Isolation and characterization of an atypical Mycoplasma gallisepticum strain showing a new mgc2 variant.

Mycoplasma gallisepticum (MG) is an important pathogen of the poultry industry able to cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite the application of biosecurity measures and the availability of vaccines for chickens, monitoring systems routinely applied for MG detection are still essential for infection control. Pathogen isolation is time-consuming and not suitable for rapid detection, albeit it is a compulsory step for genetic typing and antimicrobial susceptibility evaluation of single strains. The mgc2 gene is a species-specific molecular target adopted by most of the PCR protocols available for MG diagnosis, which are also included in the WOAH Terrestrial Manual. We describe the case of an atypical MG strain, isolated in 2019 from Italian turkeys, characterized by an mgc2 sequence not detectable by common endpoint PCR primers. Considering the potential risk of false negative results during diagnostic screenings with the endpoint protocol, the authors propose an alternative mgc2 PCR endpoint protocol, named MG600, which should be considered as a further diagnostic tool.

Application of different laboratory techniques to monitor the behaviour of a Mycoplasma synoviae vaccine (MS-H) in broiler breeders.

BACKGROUND: Mycoplasma synoviae (MS) is a major poultry pathogen which causes severe economic losses in all the productive sectors. The prevalence of MS in European countries has increased in the last few years, leading to greater attention to the available methods to prevent its spread. The main strategy currently applied for its containment is the development and maintenance of MS-free breeder flocks. A live MS vaccine (MS-H) obtained by mutagenizing an Australian field strain has recently been introduced in Italy. The aim of the present study was to evaluate the vaccine behaviour in broiler breeder groups at different production stages and the effectiveness of the available laboratory tests in discriminating the MS-H from a field strain. RESULTS: The vaccine diffused extensively through the population, shown by the wide serological response (over 80% of positive samples in RSA and 85% in ELISA), the high serological titres, the positivity of all the tracheal samples collected during the production phase by MS PCR and the positivity by cultivation from tracheal swabs at the end-point (55 weeks after vaccination). In contrast, only one swab from a sternal bursa was positive in MS PCR, while all the joint and oviduct samples were negative. There was no evidence of vertical transmission. Different genotyping techniques were used to achieve a clear classification of the MS positive samples. The vlhA and the obg gene analysis showed that most of the strains were homologous with the vaccine, but some ambiguous samples were further investigated with the multi locus sequence typing (MLST) scheme which confirmed the homology. CONCLUSIONS: The development of a multi-technique approach to monitor vaccinated avian flocks, based both on serological and biomolecular methods, is advised as well as the use of effective genotyping techniques to analyse the MS strains circulating in high densely populated poultry areas.

Favorable effects of myo-inositol, selenomethionine or their combination on the hydrogen peroxide-induced oxidative stress of peripheral mononuclear cells from patients with Hashimoto's thyroiditis: preliminary in vitro studies.

OBJECTIVE: The aim of this study was to assess whether blood mononuclear cells (PBMC) from Hashimoto's thyroiditis (HT) and control women, were protected from in vitro H2O2-induced oxidative stress after addition of antioxidants. PATIENTS AND METHODS: PBMC, from 8 HT women and 3 healthy women (controls), were cultured in the presence of 200 µM H2O2 alone, with subsequent addition of myo-inositol (Myo) (0.25, 0.5, 1.0 µM), selenomethionine (SelMet) (0.25, 0.5, 1.0 µM), or their combination (0.25+0.25, 0.5+0.5, 1.0+1.0 µM). PBMC proliferation, vitality, genotoxicity (Comet score) and secretion in the medium of the chemokines CXCL10 [IP10], CCL2 e CXCL9 [MIG] were the indices measured. RESULTS: PBMC proliferation was decreased by H2O2 alone, and it decreased further and dose-dependently in either group (greatest decrease with Myo+SelMet in HT). H2O2 alone decreased vitality by 5% in controls and 10% in the HT group, but vitality was rescued by the three additions. The addition of H2O2 alone increased the Comet score at +505% above baseline in controls and +707% in HT women. In either group, each addition dose-dependently contrasted genotoxicity. Concentrations of chemokines in the medium were increased by H2O2 alone, and in HT women more than in controls. Each addition dose-dependently decreased these concentrations in either group, and often below baseline levels, with Myo+SelMet being the most potent addition (up to approximately -80% of baseline). CONCLUSIONS: The tested antioxidants exert beneficial effects on PBMC exposed in vitro to H2O2-induced oxidative stress in both control and HT women. Particularly, the association Myo+SelMet is the most effective. After the demonstration of a favorable in vitro outcomes in a large cohort of HT patients, we could predict favorable in vivo outcomes given by the same supplement. Thus, one can select HT patients with a high chance of benefit from supplementation.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality.

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy.

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8µg/mL for erythromycin, >16µg/mL for spiramycin, and >32µg/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32µg/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC ≤0.5µg/mL and 30 isolates >32µg/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5µg/mL) and 1-fold higher MIC90 (4 vs. 2µg/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2µg/mL, whereas tiamulin had a MIC90 of 0.5µg/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population.

Characterisation of Yersinia pseudotuberculosis isolated from animals with yersiniosis during 1996-2013 indicates the presence of pathogenic and Far Eastern strains in Italy.

Yersinia pseudotuberculosis is a pathogen that infects both animals and humans worldwide. The epidemiology of infection caused by Y. pseudotuberculosis is poorly understood; however, its outbreaks have been traced back to a probable source in wildlife. This study aimed to characterise Y. pseudotuberculosis isolates collected from animals with yersiniosis. This study included 90 isolates of Y. pseudotuberculosis collected from different animals with yersiniosis between 1996 and 2013 in Italy. The isolates were tested for antimicrobial susceptibility and were biotyped. Genes associated with virulence plasmid pYV and those encoding O-antigen, high pathogenicity island (HPI), and superantigenic toxin (YPM) were determined by performing PCR. Pulsed-field gel electrophoresis (PFGE) was performed using NotI and SpeI enzymes, and 3 dendrograms were generated. No antibiotic resistance was found. The presence of pYV was shown in 57 out of 90 isolates. Virulence profiles of majority of the isolates indicated that they belonged to O:1a and O:1b serotypes, biotype 1, and genetic type 2. Isolates belonging to O:2a serotype were detected in sheep and cattle and were found to be associated (for the first time) with septicemia in hares. Y. pseudotuberculosis isolates belonging to O:5a and O:12-O13 serotypes were also detected in hares. To our knowledge, this is the first study to detect Y. pseudotuberculosis isolates belonging to the O:12-O13 serotype from a clinical case in Europe. Results of PFGE indicated that it was a reliable method for investigating the genetic relatedness of Y. pseudotuberculosis isolates. Thus, characterisation of Y. pseudotuberculosis infection in animals should be considered a possible tool for the surveillance of pseudotuberculosis.

Management of breast cancer after Hodgkin's lymphoma and paediatric cancer.

RATIONALE: The risk of women developing a breast cancer (BC) after receiving chest radiotherapy for paediatric cancers and Hodgkin lymphomas is well established. The aim of this study was to assess these patients' clinical characteristics and clinical outcomes. METHODS: The study concerns women with a history of primary neoplasms treated with chest irradiation ± chemotherapy and subsequently diagnosed with BC. RESULTS: We identified 78 women who developed BC (invasive in 68 cases, 87%). They were a median 18 and 38 years of age when their first neoplasm and BC were diagnosed, respectively. Breast-conserving surgery was performed in 39 patients, and 32 underwent breast irradiation. Twenty of the 41 patients (49%) treated with chemotherapy received an anthracycline-containing regimen. The 5- and 11-year event free survival (EFS) and overall survival (OS) rates were 69% and 42%, respectively. Nine patients (12%) developed a third cancer and 18 (23%) a cardiovascular event. Of the 68 women with invasive BC, the first event involved contralateral BC in 55% of cases: time to progression (TTP) rates were 70% and 47% at 5 and 11 years. The 5- and 11-year BC-specific survival rates (BCSS) were 84% and 68%, respectively. CONCLUSIONS: Judging from our experience, survival rates after BC developing in women previously given chest radiotherapy are not dissimilar to those observed in other women with primary BC. Given the far from negligible risk of subsequent cancers and cardiovascular events, it is mandatory to discuss the best choice of treatment for such patients in terms of their chances of cure and quality of life, and also the risks of late sequelae.

Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.

[Differences in sports participation for children and adolescents with solitary kidney due to renal tumors across Europe. Time for harmonization]. / Partecipazione in attività sportive per bambini e adolescent con un solo rene. È tempo di armonizzare in comportamenti in Europa per I ragazzi guariti da un tumore renale.

As a result of advances in treatment, almost 90% of children diagnosed with Wilms tumor became long-term survivors, and have a sustainable quality of life. These patients' involvement in sports during their childhood is hopefully increasing too. The cornerstone of renal tumor cure remains radical nephrectomy, however, so survivors live with a solitary kidney. In most European countries and the USA, the involvement in sports of children with a solitary kidney depends on a responsible physician saying a "qualified yes", pending individual assessment. Unlike the case in the rest of Europe, in Italy having only one kidney automatically disqualifies an individual wishing to participate in any organized "competitive" sports carrying some risk of renal trauma, including basketball, soccer and sometime volleyball. This absolute restriction is based on ad hoc Ministerial rulings concerning "Health protection in sport activities". But available data do not seem to support such an absolute limitation on participation in sports based exclusively on the fact of having a single kidney. The sport-specific incidence of kidney injuries has been estimated at 2.3 injuries per million male athlete/exposures for basketball (2.5 for females), and 2.6 for soccer (6.0 for girls). Kidney injuries are significantly more rare than head or spine injuries. This article aims to provide Italian sport medicine specialists and policy-makers with the necessary background so that the current, over-protective "unquestionably no" response can be reconsidered, and converted into a still well-founded, more permissive attitude to the sports activities suitable for any children with a solitary normal kidney.

Treatment of relapsed Wilms tumour (WT) patients: experience with topotecan. A report from the SIOP Renal Tumour Study Group (RTSG).

BACKGROUND: Topotecan has been variably incorporated in the treatment of patients with relapsed Wilms tumour (WT) who failed initial treatment with three or more effective drugs. Our objective was to describe outcome and to retrospectively investigate the potential role of topotecan in relapsed WT patients. METHODS: Children who were treated with topotecan as part of their chemotherapeutic regimens for relapsed WT were identified and included in our retrospective study. Patient charts were reviewed for general patient characteristics, histology and stage at initial diagnosis, number and type of relapse, salvage treatment schedules, toxicity, response to treatment and outcome. RESULTS: From 2000 to 2012, 30 children (median age at relapse 5.5 years, range 1.6-14.5 years) were identified to have received topotecan as part of their salvage regimens (primary progressive disease n = 3, first, second and third relapse n = 13, 9 and 2 respectively, partial response n = 3). Topotecan was administered as a single agent (12 patients) or in combination with other drugs (18 patients). Sixteen patients had high-risk histology according to the SIOP classification, 15 died within 12 months because of progressive disease. Fourteen patients had SIOP intermediate-risk histology of which four patients displayed objective responses to topotecan. Overall, 6 out of 14 intermediate-risk patients survived (median follow up of 6 years), however, three of whom (stage V) had bilateral nephrectomy after topotecan treatment. CONCLUSIONS: Topotecan does not seem to show effectiveness in the treatment of relapsed WT patients with initial high-risk histology. In patients with intermediate-risk histology, the role of topotecan might deserve further attention, to prove its efficacy.

Global multilocus sequence typing analysis of Mycoplasma bovis isolates reveals two main population clusters.

Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.

Evaluation of the udder health status in subclinical mastitis affected dairy cows through bacteriological culture, somatic cell count and thermographic imaging.

Subclinical mastitis in dairy cows is a big economic loss for farmers. The monitoring of subclinical mastitis is usually performed through Somatic Cell Count (SCC) in farm but there is the need of new diagnostic systems able to quickly identify cows affected by subclinical infections of the udder. The aim of this study was to evaluate the potential application of thermographic imaging compared to SCC and bacteriological culture for infection detection in cow affected by subclinical mastitis and possibly to discriminate between different pathogens. In this study we evaluated the udder health status of 98 Holstein Friesian dairy cows with high SCC in 4 farms. From each cow a sample of milk was collected from all the functional quarters and submitted to bacteriological culture, SCC and Mycoplasma spp. culture. A thermographic image was taken from each functional udder quarter and nipple. Pearson's correlations and Analysis of Variance were performed in order to evaluate the different diagnostic techniques. The most frequent pathogen isolated was Staphylococcus aureus followed by Coagulase Negative Staphylococci (CNS), Streptococcus uberis, Streptococcus agalactiae and others. The Somatic Cell Score (SCS) was able to discriminate (p<0.05) cows positive for a pathogen from cows negative at the bacteriological culture except for cows with infection caused by CNS. Infrared thermography was correlated to SCS (p<0.05) but was not able to discriminate between positive and negative cows. Thermographic imaging seems to be promising in evaluating the inflammation status of cows affected by subclinical mastitis but seems to have a poor diagnostic value.

First isolation of Mycoplasma iowae in grey partridge flocks.

Mycoplasma iowae, an occasional pathogen of turkeys, was isolated for the first time from captive grey partridges (Perdix perdix). Clinical signs including respiratory and intestinal disorder were seen in birds of all ages but mainly in those kept housed during rearing. Mortality rates averaged over 20% during the year. Treatment with antibiotics and antiparasitic drugs produced only a transient improvement in condition. The gross pathology findings included poor body growth, lack of development of the breast muscles, abnormalities in the keel development, and bone fragility. Some birds showed infraorbital sinusitis with serous or fibrinous exudates and catarrhal tracheitis, while others presented serofibrinous airsacculitis and splenomegaly. Laboratory investigations revealed pure cultures of M. iowae in the gut as well as sinus and air sacs. While other organisms such as coccidia, Trichomonas, Escherichia coli, Clostridium perfringens, and Aspergillus spp. were detected, the similarity of the disease with that seen in turkeys infected with M. iowae strongly suggests that this mycoplasma may be the primary pathogen here. The presence of M. iowae in game birds commonly released into the wild could have serious implications particularly in areas where industrial poultry farms are concentrated.

Molecular characterization of acquired enrofloxacin resistance in Mycoplasma synoviae field isolates.

The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 µg/ml. The estimated MIC50 and MIC90 values for enrofloxacin were 2 and 8 µg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥ 2 µg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥ 1 µg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.

Exposure of human skin to benzo[a]pyrene: role of CYP1A1 and aryl hydrocarbon receptor in oxidative stress generation.

Exposure to benzo[a]pyrene (BaP) can induce inflammatory skin diseases and skin cancer, which are both associated to oxidative stress. BaP is known to bind with high specificity to the aryl hydrocarbon receptor (AhR), modifying the expression of CYP1A1, involved both in cancer and inflammation. While the current knowledge is based on murine skin and cell culture data, in this study human healthy skin has been treated with 5muM BaP in conditions simulating occupational and environmental exposure. AhR and CYP1A1 expression was evaluated by Western blotting, which revealed their presence even in control untreated skin; both enzyme and receptor increased more than twofold after exposure to BaP. AhR expression level was lower than CYP1A1 in basal conditions and following induction. Oxidative stress was evaluated in terms of MTT reduction, protein peroxidation and reactive oxygen species (ROS) formation. A significant increase in ROS and carbonyl compound production, as well as reduced tissue viability have been determined by BaP. The results of this experiment indicate that BaP, an AhR agonist, can significantly increase receptor and CYP1A1 expression and induce oxidative stress in human skin, confirming the involvement of this pathway in the pathogenesis of tissue damage due to polycyclic aromatic hydrocarbons.

Efficacy of topotecan plus vincristine and doxorubicin in children with recurrent/refractory rhabdomyosarcoma.

BACKGROUND: This study investigates the efficacy and the feasibility of a chemotherapy regimen with topotecan plus vincristine and doxorubicin (TVD) given on an individually tailored basis to patients with refractory/recurrent rhabdomyosarcoma (RMS). PATIENTS AND METHODS: Nine patients received TVD therapy at relapse, and six were assessable for response. RESULTS: All the six patients experienced objective response after two cycles of chemotherapy: one minor response, four partial response, and one complete response. CONCLUSIONS: The value of our study is severely limited by the small number of cases, the single-institutional setting and the individually tailored treatment, but we nonetheless confirmed the feasibility and tolerability of topotecan-based chemotherapy in RMS.

Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product.

Imidacloprid is a neonicotinoid insecticide combining excellent efficiency against parasites with low toxicity for mammals. Commercially, it is co-formulated with dimethyl sulfoxide, methylpyrrolidone, propylene carbonate and mineral oil, which can modify its bioavailability and toxicological profile for humans following occupational exposure. A combined in vitro approach employing the comet assay and the micronucleus test was used to assess the genotoxicity of imidacloprid in relation to formulation, metabolic activation and exposure level. Human peripheral blood lymphocytes from unexposed healthy volunteers were treated with imidacloprid (0.2, 2 and 20 µM) and with equimolar concentrations of a commercial product, with and without addition of S9 fraction. Imidacloprid significantly increased the comet score and the frequency of micronuclei only at the highest concentration tested. DNA damage was slightly more severe with the commercial product, and was increased, though not significantly, by metabolic activation. Formation of reactive oxygen species (ROS) does not seem to be involved as a mechanism of genotoxicity, but this result may be explained by the insufficient sensitivity of the 2',7'-dichlorofluorescein diacetate assay at the test concentrations of imidacloprid. These results suggest that at concentrations<20 µM imidacloprid is not genotoxic to human lymphocytes in vitro. Nonetheless, the presence of co-formulants in the commercial product and occupational exposure, along with poor safety procedures, may present an increased risk for DNA fragmentation and chromosomal aberrations.