Vincenzo Romano Spica

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.

Microarrays and high-throughput transcriptomic analysis in species with incomplete availability of genomic sequences

Microarrays produce a measurement of gene expression based on the relative measures of dye intensities that correspond to the amount of target RNA. This technology is fast developing and its application is expanding from Homo sapiens to a wide number of species, where enough information on sequences and annotations exist. Anyway, the number of species for which a dedicated platform exists is not high. The use of heterologous array hybridization, screening for gene expression in one species using an array developed for another one, is still quite frequent, even though cross-species microarray hybridization has raised many arguments. Some methods which are high throughput and do not rely on knowledge of the DNA/RNA sequence exist, namely serial analysis of gene expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and deep sequencing of full transcriptome. Although very powerful, particularly the latter, they are still quite costly and cumbersome methods. In some species where genome sequences are largely unknown, several anonymous sequences are deposited in gene banks as a result of Expressed Sequence Tags (ESTs) sequencing projects. The ESTs databases represent a valuable knowledge that can be exploited with some bioinformatic effort to build species-specific microarrays. We present here a method of high-density in situ synthesized microarrays starting from available EST sequences in, Ovis aries. Our data indicate that the method is very efficient and can be easily extended to other species of which genetic sequences are present in public databases, but neglected so far with advanced devices like microarrays. As a perspective, the approach can be applied also to species of which no sequences are available to date, thanks to high-throughput deep sequencing methods.

Forensic interlaboratory evaluation of the ForFLUID kit for vaginal fluids identification

Identification of vaginal fluids is an important step in the process of sexual assaults confirmation. Advances in both microbiology and molecular biology defined technical approaches allowing the discrimination of body fluids. These protocols are based on the identification of specific bacterial communities by microfloraDNA (mfDNA) amplification. A multiplex real time-PCR assay (ForFLUID kit) has been developed for identifying biological fluids and for discrimination among vaginal, oral and fecal samples. In order to test its efficacy and reliability of the assay in the identification of vaginal fluids, an interlaboratory evaluation has been performed on homogeneous vaginal swabs. All the involved laboratories were able to correctly recognize all the vaginal swabs, and no false positives were identified when the assay was applied on non-vaginal samples. The assay represents an useful molecular tool that can be easily adopted by forensic geneticists involved in vaginal fluid identification.

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.

Personalised Medicine in 2012: Editorial to the Special Issue of New Biotechnology on “MOLECULAR DIAGNOSTICS & PERSONALISED MEDICINE”

This special issue of New Biotechnology is focused on molecular diagnostics and personalised medicine and appears at an epochal moment in the development of the field. The practice of medicine is taking a significant and irrevocable turn towards personalisation, due to the great progress in areas such as genomics, pharmacogenomics and molecular diagnosis. It becomes increasingly apparent that to deliver the promise of personalised treatments, more and more novel medicines discovered today will be presented together with innovative companion diagnostics. The contributions to this volume touch on many disciplines, ranging from cell biology to genetics, immunology, molecular diagnostics, pharmaceutics and economic issues. The contributions of clinicians and basic scientists are synergistically presented to underline better the wide spectrum of studies that can contribute to the new field of personalised medicine. The promising perspectives of individualised treatments are related not only to higher effectiveness, but also to increased efficiency. This is relevant not only for the individual patient, but even more so for the general public, within a wider economical perspective where resources are limited and it becomes more and more mandatory to close the gap between social costs and benefits. This approach follows the steps of a stratified and individualised medicine and finds its final goal in an individualised healthcare.

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.

Compliance with herpes zoster vaccination in young and adult individuals in two regions of Italy.

BackgroundThe purpose of this work was to explore the knowledge and acceptance of Varicella Zoster Virus (VZV)-Herpes Zoster (HZ) vaccination in the general Italian population, where the HZ vaccine has not yet been distributed, using a prevalence study of subjects from two regions in Italy.MethodsA group of 3,173 individuals were interviewed using a questionnaire. The youngest age group (≤ 20 year) was composed of students interviewed at university. The middle age group (21-40 years) and the older age group (≥ 41 years) were interviewed by general practitioners in their office.ResultsIn both regions, the majority of subjects had been infected with varicella, and only 165 (5.2%) subjects reported receiving the VZV vaccination. Regarding HZ, 2,749 (86.6%) individuals stated that they knew of the virus and 2,233 (70%) were willing to be vaccinated against HZ. The majority of people willing to be vaccinated were in the middle and older age groups (36.6% and 44.7%, respectively).ConclusionCompliance versus vaccination results were satisfactory and probably, with the upcoming availability of the HZ vaccine in Italy, adults will be favourably disposed towards vaccination.

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.