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PubMed2005

Quantitative PCR-enhanced immunoassay for measurement of enteroviral immunoglobulin M antibody and diagnosis of aseptic meningitis.

A PCR-enhanced immunoassay (PIA) to detect enterovirus (EV) immunoglobulin M (IgM) for diagnosis of recent EV infection was recently developed. This test was compared with another EV IgM capture technique, the solid-phase reverse immunosorbent test (SPRIST). Fourteen of 43 serum samples from aseptic meningitis patients were positive by PIA, whereas 10 were positive by SPRIST. One of 39 control serum samples was weakly positive by PIA. A single-serum-dilution real-time PCR-based PIA for EV IgM (quantitative PIA [QPIA]) was also developed and evaluated against PIA, SPRIST, an EV IgM radioimmunoassay (RIA), and clinical data. A mixture of 12 EVs was used as the antigen. Results from investigating four groups of serum samples were as follows. (i) The nine PIA-positive serum samples in group 1 were all positive by QPIA. (ii) Group 2 consisted of 59 serum samples from aseptic meningitis patients. Nineteen of 30 serum samples (63%) taken at hospital admission were positive by QPIA. Of these, 17 were positive in EV PCR. (iii) None of the 30 control serum samples in group 3 were positive by QPIA. (iv) For the 24 serum samples in group 4, of which 11 were positive and 13 were negative by RIA, the QPIA results were completely concordant. The sensitivity and specificity of QPIA for diagnosis of EV infection were 70 and 80%, respectively. QPIA provides a rational strategy for the detection of EV IgM, allows the use of viral antigens with minimal purification, and needs no virus-specific reagents apart from those in the PCR. QPIA is a generally applicable method for the detection of viral IgM in IgM capture assays.

Antibodies, Viral↗
PubMed1995

Interleukin-6 in amniotic fluid obtained at forewater amniotomy compared with hindwater samples in women in spontaneous labour.

Using an aseptic technique, samples of amniotic fluid were obtained from the forewaters and hindwaters of fifteen women in spontaneous labour with a deeply engaged fetal head. Prior to sampling, a cervical swab was obtained to exclude infection and in 10 cases samples of amniotic fluid were sent for microscopy and culture. Forewater samples were obtained using a pudendal block needle. Hindwater samples were obtained after membrane rupture by inserting an intra-uterine pressure catheter. Batch assay for interleukin-6 (IL-6) concentrations was performed using a specific ELISA. The levels of IL-6 in forewater samples of amniotic fluid were significantly higher than those found in hindwater samples (t = 3.65, P < 0.002). This suggests that either the production of IL-6 as a result of stimulation by its cytokine 'effectors' is greater at this site, or that the loss or metabolism of IL-6 in the forewaters is less than that in the hindwaters. The concentration of IL-6 parallels the reported levels of PGE2, and it is therefore likely that if IL-6 is involved in the mechanism of labour it acts through the effects of the resultant PGE2 release.

Adolescent↗
PubMed1988

The effect of decreased portal blood flow on the biliary system.

A disturbance in the regional portal blood flow adjacent to the dilated bile duct in the liver is often observed in patients with hepatolithiasis. The effects of this disturbed portal blood flow on the biliary system, with or without cholangitis, were therefore investigated. Young rabbits were divided into the following four groups; (1) controls that had a laparotomy only (n = 3), (2) those that had a ligation of the portal branch of the right posterior lobe (RP lobe) (PL) (n = 10), (3) those that had tubing inserted into the bile duct through the duodenal papilla (BS) (n = 10), and (4) PL + BS (n = 10). Despite marked atrophy of the RP lobe, no distinct changes were seen in the biliary systems of groups 1 or 2. In groups 3 and 4, however, infiltration of inflammatory cells and glandular proliferation in the wall of the markedly dilated extrahepatic bile duct (proliferative cholangitis (PC), characteristic to hepatolithiasis) were seen. Findings of PC were also noted in the intrahepatic bile duct of the atrophied RP lobes of these 2 groups. The incidence of PC was 20 per cent in group 3 and 60 per cent in group 4, respectively, but the PC of the intrahepatic bile ducts in group 3 was more localized than in group 4. Goblet cell metaplasia was seen in the epithelial cells of PC. Bacteriologically, bile samples were aseptic in groups 1 and 2, however, samples of bile from groups 3 and 4 were all contaminated with Escherichia coli or Streptococcus Faecalis. The biliary contents of phospholipids, total cholesterol and total bile acid were significantly decreased in groups 3 and 4, when compared with groups 1 and 2. In conclusion, a disturbance in portal blood flow, accompanied by cholangitis and segmental liver atrophy, may play an important role in the clinical course of hepatolithiasis.

Animals↗
PubMed2004

Insulin-like growth factors I and II in the aseptic loosening of total hip implants.

Periprosthetic interface tissue and pseudocapsule samples surrounding aseptically loosened hip implants and control knee synovium were studied by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Endothelial cells, fibroblasts, and monocyte/macrophages contained bone formation-enhancing insulin-like growth factors (IGFs). In interface tissue we found fewer IGF-I and IGF-II positive cells than in control tissue. In pseudocapsular tissue we found fewer IGF-I positive cells and an equal amount of IGF-II positive cells compared to control tissues. Decreased bone formation may contribute to net loss of bone around aseptically loosened hip implants.

Adult↗
PubMed2003

Transient elevation of interleukin-16 levels at the initial stage of meningitis in children.

IL-16 is an immunomodulatory cytokine that is characterized by chemotactic activity and stimulation of proinflammatory cytokine expression in monocytic cells. We studied IL-16 using ELISA in children with meningitis. When meningeal symptoms existed, IL-16 levels were high in the cerebrospinal fluid (CSF) of both bacterial (939 +/- 877 ng/l, n = 20) and aseptic (341 +/- 371 ng/l, n = 23) meningitis. The values in the CSF were significantly higher than those in non-meningitis controls (29 +/- 8 ng/l, n = 22, P < 0.0001). After meningeal symptoms disappeared, IL-16 levels in bacterial (191 +/- 149 ng/l, n = 10, P = 0.0042) and aseptic (159 +/- 188 ng/l, n = 13, P = 0.0118) meningitis were lower than those during the symptomatic stage. IL-16 levels were the highest before day 5 of the illness and then gradually fell. Significant correlations were found between IL-16 levels and both G-CSF levels (r = 0.783, n = 11, p = 0.0029) and IL-6 levels (r = 0.818, n = 12, P = 0.0005) in the CSF of bacterial and aseptic meningitis. IL-16 levels in all CSF samples from non-meningitis controls were lower than those in serum. In contrast, IL-16 levels in the CSF in six of 16 samples from bacterial meningitis and two of 18 samples from aseptic meningitis were higher than those in serum. Serum levels of IL-16 did not fluctuate throughout the course of meningitis. These data indicate that IL-16 levels rise transiently in CSF at the initial stage of meningitis. We speculate that IL-16 may promote inflammatory responses during meningitis in concert with other proinflammatory cytokines.

Child↗
PubMed1996

Sterility of filgrastim (G-CSF) in syringes.

OBJECTIVE: To determine if the sterility of filgrastim (G-CSF) is maintained for up to 7 days when aseptically transferred from the vial to tuberculin syringes in a laminar air flow environment. DESIGN: The study was conducted in two phases: a validation and an experimental phase. The method was validated by inoculating samples of sterile filgrastim solution with common bacterial and fungal skin contaminants. Samples were aseptically drawn into syringes in a class 100 horizontal laminar air flow hood and refrigerated. The samples were equally divided and transferred to microbiology culture media at times 0, 24 hours, 48 hours, and 7 days; incubated; and the organisms identified and quantitated. In the experimental phase, samples of filgrastim were aseptically drawn into syringes, separated into three groups, and refrigerated. At 24 hours, 48 hours, and 7 days, the samples were transferred to broth, incubated, and observed for the development of turbidity. SETTING: A class 100 laminar air flow hood in a pediatric hospital pharmacy and a home-infusion pharmacy class 100,000 clean room. MAIN OUTCOME MEASURES: The sterility of filgrastim in syringes was determined by comparing experimental broth culture tubes to a control tube and observing for the development of turbidity. RESULTS: Filgrastim demonstrated the ability to support the growth of intentionally inoculated skin contaminants, both qualitatively and quantitatively. However, when aseptically transferred to syringes and refrigerated, all tested filgrastim samples remained sterile for at least 7 days. CONCLUSIONS: Syringes of filgrastim remain sterile for 7 days when prepared in a class 100 laminar air flow hood, using aseptic technique, and stored under refrigeration. This change in practice can result in significant cost savings.

Drug Contamination↗
PubMed1995

Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis.

Two polymerase chain reaction (RT-PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5' noncoding region of the enterovirus genome were designed. The RT-PCR product size was approximately 500 bp (479 bp for Poliovirus, 500 bp for Coxsackievirus) and was visualized using ethidium bromide-stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV-RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR were accomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10-fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis.

Acute Disease↗
PubMed1995

Subclinical mastitis and milk production in primiparous Simmental cows.

The prevalence of subclinical mastitis in beef cows was investigated and its relationship with milk production and 205-d adjusted calf weaning weights (ADJWW) determined. Primiparous Simmental cows (n = 25) were milked six times at approximately 30-d intervals. Before each milking, milk samples were aseptically collected from each quarter for bacteriological analyses. After recording milk weights, samples were obtained for determination of somatic cell counts (SCC), butterfat (BF), and protein (PRO). Somatic cell counts were transformed to Dairy Herd Improvement Association linear scores for statistical analyses. Cows were retrospectively assigned to one of two groups based on their SCC linear score being either less than (LOSCC) or more than (HISCC) 4.5, which corresponded to a SCC of 292 x 10(3) cells/mL. Milk samples from 32% (8/25) of the cows and 18% (18/100) of the quarters were culture-positive for bacteria (Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus species, Actinomyces pyogenes, and[or] coagulase-negative Staphylococcus) at one or more times. Milk production was higher in LOSCC than in HISCC cows (6.3 +/- .2 vs 5.4 +/- .3 kg/24 h; P = .004). Mean SCC linear score tended to be negatively correlated with mean milk production over the entire study (r = -.36; P = .08). Adjusted weaning weights were similar between calves suckling LOSCC and HISCC cows (243 +/- 5 vs 236 +/- 10 kg; P = .57). In summary, milk SCC concentrations in primiparous Simmental cows were highly variable. Higher SCC concentrations were associated with lower milk production in cows.

Animals↗
PubMed1991

Chemical and visual stability of amphotericin B in 5% dextrose injection stored at 4 degrees C for 35 days.

The chemical and visual stability of amphotericin B in 5% dextrose injection under refrigeration was assessed. Three admixtures of amphotericin B 0.1 mg/mL in 5% dextrose injection and three admixtures of amphotericin B 0.25 mg/mL in 5% dextrose injection were aseptically prepared in polyvinyl chloride (PVC) bags. Immediately after preparation (at time zero), six 5-mL samples were aseptically transferred from each admixture to sterile collection tubes. Three of the samples from each admixture were quick-frozen for later assay by stability-indicating high-performance liquid chromatography (HPLC), and the other three were immediately assessed for pH. Each of the six admixtures was also assessed visually under fluorescent light and 2x magnification for color change, turbidity, gas evolution, and precipitation. The admixtures were stored in PVC bags at 4 degrees C and protected from light. Six 5-mL samples were withdrawn from each admixture at 10, 21, and 35 days. Three of the samples from each admixture were assessed for pH, and three were quick-frozen for subsequent HPLC assay. There was no substantial loss or deterioration of amphotericin B during the 35-day study. At no time was the mean concentration of amphotericin B in the samples less than 96.4% of the concentrations at time zero for the 0.1-mg/mL samples or less than 96.6% of the time zero concentrations for the 0.25-mg/mL samples. There were no appreciable changes in pH, and there was no visual evidence of instability in any of the samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗
PubMed1996

Bovine mastitis due to algae of the genus Prototheca.

Protothecosis was described in many animals, with bovine mastitis being the main form. The increasing number of isolations of Prototheca spp. from bovine mastitis cases indicates the need of a detailed evaluation of this problem. Besides this, these algae do not respond to treatment with the antimicrobians most frequently applied, leading to elimination of the affected animals, as the best method to control the disease. In two dairy farms in the State of São Paulo, Brazil, a total of 155 lactating cows and 52 dry cows were examined. Milk samples were aseptically collected from lactating cows with clinical or subclinical mastitis. From dry cows, secretion samples from all quarters were collected. All samples were then taken to microbiological exams. At dairy farm 1, Prototheca spp. was isolated from 14.95% milk samples from lactating cows, (all of them cases of subclinical mastitis), and from 8.06% samples from dry cows. At dairy farm 2, Prototheca spp. was isolated from 5.1% milk samples and there were ten cases of clinical mastitis due to this agent. Although clinical mastitis has been considered the main form of occurrence of this pathology until now, it is important to consider Prototheca spp. as subclinical mastitis pathogen. Prototheca zopfii was the main specie isolated. In this study a high rate of intramammarian infections, as clinical or subclinical mastitis during lactation as well as in dry period, was observed, high lighting the importance of suitable diagnosis, so that control and preventive measures can be implemented to avoid dissemination of the agent.

Animals↗
PubMed1999

Sources and extent of microbiological contamination of beef carcasses in seven United States slaughtering plants.

This study determined microbiological loads of beef carcasses at different stages during the slaughtering to chilling process in seven (four steer/heifer and three cow/bull) plants. Potential sources of contamination (feces, air, lymph nodes) were also tested. Each facility was visited twice, once in November through January (wet season) and again in May through June (dry season). Carcasses were sampled by aseptic excision of surface tissue (100 cm2) from the brisket, flank, and rump (30 samples each) after hide removal (pre-evisceration), after final carcass washing, and after 24-h carcass chilling. The samples were analyzed individually by standard procedures for aerobic plate counts (APC), total coliform counts (TCC), Escherichia coli biotype I counts (ECC), and presence of Salmonella. Incidence of Salmonella was higher on dry feces of older compared to younger animals, fresh feces of younger compared to older animals, and on cow/bull carcasses compared to steer/heifer carcasses. Most factors and their interactions had significant (P < or = 0.05) effects on the bacterial counts obtained. Depending on plant and season, APC, TCC, and ECC were < or =10(4), < or =10(2), and < or =10(1) CFU/cm2 in 46.7 to 93.3, 50.0 to 100.0, and 74.7 to 100.0% of the samples, respectively. TCC exceeded 10(3) CFU/cm2 in 2.5% (wet season) and 1.5% (dry season) of the samples. ECC exceeded 10(2) CFU/cm2 in 8.7%, 0.3%, and 1.5% of the pre-evisceration, final carcass-washing, and 24-h carcass-chilling samples, respectively, during the wet season; the corresponding numbers during the dry season were 3.5%, 2.2%, and 3.0%, respectively. These data should serve as a baseline for future comparisons in measuring the microbiological status of beef carcasses, as the new inspection requirements are implemented.

Abattoirs↗
PubMed1970

Action of bacterial growth on the sarcoplasmic and urea-soluble proteins from muscle. I. Effects of Clostridium perfringens, Salmonella enteritidis, Achromobacter liquefaciens, Streptococcus faecalis, and Kurthia zopfi.

Comparisons of the starch-gel patterns of uninoculated aseptic control samples from rabbit and pig muscle with similar samples inoculated and incubated with Clostridium perfringens, Salmonella enteritidis, Achromobacter liquefaciens, and Kurthia zopfii were made. Results indicated that C. perfringens caused extensive alteration in the proteins or enzymes, or both, of the sarcoplasmic fraction of porcine muscle, whereas S. enteritidis and S. faecalis caused complete breakdown of only myoglobin. Neither A. liquefaciens nor K. zopfii showed any measurable amount of proteolysis in the sarcoplasmic fraction from pig muscle. Although some of the bands in the starch-gel pattern of rabbit muscle decreased in size and intensity of staining, complete proteolysis of any protein fraction was absent for all test organisms. The disc-gel patterns of the 8 m urea-soluble proteins showed that C. perfringens caused extensive proteolysis in pig muscle and a lesser extent of proteolysis in rabbit muscle. None of the other organisms utilized in this study had any measurable effect upon the urea-soluble proteins. In addition, a simple procedure for aseptic isolation of muscle samples for studying meat spoilage is outlined. Results indicate that careful sanitation and cleanliness will give suitable samples for meat spoilage investigations.

Alcaligenes↗
PubMed2006

Splenectomy changes the pattern of cytokine production in beta-thalassemic patients.

A major cause of morbidity and mortality in beta-thalassemic patients is infections, assumed to be the result of immunological changes. To determine the possible defect, we investigated the cytokine productions by blood cells of beta-thalassemic patients using in-vivo and in-vitro methods. Heparinized blood samples collected aseptically from 22 beta-thalassemic children aged 10-12yrs (half of them were splenectomized). Samples from 10 healthy children served as control group. Part of samples was used for evaluation of plasma IL-2, IL-10 and TGF-beta1. Other part were stimulated with a mixture of LPS and PHA (1 and 10 microg/ml final concentration), for different time period (4, 24, 48 and 72h). Results showed circulating TGF-beta1 of splenectomized patients was significantly higher (p<0.01) than the control group. In-vitro results showed IL-2 production of patients' groups were significantly (p<0.01) lower than corresponding value obtained for the control group. In addition, IL-10 production by splenectomized group were less than other two group (p<0.01), while their TGF-beta1 were higher (p<0.001) at all time points treated. In conclusion, multi-transfusions could be responsible for a change in the subset of circulating lymphocytes that could contribute to a state of partial immune deficiency in beta-thalassemic patients, which is more prominence among the splenectomized patient.

Cells, Cultured↗
PubMed1989

Chronic central vein catheterization for intraoperative and long-term venous access in swine.

Chronic venous access and repeated blood sampling for research purposes in large swine ideally should be possible without sedation, restraint or direct venipuncture of deep vessels. An operative technique of cranial vena cava catheterization and chronic catheter maintenance methods are described which were used successfully in the placement of 11 silicone rubber catheters in 10 animals. All were used for repeated blood sampling, as well as intraoperative infusion of medications and large fluid volumes. Long term patency was excellent with 10 catheters patent at the end of the study interval, up to 14 weeks after insertion. Serial blood sampling was accomplished easily without restraint. Catheter damage, infection or malfunction was rare. Proper maintenance and careful aseptic blood sampling render the cranial vena cava catheter a safe and reliable alternative to direct venipuncture in swine.

Animals↗
PubMed1997

Microbiological quality of cooked chicken breasts containing commercially available shelf-life extenders.

Experiments were conducted to determine the effect of various shelf-life extenders on the aerobic plate counts (APC) of cooked chicken breast meat stored at refrigeration temperatures. Fresh chicken breast meat obtained from local grocers was injected with either 0.5, 1, 1.5, or 2% sodium lactate; 0.63, 1.25, 1.88, or 2.51 g/kg of a liquid smoke flavoring; 0.33, 0.66, 1, or 1.33% Per/Lac 1901, a fermented whey product; or 0.25, 0.5, 0.75, or 1% Alta 2341, a fermented corn syrup product. The samples were cooked at 85 C dry bulb, 77.8 C wet bulb to an internal temperature of 76.7 C. The cooked chicken breasts were cut into 20-g samples and aseptically placed into Ziploc bags. Initial APC were enumerated following 2-d incubation at 30 C. Additional stored samples (2 C) were subsequently evaluated for APC every week for 5 wk. Only one of the four ingredients, Alta 2341, significantly extended cooked breast meat shelf-life over that of the controls. Using Alta 2341 would be beneficial in extending the refrigerated shelf-life of cooked chicken breast meat up to 5 wk.

Analysis of Variance↗
PubMed1998

Intrauterine viral infection at the time of second trimester genetic amniocentesis.

OBJECTIVE: To determine whether preexisting intrauterine viral infection is associated with postamniocentesis pregnancy loss. METHODS: We accessed our bank of second-trimester amniotic fluid (AF) samples obtained aseptically and stored at -20C from all 11,971 women who underwent genetic amniocentesis between 1988 and 1995. Samples were retrieved from every case of spontaneous pregnancy loss within 30 days of the amniocentesis (excluding aneuploidy and anomalies, n = 66). Sixty-six control samples were randomly chosen from subjects who delivered at term and were matched for year of test, gestational age, maternal age, and indication for amniocentesis. Investigators were blinded to the status of the samples, which were studied by polymerase chain reaction (PCR) for the presence of adenovirus, parvovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, enterovirus, influenza A virus, and beta-actin DNA. Results were compared with interleukin-6 (IL-6) levels previously measured by enzyme-linked immunosorbent assay in the same samples. RESULTS: Sixty-two study cases and 60 controls were sufficient for all PCR studies. Fourteen AF samples contained a single virus: five (8%) of 62 study cases and nine (15%) of 60 controls (P = .27). Adenovirus accounted for nine (64%) of 14 viruses identified: four of 62 cases and five of 60 controls (P = .74). Cytomegalovirus was not identified in any study cases but was found in three controls. The mean IL-6 levels in samples with and without virus were not significantly different (4.8+/-15.9 ng/mL with virus compared with 2.0+/-8.8 ng/mL without virus; P = .53). CONCLUSION: Presence of virus in second-trimester AF is not significantly associated with elevated IL-6 levels or with early postamniocentesis pregnancy loss.

Abortion, Spontaneous↗