Federica Valeriani

Molecular identification of vaginal fluid by microbial signature.

The discrimination of body fluids in forensic examinations can play an important role in crime scene reconstruction. Conventional methods rely on the detection of antigens or enzymatic activity, limiting detection sensitivity and specificity, particularly on old forensic samples. Methods based on human RNA analysis are not easily applicable to samples exposed to harsh and degrading environments. An alternative approach based on the identification of prokaryotic genomes was developed. Specific bacterial communities are characteristic typical of different human non-sterile body fluids: the molecular characterization of a microbial signature, and not the typing of single bacterial species, can effectively lead to univocal identification of these fluids. A multiplex real time PCR assay was developed using oligonucleotide mixtures targeting genomes specific for a selected group of bacteria. Microflora DNA (mfDNA) was extracted from vaginal, oral and fecal clinical swabs. In addition forensic samples were processed. Vaginal samples showed a strong specific signal for bacteria of the female genital tract. Oral samples clearly showed signal for bacteria present in saliva, and in fecal samples the main signal was from Enterococcaceae. Vaginal casework samples showed results comparable to freshly collected ones; moreover the DNA extracted was successfully used for STR typing. Also mixtures of body fluids were analyzed, providing a microbiological signature compatible with the presence of microbes of oral, fecal and vaginal origin. The presented method can be useful in identifying biological fluids, and it is based on DNA technologies already available in forensic laboratories and feasible for further high throughput automation.

The environmental biological signature: NGS profiling for forensic comparison of soils

The identification of the source of a specific soil sample is a crucial step in forensic investigations. Rapid advances in next generation sequencing (NGS) technology and the strong reduction of the cost of sequencing have recently opened new perspectives. In the present work a metabarcoding approach has been successfully applied to forensic and environmental soil samples, allowing the accurate and sensitive analysis of microflora (mfDNA), plants, metazoa, and protozoa DNA. The identification of the biological component by DNA metabarcoding is a strong element for the discrimination of samples geologically very similar but coming for distinct environments.

The SNPs in the human genetic blueprint era

The analysis of human genetic variability can lead to the comprehension of medical issues and to the development of personalized therapeutic protocols. Single nucleotide polymorphisms, are the most common type of human genetic variation and have been associated to disease development and phenotype forecasting. The recent technologies for DNA sequencing and bioinformatic analysis are now giving the opportunity to develop new diagnostic and prevention approaches also through health promotion protocols. The genetic data management is at the same time underlining technical limitations and old ethical issues.

Molecular enrichment for detection of S. aureus in recreational waters

The identification of rapid methods for the control of recreational water and of aquatic environments with similar characteristics is necessary to provide adequate levels of health safety for users. Molecular techniques have been proposed in recent years as a viable alternative to traditional microbiological methods, as they offer various advantages and are less time consuming than traditional tests. An innovative protocol based on molecular enrichment that allows the identification of low concentrations of Staphylococcus aureus in recreational water has been developed. The method is based on the specific amplification of prokaryotic genomic DNA by the usage of universal primers for 23S rDNA; subsequently, a second amplification step is performed with specific real-time polymerase chain reaction (PCR) primers and probe. This approach shows sensitivity levels similar to those observed with microbiological tests, with the additional benefits of the specificity typical of nucleic acids techniques. This methodology is easily applicable also to other microbiological parameters, representing an important milestone in hygiene monitoring by the detection of specific pollution indicators.

Evaluation of Legionella Air Contamination in Healthcare Facilities by Different Sampling Methods: An Italian Multicenter Study

Healthcare facilities (HF) represent an at-risk environment for legionellosis transmission occurring after inhalation of contaminated aerosols. In general, the control of water is preferred to that of air because, to date, there are no standardized sampling protocols. Legionella air contamination was investigated in the bathrooms of 11 HF by active sampling (Surface Air System and Coriolis®μ) and passive sampling using settling plates. During the 8-hour sampling, hot tap water was sampled three times. All air samples were evaluated using culture-based methods, whereas liquid samples collected using the Coriolis®μ were also analyzed by real-time PCR. Legionella presence in the air and water was then compared by sequence-based typing (SBT) methods. Air contamination was found in four HF (36.4%) by at least one of the culturable methods. The culturable investigation by Coriolis®μ did not yield Legionella in any enrolled HF. However, molecular investigation using Coriolis®μ resulted in eight HF testing positive for Legionella in the air. Comparison of Legionella air and water contamination indicated that Legionella water concentration could be predictive of its presence in the air. Furthermore, a molecular study of 12 L. pneumophila strains confirmed a match between the Legionella strains from air and water samples by SBT for three out of four HF that tested positive for Legionella by at least one of the culturable methods. Overall, our study shows that Legionella air detection cannot replace water sampling because the absence of microorganisms from the air does not necessarily represent their absence from water; nevertheless, air sampling may provide useful information for risk assessment. The liquid impingement technique appears to have the greatest capacity for collecting airborne Legionella if combined with molecular investigations.

Swimming attendance during childhood and development of asthma: a meta-analysis

The association between asthma and swimming pool attendance has not been demonstrated and currently there are conflicting results. In order to clarify the association between asthma diagnosis in children and swimming pool attendance, and to assess the consistency of the available epidemiological studies, we completed a literature analysis on the relationship between the exposure to disinfection by‐products in indoor swimming pools during childhood and asthma diagnosis.

Informativeness of NGS Analysis for Vaginal Fluid Identification

The identification of vaginal fluids in forensic examinations plays an important role in crime scene reconstruction. Molecular detection of vaginal bacterial communities can lead to the correct discrimination of body fluids. These kinds of studies can be performed through multiplex real‐time PCR using primers for a specific selection of bacteria. The availability of next‐generation sequencing (NGS) protocols provided for the extension of the analysis to evaluate the prokaryotes present in specimens. In this study, DNA was extracted from 18 samples (vaginal, oral, fecal, yoghurt) and analyzed by real‐time PCR and NGS. The comparison between the two approaches has demonstrated that the information developed through NGS can augment the more conventional real‐time PCR detection of a few key bacterial species to provide a more probative result and the correct identification of vaginal fluid from samples that are more forensically challenged.

Pseudomonas aeruginosa in Swimming Pool Water: Evidences and Perspectives for a New Control Strategy.

Pseudomonas aeruginosa is frequently isolated in swimming pool settings. Nine recreational and rehabilitative swimming pools were monitored according to the local legislation. The presence of P. aeruginosa was correlated to chlorine concentration. The ability of the isolates to form a biofilm on plastic materials was also investigated. In 59.5% of the samples, microbial contamination exceeded the threshold values. P. aeruginosa was isolated in 50.8% of these samples. The presence of P. aeruginosa was not correlated with free or total chlorine amount (R2 < 0.1). All the isolates were moderate- to strong-forming biofilm (Optical Density O.D.570 range 0.7–1.2). To control biofilm formation and P. aeruginosa colonization, Quantum FreeBioEnergy© (QFBE, FreeBioEnergy, Brisighella, Italy), has been applied with encouraging preliminary results. It is a new, promising control strategy based on the change of an electromagnetic field which is responsible for the proliferation of some microorganisms involved in biofilm formation, such as P. aeruginosa.

Family-based social determinants and child health: Cross-sectional study

The aim of this study was to evaluate the association between urbanization of residential area, parent education level, employment status, overweight/obesity, physical activity, and exposure to environmental tobacco smoke (ETS), in a sample of healthy Italian school‐age children (5–11 years).

River water quality assessment: comparison between old and new indices in a real scenario from Italy

ABSTRACT According to the European Water Framework Directive, Italy introduced two aggregate indices for evaluating the ecological status of rivers, called respectively LIM (Pollution Level of the Macro-descriptors) from 2000 to 2008, and LIMeco from 2010 onwards. The LIM index considered the % of Dissolved Oxygen (DO%), ammonium nitrogen (NH4-N), nitrate nitrogen (NO3-N), total phosphorus (P), Biochemical Oxygen Demand (BOD5), Chemical Oxygen Demand (COD), and Escherichia (E.) coli, while LIMeco considered only DO%, NH4-N, NO3-N, and total P. The aim of this study was to evaluate differences between LIM and LIMeco for defining quality status of freshwaters. For this purpose, data obtained during an annual monitoring campaign of four main rivers of southern Italy were used to calculate both indices. LIMeco seems to return the quality status of a river better than LIM when the state of the water body is more compromised, while there is a substantial consistency when the river quality is at a medium/high level. Indeed, LIM intended to give a ‘traditional’ classification of freshwaters, integrating environmental and ecological descriptors, while LIMeco focuses on the trophic status of freshwaters, considering only nutrients and oxygen, essential for the aquatic biological communities and it does not consider organic load and microbiological contamination.