DIP-STR: Highly Sensitive Markers for the Analysis of Unbalanced Genomic Mixtures.

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP-STR that relies on pairing deletion-insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP-STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP-STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures

Touch DNA collection - Performance of four different swabs.

A collaborative study conducted by three police forensic units, a DNA laboratory, and a forensic academic institute was undertaken in order to compare the performance of four different swabs in controlled and quasi-operational conditions. For this purpose, a reference swab (Prionics cardboard evidence collection kit) currently used within the police forensic units and 3 challenger swabs (COPAN 4N6FLOQSwabs™ (Genetics variety), Puritan FAB-MINI-AP and Sarstedt Forensic Swab) were used for collecting DNA traces from previously used items (referred as "touch DNA" in this article) including on 60 collars, 60 screwdrivers and 60 steering wheels obtained from volunteers. For each comparison, the surface considered was divided into two equal components; one was sampled with the reference swab and the other with one of the three challenger swabs. This lead to a total of 360 samples. Conclusions were consistent within the four operational partners. From a practical point of view, the COPAN 4N6FLOQSwabs™ (Genetics variety) was judged the most convenient to use. Furthermore, it allowed the recovery of significantly more DNA from collars (0.65 vs 0.13 ng/μL) and steering wheels (2.82 vs 1.77 ng/μL), and a similar amount of DNA from screwdrivers (0.032 vs 0.026 ng/μL) compared with the Prionics reference swab. The two other challenger swabs provided results that were not significantly different from the reference swab, except for the Puritan swab, whose performance was significantly lower for steering wheels (0.37 vs 0.58 ng/μL). As part of a conservation study, 50 μL of a blood dilution (1/4 with PBS) was deposited on a total of 105 COPAN (Genetics and Crime Scene varieties), Prionics and Sarstedt swabs. They were stored within a cupboard at room temperature. The integrity of the recovered DNA was evaluated with NGM SElect™ DNA profiles after different time-spans ranging from 1 day to 12 months by comparing the height difference of the peaks occurring at the shortest and longest loci, respectively. DNA seemed to remain stable, except when using the COPAN 4N6FLOQSwabs™ treated with an antimicrobial agent (Crime scene variety), which resulted in significant DNA degradation. Following these tests, the COPAN 4N6FLOQSwabs™ (Genetics variety), a model with a desiccant, was selected for further testing in fully operational conditions

Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples.

Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family

Prevalence and diversity of Chlamydiales and other amoeba-resisting bacteria in domestic drinking water systems.

A growing number of human infections incriminate environmental bacteria that have evolved virulent mechanisms to resist amoebae and use them as a replicative niche. These bacteria are designated amoeba-resisting bacteria (ARB). Despite the isolation of these ARB in various human clinical samples, the possible source of infection remains undetermined in most cases. However, it is known that the ARB Legionella pneumophila, for instance, causes a respiratory infection in susceptible hosts after inhalation of contaminated water aerosols from various sources. The Chlamydiales order contains many ARB, such as Parachlamydia acanthamoebae or Simkania negevensis, previously implicated in human respiratory infections with no identified contamination sources. We thus investigated whether domestic water systems are a potential source of transmission of these Chlamydiales to humans by using amoebal culture and molecular methods. Other important ARB such as mycobacteria and Legionella were also investigated, as were their possible amoebal hosts. This work reports for the first time a very high prevalence and diversity of Chlamydiales in drinking water, being detected in 35 (72.9%) of 48 investigated domestic water systems, with members of the Parachlamydiaceae family being dominantly detected. Furthermore, various Legionella and mycobacteria species were also recovered, some species of which are known to be causal agents of human infections

Undressing of Waddlia chondrophila to enrich its outer membrane proteins to develop a new species-specific ELISA.

Waddlia chondrophila, an obligate intracellular bacterium of the Chlamydiales order, is considered as an agent of bovine abortion and a likely cause of miscarriage in humans. Its role in respiratory diseases was questioned after the detection of its DNA in clinical samples taken from patients suffering from pneumonia or bronchiolitis. To better define the role of Waddlia in both miscarriage and pneumonia, a tool allowing large-scale serological investigations of Waddlia seropositivity is needed. Therefore, enriched outer membrane proteins of W. chondrophila were used as antigens to develop a specific ELISA. After thorough analytical optimization, the ELISA was validated by comparison with micro-immunofluorescence and it showed a sensitivity above 85% with 100% specificity. The ELISA was subsequently applied to human sera to specify the role of W. chondrophila in pneumonia. Overall, 3.6% of children showed antibody reactivity against W. chondrophila but no significant difference was observed between children with and without pneumonia. Proteomic analyses were then performed using mass spectrometry, highlighting members of the outer membrane protein family as the dominant proteins. The major Waddlia putative immunogenic proteins were identified by immunoblot using positive and negative human sera. The new ELISA represents an efficient tool with high throughput applications. Although no association with pneumonia and Waddlia seropositivity was observed, this ELISA could be used to specify the role of W. chondrophila in miscarriage and in other diseases

The Accuracy of Automatic Photogrammetric Techniques on Ultra-light UAV Imagery

This paper presents an affordable, fully automated and accurate mapping solutions based on ultra-light UAV imagery. Several datasets are analysed and their accuracy is estimated. We show that the accuracy highly depends on the ground resolution (flying height) of the input imagery. When chosen appropriately this mapping solution can compete with traditional mapping solutions that capture fewer high-resolution images from airplanes and that rely on highly accurate orientation and positioning sensors on board. Due to the careful integration with recent computer vision techniques, the post processing is robust and fully automatic and can deal with inaccurate position and orientation information which are typically problematic with traditional techniques

Further investigation of confirmed urinary tract infection (UTI) in children under five years: a systematic review.

Background: Further investigation of confirmed UTI in children aims to prevent renal scarring and future complications. Methods: We conducted a systematic review to determine the most effective approach to the further investigation of confirmed urinary tract infection (UTI) in children under five years of age. Results: 73 studies were included. Many studies had methodological limitations or were poorly reported. Effectiveness of further investigations: One study found that routine imaging did not lead to a reduction in recurrent UTIs or renal scarring. Diagnostic accuracy: The studies do not support the use of less invasive tests such as ultrasound as an alternative to renal scintigraphy, either to rule out infection of the upper urinary tract (LR- = 0.57, 95%CI: 0.47, 0.68) and thus to exclude patients from further investigation or to detect renal scarring (LR+ = 3.5, 95% CI: 2.5, 4.8). None of the tests investigated can accurately predict the development of renal scarring. The available evidence supports the consideration of contrast-enhanced ultrasound techniques for detecting vesico-ureteric reflux (VUR), as an alternative to micturating cystourethrography (MCUG) (LR+ = 14.1, 95% CI: 9.5, 20.8; LR- = 0.20, 95%CI: 0.13, 0.29); these techniques have the advantage of not requiring exposure to ionising radiation. Conclusion: There is no evidence to support the clinical effectiveness of routine investigation of children with confirmed UTI. Primary research on the effectiveness, in terms of improved patient outcome, of testing at all stages in the investigation of confirmed urinary tract infection is urgently required

How does study quality affect the results of a diagnostic meta-analysis?

Background: The use of systematic literature review to inform evidence based practice in diagnostics is rapidly expanding. Although the primary diagnostic literature is extensive, studies are often of low methodological quality or poorly reported. There has been no rigorously evaluated, evidence based tool to assess the methodological quality of diagnostic studies. The primary objective of this study was to determine the extent to which variations in the quality of primary studies impact the results of a diagnostic meta-analysis and whether this differs with diagnostic test type. A secondary objective was to contribute to the evaluation of QUADAS, an evidence-based tool for the assessment of quality in diagnostic accuracy studies. Methods: This study was conducted as part of large systematic review of tests used in the diagnosis and further investigation of urinary tract infection (UTI) in children. All studies included in this review were assessed using QUADAS, an evidence-based tool for the assessment of quality in systematic reviews of diagnostic accuracy studies. The impact of individual components of QUADAS on a summary measure of diagnostic accuracy was investigated using regression analysis. The review divided the diagnosis and further investigation of UTI into the following three clinical stages: diagnosis of UTI, localisation of infection, and further investigation of the UTI. Each stage used different types of diagnostic test, which were considered to involve different quality concerns. Results: Many of the studies included in our review were poorly reported. The proportion of QUADAS items fulfilled was similar for studies in different sections of the review. However, as might be expected, the individual items fulfilled differed between the three clinical stages. Regression analysis found that different items showed a strong association with test performance for the different tests evaluated. These differences were observed both within and between the three clinical stages assessed by the review. The results of regression analyses were also affected by whether or not a weighting (by sample size) was applied. Our analysis was severely limited by the completeness of reporting and the differences between the index tests evaluated and the reference standards used to confirm diagnoses in the primary studies. Few tests were evaluated by sufficient studies to allow meaningful use of meta-analytic pooling and investigation of heterogeneity. This meant that further analysis to investigate heterogeneity could only be undertaken using a subset of studies, and that the findings are open to various interpretations. Conclusion: Further work is needed to investigate the influence of methodological quality on the results of diagnostic meta-analyses. Large data sets of well-reported primary studies are needed to address this question. Without significant improvements in the completeness of reporting of primary studies, progress in this area will be limited

Bacterial Signatures of Paediatric Respiratory Disease : An Individual Participant Data Meta-Analysis

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching patterns of bacterial association with acute and chronic paediatric respiratory disease in an individual participant data (IPD) meta-analysis of 16S rRNA gene sequences from published respiratory microbiota studies.Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha- and beta-diversity approaches, machine learning, and biomarker analyses.Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively.Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.Peer reviewe