Interlaboratory reproducibility of a single-locus sequence-based method for strain typing of Aspergillus fumigatus.

Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

Molecular typing of Aspergillus species.

Aspergillus species are widely distributed fungi that release large amounts of airborne conidia, which are dispersed in the environment. Several Aspergillus species have been described as human pathogens. Molecular techniques have been developed to investigate the epidemiological relation between environmental and clinical isolates. Several typing methods have been described for Aspergillus species, most of them with reference to Aspergillus fumigatus. Here, we summarise all the different available molecular typing techniques for Aspergillus. The performance of these techniques is evaluated with respect to their practical feasibility, and their interpretation and discriminatory power assessed. For A. fumigatus isolates, a large extent of genetic variability is demonstrated and therefore fingerprinting techniques with high discriminatory power and high reproducibility are required for this species. Afut1-restriction fragment length polymorphism and microsatellite typing showed the highest discriminatory power. In addition, the microsatellites show excellent reproducibility. Other typing techniques are still useful for smaller epidemiological problems and for less well-equipped laboratories.

Baseline human papillomavirus status of women with abnormal smears in cervical screening: a 5-year follow-up study in The Netherlands.

OBJECTIVE: To determine in a screening population the human papillomavirus (HPV) status in those with cytological abnormalities and to evaluate the presence of high-risk (HR) HPV with a minimum of 5-year follow up. DESIGN: Retrospective examination of HPV status on prospectively collected and cytologically screened cervical smears. SETTING: Canisius-Wilhelmina Hospital in Nijmegen, The Netherlands. POPULATION: Three hundred and fifty-seven women aged 30-60 years, from the population screened. METHODS: Three hundred and fifty-seven women with borderline or higher cytological abnormalities were retrospectively examined for HPV with DNA microarray typing. Follow up was through the nationwide Dutch Pathology database (PALGA). MAIN OUTCOME MEASURES: For the cytological abnormalities, the CISOE-A classification was used. HPV was scored as negative or positive. In case of positive HPV polymerase chain reaction, the HPV genotype was determined. The occurrence of cervical intraepithelial neoplasia lesions of grade 3 or higher was considered as endpoint for follow up. RESULTS: The majority of the women with borderline cytology in this study were HPV negative (87%). Among the HPV-positive women in borderline cytology group, 74% had HR-HPV or probable high-risk types. The overall percentage of HR-HPV types increased with progressive cytological abnormalities. The cytological classifications of borderline dyskaryosis and moderate dyskaryosis contain all types of HPVs, e.g. low risk, HR and unknown risk. The samples with severe dyskaryosis or higher contain only HR types. The negative predictive value for HR-HPV typing in the group with borderline cytological abnormalities is more than 99%. CONCLUSIONS: In cervical screening with an interval of 5 years, HPV can be reliably used as triage point in cases of borderline cytological abnormalities.

Prospects of labour migration pressure in Algeria, Morocco, Tunisia and Turkey.

Gaining control over refugee flows and undocumented migrants currently dominate the media and political arenas in Europe. Underlying driving and enduring forces, such as employment-related migration pressure, tend to be relegated to the background. In this article, we explore migration pressure prospects up to 2035 in four countries with a tradition of emigration to Europe: Algeria, Morocco, Tunisia and Turkey. More specifically, we first derive a simple decomposition model based on the relationship between working-age population (WAP) growth and growth of gross domestic production (GDP) and worker productivity (GDP/W). From this model, we derive an indicator of migration pressure: size of the non-employed population in a country. This model is then used as framework for deriving storylines for three different scenarios of economic and demographic change up to 2035. Subsequently, storylines are operationalized, leading to scenario estimates of migration pressure up to 2035. The implications of the results are then discussed. Time series of macro-level economic and demographic data are used to underpin scenario assumptions. Scenario results suggest that in all countries employment ratios are expected to increase, but only in Tunisia is the size of the non-employed population-our indicator of migration pressure-expected to decline, irrespective of the scenario. Depending on the scenario, migration pressure remains high in Turkey and Morocco and may even become somewhat higher. The general conclusion is that in the long term, after 2035, labour migration pressure can be expected to decrease because the growth and size of the working-age population is decreasing while employment ratios are rising.

Interlaboratory reproducibility of a microsatellite-based typing assay for Aspergillus fumigatus through the use of allelic ladders: proof of concept.

An interlaboratory study was performed with the aim of investigating the reproducibility of a multiplex microbial microsatellite-based typing assay for Aspergillus fumigatus in different settings using a variety of experimental and analytical conditions and with teams having variable prior microsatellite typing experience. In order to circumvent problems with exchange of sizing data, allelic ladders are introduced as a straightforward and universally applicable concept for standardization of such typing assays. Allelic ladders consist of mixtures of well-characterized reference fragments to act as reference points for the position in an electrophoretic trace of fragments with established repeat numbers. Five laboratories independently analysed six microsatellite markers in 18 samples that were provided either as DNA or as A. fumigatus conidia. Allelic data were reported as repeat numbers and as sizes in nucleotides. Without the use of allelic ladders, size differences of up to 6.7 nucleotides were observed, resulting in interpretation errors of up to two repeat units. Difficulties in interpretation were related to non-specific amplification products (which were resolved with explanation) and bleed-through of the different fluorescent labels. In contrast, after resolution of technical or interpretive problems, standardization of sizing data by using allelic ladders enabled all participants to produce identical typing data. The use of allelic ladders as a routine part of molecular typing using microsatellite markers provides robust results suitable for interlaboratory comparisons and for deposition in a global typing database.