Saverio Giampaoli

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.

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.

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.

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.

Health and safety in recreational waters.

The recreational use of waters has increased in recent decades all over the world. Swimming pools and other recreational water facilities offer opportunities for enjoyment and health promotion, but they also may involve health risks, despite the fact that better management and modern technologies for water treatment and quality monitoring (e.g. real-time protocols for detecting bacteria), have made such facilities safer.1,2 Several communicable diseases – cryptosporidiosis, giardiasis, leptospirosis, legionellosis, bacterial and viral gastroenteritis – are commonly associated with bathing in recreational waters.3,4 According to field study surveys in different countries, the rate of diarrhoeal illness among swimmers ranges from 3 to 8%.5,6 Injuries are also common. In the United Kingdom of Great Britain and Northern Ireland alone, more than 13 000 injuries are estimated to occur annually, on average, among bathers.3,4 Preventing injuries, drownings, the toxic effects of chemical contaminants and outbreaks of waterborne diseases in people who swim in recreational waters calls for a sustained, concerted, multisectoral effort involving epidemiologists, physicians, microbiologists, toxicologists, builders, public opinion leaders and public health authorities. Swimming pools and spas are used by millions of people seeking recreation, rehabilitation, wellness and other health benefits. According to 2011 market data issued by the European Union of Swimming Pool and Spa Associations, 5.7 million pools exist in Europe, or about one for every 150 inhabitants.7 A large part of the world’s population is exposed to the health risks posed by swimming pools, which account for much of the world’s water consumption.8 This includes children, the elderly, pregnant women and people with immunodeficiency and disabilities. People in certain occupations, such as swimming coaches, water sport professionals and pool operators, are also exposed to such risks. In the twenty-first century, international safety standards or harmonized national safety laws are essential in light of the booming tourist industry – now the third largest in the world. From a global health perspective, water facilities must be made safe not just for resident populations, but also for travellers. The absence of an international regulatory framework for pool and spa safety and the presence of incomplete national regulations in many countries put local populations and visitors at risk. For example, Europe’s lack of a directive on safety in swimming pools and other recreational water facilities limits standardization and harmonization throughout the continent. Several institutions have recognized the need for international pool and spa safety regulations. This point was discussed in Rome last April, at the Fifth International Conference on Swimming Pool and Spa.9 The Conference focused on the role of aquatic recreational environments in health promotion but also called attention to the need for good surveillance and prevention to reduce safety hazards. Engineers, chemists, biologists, public health experts, architects, sociologists, occupational medicine specialists and ecologists were in attendance. This large and committed panel of experts can update safety guidelines periodically and support their local dissemination through universities and research centres, public health authorities, swimming pool managers and sports clubs and organizations catering to tourists and local residents. Conference attendees also discussed new technologies for monitoring the microbiological and chemical safety of recreational waters. Technological innovations can play a pivotal role in prevention. Rapid techniques for monitoring water quality can allow for quick measures in response to contamination. The new technologies are gradually shifting the focus from mandatory safety requirements towards expectations of good water quality and towards the implementation of collaborative surveillance strategies based on the hazard analysis and critical control point model. Use of the new technologies must, of course, be harmonized and adopted in consultation with public health institutions to avoid any risk to bathers from the application of unvalidated procedures. Appropriate water sanitation and safety measures are not the only priorities, however. Water is a scarce commodity. The conservation of this most precious resource and its rational use in recreation calls for advanced water management in the context of sustainable development. This and other issues will be discussed in Amsterdam in 2015 at the Sixth International Conference on Swimming Pool and Spa,10 which scientists are currently organizing, together with World Water Day (22 March 2014) and other activities promoted by agencies such as UN-Water and the United Nations Educational, Scientific and Cultural Organization.

Draft Genome Sequence of Tepidimonas taiwanensis Strain VT154-175

ABSTRACT The slightly thermophilic bacterium Tepidimonas taiwanensis strain VT154-175 has been isolated from a hot spring in the area of Viterbo, Italy. The whole draft genome of 2.9 Mb obtained by paired-end next-generation sequencing and divided into 60 scaffolds is presented.

Allergic risk in sport: public health issues

The allergic risk in sport is an emerging issue of relevance for public health. Several natural and synthetic allergens are present in indoor and outdoor sport environments. Epidemiological data show the widespread high incidence of allergies in the population. Elite athletes have been observed to have a high prevalence of allergic reactions and exposure to allergens during training and competitions is a concern for environmental occupational medicine. The connection between exercise-induced bronchoconstriction, genetics and environment is still debated. In recent decades, interest in this field has been growing as shown by the increasing number of ISI/PubMed publications. The main allergens present in the sport and physical activity environments are classified and reported here.

Environmental microbiology: perspectives for legal and occupational medicine

The analysis of microorganism population is crucial in several medical fields. This is especially true in legal and occupational medicine, where the specialist can be asked to perform an evaluation of several environmental matrices. In these two medical fields an accurate microbiological analysis is part of a wide process aimed to the definition of the interactions between human beings and environment. In legal medicine it is important to deserve attention to the identification of microbiological traces in order to better understand past events, while in occupational and preventive medicine the microbiological evaluation of environmental samples is crucial for an effective risk management and the definition of safety procedures. The achievement of these objectives requires the comprehension of microbial biodiversity and not only the identification of few biomarkers. In the present paper, the complexity of this process is highlighted through the presentation of typical scenarios where microorganism population analyses are relevant in legal medicine and occupational medicine. The similarities between the microbiological approach in legal and occupational medicine lead to the sharing of laboratory approaches. A description of technological evolution shows how new protocols and procedures are supporting a wider microbiological comprehension of specimens. The development of molecular tools has opened new opportunities, but it has underlined the need for the implementation of new standardized procedures dedicated to these medical fields, where science and medicine interact with the law. In addition, the rapid evolution of massive parallel sequencing technologies requires the implementation of new bioinformatic tools with a user-friendly interface.