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PubMed2003

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death.

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.

Animals↗
PubMed2004

Control of Burkholderia (Pseudomonas) bacteraemia in the intensive care and paediatric units.

OBJECTIVE: Investigation and control of nosocomial bacteraemia caused by Burkholderia (Pseudomonas) cepacia in the Intensive Care and Paediatric Units of a general care hospital. DESIGN: A cross-sectional analytical study. PLACE AND DURATION OF STUDY: Departments of Pathology, Intensive Care and Paediatrics, Kahota Research Laboratory Hospital, Islamabad from January 1998 to June 2002. SUBJECTS AND METHODS: Blood cultures from patients admitted to Intensive Care Unit and Paediatric Ward were inoculated into brain heart infusion broth and incubated for upto 10 days. Any Gram-negative rods isolated were characterized by API-20E. Environmental samples were inoculated on blood and MacConkey's agars and isolates, if any, were identified as above. Intensive intervention in the form of hand washing, strict adherence to aseptic practices and standard sterilization techniques were adopted and then cultures were again carried out with similar methodology. RESULTS: Cultures yielded 58 strains of Burkholderia cepacia, 52 from blood cultures and 6 from hospital environment, including 1 from the washbasin of the ICU. Thirty- four of these were isolated before intervention measures were adopted, mainly during 1998. Findings suggested a strong probability of nosocomial transmission, with washbasin as the common source. After a lapse of about a year, B. cepacia infection re-emerged in a sporadic form but remaining confined to paediatric unit. Only 18 isolates were yielded over the next two and-a-half years. CONCLUSION: The intervention measures for Burkholderia bacteraemia within the hospital, proved effective in stopping the nosocomial transmission leading to disappearance of B. cepacia from blood cultures. We emphasize the crucial role of hand hygienic practices in the hospital setting, especially in critical care units.

Bacteremia↗
PubMed1995

HIV type 1 intraperitoneal infection of rabbits permits early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA from peripheral blood mononuclear cells.

The aim of this study is the development of an animal model useful for studying HIV-1 pathogenesis, candidate vaccines, and antiviral drugs. Aseptic thioglycolate peritonitis was induced in six rabbits. After 4 days, four rabbits were infected with 1 ml of HIV-1 stock containing 100 times the MID50. Blood samples were collected every 2 weeks for 8 months. Serum antibodies were tested by ELISA, using as antigen the recombinant protein p24; synthetic peptides of highly conserved regions of p31, gp41, and gp120; and a synthetic peptide of gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Furthermore, neutralizing antibodies were tested by a microscale neutralization assay. Proviral DNA was detected by PCR, and virus isolation was performed by a cocultivation technique using primary rabbit peripheral blood mononuclear cells (PBMCs). All infected rabbits produced antibodies to HIV-1 proteins within 2 weeks and up to 8 months after virus infection. Serum antibodies were directed against the Env (gp120 and gp41), Gag (p24), and Pol (p31) proteins and against two synthetic peptides whose sequence corresponds to gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Neutralizing antibodies were also detected in the sera of infected animals. Proviral DNA was detected in PBMCs by PCR within 4 weeks and up to 8 months after HIV-1 infection. HIV-1 was also isolated from PBMCs of infected animals at 30, 60, and 120 days after infection. Results obtained indicate that HIV-1 intraperitoneal infection of the rabbit permits the early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA sequences from PBMCs.

Animals↗
PubMed1998

Storage of extemporaneously prepared ophthalmic antimicrobial solutions.

The feasibility of long-term storage of commonly used ophthalmic antimicrobial solutions was studied. Solutions of tobramycin 15 mg/mL (as the sulfate salt), cefazolin 33 mg/mL (as the sodium salt), and vancomycin 50 mg/mL (as the hydrochloride salt), each in artificial tears, were prepared with aseptic technique. Ten 15-mL portions of each solution were prepared; five of each were stored at 4 degrees C and the other five at 25 degrees C. Samples of each portion were tested before storage and 7, 14, 21, and 28 days after preparation for osmolality, pH, and antimicrobial activity. For the tobramycin solution there were no differences in osmolality or the zone of inhibition associated with temperature or time. The pH dropped between days 0 and 7 at both temperatures. For the cefazolin solution there were no differences in osmolality associated with temperature or time. The pH was higher in portions stored at 25 degrees C than at 4 degrees C and increased over time in portions stored at either temperature. The zone of inhibition was larger for portions stored at 4 degrees C than at 25 degrees C but did not change over time. For the vancomycin solution there were no differences in osmolality associated with temperature or time. The pH did not differ between portions stored at 4 and 25 degrees C but dropped sharply at both temperatures between days 0 and 7. The zone of inhibition did not differ with temperature or time. The tobramycin solution could be stored for 28 days at room temperature and the cefazolin solution for 28 days under refrigeration. The pH of the vancomycin solution changed too quickly for storage to be recommended.

Anti-Bacterial Agents↗
PubMedDate not supplied

The impact of catheter-restricted filling with cefotaxime and heparin on the lifespan of temporary hemodialysis catheters: a case controlled study.

BACKGROUND: Reduction in the rates of major complications such as infection and thrombosis that limit the lifespan of hemodialysis (HD) catheters could conceivably lead to improved survival of "temporary" non-tunneled HD catheters (NTCs). This study was designed to evaluate the impact of the "locking"' of a broad-spectrum antibiotic-cefotaxime with heparin, on the incidence of catheter thrombosis, catheter-related bloodstream infections (CRBSI) and the NTC lifespan. METHODS: This prospective study included 208 (109 males and 99 females) end-stage renal disease (ESRD) patients of diverse etiology enrolled for long-term HD from July 2002 to June 2003 at our tertiary care hospital. Those patients requiring NTC insertion for the maintenance or commencement of HD were eligible for the study. Cefotaxime-heparin "lock" solutions were freshly prepared by dissolving sterile cefotaxime sodium powder for injection directly in the heparin sodium to reach a concentration of 10 mg/mL for cefotaxime and 5000 U/mL for heparin. A final volume of 5 mL was prepared in a syringe using aseptic precautions to fill 1.3 mL in the venous and 1.2 mL in the arterial lumen of the catheter with a combined volume of approximately 2.5 mL. Blood samples were collected for culture and sensitivity, simultaneously, from the catheter hub and the peripheral vein, on clinical suspicion of CRBSI. Thrombosis was defined as the inability to use the catheter at a blood flow of 200 ml/min, which did not respond to catheter repositioning and/or intraluminal thrombolysis. RESULTS: An overall relative risk reduction (RRR) of 56.5% for catheter thrombosis and separately for all types of NTCs (femoral (FC) - 52.7%, subclavian (SC) - 55.9%, and internal jugular (IJC) - 53.7%), were observed in group II having a cefotaxime-heparin lock solution. A lower CRBSI incidence (1.65 vs. 3.13/1000 catheter days) compared with group I, leading to a RRR of 50.5% was also recorded in group II. Significantly higher percentage catheter survival rates at 7, 14 and at 28 days for FC and at 14, 28 and 56 days for SC and IJC were observed among patients in group II in comparison to group I. CONCLUSIONS: Cefotaxime-heparin locks led to a significant reduction in catheter thrombosis and CRBSI incidence. The enhanced NTC lifespan thus achieved could help physicians in better assessment of the patient's vasculature prior to the placement of permanent vascular access.

Adolescent↗
PubMed1986

[Mycoplasma pneumoniae infection in childhood respiratory pathology. Respiratory, extrarespiratory manifestations and immunological findings. A 2-year study].

"Mycoplasma pneumoniae" respiratory infection was diagnosed by completed fixation assay in 81 ages 8 months to 14 years. Seventy had pneumonia and ten had bronchitis. The "Mycoplasma pneumoniae" represented the 25% of all pneumonia of any aetiology. Seventy per cent of cases occurred during spring and summer months. Rashes developed in 13 (16%) patients (maculo-papular, 6; Stevents-Johnson, 2; purpuric-acular, 2; petechial, 1; urticarial, 1; erythema nodosa, 1). Other nonrespiratory complications were: hemolytic anemia, 2 cases; hepatitis, 5 cases; aseptic meningitis, 2 cases, and Guillain-Barré syndrome, 1 case. The white blood cell counts and ESR were normal or slightly raised in most of patients. Serum samples from 46 children were tested for cold agglutinins and positive titers were found in 19 (41%). The roentgenographic pattern of "Mycoplasma pneumoniae" was alveolar and/or mixed in 57 patients, and interstitial in the remainder. The lower lobes were more frequently involved. Twenty one per cent of patients with pneumonia had mediastinal lymphadenopathy. The outcome was favorable in all children except in one who developed irreversible pulmonary damage.

Adolescent↗
PubMed1982

Method for testing aseptic technique of intravenous admixture personnel.

A method of checking the aseptic technique of personnel that prepare i.v. admixtures is described. The following sterile dosage forms of trypticase soy broth (TSB) were prepared: (1) single-strength ampuls 5 ml, (2) six-times (6X) concentrated broth ampuls 10 ml, (3) lyophilized broth sufficient to make 5 ml single-strength broth in 10-ml vials, and lyophilized broth sufficient to make 10.6 ml 6X concentrated broth in 20-ml vials. Admixture personnel were told these were investigational products, and they were given simulated orders to prepare syringes and 50-ml piggyback admixtures of the products. The specimens containing the broth were intercepted by an investigator, incubated for seven days at 37 degree C, and observed for turbidity on days 2 and 7. To verify that the broth supported the growth of bacteria, two common contaminants were inoculated into the broth in the syringes and admixtures to serve as positive controls. At least 100 samples from each of four procedures were processed during the three-month study period. A total of 405 samples prepared by 13 individuals were tested. None of the samples became turbid; hence, the bacterial contamination rate was 0%. Among the positive controls, 98% inoculated with one species and 100% inoculated with the other became turbid. This method is now used for monitoring the aseptic technique of new admixture personnel. This unique method of checking the aseptic technique of personnel is accurate and cost effective, and it avoids the risk of adventitious contamination.

Antisepsis↗
PubMed2002

Bacterial populations on high-Dk silicone hydrogel contact lenses: effect of length of wear in asymptomatic patients.

BACKGROUND: Ocular infection and inflammation during extended wear with hydrogel lenses are often associated with contamination of the lenses with bacteria. This study examines the colonisation of a high-Dk silicone hydrogel contact lens worn on a 30-night extended wear basis (continuous wear) during asymptomatic lens wear. METHODS: Worn lenses were collected aseptically and placed in sterile vials. Microbial growth on various media was enumerated and the number of colony-forming units per whole lens was calculated. The proportions of samples contaminated with the bacteria and the extent of contamination were compared after one month, six months and two years of continuous wear with monthly replacement. The proportion of lenses that were sterile was calculated. RESULTS: The most frequently isolated and most numerous microbes that colonised the contact lenses during wear were the coagulase-negative staphylococci (in particular Staphylococcus epidermidis), followed by Propionibacterium sp. The frequency of isolation of the majority of bacterial isolates did not increase over time. Generally, lenses were sparsely colonised during asymptomatic wear. CONCLUSION: Up to two years continuous wear of silicone hydrogel lenses of the type used in this study does not appear to alter the types and number of bacteria that colonise the eye during wear, when lenses are replaced on a monthly schedule and the patients remain asymptomatic.

Adult↗
PubMed1988

A radiolabel ratio method for measuring pulmonary clearance of intratracheal bacterial challenges.

Calculation of bacterial clearance is a fundamental step in any study of in situ lung antibacterial defenses. A method is described whereby about 85% of a radiolabeled bacterial inoculum was consistently introduced into the bronchopulmonary tree of a mouse by the intratracheal route. Mice were then killed 1 and 4 hours later; their lungs were removed aseptically and homogenized, and viable bacteria and radiolabel counts were determined. Radiolabel counts fell slowly, and more than 80% of the original radiolabel was still present in homogenized lung samples from animals sacrificed 4 hours after challenge. Bacteria/isotope ratios for the bacterial inoculum and homogenized lung samples from animals sacrificed immediately after challenge were very similar. Bacterial clearance values were the same whether computed from bacterial counts alone or according to a radiolabel ratio method whereby the change in the bacteria/isotope ratio in ground lung aliquots was divided by a similar ratio from bacteria used to inoculate animals. Some contamination resulted from oral streptococci being swept into the bronchopulmonary free during the aspiration process. This contamination was not a problem when penicillin was incorporated into the agar and penicillin-resistant strains were used for the bacterial challenges.

Animals↗
PubMed1996

Streptozotocin induction of diabetes in rats leads to increased insulin-like growth factor-II/mannose-6-phosphate receptor mRNA expression in kidney but not in lung or heart.

Insulin and glucose are thought to act as important modulators of the expression of the IGFs, their binding proteins and their receptors. It has been postulated that changes of the IGF system after diabetes onset contribute to the development of diabetes late complications. We have measured the expression of IGF-II/M6P receptor mRNA in rat kidney, lung and heart after streptozotocin induction of diabetes. Adult rats were injected with streptozotocin, and, after the onset of diabetes, were treated with either insulin or vehicle. The rats were sacrificed on days 1, 2, 3, 4 and 9. Kidneys, lungs and hearts were removed aseptically and RNA was extracted from the tissues. In solution hybridization/RNAse protection experiments, specific IGF-II/M6P receptor and beta-actin transcripts were detected in the RNA samples from all tissues examined. To gain additional evidence for the expression of IGF-II/M6P receptor RNA in the tissues examined, Northern blotting experiments were performed: a major 9 kb RNA species was detected on the blots. Interestingly, streptozotocin-induced onset of diabetes led to a significant increase in the expression of IGF-II/M6P receptor mRNA in the kidney but not in lung and heart whereas no change in actin mRNA expression was measured. Insulin treatment did not prevent the increase of IGF-II/M6P receptor mRNA expression during short-term treatment (1-9 days). Alterations of the IGF system during diabetes onset might be of relevance for the development of early renal changes during the course of the disease.

Animals↗
PubMed1995

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid.

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.

Base Sequence↗
PubMed1991

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction.

The polymerase chain reaction (PCR) in cerebrospinal fluid was compared with conventional bacteriology and an enzyme-linked immunosorbent assay (ELISA) for cerebrospinal fluid antibodies in the diagnosis of tuberculous meningitis (TBM). PCR was the most sensitive technique; it detected 15 (75%) of 20 cases of highly probable TBM (based on clinical features), 4 (57%) of 7 probable cases, and 3 (43%) of 7 possible cases. ELISA detected 11 (55%) of the highly probable cases and 2 each of the probable and possible cases. Culture was positive in only 4 of the highly probable cases. Among the controls (14 pyogenic meningitis, 3 aseptic meningitis, 34 other neurological disorders), 6 subjects tested early in the study (2 pyogenic meningitis, 4 other disorders) were PCR positive. Second DNA preparations from their stored cerebrospinal fluid samples were all PCR negative, suggesting that the false-positive results were due to cross-contamination. 18 PCR-positive TBM samples retested were all still PCR positive. The antibody ELISA was positive in 3 controls despite the use of a high cutoff value.

Bacteriological Techniques↗
PubMed2003

[Detection of nonpoliomyelitic enteroviruses in central nervous system infections in Poland in 1995-2000; viral isolation data].

The aim of this study was evaluation of enterovirus diagnosis based on isolation of the virus in cell culture from subjects with aseptic neuroinfection conducted in six Provincial Sanitary-Epidemiological Stations in 1995-2000 years. Available data from six Sanitary-Epidemiological Stations indicated that 1429 isolation of enteroviruses from fecal samples of those patients were performed. The most frequently isolated enterovirus serotype was echovirus type 30: 138/203 isolated enteroviruses, but also others enterovirus serotypes were isolated in a study period. In the years 1995 and 1996 the percentage of positive results of isolation was highest with the numbers of 31% and 28.27% respectively. The decrease in positive isolation results has been observed from 1997 year. Negative results of isolation come up to 90% of investigated cases. Only 1.5% of enterovirus isolation attempts finished successfully in 2002 year. The situation when percentage of positive results of isolation is on so low level, the natural alterations in a profile of enterovirus serotype infected population cannot be observed, and serological investigations can be conducted for enterovirus serotypes others than really circulating and causing infections in population.

Antibodies, Viral↗
PubMed1986

[5-year evaluation of bacteriological samples (CSF, blood cultures, urine) in neonatology].

We report the result of the bacteriological samples performed for 5 years (1980-1984) in neonates, referred to the neonate unit of the hospital of Clermont-Ferrand, for infection (2,894 infants). The CSF tests show aseptic meningitis in 4% of the cases and in 0.4% meningitis with bacteria on the direct examination and in culture. We emphasize the interest of carrying out the soluble bacterial antigen assay (Strepto B, Coli K1) to find out the bacteria involved in the meningitis. 13.7% of the infants have one or several positive blood culture. The germ is a E. Coli in 30% of the cases, and less frequently a streptococcus D. They are few listeria. In 1984, the frequency of streptococcus B is increasing. Septicemia due to anaerobic germs or Candida Albicans emerge during the stay in the unit. Among 12,704 bacteriological urine analysis, we compare the real urinary infections (in which the enterobacteria are the main strain: 75.3%) and the significant bacteriuria.

Bacterial Infections↗
PubMed1993

[Acute human immunodeficiency virus infection associated with facial diplegia].

We describe the case of a 21-year-old parenterally drug-addict man, who presented an acute and self-limited disease, temporarily associated to seroconversion against the human immunodeficiency virus. During his evolution, the patient developed aseptic meningitis and facial diplegia. Two lymphatic ganglions were histopathologically and sequentially studied during the third and sixth week of the disease, showing an intense follicular depletion in the first sample, which reverted towards a pattern of follicular hyperplasia in the second one.

Acute Disease↗
PubMed2002

Evaluation of compressed air used in the dental operatory.

BACKGROUND: The authors report the findings obtained when they quantitatively examined compressed air samples from air-water syringes located in different dental operatories at the Louisiana State University School of Dentistry fo r the presence of microbial contaminants. METHODS: Streams of air of 30 seconds' duration from air-water syringes were forced through sterile modified stainless steel membrane filter holders (Millipore, Millipore Corp., Bedford, Mass.), each containing a membrane filter (average pore diameter = 0.45 micrometers). Each filter was aseptically removed, placed onto the surface of a Petri dish containing sheep blood agar and incubated under increased carbon dioxide tension at 37 C for 48 hours. The authors performed a count of the resultant microbial colonies, after which they microscopically examined the gram-stained organisms. RESULTS: Bacteria were detected in 24 percent of the samples. The number of colonies observed on the filters varied among the dental units. The air from only one of the dental units sampled repeatedly was found to be free of bacterial contaminants. This contrasted with other units for which one or more samples were found to be positive for microorganisms. The majority of colonies observed were pigmented. Microscopic examination of organisms from representative colonies revealed that most were either gram-positive cocci or gram-negative diplococci and tetrads. The results of the one-sample t test were found to be significant (t = 5.6, df = 98, P = .0001). The 95 percent confidence interval was 0.15 to 0.32. CONCLUSION: The results suggest that, at least statistically, a percentage of air lines will have bacteria present.

Air Microbiology↗
PubMed1994

Human spumaretrovirus antibody reactivity in multiple sclerosis.

The role of human spumaretrovirus (HSRV) infections in the pathogenesis of multiple sclerosis (MS) was investigated with recombinant HSRV env-specific enzyme-linked immunosorbent assay. The presence of HSRV antibodies was determined in pairs of serum and cerebrospinal fluid (CSF) samples from 60 MS patients. In 7 of these patients serial serum and CSF samples were obtained in relation to the clinical activity of the disease during a period of 2 years. No increased antibody reactivity was demonstrable in the MS population compared with 14 aseptic meningitis patients, 50 blood donors and 16 healthy controls. Slightly elevated levels of antibodies were demonstrable in serum and/or CSF in 4 MS patients but also in 1 patient with aseptic meningitis, 1 blood donor and 1 child. No marked serum or CSF HSRV antibody fluctuation was observed in the MS patients followed longitudinally. Thus, this study does not support the involvement of HSRV in the pathogenesis of MS.

Adolescent↗
PubMed1975

Endotoxin as a cause of aseptic meningitis after radionuclide cisternography.

The role of pyrogens in aseptic meningitis after radionuclide cisternography was studied by means of the Limulus test, a sensitive detector of endotoxin. During a 15-month period, 39 reactions associated with cisternography were reported. Ten samples of specific lots of the radioactive drugs implicated in 20 of these reactions were tested and all reacted strongly positive to the Limulus test. The less sensitive rabbit pyrogen test was negative for these preparations when tested on a dose-per-weight basis. Our findings apparently provide clinical evidence for the observation made in animals that endotoxin is at least 1,000 times more toxic intrathecally than intravenously. The data implicate endotoxin contamination as a cause of adverse reactions to radionuclide cisternography. We conclude that the USP pyrogen test is insufficiently sensitive for intrathecal injectables and should be supplemented by the Limulus test.

Arachnida↗