Effects of Influenza Vaccine on the Immune Responses to SARS-CoV-2 Vaccination.

A number of studies have suggested that influenza vaccination can provide protection against COVID-19, but the underlying mechanisms that could explain this association are still unclear. In this study, the effect of the 2021/2022 seasonal influenza vaccination on the immune response to the booster dose of anti-SARS-CoV-2 vaccination was evaluated in a cohort of healthy individuals. A total of 113 participants were enrolled, 74 of whom had no prior COVID-19 diagnosis or significant comorbidities were considered for the analysis. Participants received the anti-influenza tetravalent vaccine and the booster dose of the anti-SARS-CoV-2 vaccine or the anti-SARS-CoV-2 vaccine alone. Blood was collected before and 4 weeks after each vaccination and 12 weeks after SARS-CoV-2 vaccination and analyzed for anti-flu and anti-spike-specific antibody titers and for in vitro influenza and SARS-CoV-2 neutralization capacity. Results indicated an increased reactivity in subjects who received both influenza and SARS-CoV-2 vaccinations compared to those who received only the SARS-CoV-2 vaccine, with sustained anti-spike antibody titers up to 12 weeks post-vaccination. Immune response to the influenza vaccine was evaluated, and individuals were stratified as high or low responders. High responders showed increased antibody titers against the SARS-CoV-2 vaccine both after 4 and 12 weeks post-vaccination. Conversely, individuals classified as low responders were less responsive to the SARS-CoV-2 vaccine. These data indicate that both external stimuli, such as influenza vaccination, and the host's intrinsic ability to respond to stimuli play a role in the response to the vaccine.

The use of cell-mediated immunity for the evaluation of influenza vaccines: an upcoming necessity.

Influenza vaccines are a fundamental tool for preventing the disease and reducing its consequences, particularly in specific high-risk groups. In order to be licensed, influenza vaccines have to meet strict criteria established by European Medicines Agency. Although the licensure of influenza vaccines started 65 years ago, Hemagglutination Inhibition and Single Radial Hemolysis are the only serological assays that can ascertain correlates of protection. However, they present evident limitations. The present review focuses on the evaluation of cell-mediated immunity (CMI), which plays an important role in the host immune response in protecting against virus-related illness and in the establishment of long-term immunological memory. Although correlates of protection are not currently available for CMI, it would be advisable to investigate this kind of immunological response for the evaluation of next-generation vaccines.

Epidemiology and prevention of Human Papillomavirus.

Human papillomavirus is the most common sexually transmitted infection, and skin-to-skin genital contact is sufficient for virus transmission. Cervical cancer is the second-most common cancer in women living in less developed regions, with an estimated 445,000 new cases in 2012 and 230,000 deaths every year. Until now, more than 200 types of HPV have been identified, and about 15 types (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, -82) have been shown to cause cervical cancer because they are able to transform infected cells into malignant tumor cells. The bivalent vaccine containing the serotypes 16 and 18 and the quadrivalent vaccine containing the serotypes 16, 18, 6 and 11, have been used in Italy for many years. The European Medicines Agency authorized marketing of the Gardasil 9 vaccine in the European Union on June 2015. Today, Public Health targets the immunization of adolescents of both genders based on new and important scientific evidence for maximum protection from all HPV related pathologies directly preventable with vaccination.

Impact of erythrocyte species on assays for influenza serology.

The influenza viruses have the ability to agglutinate erythrocytes by binding to sialic acid receptors on the host cell. Human influenza viruses preferentially bind to sialic acid linked to galactose by α 2.6 linkage, while avian influenza viruses preferentially bind to sialic acid linked to Gal by α 2.3 linkage. There is a close correlation between the ability of influenza A viruses to agglutinate erythrocytes from different animal species and their receptor specificity. The haemagglutination and haemagglutination inhibition assays are influenced by the species of erythrocytes. To provide an overview of the expression of sialic acid receptors on different erythrocytes, avian (turkey, chicken, pigeon) and mammalian (sheep, horse, human) species have been analysed by flow cytometry. Chicken, turkey and human erythrocytes display both types of linkages. Horse and sheep erythrocytes show almost exclusively α 2.3 Gal linkages, while pigeon erythrocytes express almost exclusively α 2.6 Gal linkages. The erythrocytes from the same avian and mammalian species have been evaluated by haemagglutination and haemagglutination inhibition assays with seasonal and avian strains. Chicken and turkey erythrocytes seem to be the most appropriate for both assays with seasonal influenza strains, in addition to pigeon erythrocytes, particularly for the B strains. In the case of the avian strain, chicken erythrocytes are suitable for haemagglutination assay and horse erythrocytes for haemagglutination inhibition assay. The choice of erythrocytes has a significant impact on the titres measured by both assays.

Epidemiology of herpes simplex virus type 1 and 2 in Italy: a seroprevalence study from 2000 to 2014.

Herpes simplex viruses (HSV) are among the most widespread causative agents of human viral infections. HSV-2 is one of the commonest causes of genital disease, while HSV-1 is associated primarily with orolabial ulceration; however, recent changes in HSV epidemiology showed an increase in genital and neonatal herpes particularly caused by HSV-1. The main purpose of this study was to assess the seroprevalence of HSV-1 and HSV-2 in a random population in Siena (central Italy) in 2000, 2005 and 2013-2014 and in Bari (southern Italy) in 2005. Moreover, a preliminary study was conducted to investigate the spread of HSV infection in a population of pregnant women and infants in Bari in 2003, 2004 and 2005. Human serum samples were tested for the presence of specific anti-HSV-1 and anti-HSV-2 IgG antibodies using a commercially available ELISA test. For the primary purpose, seroprevalence rates observed in Siena were compared over the years sampled and with the seroprevalence rate found in Bari. Results of seroprevalence in Siena show a decreased trend for both viruses, especially in adolescents and young adults; moreover, HSV-2 seroprevalence rates found in the two cities suggest geographical differences. For the secondary purpose, prevalence rates among pregnant women were compared with the seroprevalence found in women of the general population. No significant difference in prevalence rates were found among pregnant women, while results indicate both viruses are a source of infection in infants.

Water and air ozone treatment as an alternative sanitizing technology.

AIMS: We investigated the effectiveness of ozone (aqueous and gaseous) treatment as an alternative sanitizing technology to common conventional disinfectants in reducing the microbial contamination of both water and air. METHODS: Ozone was added for 20 minutes to a well-defined volume of water and air by the system named "Ozonomatic®". The effectiveness of ozonation was determined by counting CFU/ m3 or ml of bacteria present in samples of air or water collected before (T0) and after (T1) the addition of ozone and comparing the microbial load of different bacteria present in ozonized and nonozonized samples. RESULTS: When the ozonisation equipment was located at 30 cm from the surface of the water in the bath tub in which the bacteria investigated were inoculated, the treatment was able to reduce the total microbial load present in the aerosol by 70.4% at a temperature of 36°C for 48 hours. Conversely, at 22°C for 5 days, only a modest decrease (9.1%) was observed. Escherichia coli and Pseudomonas aeruginosa were completely eliminated. A 93.9% reduction was observed for Staphylococcus aureus, followed by Streptococcus faecalis (25.9%). The addition of ozone to water was able to almost eliminate Staphylococcus aureus (98.9% reduction) and also to exert a strong impact on Legionella pneumophila (87.5% reduction). Streptococcus faecalis and Pseudomonas aeruginosa showed a decrease of 64.2% and 57.4%, respectively. Conversely, only a 26.4% reduction was observed for the bacterium Escherichia coli. This study showed that the addition of ozone in the air exerted a modest reduction on microbial load at 36°C, whereas no effect was observed at 22°C. CONCLUSIONS: Aqueous and gaseous ozone treatments were effective against microbial contaminants, reducing the CFU of the microorganisms studied. These results confirm the efficacy of the ozone disinfection treatment of both water and air; particularly, it constitutes an extremely promising alternative, allowing the possibility to reuse contaminated water.

Retrospective Study Evaluating Seroprevalence of Hepatitis E Virus in Blood Donors and in Swine Veterinarians in Italy (2004).

Hepatitis E is an emerging viral disease in developed countries, with sporadic cases occasionally linked to the consumption of raw or undercooked pork, wild boar or deer meat. Cases due to transfusion or transplantation have also been reported. In developed countries, hepatitis E is considered a zoonosis and pig is the main reservoir. In the last few years, several studies conducted in Europe reported variable seroprevalence rates among the general population, ranging between 0.26% and 52.5%. A higher seroprevalence was described among workers who come in contact with pigs. The aim of this retrospective study was to evaluate the seroprevalence of anti-HEV IgG and IgM antibodies in blood donors (170) and in pig veterinarians (83). Archival sera were collected in Italy in 2004. The observed seroprevalence was 9.64% and 8.82% in veterinarians and blood donors, respectively. Overall, only three sera from blood donors were positive for IgM, but no HEV-RNA was detected.

Sea buckthorn bud extract displays activity against cell-cultured Influenza virus.

INTRODUCTION: Vaccines and antiviral drugs are the most widely used methods of preventing or treating Influenza virus infection. The role of sea buckthorn (SBT) bud dry extract as a natural antiviral drug against Influenza was investigated. METHODS: Influenza virus was cultured in the MDCK cell line, with or without SBT bud extract, and virus growth was assessed by HA and TCID50 virus titration in terms of cytopathic effect on cells. Several concentrations of extract were tested, the virus titer being measured on day 4 after infection. RESULTS: After infection, the virus titer in the control sample was calculated to be 2.5 TCID50/ml; treatment with SBT bud extract reduced the virus titer to 2.0 TCID50/ml at 50 µg/ml, while the HA titer was reduced from 1431 (control) to 178. Concentrations lower than 50µg/ml displayed an inhibitory effect in the HA assay, but not in the TCID50 virus titration; however, observation of the viral cultures confirmed a slowdown of viral growth at all concentrations. DISCUSSION: Natural dietary supplements and phytotherapy are a growing market and offer new opportunities for the treatment of several diseases and disorders. These preliminary experiments are the first to show that SBT bud extract is able to reduce the growth of the Influenza A H1N1 virus in vitro at a concentration of 50 µg/ml. This discovery opens up the possibility of using SBT bud extract as a valid weapon against Influenza and, in addition, as the starting-point for the discovery of new drugs.

Thymosin-alpha 1 (Zadaxin) enhances the immunogenicity of an adjuvated pandemic H1N1v influenza vaccine (Focetria) in hemodialyzed patients: a pilot study.

BACKGROUND: Although influenza vaccination is widely recommended for immunosuppressed people, the same immune dysfunction that can increase the risk of contracting influenza might also compromise vaccine effectiveness, especially during pandemics. Clinical data have highlighted the role of adjuvants in improving vaccine efficacy. As uremic patients are especially vulnerable to infections, it is recommended that they should be vaccinated yearly against influenza. This paper presents the results of a pilot clinical trial, conducted in hemodialyzed patients with an adjuvated pandemic H1N1v influenza vaccine alone and combined with Thymosin-alpha 1. METHODS: Subjects were subdivided into 3 treatment groups receiving: the adjuvated pandemic influenza vaccine (Focetria) only (first treatment group), and the Vaccine+Thymosin alpha 1 (Zadaxin) at a dose of 3.2 and 6.4 mg (second and third treatment groups respectively). The immunoresponse was assessed on days 0, 21, 42, 84 and 168 after vaccine administration by means of Hemagglutination Inhibition (HI), Microneutralization (MN) and Single Radial Hemolysis (SRH) assays. The CHMP regards HI as the gold standard test to evaluate the immune response to influenza vaccines before influenza vaccines are licensed. The CHMP criteria are slightly different in adults (18-60-year-old subjects) and the elderly (>60 years old). Indeed, 40% of seroconversion, 70% of subjects seroprotected 21 days after vaccination, and a 2.5-fold increase in GMR (Geometric Mean Ratio) are required in adults, while in the elderly, the corresponding threshold values are: 30%, 60% and a 2-fold increase. All these criteria must be met for the licensing of a pandemic influenza vaccine. Safety evaluation was performed by means of Adverse Event (AE) recording, laboratory assays (hematology and chemistry), electrocardiogram, and assessment of vital signs. RESULTS: Three populations were considered: Intention-To-Treat (ITT) (94 patients), Per Protocol (PP) (82 patients), and Safety population (99 patients). With regard to the Geometric Mean Titer (GMT) and the Geometric Mean Ratio (GMR) of HI on Day 21 in the ITT population, both "Vaccine+Thymosin alpha 1" groups presented better results than the "Vaccine only" group. A large proportion of ITT patients in the two Vaccine+Thymosin alpha 1 groups achieved seroconversion by Day 21. On Day 42, the decrease in the GMT of HI was greater in the Vaccine+Thymosin alpha 1 groups than in the vaccine only group. Similar results were obtained in the PP population. The CHMP criteria were fully met in the groups treated with Vaccine+Thymosin alpha 1. No AE was found to be related to Thymosin alpha 1 nor to the Focetria vaccine. CONCLUSIONS: Although further studies in larger hemodialyzed populations are necessary, it can be concluded that Thymosin alpha 1 enhanced the immunogenicity of the pandemic influenza vaccine used. Moreover, it proved safe and well tolerated, and did not affect hematology or blood-chemistry values.

Collaborative study on influenza vaccine clinical trial serology - part 2: reproducibility study.

A collaborative study was run by the Biological Standardisation Programme (BSP) under the aegis of the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission, to address the issue of the poor standardisation of serological assays used for the evaluation of seasonal influenza vaccines in Europe. The Phase 1 of the study focused on the compliance to Committee for Human Medicinal Products (CHMP) criteria by 6 manufacturers and 5 public laboratories. It confirmed the poor inter-laboratory correlation of haemagglutination inhibition (HI) test results. Phase 2 consisted in a reproducibility study examining the impact of extended method standardisation and the use of reference sera on inter-laboratory variation. Six manufacturers and 5 public laboratories contributed HI results, while the 5 public laboratories also performed single radial haemolysis (SRH) tests on the same sample panels. Results showed that method standardisation failed to significantly improve the inter-laboratory variation. Correction for pre-vaccination titres (Beyer correction) was found to have limited effect to improve the bias constituted by the Protection Rate (PR) criterion. The reasons underlying the difficulty in standardisation of HI and SRH tests are discussed and improved approaches for the compliance testing to CHMP criteria are suggested.

Hepatitis A seroprevalence in Tuscany, Italy.

Information regarding the current seroprevalence of hepatitis A virus (HAV) is useful for the control of HAV infections. The objective of our study was to evaluate the prevalence of anti-HAV antibodies among children (1-5 years old) and young adults (15-20 years old) in Tuscany, in central Italy. A total of 565 sera were collected in three years 1992, 1998 and 2004, equally distributed between the two age groups. The overal proportion of those that tested positive for anti-HAV antibodies was 8.3%. The proportion of immune children (1-5 years old) statistically significantly increased over the years. The percentage of immune subjects among 15-20-year-old young adults varied over the years, not showing a significant statistical trend, nevertheless our findings indicate that in a low endemicity area, adolescents and young adults are becoming increasingly susceptible to HAV infection. On-going monitoring of immunity to HAV is necessary for detecting trends over time.

How much ozone bactericidal activity is compromised by plasma components?

AIMS: Evaluation of bactericidal effect of different concentrations of ozone when used (a) as a gas, or (b) dissolved in saline. The addition of hydrogen peroxide or 4-hydroxynonenal dissolved in saline was also tested, as well as the effect of human plasma. METHODS AND RESULTS: Staphylococcus aureus, methicillin-resistant Staph. aureus (MRSA), and Pseudomonas aeruginosa, suspended in their culture media were tested. While all bacteria suspended in protein-free saline were killed at high ozone concentrations, they survived when as little as 5% human plasma was present. Hydrogen peroxide was 100-fold less active than ozone and needed to remain in contact with bacteria for at least 60 min. 4-hydroxynonenal (2 micromol l(-1)) was inhibitory for proliferation of both Staph. aureus and MRSA, but not for Ps. aeruginosa. CONCLUSIONS: Ozone and the cascade of its derivative products are potent bactericidal agents, but even small amounts of human plasma, hence of hydro- and liposoluble antioxidants, in bacterial suspensions inhibit oxidation and protect bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Any substantial in vivo cytocidal effect of ozone and its derivatives can be excluded. On the other hand, topical and continuous action of various ozone preparations remains valuable in a variety of skin and mucosal infections.

Safety, tolerability and immunogenicity of a mammalian cell-culture-derived influenza vaccine: a sequential Phase I and Phase II clinical trial.

This sequential, observer-blind, randomised, single-centre, combined Phase I and Phase II clinical trial compared the tolerability and immunogenicity of a single intramuscular dose of a novel cell-culture-derived influenza vaccine (CCIV), produced in Madin-Darby canine kidney cells, with a conventional egg-based vaccine. The immunogenicity of both vaccines was assessed by SRH assay, a well-recognized test by EMEA, in compliance with the requirements of the EU Committee for Medicinal Products for Human Use (CHMP). The Phase I part of the trial comprised 40 healthy adults (18-40 years of age); the subsequent Phase II part involved 200 healthy adult (n=80, 18-60 years of age) and elderly (n=120, > or =61 years of age) subjects. Both vaccines showed similar reactogenicity and any solicited local or systemic reactions were mostly mild or moderate. Regarding immunogenicity, both the CCIV and the control vaccine met all of the EU Committee for Medicinal Products for Human Use criteria for influenza vaccines for each strain and in both age groups. In conclusion, the CCIV produced in mammalian cell-culture is as well tolerated and as immunogenic as the control egg-based vaccine in non-elderly and elderly adults.

Serological response to hepatitis B virus vaccine in HIV-infected children in Tanzania.

BACKGROUND: HIV-infected children have a lower seroconversion rate to hepatitis B virus (HBV) immunization than healthy children. Previous studies have produced conflicting results on CD4 cell counts as predictors of vaccine response. No study has evaluated the response rate to HBV vaccination in HIV-infected children receiving highly active antiretroviral therapy (HAART). Our aim was to vaccinate HIV-infected children living in a close community and to investigate the anamnestic response rate after vaccination with its predictors. METHODS: Eighty-four HIV-positive children aged 1-10 years who were negative for antibodies to the HBV core antigen (anti-HBc) completed immunization with three doses of 5 microg HBVAXPRO (Aventis, Milan, Italy). Quantitative testing for antibodies to the HBV surface antigen (anti-HBs) was performed: a seroprotective titre was defined as anti-HBs>10 mUI/mL. RESULTS: After the vaccination, the anti-HBs seroconversion rate was 59.5%. It was higher in individuals in Centers for Disease Control and Prevention (CDC) immune category 1 than in those in CDC categories 2 and 3. Seroconversion was found in 70.8% of HAART-treated and 44.4% of treatment-naïve children. In multivariable models, HAART use and absolute CD4 cell counts were independently associated with probability of seroconversion and with higher anti-HBs titres. CONCLUSIONS: We found a higher seroconversion rate compared with previous studies in HIV-infected children. In children who are candidates to receive antiretroviral therapy, it may be advisable to defer HBV vaccination until after treatment initiation.

A new European perspective of influenza pandemic planning with a particular focus on the role of mammalian cell culture vaccines.

Sixteen EU scientists and doctors were interviewed about pandemic planning using psychometric methods applied to a scientific problem for the first time. Criticism was aimed at countries which have no plan whatsoever, the majority of nations. Many such countries have not invested in scientific infrastructure and public health. Amongst the 15 or so published pandemic plans a lack of detail was identified. Of particular need was investment into avian virus vaccine stocks (H1-15), prepared licenses of vaccine and pre purchase and agreed distribution, investment into stocks of antivirals, antibiotics and masks. Most but not all members of the group predicted a global outbreak within 5 years, most probably starting in SE Asia. However it was recognised that a pandemic could start anywhere in the world which had juxtaposition of young people, chickens, ducks and pigs. Mammalian cell culture production using wild type virus with the production factory at category III levels of security was exemplified. Antivirals would be essential to ameliorate the first wave of infection although significant quantities of cell grown vaccine could be produced if, as in 1918, 1957 and 1968 there is a long period between the first virus isolation and person to person spread. The wider scientific community is more energised than previously for very serious preparations to be in place way before the outbreak begins as this is a major public health problem, completely dwarfing concerns about bioterrorism.

Increased immunogenicity of the MF59-adjuvanted influenza vaccine compared to a conventional subunit vaccine in elderly subjects.

Three-hundred and eight outpatient elderly subjects (> or = 65 years) were randomly assigned to receive the MF59-adjuvanted influenza vaccine (FLU-AD; n = 204) or a conventional subunit influenza vaccine (AGRIPPAL S1; n = 104) in order to compare the safety and immunogenicity of the two vaccines. Although mild pain at the injection site was reported more frequently by subjects immunised with the adjuvanted vaccine, both vaccines were shown to be safe and well tolerated. The adjuvanted vaccine was more immunogenic as indicated by higher post-immunisation geometric mean titres (GMTs) and by higher proportions of subjects with post-immunisation > or = four fold increases of antibody titres or subjects with > or = 1/160 post-immunisation HI titres. These differences, statistically significant for all three strains after immunisation, indicated that, by addition of the MF59 adjuvant emulsion, conventional subunit influenza antigens acquire an enhanced immunogenicity without any clinically significant increase of their reactogenicity.

Impact of routine infant and adolescent hepatitis B vaccination in Tuscany, Central Italy.

BACKGROUND: In Italy routine infant and adolescent immunization against hepatitis B was introduced in 1991. OBJECTIVE: Evaluation of (1) coverage with three doses of hepatitis B vaccine in infants and adolescents; (2) seroconversion to anti-hepatitis B surface antigen antibody (anti-HBs) in adolescents receiving three doses of vaccine; (3) concordance of coverage rates in infants with prevalence of neutralizing antibodies in sera from anonymous children; (4) trend of notified cases of acute hepatitis B. METHODS: A sample of infants and adolescents living in Tuscany was studied during 6 years (1992 through 1997) by matching birth records and immunization certificates. Sera from 139 adolescents who completed the vaccination course and from 159 anonymous children belonging to immunized cohorts (1 to 5 years) were tested with a quantitative anti-HBs assay. Incidence of acute hepatitis B by age was calculated from regional statistics on notified infectious diseases between 1992 and 1996. RESULTS: Overall 10,606/11,164 (95%) infants and 10,599/11,100 (95%) adolescents received 3 doses of vaccine. Seroconversion to anti-HBs was detected in 98% of adolescent vaccinees. Anti-HBs titers > or =10 IU/l were detected in 87% of children. A 49% decline of acute hepatitis B cases was registered between 1992 and 1996 in 15- to 24-year-olds living in Tuscany. No case occurred in vaccinated adolescents. CONCLUSIONS: Coverage against hepatitis B is excellent in cohorts subject to mandatory immunization. If efforts to vaccinate are maintained at these levels, elimination of hepatitis B virus transmission could occur within few decades in Italy.

Chlamydia pneumoniae antibodies and angiographically demonstrated coronary artery disease in a sample population from Italy.

Recent reports suggest an association between Chlamydia pneumoniae and chronic coronary heart disease. This case-control study investigates the relationship between the presence of immunoglobin G (IgG) and immunoglobin A (IgA) when measured by means of microimmunofluorescence (MIF) and angiographically diagnosed coronary disease. Cases (n = 150) were angiography patients with at least one coronary artery lesion occupying at least 50% of the luminal diameter. Controls (n = 49) were angiography patients with no detectable signs of coronary artery disease and patients (n = 56) without signs or symptoms of coronary disease and with normal ECG results. No significant differences were revealed between the seroprevalence of IgG and IaA and geometric mean titers (GMT) as measured in cases and controls. When cases were compared with controls whose angiographic results were normal, after adjusting for established risk factors (cholesterol, smoking, hypertension, diabetes, age, gender and family history), the estimated risk of coronary artery disease was 0.79 (95% confidence interval (C.I.), 0.31-1.99) for the presence of IgG and was 0.94 (95 C.I., 0.37-2.39) for IgA. When cases were compared with controls with normal ECG results, the adjusted odds ratio (O.R.) for coronary artery disease was 1.17 (95%, C.I., 0.52-2.62) for the presence of IgG and 0.82 195% C.I., 0.36-1.86) for the presence of IgA. These results do not support an association between C. pneumoniae infection and coronary disease.

Morpho-structural variations of bacterial spores after treatment in steam vacuum assisted autoclave.

This study intended to verify, through microbiological techniques and TEM investigations, the killing of bacterial spores after treatment in steam autoclave, and to propose strictly morphological considerations about the target of this sterilisation process. Autoclave is the most common device for sterilising instruments in order to prevent cross infections in dental offices. The autoclave efficiency has been improved in the last years and part of this improvement is related to both a better and more correct use of the autoclave system and to the technological innovations introduced in the last generation of devices. However, associations as ADA or CDC suggest to regularly verify the process of 'autoclaving' through biological indicators (BI). The most commonly used BI are made of spores strips or suspensions of Bacillus Subtilis (pb 168) and Bacillus Stearothermophilus (ATCC 10149). They visually prove, changing colours on enzymatic base, the death of micro-organism and if the physical parameters, necessary for sterilisation, have been achieved. These two strains of endospore-forming bacteria were processed and prepared following two different techniques: Karnovsky fixed and epon embedded--phosphotungstic acid fixed for direct observation. The kind and the extent of analysed modifications are extremely various: from deep lacerations, which changed the spore structure, to little clefts which let the cytoplasm go out.