2) (sPLA(2)-X), the sPLA(2) with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA(2)-X(-/-) mice, compared with those of sPLA(2)-X(+/+) littermates, had significant reduction in ovalbumin-induced infiltration by CD4(+) and CD8(+) T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA(2)-X as a novel therapeutic target for asthma.     

ELECTRON MICROSCOPIC STUDIES ON STREPTOCOCCI : I. M ANTIGEN

The presence of M antigens on group A streptococci is associated with hairlike fimbriae that cover the surface of the streptococcal cell wall and are demonstrable by electron microscopy. These fimbriae also may be associated with R antigen. Like M protein, the surface fimbriae are destroyed by trypsin treatment and reappear when "trypsinized" streptococci are reincubated in fresh, trypsin-free broth. Ferritin-conjugated, type-specific antibodies localize on homologous M+ cells in a pattern suggestive of several M antigenic sites along the length of individual surface fimbria. The M-associated fimbriae remain on the residual cell wall after removal of the bulk of group-specific polysaccharide through nitrous acid extraction. This suggests attachment of the fimbriae to the mucopeptide and minor polysaccharide components remaining in the nitrous acid-extracted wall. The pattern of localization of ferritin-conjugated antibodies on homologous streptococci before and after trypsin exposure and upon reincubation of the trypsinized cells in fresh medium suggests the following hypothesis: M antigen is secreted by the cell, is partially excreted through the otherwise intact cell wall, and is bound by the wall so that M protein occupies a peripheral, exposed position on the surfaces of the streptococcal cell wall.     

Prostate‐specific antigen testing in older men in the USA: data from the behavioral risk factor surveillance system

Study Type – Diagnosis (cohort) Level of Evidence 2a What's known on the subject? and What does the study add? Previous studies from the USA reported that, in recent years, prostate cancer screening with PSA was very common among the elderly men, including those whose life expectancy was substantially limited by advanced age and known comorbidities. In 2008, the US Preventive Services Task Force (USPSTF) recommended against PSA screening after age 75. The purpose of this study was to examine the impact of the USPSTF 2008 recommendation on the frequency of PSA testing among elderly men in the USA. Our findings suggest that the USPSTF recommendation had no major impact on clinical practice.

OBJECTIVE

• ? To examine the frequency of PSA testing in men aged ≥75 years before and after the 2008 US Preventive Services Task Force (USPSTF) recommendation to stop prostate‐specific antigen (PSA) screening at age 75.

MATERIALS AND METHODS

• ? Data were obtained from the Behavioral Risk Factor Surveillance System (BRFSS) surveys completed in 2006, 2008 and 2010.
• ? Men aged ≥ 76 years at the time of survey and without a prostate cancer diagnosis were included in the study.
• ? The percentage of men who had a PSA test in the year before the survey was computed separately for survey years 2006, 2008 and 2010.

RESULTS

• ? The estimated percentages of men with a PSA test in the year before the survey were 60% (95% CI: 58–62%), based on 9033 respondents interviewed in 2006, 63% (95% CI: 62–65%), based on 12 063 respondents interviewed in 2008, and 60% (95% CI: 59–61%), based on 14 782 respondents interviewed in 2010.

CONCLUSION

• ? No substantial reduction in the frequency of PSA testing was observed in the BRFSS 2010 survey data compared with the earlier years, suggesting that the USPSTF 2008 recommendation had no major impact on the frequency of PSA testing in older men in the USA.