Deciphering the Broad Antimicrobial Activity of Melaleuca alternifolia Tea Tree Oil by Combining Experimental and Computational Investigations.

Tea Tree Oil (TTO) is an essential oil obtained from the distillation of Melaleuca alternifolia leaves and branches. Due to its beneficial properties, TTO is widely used as an active ingredient in antimicrobial preparations for topical use or in cosmetic products and contains about 100 different compounds, with terpinen-4-ol, γ-terpinene and 1,8-cineole (or eucalyptol) being the molecules most responsible for its biological activities. In this work, the antimicrobial activity of whole TTO and these three major components was evaluated in vitro against fungi, bacteria and viruses. Molecular dynamics simulations were carried out on a bacterial membrane model and a Coxsackievirus B4 viral capsid, to propose an atomistic explanation of their mechanism of action. The obtained results indicate that the strong antimicrobial activity of TTO is attributable to the induction of an altered membrane functionality, mediated by the incorporation of its components within the lipid bilayer, and to a possible ability of the compounds to bind and alter the structural properties of the viral capsid.

Circulation of Enteric Viruses in Tirana, Albania: Situation Ten Years After the First Study.

A variety of enteric bacteria, viruses and protozoa are the leading causes of morbidity and mortality worldwide. To understand the evolution of gastroenteritis in Albania, in terms of distribution of aetiological pathogens, a one-year observational study was repeated in 2017, ten years after the first study performed in 2007. The data still show a clear circulation of viruses that cause gastroenteritis. Compared to the previous study in 2007, the data from the 2017 study showed the incidence of Norovirus and Adenovirus were significantly higher (p value <0.05), while Rotavirus was verified at a similar incidence rate.

Potential Use of Tea Tree Oil as a Disinfectant Agent against Coronaviruses: A Combined Experimental and Simulation Study.

The COVID-19 pandemic has highlighted the relevance of proper disinfection procedures and renewed interest in developing novel disinfectant materials as a preventive strategy to limit SARS-CoV-2 contamination. Given its widely known antibacterial, antifungal, and antiviral properties, Melaleuca alternifolia essential oil, also named Tea tree oil (TTO), is recognized as a potential effective and safe natural disinfectant agent. In particular, the proposed antiviral activity of TTO involves the inhibition of viral entry and fusion, interfering with the structural dynamics of the membrane and with the protein envelope components. In this study, for the first time, we demonstrated the virucidal effects of TTO against the feline coronavirus (FCoVII) and the human coronavirus OC43 (HCoV-OC43), both used as surrogate models for SARS-CoV-2. Then, to atomistically uncover the possible effects exerted by TTO compounds on the outer surface of the SARS-CoV-2 virion, we performed Gaussian accelerated Molecular Dynamics simulations of a SARS-CoV-2 envelope portion, including a complete model of the Spike glycoprotein in the absence or presence of the three main TTO compounds (terpinen-4-ol, γ-terpinene, and 1,8-cineole). The obtained results allowed us to hypothesize the mechanism of action of TTO and its possible use as an anti-coronavirus disinfectant agent.

Cytotoxicity and internalization analysis of silicon nanowires in Buffalo Green Monkey cells: a preliminary study to evaluate the possibility of carrying viruses inside the cells.

Silicon nanowires (SiNWs) are attractive functional nanomaterials for biomedical applications. The ability to easily tune their size and density, potential biocompatibility, and knowledge of the chemical activation of SiNWs surface make them natural tools to interact with biological materials. We evaluated the possibility of exploiting SiNWs as carriers to introduce organic compounds into cells. The cellular toxicity and the internalization capacity of free-standing and label-free SiNWs were tested on Buffalo Green Monkey cells (BGM). Confocal fluorescent observation of SiNWs conjugated with fluorescein-polyethylene imine (PEI) confirmed the internalization of the NWs into the Buffalo Green Monkey Cells (BGM).

Infection control in healthcare settings: perspectives for mfDNA analysis in monitoring sanitation procedures.

BACKGROUND: Appropriate sanitation procedures and monitoring of their actual efficacy represent critical points for improving hygiene and reducing the risk of healthcare-associated infections. Presently, surveillance is based on traditional protocols and classical microbiology. Innovation in monitoring is required not only to enhance safety or speed up controls but also to prevent cross infections due to novel or uncultivable pathogens. In order to improve surveillance monitoring, we propose that biological fluid microflora (mf) on reprocessed devices is a potential indicator of sanitation failure, when tested by an mfDNA-based approach. The survey focused on oral microflora traces in dental care settings. METHODS: Experimental tests (n = 48) and an "in field" trial (n = 83) were performed on dental instruments. Conventional microbiology and amplification of bacterial genes by multiple real-time PCR were applied to detect traces of salivary microflora. Six different sanitation protocols were considered. A monitoring protocol was developed and performance of the mfDNA assay was evaluated by sensitivity and specificity. RESULTS: Contaminated samples resulted positive for saliva traces by the proposed approach (CT < 35). In accordance with guidelines, only fully sanitized samples were considered negative (100 %). Culture-based tests confirmed disinfectant efficacy, but failed in detecting incomplete sanitation. The method provided sensitivity and specificity over 95 %. CONCLUSIONS: The principle of detecting biological fluids by mfDNA analysis seems promising for monitoring the effectiveness of instrument reprocessing. The molecular approach is simple, fast and can provide a valid support for surveillance in dental care or other hospital settings.

Oxidative Stress, Cytotoxicity, and Genotoxicity Induced by Methyl Parathion in Human Gingival Fibroblasts: Protective Role of Epigallocatechin-3-Gallate.

Organophosphorous (OP) compounds are pesticides frequently released into the environment because of extensive use in agriculture. Among these, methyl parathion (mPT) recently received attention as a consequence of illegal use. The predominant route of human exposure to mPT is via inhalation, but inadvertent consumption of contaminated foods and water may also occur. The goal of this study was to investigate the in vitro effects of mPT on cells in the oral cavity and evaluate the potential protective role of epigallocathechin-3-gallate (EGCG) on these effects. Human gingival fibroblasts (HGF) were exposed to 10, 50, or 100 µ g/ml mPT for 24 h and assessed for oxidative stress, as evidenced by reactive generation of oxygen species (ROS), induction of apoptotic cell death, DNA damage (comet assay and cytochinesis-block micronucleus test), and nitric oxide (NO) production. The results showed that mPT produced significant oxidative stress, cytotoxicity, and genotoxicity and increased NO levels through stimulation of inducible NO synthase expression. Finally, data demonstrated that EGCG (10, 25, or 50 µ M) was able to inhibit the pesticide-induced effects on all parameters studied. Data indicate that cytotoxic and genotoxic effects may be associated with oxidative stress induced by mPT observed in HGF cultures and that EGCG plays a protective role via antioxidant activities.

Oral health conditions in an Albanian adolescent population: an epidemiological study.

BACKGROUND: The aim of this study was to determine the oral health conditions of an adolescent population of Tirana. METHODS: A cross-sectional epidemiological study was carried out in a sample (n = 1885), aged 16-19, mean age 17.4 (SD = 1.0), attending public schools in Tirana and province; 1200 adolescents were included into the analysis. A clinical observation without radiographs was conducted in the medical room of the schools during the 2012-2013 school year. RESULTS: Very severe and severe orthodontic treatment need, grade 5 and 4 of IOTN (Index of Orthodontic Treatment Need), were found in 17.0 % of the sample. DMFT (Decayed, Missing and Filled Teeth) was 4.9, whereas OHI (Oral Hygiene Index) was documented in the highest number of subjects (n = 384), 32 % of the total sample possessed "good" grade of oral hygiene. CPI (Community Periodontal Index) was observed at score 0 (healthy gingival condition) in most of the subjects (53.1 %), score 1 (gingival bleeding) in 33.4 % of the total sample. PI (Plaque Index) results showed 43.9 % of the sample (527 subjects) with score 0. CONCLUSIONS: The study findings highlight the need for preventive care programs to improve oral health conditions as well as to reduce oral pathology risk factors in Albania.

Molecular characterization of norovirus GII strains identified in Albania.

Noroviruses (NoVs) are considered as the leading cause of diarrheal diseases in all groups of age. In the last decade the number of NoV outbreaks worldwide is increasing. Data published by the systems of NoV surveillance show the GII.4 strain as the human predominant genotype circulating worldwide and new genetic variants of GII.4 were associated with epidemic events. In Albania the economy transformation has damaged significantly the environment and a large circulation of enteric viruses was reported in the past with the presence of NoV among the genotyped strains. This study aimed to characterize, by molecular analysis, the NoV GII strains detected in Albania during two time periods: in 2010 from the outbreak occurred in Ballsh and in 2002-2003 from sporadic cases of diarrhoea. A total of 21 Nov GII strains were characterized. The NoV GII.4 was genotyped more frequently and it was related closely to the pandemic variants recorded in GenBank. During 2002-2003, six NoV GII recombinant strains have been characterized.

Analytical solution for the modeling of the natural time-dependent reduction of waterborne viruses injected into fractured aquifers.

We propose an analytical solution in order to explain the processes that determine the fate and behavior of the viruses during transport in a fractured aquifer at Salento (Italy). The calculations yield the efficiency of filtration in fractures at a site near Nardò (Southern Italy) in reducing the numbers of enteric viruses (i.e., Enteroviruses and Norovirus) in secondary municipal effluents that have been injected in the aquifer over the period 2006-2007. The model predicted, by a theoretical expression, the time-dependent rate of virus reduction, which was in good agreement with field data. The analytical solution yields the achievable "Log reduction credits" for virus reduction in wells located at the setback distances that are usually adopted in local drinking water regulations. The resulting new analytical formula for the time-dependent reduction of viruses during subsurface transport can easily be applied in health risk-based models used to forecast the spread of waterborne diseases and provides appropriate criteria (i.e., distances) needed to meet standards for the quality of drinking water derived from undisinfected groundwater.

An ELIME assay for hepatitis A virus detection.

An Enzyme Linked ImmunoMagnetic Electrochemical assay (ELIME) was developed for the detection of the hepatitis A virus (HAV). This system is based on the use of new polydopamine-modified magnetic nanobeads as solid support for the immunochemical chain, and an array of 8 screen-printed electrodes as a sensing platform. Enzymatic-by-product is quickly measured by differential pulse voltammetry. For this purpose, all analytical parameters were optimized; in particular, different blocking reagents were evaluated in order to minimize the nonspecific interaction of bioreagents. Using the ELIME assays, a quantitative determination of HAV can be achieved with a detection limit of 1·10-11 IU mL-1 and a working range between 10-10 - 5 × 10-7 IU mL-1. The cross-reactivity of the commercial monoclonal antibodies against HAV used in ELIME assays was tested for Coxsackie B4, resulting very low. The sensitivity was also investigated and compared with spectrophotometric sandwich ELISA. The average relative standard deviation (RSD) of the ELIME method was less than 5% for the assays performed on the same day, and 7% for the measurements made on different days. The proposed system was applied to the cell culture of HAV, which title was quantified by Real-Time Quantitative Reverse Transcription PCR (RT¬qPCR). To compare the results, a correlation between the units used in ELIME (IU mL-1) and those used in RT¬qPCR (genome mL-1) was established using a HAV-positive sample, resulting in 1 IU mL-1-10-4 gen mL-1 (R2 = 0.978). The ELIME tool exhibits good stability and high biological selectivity for HAV antigen detection and was successfully applied for the determination of HAV in tap water.

Detection and molecular characterization of human rotaviruses isolated in Italy and Albania.

Rotaviruses are one of the most important causes of gastroenteritis in children under 5 years old. Analysis of G and P rotavirus genotypes in circulation is crucial in evaluating the appropriacy of mass vaccination of children worldwide. Overall, 592 stool samples were collected in Tirana (Albania), the Salento peninsula (South Italy), and three different hospitals in Rome (Central Italy). Of the total samples, 31.3% were rotavirus positive in Albania, 78.3% in the Salento, and 40.3% in Rome. The samples collected in Tirana and Rome were G-P typed, whereas the samples collected in the Salento were only G typed. Overall, in Italy the most frequent combinations were G4 P[8] (54.5%), G1 P[8] (27.3%), and G2 P[4] (18.2%); in Albania they were G9 P[8] (72.1%), G4 P[8] (8.8%), G1 P[8] (5.9%), and G2 P[4] (2.9%). The prevalence in Albania of atypical combinations was 7.4% for G4 P[4] and 2.9% for G9 P[4]. Phylogenetic analysis was also performed to assess the genetic relatedness of the strains. J. Med. Virol. 82:510-518, 2010. (c) 2010 Wiley-Liss, Inc.

A strategy to increase the specificity of Syber Green I qRT-PCR in hepatitis A detection.

Various methods for the recovery and detection of HAV have been suggested, and molecular tests have recently provided an effective replacement for the traditional methods. Real-time RT-PCR technology offers many advantages over conventional RT-PCR in terms of rapidity and specificity. Most procedures are based on the TaqMan chemistry, but some researchers have used the SYBR Green I approach, which is less expensive and simpler to carry out. However the formation of primer-dimers needs to be distinguished from specific products through a melting curve analysis. This study focused on a strategy to increase the specificity of Syber Green I chemistry, thus nullifying the primer-dimers interference. To this end, forward and reverse primers were specially designed for hairpin loop formation, a strategy widely used to improve the specificity and the efficiency of PCR. Two different concentrations of primers were assayed (200 nM and 400 nM) in a one-step, real-time RT-PCR procedure, evaluating the specificity of the amplicons and the optimization of the real-time protocol. We demonstrated that this approach can increase the specificity of the Syber Green I qRT-PCR performance with a good reproducibility of the method. Because of the simplicity of the assay and the lower costs involved, this procedure could be a valid alternative to HAV monitoring from environmental matrices.

[The birth of nanobiotechnologies: new nanomaterials, potential uses, toxic effects and implications for public health]. / La nascita delle nanobiotecnologie: I nuovi nanomateriali, i potenziali applicativi, gli effetti tossici e le implicazioni per la Sanità Pubblica.

Nanotechnologies hold considerable promise of advances in many sectors especially the biomedical field, since the materials used are of the appropriate dimensions to interact with important biological matter such as proteins, DNA and viruses. In this field the use of nanotechnologies will probably be second in importance only to biotechnologies. However many characteristics of nanomaterials that make them so promising from a technological point of view may also lead to negative effects on the environment and human health. It is important therefore that the environmental and work-related exposure effects to these materials be evaluated. In this article the potential uses, toxic effects and public health implications of nanobiotechnologies are discussed.

Laser-induced fluorescence (LIF) as a smart method for fast environmental virological analyses: validation on Picornaviruses.

Virological analysis is time-consuming and expensive. The aim of this work is to demonstrate the applicability of laser-induced fluorescence (LIF) to the classification of viruses, reducing the time for this analysis and its costs. Experimental tests were performed in which different viruses were irradiated with a UV laser emitting at 266 nm and the emitted spectra were recorded by a spectrometer. The classification techniques show the possibility of discriminating viruses. Although the application of the LIF technique to biological agents has been thoroughly studied by many researchers over the years, this work aims at validating for the first time its applicability to virological analyses. The development of a fast virological analysis may revolutionize this field, allowing fast responses to epidemiologic events, reducing their risks and improving the efficiency of monitoring environments. Moreover, a cost reduction may lead to an increase in the monitoring frequency, with an obvious enhancement of safety and prevention.

Sewage workers: risk of acquiring enteric virus infections including hepatitis A.

To determine if sewage workers have an increased risk of acquiring viral infections, 66 workers at a small wastewater plant in north-eastern Italy and 72 control subjects recruited from blood donors were enrolled in a seroprevalence study to determine whether sewage workers are at increased risk of acquiring viral infections. In order to evaluate various risk factors, a questionnaire was filled out by each worker whereas seropositivity to Hepatitis A virus, Coxsackievirus B2 - B3 - B4 - B5, and Echovirus types 1 and 9 was determined in the laboratory. Anti-HAV antibodies were present in 37.8% of sewage workers and 36.1% of subjects in the control group. The difference was not statistically significant in the two groups, whereas a significant association was observed regarding age (P < 0.3). No association was observed with the occupational age, or with number and duration of contacts per day. The lack of evident occupational risk for hepatitis A among sewage workers may be explained by the adult age of the workers (mean age 41.3 years, range 22-58 years), and thus the antibody titre against different enteroviruses was determined. No statistically significant differences were evident with the raw values, but considering the 90 degrees percentile as a dichotomic value for the antibody levels a strong and significant association was present with Coxsackievirus B3 (O.R. 22.85, C.I. 95% 2.93-178.08) and Coxsackievirus B2 (O.R. 14.25, C.I. 95% 1.78-113.87). Analysis of the data confirms a limited risk of acquiring infection and/or disease but also the evident possibility of silent exposure to the viruses. The shift in HAV epidemiology and increased morbidity and mortality in adult age suggest that active immunization against hepatitis A should be considered.

[Virus removal in conventional wastewater treatment process]. / Rimozione dei virus nei processi convenzionali di trattamento delle acque reflue.

Little has been published in the scientific literature on the distribution of pathogenic viruses and on their removal in the different treatment phases in wastewater treatment plants in Italy. Recent advances in molecular biology have reduced analytic costs and allowed the detection of virus directly in the environment. This study evaluates the presence of enteric viruses and their removal in three treatment plants of different sizes and which use different treatment methods. Overall 92 samples were collected during three seasonal campaigns (winter, spring-summer, and autumn). Samples were collected at different phases of the treatment process. Chemical and microbiological analyses were performed. Samples were concentrated by ultrafiltration and tested for the presence of hepatitis A virus, adenovirus, rotavirus, astrovirus, norovirus and enteroviruses. All viruses, excluding the Enteroviruses were detected through biomolecular testing. Enteroviruses, were isolated on BGM cells and subsequently genotyped at the VP1 region. Sixty-two of the 92 samples tested positive for viruses; of these, 21 tested positive for HAV, 4 for adenovirus, 9 for rotavirus, 21 for astrovirus, and 7 for norovirus. Enteroviruses were present on average in 44% of samples collected during the three sampling campaigns. Hepatitis A virus was isolated during the spring-summer period while Astroviruses were identified in all three sampling campaigns. Different enteroviruses were isolated, including four types of Coxsackie B virus variably isolated in all three campaigns, and type 1 Echovirus isolated in the second and third campaigns. All detected polioviruses were of the vaccine-type and were identified as types 1 and 3. Both viral genotypes of Norovirus were identified. Adenoviruses were only sporadically isolated and rotaviruses were identified as type A. As expected, results show a greater prevalence of Enteroviruses and this reflects viral circulation in the population. Regarding the efficacy of the different treatment stages, primary treatment was found to remove only small percentages of viruses, while activated sludge e subsequent sedimentation were found to be more effective.

Enteric viruses in molluscan shellfish.

One hundred and thirty-seven bivalves were collected for environmental monitoring and the market; all the samples were analysed by RT-PCR test. Bacteriological counts meeting the European Union shellfish criteria were reached by 69.5% of all the samples, whereas the overall positive values for enteric virus presence were: 25.5%, 18.2%, 8.0% and 2.1% for Rotavirus, Astrovirus, Enteroviruses, Norovirus, respectively. Mussels appear to be the most contaminated bivalves, with 64.8% of positive samples, 55.7% and 22.7% respectively for clams and oysters, whereas in the bivalves collected for human consumption 50.7% were enteric virus positive, as compared to 56.4% of the samples collected for growing-area classification. The overall positive sample was 54.0%.

Identification and sequence analysis of hepatitis A virus detected in market and environmental bivalve molluscs.

In Italy in 1998, hepatitis A virus (HAV) was responsible for an infectious disease transmitted by contaminated bivalve molluscs. To determine the presence of HAV in the bivalves collected during a 1-year follow-up study, hepatitis A RNA was extracted and amplified by a nested reverse transcriptase-PCR method overlapping the VP1/2A region. The HAV genome was detected in 24 (14.1%) of 170 samples: 19 clams (Tapes decussates and Tapes semidecussatus), 1 oyster (Crossostea gigas), and 4 mussels (Mytillus galloprovincialis). Eleven positive samples were collected from marketing areas, and 13 positive samples were collected from growing areas. Seventeen of the 24 positive samples had been taken from domestic products, and 7 had been imported. Sequence analysis showed the presence of genotypes IA and IB. Our results suggest significant presence of HAV in bivalves from both marketing (public consumption) and environmental (growing) areas.

Analysis of the shift of the transmission pattern for hepatitis C in a community in Central Italy.

Anti-HCV and HCV-RNA prevalence among adults from an isolated Central Italian community were 16.3% and 9.0%; 3.0%, 2.3% among 25-to-49-year-olds, 27.6%, 14.7% among older subjects. 1b genotype prevailed (88.5%). 1a, la/1b, 4 were also isolated. Seropositivity was associated with age > 50 years, public dental health care, glass syringe use, surgical interventions. The transmission pattern seems to be shifting from that typical of Southern Italy (high prevalence, particularly in the elderly, 1b genotype predominant, various routes of transmission involved, cohort effect), to that typical of Europe (lower prevalence, particularly among younger adults, other genotypes involved, association with intravenous drug use and immigrants).

[Problems related to the use of real-time RT-PCR in environmental analysis]. / Problematiche connesse con l'applicazione della RT-PCR real-time nelle analisi ambientali.

Molecular biology techniques allow high sensitivity and specificity in the detection of enteric viruses in various environmental samples, and are considerably less costly and more rapid than traditional analytical methods. Real time RT-PCR technology allows accurate, efficient, and reproducible quantification of viral genes, by amplifying enteroviral RNA directly from an adequately treated environmental sample. It uses different chemical systems, including TaqMan and Syber Green probes, for detection of the amplificon. Both systems allow quantification of the initial number of copies in each cycle by comparing values with those of an external calibration curve (standard curve), generated by serial dilutions of a reference RNA sample with a known concentration. Difficulties in generating a standard curve for each enteric virus however, make standardization of the system time consuming. In an attempt to overcome this obstacle, we used an internal standard with a known concentration, to obtain a valid calibration curve for the quantification of environmental enteroviruses. A comparative analysis was performed with various commercially available extraction and amplification systems to evaluate the method's efficiency and reproducibility.