Simple method for collection of samples from a frozen food.
The conical end of a plastic funnel can be sterilized and used for aseptic collection, transfer, and distribution of shaving samples drilled from a frozen-food product.
Eggs↗
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[Development of cell content and shedding of Prototheca spp. in milk from infected udder quarters of cows].
It was the objective of this study to analyse shedding patterns and somatic cell counts in cows and quarters infected with Prototheca spp. and to evaluate two approaches to identify infected animals by somatic cell count (SCC) or by bacteriological analysis of pooled milk samples. Five lactating dairy cows, chronically infected with Prototheca spp. in at least one quarter were studied over 11 weeks to 13 months. Quarter milk samples and a pooled milk sample from 4 quarters were collected aseptically from all quarters of the cows on a weekly basis. Culture results of quarter milk and pooled samples were compared using cross tabulation. SCC of quarter milk samples and of pooled samples were related to the probability of detection in the infected quarters and cows, respectively. Shedding of Prototheca spp. was continuous in 2 of 8 quarters. In the other quarters negative samples were obtained sporadically or over a longer period (1 quarter). Overall, Prototheca spp. were isolated from 83.6% of quarter milk samples and 77.0% of pooled milk samples of infected quarters and cows. Somatic cell counts were higher in those samples from infected quarters that contained the algae than in negative samples (p < 0.0001). The same applied for composite samples from infected cows. Positive samples had higher SCC than negative samples. However, Prototheca spp. were also isolated from quarter milk and pooled samples with physiological SCC (i.e. < 10(5)/ml). Infected quarters that were dried off did not develop acute mastitis. However, drying off had no effect on the infection, i.e. samples collected at calving or 8 weeks after dry off still contained Prototheca spp. Results indicate that pre-selection of cows to be sampled for Prototheca spp. by SCC and the use of composite samples are probably inadequate in attempts to eradicate the disease. However, due to intermittent shedding of the algae in some cows, single herd sampling using quarter milk samples probably also fails to detect all infected cases. Therefore, continuous monitoring of problem cows with clinical mastitis or increased SCC in herds during eradication programs is recommended.
Animals↗
Cultivation-independent assessment of the bacterial diversity of breast milk among healthy women.
Breast milk has been shown to be an excellent and continuous source of commensal and potentially probiotic bacteria to the infant gut. Our aim was to evaluate the dominant bacteria existing in breast milk of healthy women and the potential role of transit through the vagina in the acquisition of breast milk microbiota using the 16S rRNA amplified gene approach. Samples of breast milk were aseptically collected, at day 7 after delivery, from five mothers whose neonates were born by vaginal delivery and from five others who had had their babies by programmed elective cesarean section. All mothers were healthy, had a full-term pregnancy and breastfed their infants. DNA extracted from biological samples was used as a template for PCR amplification of 16S rRNA gene sequences with universal bacterial primers; then the PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE); finally, clone libraries of 16S rRNA gene sequences from 4 mothers (2 from each group) were constructed. PCR DGGE patterns and clone libraries suggest that each woman had a specific bacterial pattern in her breast milk, and confirm, at the molecular level, that breast milk of healthy women is a source of commensal bacteria to the infant gut. They also reinforce recent molecular studies which have shown that lactic acid bacteria colonization is not significantly related to the delivery method.
Bacteria↗
Detection of septicemia in chicken livers by spectroscopy,.
To establish a procedure for differentiating normal chickens from chickens with septicemia/toxemia (septox) by machine inspection under the Hazard Analysis and Critical Control Point-Based Inspection Models Project, spectral measurements of 300 chicken livers, of which half were normal and half were condemned due to septox conditions, were collected and analyzed. Neural network classification of the spectral data after principal component analysis (PCA) indicated that normal and septox livers were correctly differentiated by spectroscopy at a rate of 96%. Analysis of the data established 100% correlation between the spectroscopic identification and the subset of samples, both normal and septox, that were histopathologically diagnosed. In an attempt to establish the microbiological etiology of the diseased livers, isolates from 30 livers indicated that the poultry carcasses were contaminated mostly with coliforms present in the environment, hindering the isolation of pathogenic microorganisms. Therefore, to establish the cause of diseased livers, a strictly aseptic environment and procedure for sample collection is required.
Animals↗
The release of prion protein from platelets during storage of apheresis platelets.
BACKGROUND: Recent studies using a time-resolved fluoroimmunoassay method (dissociation-enhanced lanthanide fluoroimmunoassay) showed that platelets and plasma are the main reservoir of the normal isoform of cell-associated prion protein (PrPc) in human blood. The aims of the present study were to monitor PrPc levels in various fractions of apheresis platelets during storage by using the DELFIA method and to assess the association of this release with alpha-granule protein ss-thrombo-globulin and cytoplasmic LDH. STUDY DESIGN AND METHODS: Units of apheresis platelets (n = 6) were obtained from volunteer donors by the use of a cell separator and stored up to 10 days. Samples (7-9 mL) were aseptically collected from each unit on storage Days 1, 2, 3, 4, 5, 8, and 10. Platelet-poor plasma and apheresis platelets were prepared and the former split into two fractions, one centrifuged at 40,000 x g for 2 hours at 4 degrees C to remove microparticles. The spun microparticles, apheresis platelets and platelet samples, platelet-poor plasma, and high-spun plasma fractions were stored in a frozen state until they were tested. RESULTS: The results showed that the mean overall levels of PrPc throughout storage remained within 15 percent of Day 1 levels. In contrast, the mean cellular levels in platelets significantly decreased to 46 percent of Day 1 levels by Day 10 of storage (p<0.01), while the corresponding levels in plasma significantly rose as much as 329 percent (p<0.01). Moreover, although microparticle-bound PrPc was released during storage, it was increasingly superseded by soluble protein. PrPc and ss-thrombo-globulin release exhibited very similar patterns (p<0.01). In contrast, LDH showed a significant increase in high-spun plasma only toward the end of the storage period (p<0.01). CONCLUSION: These results indicate that PrPc is released from platelets during the storage of apheresis platelets and that this release is probably due mainly to platelet activation and alpha-granule release in the first few days of storage. Moreover, the released PrPc is increasingly composed of soluble proteins, as the storage period exceeds 5 days.
Blood Platelets↗
Sterility validity period of vials after multiple sampling under vertical laminar airflow hood.
STUDY OBJECTIVES: The aim of this study is to validate the sterility period of vials after multiple sampling under Grade A vertical laminar airflow hood. METHODS: Vials filled aseptically with a sterile culture medium have been sampled with syringes three times a week over one month under Grade A vertical laminar airflow hood and in the mean-while, keeping the vials out of the laminar airflow hoods. RESULTS: No microbial growth has been observed. On the basis of these results, it has been decided to modify our standard operating procedures, to allow keeping the vials for two weeks in a box out of the laminar airflow hoods (ambient temperature Grade B) or any controlled environment (under refrigeration). CONCLUSIONS: This study validates the multiple use of vials of small to large volumes (5-100 mL), to simplify handling and to reduce the costs in centralized cytostatic reconstitution units in hospital pharmacies, with no microbial risk.
Bacteria, Aerobic↗
Identification of oral spirochetes at the species level and their association with other bacteria in endodontic infections.
OBJECTIVES: Recent molecular approaches have revealed that fastidious organisms such as Bacteroides forsythus and oral treponemes were frequently found in root canals with apical periodontitis. The purpose of this study was to identify the isolates of oral spirochetes at the species level in endodontic infections and to determine their association with B forsythus and Porphyromonas gingivalis. STUDY DESIGN: Seventy-nine teeth with apical periodontitis were selected for this study. After sampling from the root canals aseptically, polymerase chain reaction amplification for the 16S rRNA gene was performed with eubacterial universal primers. Subsequently, dot-blot hybridization was performed with 8 species-specific oligonucleotide probes. The microbial associations were analyzed by using the odds ratio. RESULTS: The most frequently found species was P gingivalis (27.4%), followed by Treponema maltophilum (26%), B forsythus (16.4%), and Treponema socranskii (2.7%). Other treponemes, including Treponema denticola, were not detected in our samples. Significant microbial associations were identified between T maltophilum, B forsythus, and P gingivalis by performing analysis with the odds ratio. CONCLUSION: Our results indicate that T maltophilum should be included in etiologic studies of endodontic diseases.
Adolescent↗
Effect of feed withdrawal on the incidence of Salmonella in the crops and ceca of market age broiler chickens.
Previous research regarding Salmonella contamination in poultry has focused predominantly on cecal and intestinal contamination. Recently, the crop has been implicated as an important source of carcass contamination within the processing plant. In the present study, broiler chickens were orally challenged with 1 x 10(8) cfu S. enteritidis at 6 wk of age. At 7 wk of age, birds were randomly divided into two groups consisting of full access to feed, or total feed withdrawal, 18 h prior to sample collection. At the time of sample collection, crops and ceca were aseptically removed and cultured for the presence or absence of S. enteritidis by enrichment. The incidence of S. enteritidis-positive crops was consistently higher (range: 2.8- to 7.3-fold increases) following feed withdrawal than the incidence in samples collected from full-fed broilers in four experiments. Similarly, the incidence of S. enteritidis isolation was consistently higher (range: 1.4- to 2.1-fold increases) in ceca following feed withdrawal than in samples collected from full-fed broilers in these experiments. In a subsequent experiment, ceca and crops were aseptically collected and cultured for the presence of Salmonella immediately prior to or following 8 h feed withdrawal at a commercial broiler house. Similar to the laboratory experiments, the incidence of Salmonella isolation was significantly (P < 0.01) greater from crops following feed withdrawal (36/100) than from samples obtained immediately prior to withdrawal (19/100). However, the incidence of Salmonella in the ceca was not significantly higher following feed withdrawal (31/100) than in samples obtained immediately prior to withdrawal (25/ 100) in this field experiment. These studies indicate that feed withdrawal increases the incidence of Salmonella in broiler crops prior to slaughter and provide further evidence that the crop may be an important critical control point for reducing Salmonella contamination of broiler carcasses.
Animals↗
Investigation of bacteremia following orthodontic debanding.
The aim of this study was to investigate the prevalence of bacteremia after orthodontic debanding and debonding. The study group comprised 30 patients (10 men, 20 women). All were treated using the Edgewise technique. Patients with acceptable oral hygiene and fixed appliances in both jaws were included in the study group. Blood samples were obtained using a strict aseptic technique before and after removal of bands and brackets. A 6.6% bacteremia prevalence was observed in both preoperative and postoperative blood samples.
Adolescent↗
Detection of enteroviral RNA by polymerase chain reaction in cerebrospinal fluid from patients with aseptic meningitis.
An assay based on a 2-step (semi-nested) polymerase chain reaction (PCR) was developed and evaluated for detection of enterovirus-specific RNA in cerebrospinal fluid (CSF) from patients with aseptic meningitis of different etiology. The limit of detectability of enteroviral RNA was equivalent to about 0.25 tissue culture infective doses 50%. In samples, stored at -70 degrees C, analyzed without repeated thawing, enteroviral RNA was demonstrable in 21/22 CSF specimens from which an enterovirus had been isolated. Enteroviral RNA was shown to be degraded during freeze-thawing of the samples. In repeatedly freeze-thawed samples from 134 consecutive patients with aseptic meningitis, a lower sensitivity (34/48 = 0.71) was observed. In the latest phase of the study, comprising 35 consecutive patients, the PCR was performed in CSF stored at -20 degrees C without thawing. In this material, the PCR yielded positive results in 19 patients, whereas enteroviruses were isolated from 6 cases only. In the total clinical material of 169 patients, 67 (40%) were found positive by PCR, whereas an enterovirus was isolated from CSF in 54 (32%) cases. All the 13 isolated enterovirus serotypes found in the study were demonstrable by PCR, indicating that the assay is broad-reacting within the enterovirus group. The specificity appeared to be high, since all of 21 patients with non-enteroviral diagnoses were negative by the PCR test, except 1 with an Epstein-Barr virus infection. As serological evidence of enteroviral etiology was found in this patient, a dual infection seemed probable. This study indicates that enteroviral RNA can be detected in CSF by a 2-step PCR in meningitis caused by enterovirus and that the technique has the potential to become a screening method for routine diagnosis of enteroviral meningitis.
Adolescent↗
Bacterial and yeast flora of root surface caries in elderly, ethnic Chinese.
OBJECTIVES: Root caries is emerging as a significant problem in the middle aged and elderly. As little data is available on the microbiology of root caries in Chinese cohorts, we evaluated 30 such lesions in elderly, institutionalized, ethnic Chinese. METHODS: Samples of carious dentine were aseptically taken from root caries lesions of 18 subjects (five males and 13 females, mean age 79.67 +/- 8.57). The cultivable bacteria, both aerobic and anaerobic, were isolated and identified using standard methods and commercial identification kits. The yeasts were isolated on Sabouraud's agar and identified using the API system. RESULTS: The main findings were: (1) of the total isolates, 91.09% were Gram-positive and 8.91% were Gram-negative microorganisms; (2) the proportions of cocci and rods were 36.68 and 63.31%, respectively; (3) the predominant groups of organisms isolated were Streptococcus spp., Lactobacillus spp., Staphylococcus spp. and Actinomyces spp. with isolation frequencies of 100, 90.00, 73.33 and 63.33%, respectively; (4) the isolation frequency of yeasts belonging to Candida spp. (63.33%) was notably high although the proportion of yeasts within each sample was low (0.01%). Candida dubliniensis, a newly identified yeast species particularly prevalent in HIV infection, comprised 14.29% of yeasts. CONCLUSIONS: This study provides baseline information on the microbiologic features of root caries in the elderly, ethnic Chinese. Whilst our data on the most predominant bacteria isolated in root caries concur with those from other regions of the world the isolation of C. dubliniensis from these lesions has not been documented before.
Actinomyces↗
White blood cell counts and differential in synovial fluid of aseptically failed total knee arthroplasty.
A cell count and differential was obtained on synovial fluid samples from 79 total knee arthroplasties undergoing revision for aseptic failure over a 5-year period. The mean white blood cell count (WBC) was 782/mL (range, 11-7,200/mL). The mean percentage of monocyte cells was 87%, and the mean polymorphonuclear leukocyte (PML) cell count was 13%. Eight patients (10.4%) had leukocyte cell counts >2,000. Five knees in 4 patients with rheumatoid arthritis had PML counts >50%. There was a single postoperative infection. In patients with osteoarthritis as a primary diagnosis, a synovial WBC of <2,000 and a differential with <50% PMLs had a 98% negative predictive value for the absence of infection. Key words: synovial fluid, cell count and differential, failed total knee arthroplasty, revision total knee arthroplasty.
Adult↗
[Investigation of enterovirus in cases of aseptic meningitis syndrome of Belém, PA].
With the objective of isolating and identifying enteroviral serotypes, which are the most frequent etiologic agents of aseptic meningitis syndrome, cerebrospinal fluid samples were studied from patients referred to the health unit in the City of Belém (State of Pará, Brazil), from March 2002 to March 2003. The samples were inoculated in RD and HEp-2 cells line, and the positive were identified by neutralization test or indirect immunofluorescence. Out of 249 samples, 33 (13.2%) were positive 57.6% (n = 19) in patients under 11 years of age (p < 0.03) and predominantly (72.7%) in males (p < 0.008). The serotypes isolated were: Echovirus 30 (n = 31), Coxsackievirus B5 (n = 1) and Echovirus 30 and 4. In conclusion, this type of study also serves to determine the national picture, which is still little defined, regarding the most prevalent viral agents in AMS cases.
Adolescent↗
Antibodies to antigen A60 in cerebrospinal fluid from patients with tuberculous meningitis.
Cerebrospinal fluid (CSF) anti-mycobacterial antigen 60 (A60) IgM, IgG and IgA in patients affected by meningitis of different etiologies were assayed as a rapid diagnostic test in cases of tuberculous meningitis. A commercial EIA was used to test 127 CSF samples classified as follows: tuberculous meningitis (n = 27 CSF samples from 16 patients, 6 of them with AIDS), pyogenic meningitis (n = 13), non-tuberculous aseptic meningitis (n = 43) and 44 normal CSF samples (16 of them from HIV-positive patients, 8 of whom had extraneurological tuberculosis). Anti-A60 IgM was positive only in two cases (1 tuberculous meningitis and 1 self-resolving aseptic meningitis). Positive CSF anti-A60 IgG and IgA were observed in eight and nine out of 16 patients with tuberculous meningitis, but only in four and five out of 13 samples studied prior to or in the first ten days of treatment, respectively. Most of the patients with false-positive IgG and IgA (16%) had pyogenic meningitis, but without intrathecal synthesis of antibodies. In patients with aseptic meningitis, the finding of CSF anti-A60 IgG plus IgA, initially or during follow-up, can be used as a diagnostic criterion for tuberculous meningitis, with a specificity of 100%, a positive predictive value of 1, and a negative predictive value of 0.81. However, its sensitivity is only 50% in immunocompetent patients and 16% in patients with AIDS.
Adolescent↗
Survey of intramammary infections in ewes on the New England Tableland of New South Wales.
Samples of mammary secretion were collected aseptically from 1093 ewes in 8 separate flocks. Most of the ewes were suckling lambs 4 to 6 weeks old. Standard bacteriological tests were carried out on the samples to identify the organisms involved in intramammary infections. Data on age, breed, lactational status and clinical status of the gland and its secretion were recorded at the time of sampling. The prevalence of intramammary infection was 14% of ewes (8% of glands). There was a tendency for prevalence of intramammary infection to be positively correlated with age of the ewe (two-year-old and six-year-old ewes had, respectively, 4.4% and 14.0% of glands infected). This relationship was highly significant for Border Leicester x Merino ewes. There were also significant differences in infection prevalence between breeds. infected glands had a higher prevalence of clinical abnormalities of udder, teat and secretion than did non-infected glands. Staphylococcus aureus was overwhelmingly the most frequently isolated bacterium being responsible for 40% of all intramammary infections.
Age Factors↗
Oxidative stress in childhood meningitis: measurement of 8-hydroxy-2'-deoxyguanosine concentration in cerebrospinal fluid.
To examine the involvement of reactive oxygen species, we measured the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative stress, in cerebrospinal fluid samples from 63 children with and without meningitis. We observed that the mean concentration of 8-OHdG in samples obtained during the early phase of bacterial meningitis, but not aseptic meningitis, was significantly higher than that in control samples. Clinical and laboratory improvement was associated with a fall in the 8-OHdG concentration in the patients with bacterial meningitis. Our findings suggest the presence of enhanced oxidative stress in the central nervous system of children with bacterial meningitis.
8-Hydroxy-2'-Deoxyguanosine↗
Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000.
Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.
Adolescent↗
Is there a role for Chlamydia pneumoniae in hemodialysis vascular access thrombosis?
Significant evidence suggests that Chlamydia pneumoniae has a major role in occlusive vascular disease. Vascular access thrombosis in chronic hemodialysis patients is a frequent problem; the underlying pathological state is stenosis caused by endothelial hyperplasia. There is presently no literature concerning C pneumoniae in vascular access thrombosis. We embarked on a study to evaluate the possible role of C pneumoniae in access failure. Ten consecutive patients with thrombosed polytetrafluoroethylene (PTFE) conduit arteriovenous fistulae undergoing surgical thrombectomy and revision were studied. We sought to detect C pneumoniae using both culture and polymerase chain reaction (PCR) methods. An excisional biopsy of the stenotic vein segment just above the anastomosis with the PTFE graft was obtained at surgery. Vein samples weighing at least 30 mg were aseptically placed in transport media and stored at 4 degrees C for up to 24 hours. The samples then were sonicated, inoculated in Hep-2 cell culture vials containing confluent monolayers, and passaged three times over 2 weeks. Detection was by direct fluorescent antibody staining. Both culture and PCR were performed in an active chlamydia research laboratory. None of the 10 samples was positive for C pneumoniae by culture or PCR. Based on our preliminary pilot study, we do not believe C pneumoniae has a major role in endothelial hyperplasia and consequent graft loss in the hemodialysis patients we studied.
Aged↗