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PubMed2006

Changes in MMP-2 and -9 activity and MMP-8 reactivity after amphotericin B induced synovitis and treatment with bufexamac.

The objective here was to evaluate the acute effects of induced arthritis on synovial fluid (SF) levels of matrix metalloproteinases (MMP) -2, -8 and -9 in horses. To evaluate MMP-2 and -9 activities and the effect of non-steroidal anti-inflammatory drug (NSAID) bufexamac during remission from acute arthritis. Aseptic arthritis was induced in 24 Standardbred horses using 20 mg of amphotericin B as a single intra-articular (IA) injection in the right intercarpal joint. After 1 week and 2 weeks, horses were treated intra-articularly with 10, 20, or 40 mg of bufexamac suspension or with sterile saline solution as control. SF was sampled prior to induction and at weekly intervals for 5 weeks. Fluids were evaluated for MMP-2 and MMP-9 activity by gelatin zymography or for MMP-8 immunoreactivity by Western Blotting. IA injection of amphotericin B consistently resulted in significant increase in the immunoreactivity of MMP-8 and activity of both the latent and the active forms of MMP-2 and -9, among which the active form of MMP-2 increased the most. MMP-9 levels declined to pre-induction levels within 2 weeks, whereas levels of MMP-2 remained still high after 5 weeks. Treatment with bufexamac did not significantly affect levels of gelatinolytic MMP. Results suggest that after acute arthritis of horses, elevated MMP activity is present in the joint, for several weeks, to a degree that could promote cartilage degradation, and treatment with the NSAID bufexamac is not likely to affect that. Furthermore, analysing levels of MMP-9 activity and especially levels of active forms of MMP-2 activity may be valuable to predict the time of occurrence of arthritis in horses.

Amphotericin B↗
PubMedDate not supplied

Practical methods for the microbial validation of sterilizing-grade filters used in aseptic processing.

Microbial challenge test methods to validate sterilizing-grade filters were developed to facilitate the use of parenteral drug products as liquid vehicles. Scaled-down filtration systems were developed in response to processing parameters, e.g., flow rates, pressures, duration, and temperature, provided by customers. Accordingly, 47 mm disc filter holders were chosen to minimize the volume of product necessary for each test. A peristaltic pump was used to produce positive pressure within the test system, and a 47mm filter sampling manifold was used to assay the entire filtrate. The resultant system was able to accommodate a variety of flow rates while maintaining the viability of the retention test microorganism, Pseudomonas diminuta (ATCC Number 19146). Experiments were conducted by challenging sterilizing-grade (0.22 micron) and 0.45 micron polyvinylidene difluoride (PVDF) (Durapore) filter discs under simulated processing conditions with P. diminuta resuspended in the pharmaceutical products. The challenge concentration was at least 10(7) colony forming units per cm2 of filter area. The 0.22 micron filters were shown to be retentive in all cases, while the 0.45 micron membranes exhibited some degree of bacterial passage as expected. The results of these experiments were communicated to the customers for inclusion in their process validation documentation.

Antisepsis↗
PubMed2000

Determination of endotoxins in caries: association with pulpal pain.

AIM: The aims of this study were: (i) to determine the presence or absence of endotoxins in the superficial and deep layers of carious lesions of symptomatic and asymptomatic teeth with vital pulps; (ii) to quantify the amount of endotoxin present; and (iii) to associate the presence of endotoxins with the acute pulpal pain derived from the irreversible pulpitis. METHODOLOGY: Two specimens of carious dentine were taken under aseptic conditions from symptomatic teeth with irreversible pulpitis (n = 9) and asymptomatic teeth with reversible pulpitis (n = 11). The first specimen was taken from a layer of superficial caries and the second from a deeper one. Sound dentine was also collected from intact teeth without restoration and used as a noncarious control group (n = 4). During the patient sampling procedure an effort was made to collect an equal quantity of caries and sound dentine in all cases (approximately 6 mg). The extraction of endotoxins was performed using the Phenol-water method. The assay and quantitative determination of endotoxins was performed by the Quantitative Chromogenic test using Limulus Lysate. Data were analysed statistically using either independent or paired t-tests. RESULTS: The results indicated that endotoxins were present in the superficial and deep layers of caries of all symptomatic teeth with irreversible pulpitis (0.15078 and 0.12111 ng mL-1, respectively), with significantly greater amount (P < 0.01) in the superficial compared to the deep layer. Endotoxins were found in superficial and deep layer of caries of all asymptomatic teeth with reversible pulpitis (0.12091 and 0.07163 ng mL-1, respectively), with significantly greater amounts (P < 0.001) in the superficial compared to the deep layer. The results also demonstrated that significantly greater concentrations (P < 0.005) of endotoxins were present in the superficial carious layer of symptomatic compared with asymptomatic teeth (0.15078 and 0.12091 ng mL-1, respectively). Likewise, significantly greater amounts (P < 0.001) of endotoxins were present in the deep carious layer of symptomatic compared with asymptomatic teeth (0.12111 and 0.07163 ng mL-1, respectively). In sound dentine no endotoxins were detected. CONCLUSIONS: This study demonstrates that endotoxins are present in carious lesions of symptomatic and asymptomatic teeth. The amount of endotoxin was significantly greater in the superficial compared to the deep layer of carious dentine. More endotoxins are present in caries of painful teeth compared with those without symptoms.

Adolescent↗
PubMed2004

Is "aseptic" loosening of the prosthetic cup after total hip replacement due to nonculturable bacterial pathogens in patients with low-grade infection?

BACKGROUND: Loosening of the prosthetic cup is the limiting factor in the service life of total hip prostheses (THPs). Despite effective culture methods, the detection of low-grade infection in patients with loose implants still presents a challenge. It is crucial to distinguish between "aseptic" loosening and loosening due to periprosthetic infection, so that appropriate treatment can be administered. We investigated whether aseptic loosening of the acetabular components of THPs is due to unrecognized infection. METHODS: From October through December 2002, a total of 24 patients with acetabular cup loosening were investigated. Only patients without clinical signs of infection and with negative results of bacteriologic culture of synovial fluid (obtained by preoperative aspiration) were included in the study. Intraoperative biopsy samples obtained from the neocapsule and synovia (e.g., the interface membrane) were examined by means of routine culture methods and by polymerase chain reaction (PCR) for detection of 16S ribosomal RNA (rRNA). Control subjects included 9 patients undergoing primary hip arthroplasty. RESULTS: C-reactive protein levels and erythrocyte sedimentation rates were slightly elevated in the group with loosening, compared with the control group, but the difference was not statistically significant. PCR and routine culture showed no microorganisms in either group, with the exception of 1 patient in the loosening group. CONCLUSIONS: PCR for detection of 16S rRNA in tissue specimens obtained from hip joints is not superior to routine bacteriologic culture techniques for detection of low-grade infections. However, these results demonstrate that the loosening of cups in THPs do not usually result from nonculturable periprosthetic infection, if the microbiological processing is adequate.

Aged↗
PubMed1999

A new variant of echovirus 4 associated with a large outbreak of aseptic meningitis.

BACKGROUND: A large outbreak of aseptic meningitis which began in April 1997 involved hundreds of cases in all geographical regions of Israel and the Palestinian Authority, peaked between June and September, and lasted until December. OBJECTIVES: We have investigated the virus associated with the outbreak to determine its serotype and molecular type and to establish epidemiological links. DESIGN: Virus strains isolated from 210 clinical samples were serotyped by neutralization using LBM and WHO antiserum pools and two echovirus 4 (EV4)-specific antisera, and by immunofluorescence using a monoclonal antibody. RNA was extracted and a 435 base long fragment derived from the 5'UTR of the genome was amplified by RT-PCR using common primers, and sequenced. Sequences were compared to echoviruses 4, 6 and 7 prototypes from ATCC, and to other echoviruses sequences from the EMBL/Genbank data base. RESULTS: The outbreak isolates were identified by the EV4 type-specific antisera and the monoclonal antibody but not with the WHO pools. Very few isolates could be typed by the LBM pools. The EV4 isolates accounted for 68% of all enterovirus isolates in our laboratory in 1997. The age distribution of the patients was: 0-11 month, 11.2%; 1-4 years, 16.1%; 5-9 years, 31.8%; 10-14 years, 9.9%; 15-20 years, 9.5%; 21-44 years, 21.5%; and > 45 years, 0%. Males between 1 and 14 years of age were affected more frequently than females of the same age. The sequences of 25 of 28 EV4 isolates analyzed were closely related to each other (> 95% homology) and the remaining three isolates had < 95% homology to the others and to each other. Interestingly, the outbreak strains were less closely related to the EV4 prototype, than to several other echoviruses. Three closely related subgroups were identified which correlated with geographical distribution but the temporal distribution did not reveal links leading to the source of the outbreak. CONCLUSION: The outbreak was caused by a variant of EV4 which apparently did not circulate in the area before and thus was capable of causing a widespread infection.

Disease Outbreaks↗
PubMed1994

Detection of varicella-zoster virus-specific DNA sequences in cerebrospinal fluid from patients with acute aseptic meningitis and no cutaneous lesions.

Acute aseptic meningitis (AAM) is considered as an uncommon manifestation of varicella-zoster virus (VZV) recrudescence and is usually regarded as a complication of the cutaneous infection in patients with impaired cellular immunity. Indirect evidence suggests, however, that VZV-associated AAM may also respond to direct spread of the virus to the leptomeninges from the cells supporting the latency. The polymerase chain reaction (PCR) was used to amplify VZV-specific DNA sequences in serial cerebrospinal fluid (CSF) samples from 21 patients with AAM, who presented laboratory evidence of intrathecal production of VZV-specific antibody on follow-up. Eleven of these patients never showed cutaneous zosteriform lesions. VZV-DNA sequences were detected in the CSF from all patients with cutaneous zoster, as well as from six patients (55%) lacking skin lesions. Viral DNA sequences were present in six cases before the rise in specific antibody was seen in CSF, disappearing during follow-up in the seven positive cases. These results support the proposed involvement of VZV in the etiology of AAM seen among normal young adults and strongly suggest that the virus can reach directly and infect the CNS from the latently infected spinal ganglia.

Acute Disease↗
PubMed2001

Modification of cell perifusion for extended study of hormone release in the rat pituitary.

We use a flow-through cell perifusion system allowing continuous sampling of Luteinizing Hormone (LH) release from excised pituitary tissue. With this system, we are able to sample from viable tissue in primary culture for three days as opposed to a more typical of primary culture of 6-12 hours. Rat pituitary tissue was perifused and challenged with a physiological dose of LHRH. Samples were collected for 3.5 hours on each of 3 consecutive days in a refrigerated fraction collector and assayed for LH concentration by radioimmunoassay. In these experiments we tested two types of media, each with or without fetal calf serum supplementation, for their ability to support pituitary tissue for extended lengths of perifusion. In addition, we broadened the use of aseptic technique, sterile media and equipment to limit microbial contamination. Our results indicate that perifusion of pituitary tissue can be done successfully for 3 or more days at a modest cost, greatly increasing the amount of information that can be collected from each piece of tissue harvested.

Animals↗
PubMed1998

High human immunodeficiency virus type 1 RNA load in the cerebrospinal fluid from patients with lymphocytic meningitis.

Thirty-seven matched cerebrospinal fluid (CSF) and plasma samples from 34 human immunodeficiency virus type 1 (HIV-1)-infected patients with suspected meningitis were analyzed for levels of HIV-1 RNA and markers of inflammation. Patients with tuberculous (n = 9) or cryptococcal (n = 6) meningitis had the highest CSF virus loads, which in many cases exceeded the levels in plasma, compared with patients with meningococcal meningitis (n = 3), aseptic meningitis (n = 8), tuberculoma (n = 2), or AIDS dementia complex (n = 4) or with normal lumbar punctures (n = 3). CSF virus load correlated significantly with the number of infiltrating lymphocytes (r = .60, P < .001) but not with plasma virus load, the levels of beta2-microglobulin in the CSF, or the integrity of the blood-brain barrier. These data suggest significant intrathecal HIV-1 replication in patients with lymphocytic meningeal infections such as tuberculous and cryptococcal meningitis.

AIDS Dementia Complex↗
PubMedDate not supplied

RT-nested PCR for the detection of enterovirus in biological samples from patients with suspected enteroviral infections.

In this study, we have tested a reverse transcription (RT) nested polymerase chain reaction (nPCR) for detection of enterovirus (EV) RNA in cerebrospinal fluid (CSF), serum samples, and conjunctival swabs (CS) from patients with suspected enterovirus infections. A specific 113-bp fragment was amplified using primers designed based on 5' non coding region of the enterovirus genome. The enterovirus RT-nPCR was able to detect 0.001 plaque forming unit (pfu)/ml. Since no PCR product was detected in each of the CSF, CS and serum samples from patients with proven-non-enterovirus viral infections, this method was found to be specific. EV RNA was detected in all 30 culture-confirmed CSF samples and yielded positive results in 5 out of 7 additional cases of culture-negative CSF samples with other evidences of enterovirus infection. Overall, EV RNA was detected in 95% of the patients with clinical diagnosis of viral central nervous system (CNS) disease and confirmed enterovirus infection. Furthermore, we were able to detect EV RNA in 24 (47%) out of 51 CSF samples from patients with clinical diagnosis of viral CNS disease and negative laboratory evidence of viral infection. The percentage of positive EV RNA detection in paired CSF and serum samples from 11 patients with an enterovirus isolate in CSF was 100% (11 of 11) and 73% (8 of 11), respectively. In addition, EV-specific IgM was detected in 64% (7 of 11) of the sera tested. The method was also tested against 136 samples of CS from patients with clinical diagnosis of acute hemorrhagic conjunctivitis. Ninety nine of them resulted positive (73%), while only 27 (20%) had been positive for viral culture. In summary, our study shows the importance of enterovirus RT-nPCR for the diagnosis of enterovirus associated disease in different kind of biological samples and different types of diseases.

Acute Disease↗
PubMed1989

Cannulation of inferior vena cava for long term central venous access.

Nineteen patients had a Silastic (silicone rubber) catheter placed into the inferior vena cava (IVC) by way of a vein of a lower extremity. All patients needed long term venous access but had conditions precluding access to the superior vena cava (SVC) or access sites of the upper torso. Precautions regarding operative technique for the placement of the catheter include incisions through healthy skin, maintenance of aseptic technique at the site of access, an atraumatic subcutaneous tunnel at least 25 centimeters in length and a long acting local analgesic effect. Catheters were in situ for a total of 2,215 days (a mean of 111 days per catheter). Catheters were also used to administer intravenously medications, blood and blood products, chemotherapeutic agents, parenteral nutrition and for sampling of blood. Four complications occurred: one instance each of catheter sepsis and infection of the subcutaneous tract and two of thromboses of the IVC. No deaths occurred. The complication rate was 0.18 per cent per catheter-day. Long term access to the IVC is feasible without undue concern in conditions in which access to the SVC is precluded but long term central access is essential.

Adult↗
PubMed1994

Hydrogen gas is not oxidized by mammalian tissues under hyperbaric conditions.

Mammalian tissues, including heart, lung, liver, kidney, spleen, and skeletal muscle of guinea pig, rat, or pig, were exposed to tritium (T2) and high pressures of H2. Incorporation of the tritium label was measured to test for a latent capacity by mammalian tissues to oxidize H2 under conditions such as those experienced by deep divers breathing H2. Tissues were removed aseptically, and either minced, homogenized, or prepared as live cell cultures. The tissues were placed in a chamber to which 8 mCi T2, 1 MPa He, and either 1 or 5 MPa H2 were added. After 1 h the chamber was decompressed. The tissues were spun briefly in a vortex mixer to facilitate elimination of T2 in the gas phase. Samples were analyzed by scintillation counting for tritium incorporation in the liquid phase or in the tissues. Saline and distilled water were used as negative controls. Palladium (Pd) beads immersed in water, and cultures of the H2-metabolizing bacterium Alcaligenes eutrophus were used as positive controls. The tissues incorporated on the order of 10 nCi T2.ml-1, which implied a H2 incorporation of 10-50 nmol H2.g-1.min-1. However this incorporation was not different from that found in the water controls and was attributed to radioisotope effects. The Pd and bacterial samples incorporated over 1,000-fold more T2 than the mammalian tissues. We concluded that the mammalian tissues did not oxidize H2 under hyperbaric conditions, with a limit of detection of 100 nmol H2.g-1.min-1.

Alcaligenes↗
PubMed1998

Epidemics of aseptic meningitis due to enteroviruses following national immunization days in Bahrain.

We report two successive epidemics of aseptic meningitis due to enteroviruses (EV) observed after national immunization days against polio. Meningitis due to echovirus 30 occurred from July 1995 to the end of January 1996, mostly among children aged 0-12 years (95.1% of cases), and meningitis due to echovirus 4 occurred from May 1996 to the end of September 1996 in the same age group. There were 286 and 169 cases, respectively. Specimens from several representative cases were sent to the WHO Collaborating Center for Virus Reference and Research Laboratory for serological testing and virus detection, including polymerase chain reaction (PCR) studies. Using those tests, evidence of echovirus 30 infection was found in 44% of the children who were sampled during the first epidemic and 45.5% during the second. During echovirus 30 and echovirus 4 epidemics, a similar decline in the age-specific attack rate from 19.1/10,000 and 10.1/10,000 population aged 12 years to 2.4/10,000 and 3.6/10,000 population aged 13 years was observed, respectively.

Adolescent↗
PubMed1983

Techniques and clinical effect of aseptic procedures on patients with acute leukemia in laminar airflow rooms.

The techniques of aseptic procedures in the laminar airflow room (LAF) were evaluated in 110 adult patients undergoing antileukemic chemotherapy for remission induction. The patients were divided into three groups according to the regimens: Group A, consisting of 20 patients who stayed in the LAF and received the gown technique + sterile food + prophylactic oral and topical antibiotics; Group B, consisting of 12 patients who stayed in the LAF and received sterile food + prophylactic oral antibiotics; and Group C, consisting of 78 patients in open wards, who received prophylactic oral antibiotics alone. Species and numbers of microorganisms on the skin surface were far less in the patients in Group A than in those in Group B. Airborne microorganisms were counted by the air sampling method. No microorganisms could be detected at the time of the patient's rest and of blood collection in either Group A or B. Electrocardiography and X-ray examination caused an increase in the number of colonies to more than one colony in Group B, but Group A had a count of less than 0.5 colony. The colony counts became negative within 5 min after the cessation of each operation. The percentage of febrile days for patients with a peripheral granulocyte count of less than 100/microliter was 29% in Group A, 21% in Group B and 44% in Group C. The incidence of documented infections during the total hospital stay was 25% (5/20), 42% (5/12) and 86% (67/78), respectively. The aseptic procedures in Group B were not as strict as in Group A, but the incidence of infections in Group B was significantly lower than in Group C.

Adolescent↗
PubMed2006

Salmonella prevalence in market-age turkeys on-farm and at slaughter.

The objective of this study was to compare the prevalence of Salmonella in market-age turkeys on-farm and at slaughter (i.e., before and after feed withdrawal, catching, loading, transportation, and preslaughter holding). Thirty birds were randomly selected from each of 6 commercial turkey flocks scheduled to be loaded and shipped to the abattoir during the evening of the same day. Selected birds were euthanized on the farm, and the cloacal contents, large intestine, crop, ceca, liver and gallbladder, and spleen were aseptically collected. At the abattoir, 30 birds from the same flock were randomly selected from the slaughter line, and the crop, ceca, liver and gallbladder, and spleen were collected for subsequent culture at the laboratory. All flocks studied were positive for Salmonella at slaughter. No statistical difference was found between the overall prevalence on-farm and at slaughter. At both sampling points, the overall prevalence found was 33.3%. Diverging prevalence estimates were obtained based on the different sample types collected on-farm and at slaughter. In both cases, cecal content samples had the highest relative sensitivity (73.3% on-farm and 68.3% at slaughter). This study demonstrates that the preslaughter practices of feed withdrawal, catching, loading, transportation, and holding do not significantly alter the prevalence of Salmonella in market-age turkeys. Therefore, our results suggest that it may be possible to monitor the Salmonella status of turkey production farms based on samples collected at the abattoir.

Abattoirs↗
PubMed1980

Aseptic aspiration of rabbit bone marrow and enrichment for cycling cells.

Female adult New Zealand white rabbits were anesthetized using a combination of xylazine and ketamine administered intramuscularly. Bone marrow was aspirated aseptically from the humerus of femur using an 18-gauge Rosenthal pediatric needle. Marrow was obtained from six animals per hour using this technique. Recovery was rapid and no infection or loss of limb function was observed over the course of more than 100 aspirations. Approximately 2 x 10(8) mononuclear cells were obtained from 0.5 ml of aspirated marrow. Marrow samples were fractioned using isopynic single step density gradients. A 10-fold enrichment of blast cells was achieved using a density of 1.077 g/ml.

Animals↗
PubMed2001

[Patient surgical masks during regional anesthesia. Hygenic necessity or dispensable ritual?].

OBJECTIVE: The use of surgical face masks (SFM) is believed to minimize the transmission of oro- and nasopharyngeal bacteria to wounds and surgical instruments. However, there are disadvantages for patients undergoing regional anaesthesia and wearing masks: deficient assessment of lip cyanosis, anxiety, retention of CO2, costs. Up to now no studies have been published investigating whether or not SFMs, worn by patients during regional anaesthesia, will reduce bacterial convection. METHODS: We investigated 72 patients during aseptic operations: 24 individuals with regional anaesthesia and SFMs, 22 individuals with regional anaesthesia without SFMs and 26 patients undergoing general anaesthesia. Using an air sampler (volumetric impaction method) 100 L air were collected on blood agar over 2 min. After incubation at 37 degrees C over 60 h the colony forming units (CFU) were counted and differentiated. Airborne culturable bacteria were sampled over the operation field, on the anaesthetic side of the surgical curtain, as well as 10 cm before and to the side of the patients mouth. RESULTS: At all 4 locations there were no significant differences in the number of CFUs between patients wearing a SFM or not (e.g. over the operation field: patient with SFM 5.5 +/- 1.1; no SFM 4.8 +/- 1.2; mean +/- SEM). Significantly more CFUs were detected in patients undergoing general anaesthesia (p < or = 0.05). The extent of the operation did not correlate with the number of CFUs; however, we observed a trend that more CFUs were detected with an increasing number of persons working in the operating room. CONCLUSION: Surgical face masks worn by patients during regional anaesthesia, did not reduce the concentration of airborne bacteria over the operation field in our study. Thus they are dispensable. A higher airborne germ concentration has been detected in patients during general anaesthesia. The reasons for this finding are unknown, but it may be discussed as being a result of a higher activity and number of staff involved during general anaesthesia causing more air turbulence.

Adult↗
PubMedDate not supplied

[The role of the alloy-specific elements iron, cobalt, chromium and nickel in aseptic loosening of total hip joint prosthesis].

The cause of aseptic loosening of total hip joint prostheses is still largely unexplained. In addition to mechanical forces, material factors may also be instrumental. In 16 patients with loosened hip prostheses who underwent re-operation (8 X Huggler-Weber, 8 X McKee-Farrar), the concentration of the alloy-specific elements cobalt, chromium, nickel and iron were determined in 58 tissue samples by means of neutron activation and emission spectrum analysis. An increase in the element concentrations was found in all the tissue samples, and this was unrelated to the time elapsed since implantation. The chromium concentration in particular was significantly elevated in all the tissue samples, in some patients with McKee-Farrar prostheses up to 10,000 times the norm. In view of the known toxicity of cobalt, chromium and nickel in high doses it seems that the presence of unphysiologically high concentrations of these elements in the vicinity of total hip joint prostheses represents an explanation of some of the loosened prostheses.

Chromium Alloys↗
PubMed1981

Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys.

In nine monkeys (Macaca fascicularis) the pulps of 78 teeth were aseptically necrotized. Twenty-six of the pulp chambers were kept bacteria-free by sealing, while 52 were infected by the indigenous oral flora. The results were recorded clinically, radiographically and microbiologically at the beginning of the experiment and after 6-7 months. The final examination also included histologic recordings. The initially noninfected root canals were all sterile at the final samplings, indicating that the risk of contamination (including hematogenous) of root canals of this animal is very slight. It was shown that noninfected necrotic pulp tissue did not induce inflammatory reactions in the apical tissues. By contrast, teeth with infected pulp tissue showed inflammatory reactions clinically (12/52 teeth) and radiographically (47/52 teeth). Facultatively anaerobic streptococci, coliform rods and obligately anaerobic bacterial strains were most frequently found. In the final samples the number of obligately anaerobic strains increased. Some microorganisms which were isolated in the initial samples were not detected in the final samples. All infected teeth histologically examined showed strong inflammatory reactions in the periapical region.

Animals↗