Echocardiographic evaluation of regurgitant fraction in dogs with subclinical myxomatous mitral valve disease: Method comparison, effects of pimobendan, and reproducibility.

INTRODUCTION/OBJECTIVES: To evaluate regurgitant fraction (RF) using Simpson's method of discs to estimate total stroke volume (RFSMOD_TSV) and using Motion-mode to estimate total stroke volume (RFM-modeTSV) in dogs with subclinical myxomatous mitral valve disease (MMVD). We also sought to evaluate the effects of pimobendan on RF, and to determine the reproducibility of RFSMOD_TSV and RFM-modeTSV. ANIMALS, MATERIALS, AND METHODS: Echocardiography was performed on 57 dogs with MMVD (30 stage B1 and 27 stage B2). Ten dogs received pimobendan for 7-10 days and had a second echocardiogram. Nine dogs underwent six repeated echocardiographic examinations by two operators on three nonconsecutive days within one week for reproducibility analysis. RESULTS: Both RFSMOD_TSV and RFM-modeTSV exhibited a curvilinear relationship with left atrium-to-aortic root ratio. Both RFSMOD_TSV and RFM-modeTSV varied considerably within stage B1 (minimum-maximum: -9.1%-58.2% and -35.7%-66.2%, respectively) and B2 (13.6%-76.2% and 20.1%-85.7%, respectively). Method comparison showed RFSMOD_TSV and RFM-modeTSV were not interchangeable with proportional bias. Pimobendan significantly reduced RFSMOD_TSV (-32.0% ± 23.3%) and RFM-modeTSV (-19.2% ± 10.9%) within the same dog and relative to controls. Good inter-day and between-operator reproducibility was observed for RFSMOD_TSV and RFM-modeTSV based on intraclass correlation coefficients 0.86-0.90 and 0.83-0.90, respectively. Reproducibility coefficients were 19.6%-24.1% and 24.1%-27.0%, respectively. CONCLUSIONS: Use of RF using the total stroke volume method to aid the assessment of dogs with subclinical MMVD might be of clinical value. However, further study is warranted. Based on response to pimobendan and reproducibility analysis, RF SMOD_TSV might be a more reliable technique to quantify RF.

Aorto-left atrial fistula secondary to infective aortic endocarditis and endarteritis in a cat with valvular aortic stenosis.

A cat previously diagnosed with valvular aortic stenosis developed acute respiratory distress. A new continuous heart murmur was noted on physical exam. Echocardiographic examination revealed vegetative lesions on the aortic valve and continuously shunting blood flow from the aorta into the left atrium. Despite initial treatment for left-sided congestive heart failure, the cat died suddenly. In addition to confirming aortic valve endocarditis and an acquired aorto-left atrial shunt, pathological examination identified vegetative lesions on the luminal surface of the ascending aorta. Although antemortem aerobic blood culture, 16s bacterial ribosomal DNA PCR, and Bartonella PCR failed to identify causative organisms, Escherichia coli was identified on postmortem tissue culture of the aortic lesion. This represented a unique case of primary valvular aortic stenosis with secondary infective aortic endocarditis, infective aortic endarteritis, and aorto-left atrial fistula in a cat. It highlighted potential adverse outcomes of aortic stenosis that are more commonly recognized in humans and dogs.

Caesarean section at maternal request--the differing views of patients and healthcare professionals: a questionnaire based study.

OBJECTIVE: The number of caesarean sections at maternal request without medical indication is increasing. We aimed to explore the views of pregnant women, midwives and doctors using six hypothetical clinical scenarios and compare group views on: (a) perceived appropriateness of requests for caesarean section and (b) the reasons underlying these requests. STUDY DESIGN: A questionnaire was distributed to 166 pregnant women, 31 midwives and 52 doctors within maternity units at two hospitals in the North East region of England. Six hypothetical clinical scenarios for maternal requests were used: (1) uncomplicated first pregnancy, (2) one previous normal delivery, (3) one previous instrumental delivery, (4) one previous caesarean section, (5) one previous caesarean section with vaginal delivery since and (6) uncomplicated twin pregnancy. To highlight the differences in group responses, two main questions were asked for each scenario: 1. Should women be able to request a caesarean section? 2. What do you feel are the reasons for requesting a caesarean section? Data was analysed using Chi-squared or likelihood ratio as appropriate. RESULTS: In scenarios 1-3, professional groups were 'less likely' than pregnant women to always support a request (2.4% vs. 19.4%), (2.6% vs. 15.6%), (4.6% vs. 22%), (p<0.001). No significant differences were shown between doctors and midwives except for scenario 6 (twins), where midwives more often felt maternal requests should be declined (26.1% vs. 1.9%) (p=0.001). Multiparous women (n=95) were more likely to agree 'sometimes' to maternal requests in scenarios 1, compared to nulliparous women (n=71) (21.1% vs. 4.2%) (p=0.04). 'Safety of the baby' was ranked highly with pregnant women in scenarios 1-3 (mean 24.4%, range [15.8-38%]) compared with healthcare professionals (7.6% [3.4-12.8%]). However in scenario 3, healthcare professionals attributed 'fear of injury to self' (29.6%) as the most likely reason compared to 14.6% of pregnant women. CONCLUSION: Healthcare professionals and pregnant women's views differ significantly. Multiparous patients' views differ from those who have not had children before. We should provide clearer information on risks and benefits which encompass areas that concern women most.

Comparison of membrane filtration rates and hydrophobic grid membrane filter coliform and Escherichia coli counts in food suspensions using paddle-type and pulsifier sample preparation procedures.

Food suspensions prepared by Pulsifier contained less debris and filtered 1.3x to 12x faster through hydrophobic grid membrane filters (HGMFs) than those prepared by Stomacher 400. Coliform and Escherichia coli counts made by an HGMF method yielded 84 and 36 paired samples, respectively, positive by both suspending methods. Overall counts of pulsificates and stomachates did not differ significantly for either analysis, though coliform counts by Pulsifier were significantly higher in mushrooms and significantly lower in ground pork (P = 0.05). Regression equations for log10 counts of coliform and E. coli by Pulsifier and Stomacher were: Pulsifier = 0.12 + 0.97 x Stomacher, and Pulsifier = 0.01 + 1.01 x Stomacher, respectively.

Membrane filter method based on FC-5-bromo-4-chloro-3-indolyl-beta-D-glucuronide medium facilitates enumeration of Escherichia coli in foods and poultry carcass rinses.

Three enumeration methods for Escherichia coli in foods, the Health Protection Branch most-probable-number (MPN) method MFHPB-19, a hydrophobic grid membrane filter method MFHPB-26 (HGMF-indole), and a hydrophobic grid membrane filter method utilizing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide in a (modified) mFC agar (HGMF-FC-BCIG) were compared in 80 food samples that included naturally and artificially contaminated raw vegetables, mung bean and alfalfa sprouts, raw meats, and chicken carcass rinses. The number of samples confirmed as positive for E. coli were 44, 36, and 42 for the MPN, HGMF-indole, and HGMF-BCIG methods, respectively. By the MPN method, E. coli was detected in 3 samples at levels below the limits of detection of the HGMFs; but the MPN method was very time-consuming. With the HGMF-indole procedure E. coli was missed in 4 artificially contaminated samples. With the HGMF-FC-BCIG method E. coli was enumerated in 1 sample of bean sprouts missed by both the MPN and HGMF-indole procedures. High levels of indole-positive Klebsiella spp. in bean sprouts interfered with the HGMF-indole method, but the blue colonies of E. coli were easily observed in the HGMF-FC-BCIG method. Specificity of the HGMF-FC-BCIG method is high enough that routine confirmation should be unnecessary; however, confirmation of presumptive E. coli is easier since no lethal indole-staining step is involved. It appears to be a very simple method for quantifying E. coli in foods or carcass rinses.

Measurement of antimicrobial-resistant Escherichia coli in pig feces with a hydrophobic grid membrane filter interpreter system.

Hydrophobic grid membrane filter technology was used to measure resistance among Escherichia coli in pig fecal samples to ampicillin, sulfisoxazole, and tetracycline. The method accurately measured resistance, with sensitivities ranging from 96.5 to 99.5% and specificities ranging from 87.0 to 98.3%, and it identified E. coli with 96% confidence.

Comparison of the hydrophobic grid-membrane filter DNA probe method and the Health Protection Branch standard method for the detection of Listeria monocytogenes in foods.

The standard Health Protection Branch (HPB) method for the detection of L. monocytogenes in foods involves lengthy enrichment, selection and biochemical testing, requiring up to 8 days to complete. A hydrophobic grid-membrane filter (HGMF) method employing a digoxigenin-labelled listeriolysin O probe required 5 days to complete, and included an image-analysis system for electronic data acquisition. A total of 200 food samples encompassing 8 high-risk food groups (soft and semi-soft cheeses, packaged raw vegetables, frozen cooked shrimp, ground poultry, ground pork, ground beef, jellied meats, and pâté) were screened for the presence of L. monocytogenes by the two methods. Overall, 32 (16%) and 30 (15%) of the naturally-contaminated food samples tested positive for L. monocytogenes by the HPB and DNA methods, respectively. The DNA probe method was highly specific in discriminating L. monocytogenes from other Listeria spp. present in 50 of the samples tested. Results showed 94% sensitivity and 100% specificity between the two methods. The HGMF DNA probe method is an efficient and reliable alternative to the HPB standard method for detecting L. monocytogenes in foods.

Efficient Nondestructive Sampler for Carcasses and Other Surfaces.

Two versions of an electrically powered device (Rotorinser) to sample carcasses or other surfaces in situ for microbiological analysis and several different sampling protocols were evaluated against excision plus stomaching for ability to remove bacteria from pig skin and beef carcass tissue. Both devices sampled circular areas of approximately 14 cm2. Ten tissue samples were used for each set of conditions. Rotorinser bacterial removal efficiency was calculated as R/(R + S), where R is the Rotorinser count (CFU cm-2) and S is the count on stomached excised tissue after rotorinsing. Stomacher efficiencies were calculated as S1/(S1 + S2), where S1 is the first stomacher count of excised tissue and S2 is the count from a second stomaching. Both Rotorinsers were much better than traditional swabs. Rotorinser 1 gave removal efficiencies of 0.79 to 0.88 for beef, and 0.79 to 0.95 for pork. Prewetting surfaces for 5 min improved removal, but mixtures of enzymes did not. Rotorinser 2 applied with NaCl or NaCl-Tween 80 diluent for either 30 or 60 s was significantly better (0.93 and 0.98) than the stomacher (0.86) at removing aerobic mesophilic bacteria from pork skin. The Rotorinser causes negligible tissue damage and can be used on surfaces at any angle.

Improved aerobic colony count technique for hydrophobic grid membrane filters.

The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial growths are various shades of green on a pale green background, which can cause problems for automated as well as manual counting. HGMFs which had been incubated 24 or 48 h at 35 degrees C on Trypticase soy agar were flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were counted after color had been allowed to develop for 15 min at room temperature. With representative foods, virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official action procedure. Manual counting was easier as well because of increased visibility of the microbial growths. Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts, whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.

Rapid hydrophobic grid membrane filter-enzyme-labeled antibody procedure for identification and enumeration of Escherichia coli O157 in foods.

An O-antigen-specific monoclonal antibody, labeled by horseradish peroxidase-protein A, was used in a hydrophobic grid membrane filter-enzyme-labeled antibody method for rapid detection of Escherichia coli O157 in foods. The method yielded presumptive identification within 24 h and recovered, on average, 95% of E. coli O157:H7 artificially inoculated into comminuted beef, veal, pork, chicken giblets, and chicken carcass washings. In food samples from two outbreaks involving E. coli O157:H7, the organism was isolated at levels of up to 10(3)/g. The lower limit of sensitivity was 10 E. coli O157 per g of meat. Specific typing for E. coli O157:H7 can be achieved through staining with labeled H7 antiserum or tube agglutination.

ICMSF methods study. XVII. An international comparative study of the direct plate and hydrophobic grid-membrane filter methods for enumeration of Escherichia coli in foods. International Commission on Microbiological Specifications for Foods.

Eight laboratories compared counts of Escherichia coli from naturally or artificially contaminated ground beef, other meats and poultry, vegetables, fish and shellfish, cheese, and diverse sources such as swabs, by the Anderson-Baird-Parker direct plate (DP) and a hydrophobic grid-membrane filter (HGMF) method. For five of the eight laboratories overall counts by HGMF were significantly low (51-83%) compared with those by DP. Counts by HGMF tended to be lower for naturally contaminated samples; several possible causes were investigated. In a subsidiary study, analyst variation in counting HGMF ranged from 0.8-7.3%, with little evidence of effects from counting positive versus negative grid cells or from the fullness of growth or staining intensity.

Comparison of bacterial counts obtained from naturally contaminated foods by means of Stomacher and blender.

Four Regional Health Protection Branch laboratories each compared aerobic colony counts obtained after "stomaching" and blending, for a minimum of 10 samples in each of the seven food groups: dry pastas; chocolate and cocoa powders; frozen entrees (macaroni and cheese, chow mein, chop suey, fried rice, seafood casseroles, and Salisbury steak); nonfat dry milk; shrimp and crabmeats; spices; and breakfast sausages. Overall, counts obtained after using the Stomacher were equivalent to or higher than counts obtained after using the blender in 73% of the comparisons (alpha = 0.05). Where differences existed, counts obtained after using the Stomacher tended to be higher than counts obtained after using the blender from milk powder and lower from sausage. Aerobic colony counts from these foods are not unacceptably biased when obtained by Stomacher.

Improved immunological membrane filter method for detection of food-borne Salmonella strains.

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.

Effect of homogenization procedures on Escherichia coli counts in naturally contaminated cheese.

A comparison was made between blending in 2% sodium citrate and stomaching in 0.1% peptone or 0.1% peptone - 1% Tween 80 for the enumeration of Escherichia coli in naturally contaminated cheeses. Statistical analysis of the results from 25 samples of cheese showed that there were no significant differences in recovery by the three methods at the 95% confidence level.

Improved recovery of fecal coliforms from the Ottawa River by membrane filters in the presence of food debris.

In the absence of food debris, Sartorius and Millipore HA filters recovered substantially fewer fecal coliforms from Ottawa River water than did Millipore HC. On addition of a small quantity of sterile blended carrot to water samples, recovery by the poorer filters equalled that on Millipore HC. Scanning electron microscopy revealed bacteria sheltered in crevices formed by carrot fibres and thus protected from the normal stresses of exposure. Addition of carrot debris (e.g., 0.03 g carrot to 100 mL of sample) thus provides a convenient and inexpensive means of reducing variations in fecal coliform recovery between brands of membrane filters.

Improved bacterial recovery by membrane filters in the presence of food debris.

In the absence of food debris, m-FC agar counts of Escherichia coli on Oxoid Nuflow membrane filters (Oxoid Canada Inc., Nepean, Ontario, Canada) were lower than the corresponding surface plate counts. For seven food types tested, recovery of E. coli improved with increasing thickness of food debris on the membrane filter, and mats thicker than 0.5 micron protected the organism completely.

Rapid enumeration of Staphylococcus aureus in foods by direct demonstration of enterotoxigenic colonies on membrane filters by enzyme immunoassay.

Based on enzyme-linked immunosorbent assay, a convenient method has been devised for the direct demonstration of enterotoxin B production by Staphylococcus aureus colonies grown for 24 h on membrane filters. The problem of false-positive reactions due to binding of immunoglobulin G to protein A was turned to advantage by conjugating horseradish peroxidase directly to protein A, which then mediated the labeling of the antitoxin. The test requires 3 h to complete and yields a purple stain at the site of enterotoxin B-producing colonies, thus allowing direct enumeration of confirmed S. aureus in foods within 27 h. The method should be applicable to other enterotoxins of S. aureus.

Collaborative study of the MPN, Anderson-Baird-Parker direct plating, and hydrophobic grid-membrane filter methods for the enumeration of Escherichia coli biotype I in foods.

Five Health Protection Branch laboratories compared two membrane filter methods (the Anderson-Baird-Parker direct plating, and a hydrophobic grid-membrane filter method) against the most probable number procedure (MPN) for enumerating Escherichia coli biotype I in foods. Results were available in 24 h by both membrane filter methods, compared with 10-14 days by the MPN procedure. For ground beef, Parmesan cheese, and cut green beans, the hydrophobic grid method generally gave the highest recovery, although the two membrane filter methods were not significantly different. Both these methods gave significantly higher recoveries than the MPN procedure, and for most foods, either method would be preferable. Further work is required before either membrane filter method can be recommended for bean and alfalfa sprouts, which may contain very high levels of Klebsiella spp.

Colony counting on hydrophobic grid-membrane filters.

A device to facilitate manual scoring of hydrophobic grid-membrane filters (HGMF) is described. Variations in scores were generally less than 2.5% between 41 analysts from six laboratories, who, using the apparatus, scored a set of five specimen HGMF in different ways, and there was good agreement between scores from positive and negative grid-cell counts by each analyst. A scoring procedure for use in routine microbiological analysis, suitable for HGMF at various degrees of saturation, is recommended.