Microbiological surveillance of endoscopes and implications for current reprocessing procedures adopted by an Italian teaching hospital.

BACKGROUND: Hospital acquired infections have been associated with the contamination of flexible endoscopes caused by a failure of the reprocessing procedure. Microbiological surveillance of endoscope reprocessing is valuable for assessing contamination by pathogens. The aim of this study is to evaluate microbiological contamination of endoscopes after reprocessing, and the involvement of reprocessing procedures adopted in endoscopy units of an Italian teaching-hospital. METHODS: The study was carried out, on several dates in 2014, in 11 endoscopic operation units equipped with 100 endoscopes (18 bronchoscopes, 41 gastroduodenoscopes, 29 colonoscopes, 12 laryngoscopes) and 9 Automated Endoscope Reprocessors. Presence/absence of common pathogens and indicator micro-organisms (including multi-drug resistant bacteria) and Total Microbiological Count (TMC) were obtained from the biopsy channels of endoscopes after reprocessing, from final rinse water of automated endoscope reprocessors and from tap water applying standard microbiological culture methods. Following the European Guidelines for quality assurance in reprocessing, the post-reprocessing criteria were "absence of indicator micro-organisms and absence of TMC in samples obtained from endoscopes' channels". RESULTS: A total of 180 samples were collected (143 endoscopes, 25 Automated Endoscope Reprocessors and 12 water supply). Compliance to the European Guidelines was achieved for 112 out of the 180 (62.2%) samples analyzed. Presence of indicator micro-organisms (mainly Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and other Gram-negative non-fermenting bacteria) was found in 51 out of 143 endoscopes (35.7%). Multi-drug resistant bacteria were also found. Presence of pathogen micro-organisms was statistically associated with the increase of TMC level, but not with time after reprocessing. CONCLUSION: The study provides information about the microbiological quality of endoscope reprocessing procedures adopted by different endoscopic operation units. The high prevalence of contaminated endoscopes provides evidence of the need to improve the quality of reprocessing.

Multimodal surveillance of healthcare associated infections in an intensive care unit of a large teaching hospital

BACKGROUND: Healthcare-associated infections (HAIs), or nosocomial infections, represent a significant burden in terms of mortality, morbidity, length of stay and costs for patients hospitalized in intensive care units (ICUs). Surveillance systems are recommended by national and international institutions to gather data on HAIs in order to develop and evaluate interventions that reduce the risk of HAIs. STUDY DESIGN: Here we describe the methodology and the results of the surveillance system implemented in the ICU of the Policlinico Umberto I, a large teaching hospital in Rome, from April 2016 to October 2018. METHODS: The multimodal infection surveillance system integrates four different approaches: i) active surveillance of inpatients; ii) environmental microbiological surveillance; iii) surveillance of isolated microorganisms; and iv) behavioral surveillance of healthcare personnel. Data were collected on catheter-related bloodstream infections, ventilation-associated pneumonia, catheter-associated urinary tract infections and primary bloodstream infections that developed in patients after 48 h in the ICU. For environmental surveillance 14 points were selected for sampling (i.e. bed edges, medication carts, PC keyboards, sink faucets). The system of active surveillance of HAIs also included surveillance of microorganisms, consisting of the molecular genotyping of bacterial isolates by pulsed-field gel electrophoresis (PFGE). From 1 November 2016, monitoring of compliance with guidelines for hand hygiene (HH) and proper glove or gown use by healthcare personnel was included in the surveillance system. After the first six months (baseline phase), a multimodal intervention to improve adherence to guidelines by healthcare personnel was conducted with the ICU staff. RESULTS: Overall, 773 patients were included in the active surveillance. The overall incidence rate of device-related HAIs was 14.1 (95% CI: 12.2-16.3) per 1000 patient-days. The monthly device-related HAI incident rate showed a decreasing trend over time, with peaks of incidence becoming progressively lower. The most common bacterial isolates were Klebsiella pneumoniae (20.7%), Acinetobacter baumannii (17.2%), Pseudomonas aeruginosa (13.4%) and Staphylococcus aureus (5.4%). Acinetobacter baumannii and Klebsiella pneumoniae showed the highest proportion of isolates with a multidrug-resistant profile. A total of 819 environmental samples were collected, from which 305 bacterial isolates were retrieved. The most frequent bacterial isolates were Acinetobacter baumannii (27.2%), Staphylococcus aureus (12.1%), Enterococcus faecalis (11.1%), Klebsiella pneumoniae (5.2%) and Pseudomonas aeruginosa (4.7%). All Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae environmental isolates were at least multidrug-resistant. Genotyping showed a limited number of major PFGE patterns for both clinical and environmental isolates of Klebsiella pneumoniae and Acinetobacter baumannii. Behavioral compliance rates significantly improved from baseline to post-intervention phase. CONCLUSIONS: By integrating information gathered from active surveillance, environmental microbiological surveillance, surveillance of bacterial isolates and behavioral surveillance of healthcare personnel, the multimodal infection surveillance system returned a precise and detailed view of the infectious risk and microbial ecology of the ICU.

Evaluation of timing of re-appearance of VBNC Legionella for risk assessment in hospital water distribution systems.

BACKGROUND: In this study we estimated the presence of Legionella species, viable but non-culturable (VBNC), in hospital water networks. We also evaluated the time and load of Legionella appearance in samples found negative using the standard culture method. METHODS: A total of 42 samples was obtained from the tap water of five hospital buildings. The samples were tested for Legionella by the standard culture method and were monitored for up to 12 months for the appearance of VBNC Legionella. RESULTS: All the 42 samples were negative at the time of collection. Seven of the 42 samples (17.0%) became positive for Legionella at different times of monitoring. The time to the appearance of VBNC Legionella was extremely variable, from 15 days to 9 months from sampling. The most frequent Legionella species observed were Legionella spp and L. anisa and only in one sample L. pneumophila srg.1. CONCLUSIONS: Our study confirms the presence of VBNC Legionella in samples resulting negative using the standard culture method and highlights the different time to its appearance that can occur several months after sampling. The results are important for risk assessment and risk management of engineered water systems.

Non Tuberculous Cutaneous Mycobacteriosis in a primary school in Rome: epidemiological and microbiological investigation.

During the school years 2009-2010 and 2010-2011 a total of 25 cases of Non Tuberculous Cutaneous Mycobacteriosis (NTCM) were notified in children attending the same school with a swimming pool in Rome. Environmental microbiological and epidemiological investigations (only for suspected outbreaks in 2009-2010) were conducted. We screened students with skin lesions, and environmental samples were collected from the school area and the swimming pool. During the school year 2009-10 18 cases were clinically identified among 514 primary school children (3.50%) and all cases attended the swimming pool. Only 2 out of 18 cultures were positive for Mycobacterium chelonae complex (Group III, M. abscessus). Attack Rate for swimming pool use was 13,10% (17/130), with a Relative Risk 54,70 (95% CI: 9,4 - ∞). In February 2011 additional 7 cases of cutaneous NTM among children - who attended the same primary school and swimming pool were notified to the local public health authority followed by environmental microbiological investigation. Environmental samples were positive for NTM but not for M. abscessus. Mycobacteria are not included in water-quality criteria in Italy for this reason it is important to collect evidences of NTM cases caused by these infrequent pathogens, to be able to perform rapid risk assessment and to identify the best practices in prevention and management of such a risk.

Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

In vivo effect of an immunostimulating bacterial lysate on human B lymphocytes.

The aim of the present study is to investigate in humans the mechanism by which the oral vaccine Polyvalent Mechanical Bacterial Lysate (PMBL) can rapidly mobilize specific immune response and evaluate the efficacy of its immunostimulating activity in preventing recurrent infections of the upper respiratory tract (URTIs) in a group of patients with a medical history of URTI recurrence. Patients received, by sublingual route, PBML, an immunostimulating lysate obtained by mechanical lysis of the most common bacteria responsible for upper respiratory tract infections. The treatment was administered for 10 consecutive days/month for 3 consecutive months. After the end of the treatment period the patients were followed up for an additional 3 months. The frequency of IgM memory B cells and the expression of the activation marker CD25 in peripheral blood lymphocytes were measured using the flow cytometric method before the start and at days 30 and 90 of the treatment cycle. To correlate clinical results to immunological parameters, the patients were monitored at different time-points during the treatment and at the end of follow-up period. The results showed that PMBL exerts a therapeutic and preventing effect in acute and recurrent infections of the upper respiratory tract and that this effect correlated with the activation and enhancement of both IgM memory B lymphocytes (CD24+/CD27+ cells) and IL2 receptor-expressing lymphocytes (CD25+ cells) involved either in humoral or cellular immunity.

Effect of resveratrol on proliferation and telomerase activity of human colon cancer cells in vitro.

A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.

Effect of morphine on cell-mediated immune responses of human lymphocytes against allogeneic malignant cells.

Opioid drugs, including morphine, are largely used as pain control in cancer patients at different stages of neoplastic growth and progression. Therefore, the possible influence of these drugs on host immunity appears to be of considerable interest. We have examined in vitro the effect of morphine on the generation of human cytotoxic T lymphocytes (CTL) against HTLV-I induced T-cell leukemia cells (MT-2 line). The results show that the drug, at graded concentrations (from 3 pg/ml to 32 microg/ml), that include those detectable in treated patients, enhances CTL activity whereas natural killer cell activity was unaffected. The enhancing effect is particularly evident when morphine was present at the onset of lymphocyte/MT-2 co-culture. On the contrary, the drug was ineffective when added on the last day of co-culture, thus indicating that morphine operates during the generation phase of CTL, but not on mature CTL. Flow cytometric analysis of intracellular cytokine expression showed that morphine increases the percentage of interferon gamma-producing CD8+ T cells in co-culture assay. Collectively, these results suggest that in our experimental model morphine enhances CTL responses by directly affecting the induction phase of T-dependent cell-mediated immunity.

Preclinical studies on detection of circulating melanoma cells in patients: telomerase as a recognition marker of malignancy.

Preclinical studies based on a "simulation design", were performed with cultured melanoma cells prelabeled with 51Cr, added to normal blood and subjected to separation and recognition steps. Mononuclear cells (MNC) were isolated on ficollhypaque gradient, and melanoma cells were separated from lymphocytes using anti-CD45 immunomagnetic beads. Malignant cells were then recognized by measuring telomerase activity (TRAP and TRAP-ELISA assays). It was found that: (a)recovery of prelabeled cells present in MNC did not exceed 75%; (b) further recovery of prelabeled cells after separation from lymphocytes did not exceed 68%. Therefore, the overall recovery of prelabeled cells did not exceed 48%; (c) the entire procedure was able to reliably detect as few as 30 malignant cells added to normal blood, providing a telomerase signal significantly higher than that found in absence of melanoma cells. These results furnish the technical bases for developing a tumor detection assay in the blood of melanoma patients.

Drug-induced modulation of carcinoembryonic antigen (CEA) expression in neoplastic cells from a patient with rectal cancer.

Treatment with 5-fluorouracil (5-FU) or recombinant interferon-gamma (IFN), alone or in combination, was found to increase carcinoembryonic antigen (CEA) expression in several carcinoma cell lines. In this study we examined the in vitro effect of these agents on CEA expression of tumor cells, obtained from a patient operated for rectal cancer. The results showed that exposure of cancer cells to 5-FU or to IFN resulted in increased CEA levels in terms of percentage of CEA-positive cells and mean fluorescence values, as indicated by FACS analysis. However, drug combination did not induce CEA expression higher than that provided by single agents alone. Treatment with 5-FU or with IFN produced a reduction of the total number of viable cells. Moreover, Western blot analysis revealed that exposure of cancer cells to each drug was followed by a substantial increase of the total cellular CEA content. On the contrary, 5-FU in combination with IFN did not increase the expression of the antigen more than that obtained by single agents. Noteworthy, exposure of CEA-negative cells from adjacent normal rectal tissue to both agents alone or in combination, did not result in CEA induction. In conclusion, the present results suggest new approaches aimed at (a) increasing the sensitivity of diagnostic procedures based on detection of CEA-positive tumor cells; (b) facilitating the recognition of CEA-positive cancer cells by immune responses induced by anti-CEA peptide vaccines.