A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid.

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

How to manage the biological risk in a dental clinic: current and future perspectives.

Dental personnel (DP) may be exposed to pathogens during dental treatment, either through contact contaminated equipment, or with blood and respiratory secretion. On the other hand, health care professionals are constantly exposed to pathogens and opportunists in their work environment. Consequently, the dental healthcare environment is connected with the risk of exposure to biological agents both for patients and dental workers, and involves a wide number of microorganisms that can be present in biological matrices (gingival fluids, saliva, blood), contaminated and/or non-sanitized surfaces, water used in the dental unit, or emitted by patients suffering or carrier of a transmissible disease. The main determinants of exposure to biological agents in dentistry are related, therefore, to several factors, such as the lack in the application of disinfection/sterilization procedures for surfaces, reusable tools, water, etc.; the lack in the use of protective equipment by workers; an insufficient or inefficient training of personnel; the use of non-targeted, too diluted, or expired biocides. Therefore, each single patient needs to be treated as a potential communicable infectious disease carrier and each case must receive high level of attention in compliance with preventive and hygiene standards, following disinfection and sterilization procedures, and always wearing personal protective equipment. The goal of this article was to discuss on the infection risks related to dental practice both for patients and workers, and to evaluate the state of the art and future perspectives, with particular attention to disinfection procedures, for occupational biological hazards and HAIs prevention in this setting.

[Antibiotic prophylaxis and therapy of pancreatonecrosis in multifield surgical hospital].

The article is based on an analysis of results of complex treatment of 497 patients with pancreatonecrosis at the period from 2010 to 2014. All patients were admitted to the surgical departments of Republican hospital No 2 and Centre of Emergency Medicine of Republic of Sakha (Yakutia). The investigation allowed adaptation and development of antibiotic prophylaxis and therapy management in pancreatonecrosis in multifield surgical hospital. More than 80% of patients avoided a contamination of necrotic destruction zones. The level of lethality was reduced in group of patients with infectious complications of pancreatonecrosis from 45.8% to 37.7%.

Assessment of the usefulness of performing bacterial identification and antimicrobial susceptibility testing 24 h a day in a clinical microbiology laboratory.

The impact of inoculating agar media with positive blood cultures and of performing bacterial identification and antimicrobial susceptibility testing (AST) for positive urine cultures, blood cultures and certain fluid cultures after day hours (night service (NS)) was evaluated in a clinical microbiology laboratory. The impact of the NS was assessed in terms of decreases in the delays from the time of sampling to the time at which results became available and of the consequences for patient management and antimicrobial treatment. Two major benefits were obtained: initiation of earlier appropriate treatment, and change to a reduced-spectrum but still efficient regimen. The hours of laboratory testing and the availability and transmission of results to the clinical staff were recorded. Concurrently, these hours were estimated as though laboratory tests had been performed in the absence of NS. Reductions in delay were defined as the differences between the hours actually spent and the estimated hours. Economic concerns were also considered. Overall, 430 samples for which an identification and/or AST were performed during the NS were included in the study. The NS led to the implementation of earlier appropriate therapy in 97 cases (22.6%), and to the change to reduced-spectrum but still efficient regimens in 23 additional cases (5.3%). In conclusion, there appeared to be benefits from a system providing bacterial identification and AST overnight, but a study of the cost-effectiveness of the NS would be useful to back up this observation.

Percutaneous and mucocutaneous exposures in nursing students: an Italian observational study.

PURPOSE: To investigate occupational exposures to biological material potentially infected by blood-borne viruses in nursing student population during the course years. DESIGN AND METHODS: An observational retrospective study was designed. Data were collected in May 2007. Two-thousand-two-hundred-fifteen nursing students from the 3 years of degree course were enrolled in the four Italian universities. A structured questionnaire was constructed and was given out unannounced to nursing students in four universities on a randomly chosen day. The likelihood of association between nursing student exposure and certain assumed risk factors was measured. FINDINGS: The exposure risk is associated with each study year of nursing students. Specifically, the probability of accidental exposure is reduced significantly with the increase of clinical skills during the training period. The risk for exposure in the 1st year students appears significantly higher than in those of the next years (odds ratio [OR] 1.465; 95% confidence interval [CI] 1.105-1.943). Data highlighted a gradual increase of bio-safety knowledge in nursing students from the 1st to the 3rd years of study. However, a statistically significant association exists only between awareness of a correct use of gloves and exposure risk (OR 0.435; 95%CI 0.227-0.834). Mucocutaneous exposures are more frequent than percutaneous exposures (62.2%), and the hollow-bore needle is the device most often involved. In 42.5% of cases, accidental exposures occurred when nursing students are working alone in a medical ward or surgery area. CONCLUSIONS: During their clinical training, nursing students can encounter a real risk for percutaneous and mucocutaneous exposures to blood potentially infected with blood-borne viruses. However, this risk is reduced with an increase in clinical skills. CLINICAL RELEVANCE: Results show that some new strategies are necessary for exposure risk reduction such as development of simulation laboratories for nursing practice and the adequate presence of tutors in clinical training education.

[Assessment of a quantitative PCR method for clinical diagnosis of imported histoplasmosis]. / Evaluación de una técnica de PCR cuantitativa para el diagnóstico clínico de la histoplasmosis importada.

OBJECTIVE: Evaluation of the usefulness of a quantitative real-time polymerase chain reaction-based (RT-PCR) technique for clinical diagnosis of histoplasmosis. METHODS: Primers and probes were designed on the basis of sequences from the ITS regions of ribosomal DNA of 20 clinical strains of Histoplasma capsulatum. LightCycler procedures (Roche Applied Science) were used with probes marked by fluorescence resonance energy transfer (FRET). Reproducibility, sensitivity, and specificity were analyzed. In addition, an internal control was designed to identify false negative results by PCR inhibition. The RT-PCR assay was tested in 22 clinical samples from 14 patients with proven histoplasmosis. In addition, 30 samples from patients with febrile neutropenia or mycoses other than histoplasmosis, and from healthy volunteers were analyzed as controls. RESULTS: The limit of detection of the assay was 1 fg of genomic DNA per microl of sample. The PCR-based technique was reproducible and highly specific. Positive results were obtained in 11/14 (78.6%) patients and in 17/22 (77.3%) clinical samples. RT-PCR was positive in 100% of respiratory secretions and bone marrow samples, but only 70% of sera (p < 0.01). Mean fungal DNA value was 23.1 fg/microl in serum and 4.85 x 10(3) fg/microl in respiratory and bone marrow samples. RT-PCR results were positive in serum from three HIV patients for which antibody detection by immunodiffusion was negative. Specificity was 100%, since PCR results were negative for all the control samples. CONCLUSION: Thes RT-PCR technique is a sensitive, specific method for early diagnosis of histoplasmosis, particularly when respiratory secretions or bone marrow samples are analyzed. The reliability is lower in serum, but it can be used as an additional, complementary technique to culture and serology in HIV patients.

[Infectious disease assessment in solid organ transplant candidates]. / Evaluación de las enfermedades infecciosas en el candidato a un trasplante de órgano sólido.

BACKGROUND: Infections are one of the leading causes of morbidity and mortality in solid organ transplant recipients. Many of these infections can be prevented or their effects reduced by accurate preoperative evaluation of risk in the transplantation candidate. The elaboration of guidelines using a multidisciplinary approach can help to establish more rational diagnostic, therapeutic, and preventive measures in this setting. OBJECTIVE: To elaborate guidelines for the assessment of infectious diseases in transplant candidates, based on consensus among professionals in this field and under the auspices of Spanish scientific societies. MATERIAL AND METHODS: The Infections in Transplant Patients Group (GESITRA), within the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), appointed a panel of four microbiologists and infectious disease specialists to elaborate a draft of the guidelines, which was subsequently approved by all the members of this Group. With the support of the National Transplant Organization, the GESITRA document was then presented to various professionals in this field so they could provide their comments and suggestions. RESULTS: The final document, after incorporation of all appropriate modifications and suggestions, is presented herein. The guidelines focus on the following: a) diagnosis of active and latent infections, and identification of risk factors in the candidate; b) recommended approach for infections diagnosed during the evaluation process and their corresponding treatment; c) definition of infections contraindicating transplantation; and d) prevention of post-transplantation infectious complications by systematic vaccination and instruction on preventive measures provided to patients, their relatives, and persons living with them. DISCUSSION: Using a multidisciplinary approach that included the efforts of experts in the field and the collaboration of scientific societies, a comprehensive document containing specific recommendations was elaborated. Systematic review of the guidelines in the future is considered worthwhile by both the authors and supporters of this document.

[Clinical evaluation of the Amplified Mycobacterium Tuberculosis Direct 2 test]. / Valoración clínica de la prueba Amplified Mycobacterium tuberculosis Direct 2 (AMTD-2, GenProbe) en el diagnóstico rápido de la tuberculosis.

OBJECTIVE: To evaluate the performance of the Amplified Mycobacterium tuberculosis Direct Test 2- Gen Probe (AMTD- 2) for direct detection of Mycobacterium tuberculosis in smear-negative samples. PATIENTS AND METHODS: From January to December 1999, 683 specimens, 333 respiratory and 350 non-respiratory ones collected from 457 patients, were included in the study. All the samples of HIV-positive patients, the respiratory samples from patients suspected of having pulmonary tuberculosis (at least two by patient) and all non-respiratory samples were included. As diagnosis method of reference, the culture isolation was considered. Clinical data were analyzed in case of discrepant results and clinical diagnosis was considered the reference criteria. The technique was performed once a week. RESULTS: The sensitivity, specificity, and positive and negative predictive values of this assay were 58.9%, 93.9%, 37.1% and 97.4% respectively related to the standard culture. When referred to clinical diagnosis of active tuberculosis, these values improved to 70.4%, 97.7%, 73.1% and 96.8% respectively (in respiratory samples were 67.6%, 98.6%, 86.2% and 95.9% and in nonrespiratory ones 76.5%, 96.9%, 56.5% and 98.7% respectively). The mean time of diagnosis by culture and by AMTD-2 were 20.3 days (range 10-63) and 5.7 days (range 2-20) respectively. DISCUSSION: It is concluded that AMTD-2 is a rapid diagnosis method when clinical data are sugestive with active tuberculosis. However, due to the low positive predictive value, it would be convenient to obtain successive samples to confirm the result in patients without clinical evidence of tuberculosis.

[Evaluation of the efficacy of a program to control nosocomial spread of methicillin-resistant Staphylococcus aureus]. / Valutazione dell'efficacia di un sistema di controllo della diffusione intraospedaliera di Staphylococcus aureus meticillino-resistente.

OBJECTIVE: To evaluate the efficacy of a program to control nosocomial spread of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Analysis of the incidence of infection and contamination due to MRSA in patients admitted to the hospital of Cremona 6 months before and 3 years after the introduction of the guidelines (July 1997). RESULTS: During the 42 months of the study period, on 80705 admissions, 511 cases of MRSA contamination/infection were identified, the incidence being 0.57 cases per 100 admissions. The infection rate dropped from 0.34 (IC95%: 0.25-0.45) in the first 6 months of the study, before the introduction of guidelines, to 0.17 (IC95%: 0.14-0.20) in the following 3 years (p=0.01). Severe infection decreased from 0.18 to 0.1 per 100 admissions, with a 44% decrease (p=0.058), while mild infections diminished from 0.16 to 0.07 per 100 admissions (p=0.045). Methicillin resistance among nosocomial isolates of Staphylococcus aureus was reduced from 53 % to 35 % (p<0.0001). CONCLUSIONS: The introduction of a program to control the nosocomial spread of MRSA proved effective in reducing both the incidence of infection and the methicillin-resistance of Staphylococcus aureus isolates. The cost effectiveness of the program seems very favourable.

Comparison of BACTEC plus Aerobic/F, Anaerobic/F, Peds Plus/F, and Lytic/F media with and without fastidious organism supplement to conventional methods for culture of sterile body fluids.

We compared the BACTEC 9240 continuous-read instrument using Peds Plus/F, Lytic/F, Aerobic/F, and Anaerobic/F media (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) with and without fastidious organism supplement to conventional centrifugation preparation and plating for the recovery and speed of detection of microorganisms. A total of 908 sterile body fluid specimens were collected and processed, yielding 116 (13%) positive cultures. Of the 80 isolates considered clinically significant, 48 (60%) were recovered by both the BACTEC system and conventional culture, whereas 32 (40%) were recovered by BACTEC only. No clinically significant isolates were recovered only by conventional culture methods. The time to detection for isolates recovered from both sets was faster for BACTEC. It was found that BACTEC, with or without the addition of fastidious organisms supplement, exhibited improved sensitivity for the recovery of microorganisms.

[Detection of Mycobacterium tuberculosis in clinical specimens other than sputum by the Mycobacterium Tuberculosis Direct Test (MTD)--assessment of sample preparation methods and clinical evaluation].

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD) has been widely used as a rapid test for the identification of Mycobacterium tuberculosis complex in clinical samples, and several research groups have verified its clinical usefulness. However, most of the specimens they tested were sputum, and there have been few reports on other specimens. In particular, there have been no reports on assessments of methods of preparing samples other than sputum for the MTD. We assessed methods of preparing samples other than sputum and the influence of a local anesthetic and an anticoagulant that may be present in samples, and also evaluated the MTD as a means of detecting M. tuberculosis in pleural fluid, bronchial lavage cerebrospinal fluid, urine and ascitic fluid. 1. Assessment of three sample preparation methods, i.e., the NALC-NaOH method GuSCN-Diatom nucleic acid extraction method, and the ultrasonication method, revealed that the combination of the NALC-NaOH method and the ultrasonication method, widely used to prepare sputum samples, is also a valid method of preparing other samples. 2. The local anesthetic and the anticoagulant used clinically and remained in specimens did not affect the results of the MTD. 3. Seven (36.8%) of 19 pleural fluid samples from patients diagnosed as tuberculous pleurisy were positive of M. tuberculosis by the MTD, while five (27.8%) of 18 pleural fluid samples cultured for bacteria were positive for M. tuberculosis complex. None of the 20 pleural fluid samples from patients diagnosed as non-tuberculous pleurisy were positive for M. tuberculosis complex either by MTD or culture. 4. Eight (32.0%) of 25 bronchial lavage samples from patients diagnosed as pulmonary tuberculosis were positive for M. tuberculosis complex by the MTD, while 3 (12.0%) were positive by culture. None of the 18 bronchial lavage samples from patients diagnosed as non-tuberculous disease were positive for M. tuberculosis complex either by the MTD or culture. Based on these results, it is concluded that the MTD is a very useful method of detecting M. tuberculosis in clinical samples other than sputum because it is more sensitive than culture on Ogawa's egg medium in detecting M. tuberculosis complex in pleural fluid samples, bronchial lavage samples, and so on, with the same preparation method as used for sputum.