Evaluation of the Cult-a-Box gas generator system for isolation of Campylobacter spp. from stool specimens.

The Cult-a-Box gas generator tablet method for isolating Campylobacter spp. from stool cultures was evaluated by comparison with a standard gas mixture of 5% oxygen, 10% carbon dioxide, and 85% nitrogen. Of the 186 positive cultures, 90.3% were found by both methods. Of the remaining 9.7% which were positive in one system only, 11 were found by the tablet method and 7 by the standard gas method.     

Incidence of Campylobacter spp. in broiler flocks monitored from hatching to slaughter

The incidence of Campylobacter jejuni/coli was examined in five flocks of broilers monitored from hatch to slaughter, in feed and water and in litter samples. A total of 1440 samples from 720 broilers was examined. Campylobacter spp. were not isolated from broiler chicks at 1 day of age and were only isolated from one broiler chick in one flock at 1 week of age. In three flocks Campylobacter spp. were isolated from all chicks sampled at 4 weeks of age. In the fourth flock all chicks sampled were negative until 8 weeks of age when all were positive. The fifth flock remained negative throughout the 8 weeks of its life. Campylobacter spp. were not found in 20 samples of food and water. Of 20 litter samples they were found in only two. Eleven broiler flocks were examined only at slaughter. Twenty-four caecal samples were examined from each flock. In three flocks no Campylobacter spp. were isolated, in one flock one broiler chick was positive and in seven flocks from 58% to 100% of sampled broilers were positive. A total of 146 isolates was typed; 123 were C. jejuni and 23 C. coli, these belonged to 46 serotypes. In some flocks several serotypes were identified, some of them were not found again on further examinations of the same flocks during the growing period.     

Application of the Fortner principle to isolation of Campylobacter from stools.

A simple biological technique for reducing oxygen tension which does not necessitate the use of conventional anaerobic equipment is described. We successfully applied this method to the isolation of campylobacters from stools.     

Midtrimester genetic amniocentesis in eastern Ontario: a review from 1970 to 1985.

Greater use of genetic amniocentesis in the Eastern Ontario region occurred once the results from the various national trials were published. Acceptance also paralleled an increase in the number of low parity older women having children and the centralisation and greater publicity given to our programme in the late 1970s. A centralised approach has had the positive effects of preventing unnecessary procedures, assuring appropriate patient counselling, follow up, and review, and of increasing obstetric and laboratory expertise. Advanced maternal age has been largely responsible for the increased demand for the service and accounted for an increasing proportion of tests performed, while the absolute number for several other diagnostic categories remained unchanged. We found no evidence that women with a history of previous miscarriage had a higher rate of pregnancy loss following the procedure, and comparison with a group of women who declined amniocentesis did not show that the test increased the risk of miscarriage.     

Rapid isolation of Yersinia spp. from feces.

Direct plating or cold enrichment or both have been used to isolate Yersinia spp. from feces. Freeze-shock double enrichment and KOH treatment have been recommended for recovery of Yersinia enterocolitica from surface waters and food, respectively. These techniques were evaluated as alternatives for rapid recovery of Yersinia spp. from feces. Stool samples were homogenized in buffered saline and autoclaved. Escherichia coli. Klebsiella pneumoniae, and Pseudomonas aeruginosa were each added to the suspension at a final concentration of 1.5 x 10(6) colony-forming units per ml. Yersinia cells were then added to a final concentration of 1.5 x 10(3), 1.5 x 10(4), 1.5 x 10(5), or 1.5 x 10(6) colony-forming units per ml. A total of 21 strains of Y. enterocolitica, 2 of Yersinia kristensenii, and 1 each of Yersinia intermedia and Yersinia fredriksenii were tested. For freeze-shock double enrichment, seeded stool samples were frozen overnight (-70 degrees C), transferred successively to m-tetrathionate broth (6 h. 37 degrees C) and selenite broth (2 h 37 degrees C), and plated on MacConkey, salmonella-shigella, and cellobiose-arginine-lysine agars for quantitation. For KOH treatment, seeded stool samples were mixed with 0.5% KOH at a ratio of 1:2 for 2 min and plated as described above. E. coli, K. pneumoniae, and P. aeruginosa were virtually eliminated after either method was used. All Yersinia strains were recovered after KOH treatment even at the lowest initial concentration (1.5 x 10(3) colony-forming units per ml). However, after freeze-shock double enrichment, not all strains were retrievable, and those isolates which were recovered were grown only from samples containing the highest number of Yersinia strains (1.5 x 10(6) colony-forming units per ml). KOH treatment of stool samples seems to be a viable substitute for more protracted methods of recovering Yersinia spp.     

The use of a combined enrichment–filtration technique for the isolation of Campylobacter spp. from clinical samples

A combined non-selective enrichment-filtration technique was investigated for the isolation of Campylobacter spp. from clinical samples. In total, 479 samples were tested by direct culture, enrichment subculture and enrichment-filtration. The enrichment-filtration technique was used with both selective and non-selective media. Direct culture and enrichment subculture yielded 13 and seven isolates, respectively, while enrichment-filtration yielded 18 and 14 isolates on selective and non-selective agar, respectively. Thus, the combination of enrichment-filtration with selective agar produced a 38.5% increase in the number of isolates (p <0.05). All isolates were identified as Campylobacter jejuni.     

Coinfection of enteric Helicobacter spp. and Campylobacter spp. in cats

During a 6-year period, 64 of 227 commercially reared cats had microaerobic bacteria isolated from their feces. All the isolates were initially identified as Campylobacter-like organisms based on biochemical and phenotypic characteristics. DNA extractions from 51 of these isolates were subjected to PCR using primers specific for Helicobacter spp. and Campylobacter spp. Of the isolates, 92% (47 of 51 isolates) were positive for Campylobacter spp., 41% (21 of 51 isolates) were positive for Helicobacter spp., 33% (17 of 51 isolates) were positive for both genera, 59% (30 of 51 isolates) were positive only for Campylobacter spp., and 8% (4 of 51) were positive only for Helicobacter spp. Sixteen of the 47 Campylobacter-positive cultures were positive for more than one Campylobacter spp. Based on a species-specific PCR assay, 83% of the isolates were identified as Campylobacter helveticus, 47% of the isolates were identified as Campylobacter upsaliensis, and 6% of the isolates were classified as Campylobacter jejuni. The 1.2-kb PCR products of the 16S rRNA genes of 19 Helicobacter species isolates were subjected to restriction fragment length polymorphism (RFLP) analysis. Of the five different RFLP patterns obtained, two clustered with Helicobacter ("Flexispira") taxon 8, one clustered with Helicobacter bilis, one clustered with Helicobacter canis, and the remaining pattern was closely related to a novel Helicobacter sp. strain isolated from a woodchuck. The sequence data for the 16S rRNA genes of 10 Helicobacter spp. validated the RFLP-based identification of these isolates. This study demonstrated that biochemical and phenotypic characteristics of microaerobic organisms in cat feces were insufficient to characterize mixed Helicobacter and Campylobacter infections. Molecular structure-based diagnostics using genus- and species-specific PCR, RFLP analysis, and 16S rRNA sequence analysis enabled the identification of multiple microaerobic species in individual animals. The clinical relevance of enteric Helicobacter and Campylobacter coinfection in cats will require further studies.     

Isolation of Blastomyces dermatitidis from an earthen floor in southwestern Ontario, Canada.

The first Canadian isolation of Blastomyces dermatitidis from nature is recorded. This was from the earthern floor of a petroleum filtering shed located in southwestern Ontario. A technician working in this environment became infected and was diagnosed as having systemic blastomycosis with meningeal involvement.     

Growth of Aeromonas spp. on Butzler Campylobacter selective agar and evaluation of the agar for the primary isolation of Aeromonas spp. from clinical specimens

The fortuitous finding that Aeromonas spp. grew well on Butzler Campylobacter selective medium (BCSA) in a microaerobic atmosphere at 42 degrees C prompted us to evaluate the performance of BCSA for selective isolation of aeromonads in comparison with ampicillin (30 micrograms/ml) sheep blood agar (ASBA30). Overall recovery rates of aeromonads from 563 stool samples from patients with acute diarrhea were higher on ASBA30 (70.4%) than on BCSA (56.3%); however, 21 (29.5%) grew only on BCSA. The three human-associated Aeromonas spp. could be recovered on BCSA and ASBA30. We recommend the use of BCSA to laboratories reluctant to include a specific selective medium for aeromonads.     

Characterization of Clostridium difficile strains isolated from patients in Ontario, Canada, from 2004 to 2006

Clostridium difficile is the bacterium most commonly surmised to cause antimicrobial- and hospital-associated diarrhea in developed countries worldwide, and such infections are thought to be increasing in frequency and severity. A laboratory-based study was carried out to characterize C. difficile strains isolated from persons in Ontario, Canada, during 2004 to 2006 according to toxin type (enterotoxin A, cytotoxin B, and binary toxin [CDT]), tcdC gene characterization, ribotyping, pulsed-field gel electrophoresis, and toxinotyping. Clostridium difficile was isolated from 1,080/1,152 (94%) samples from 21 diagnostic laboratories. Isolates with toxin profiles A⁺ B⁺ CDT⁻, A⁺ B⁺ CDT⁺, A⁻ B⁺ CDT⁻, and A⁻ B⁺ CDT⁺ accounted for 63%, 34%, 2.4%, and 0.6% of isolates, respectively. Alterations in tcdC were detected in six different ribotypes, including ribotype 027. A total of 39 different ribotypes were identified, with ribotype 027/North American pulsotype 1 (NAP1), an internationally recognized outbreak strain associated with severe disease, being the second most common ribotype (19% of isolates). Transient resistance to metronidazole was identified in 19 (1.8%) isolates. While a large number of ribotypes were found, a few predominated across the province. The high prevalence and wide distribution of ribotype 027/NAP1 are disconcerting in view of the severity of disease associated with it.     

Modified selective medium for isolation of Campylobacter spp. from feces: comparison with Preston medium, a blood-free medium, and a filtration system.

Our previously described (H. Goossens, M. De Boeck, and J. P. Butzler, Eur. J. Clin. Microbiol. 2:389-393, 1983) selective medium, consisting of cefoperazone (15 mg/liter), rifampin (10 mg/liter), colistin (10,000 IU/liter), and amphotericin B (2 mg/liter) (medium M1), for the isolation of Campylobacter jejuni and Campylobacter coli from stool specimens was modified as follows: cefoperazone (30 mg/liter), rifampin (10 mg/liter), and amphotericin B (2 mg/liter) (medium M2). A comparative study of the isolation of Campylobacter spp. from stool specimens was carried out with medium M1; medium M2; a selective blood-free medium consisting (per liter) of charcoal (4 g), ferrous sulfate (0.25 g), sodium pyruvate (0.25 g), casein hydrolysate (3 g), sodium deoxycholate (1 g), nutrient broth no. 2 (25 g), agar (12 g), and cefoperazone (32 mg) (medium M3); and Preston medium containing (per liter) trimethoprim (10 mg), rifampin (10 mg), polymyxin B (5,000 IU), and cycloheximide (100 mg) (medium M4). We also included a filtration system in which membrane filters were applied directly to the surface of the nonselective blood-free medium distributed in small petri dishes. A total of 5,276 stool specimens were tested: 2,788 stool specimens were tested on M1 and M3 in study 1; 2,488 stool specimens were inoculated on the four selective media in study 2, and the last 986 specimens of the 2,488 were tested in parallel with the filtration system. In study 2, 128 Campylobacter strains were isolated from 126 different patients; 85.0, 88.3, 82.5, and 66.6% of these strains were isolated on M1, M2, M3, and M4, respectively. No contaminating fecal flora was found on 65.4, 70.7, 62.4, and 40.3% of the M1, M2, M3, and M4 plates, respectively. Furthermore, C. coli was found to be more susceptible to antibiotics present in the selective media, particularly colistin and polymyxin B, than was C. jejuni. We therefore recommend M2 for the isolation of Campylobacter spp. Finally, the filtration method was found to be easy and cheap; although the sensitivity was low, this method allowed the isolation of new Campylobacter spp. which seem to be associated with diarrhea.     

Epidemiological aspects of enteritis due to Campylobacter spp. in Norway

Data pertaining to 249 patients with stool cultures positive for thermophilic campylobacters are presented. Campylobacters were isolated from about 3% of all cases of acute enteritis and occupied second place in the bacterial etiology of this syndrome following Salmonella spp. Concomitant isolation of salmonellae or shigellae or both was achieved in 40 (16.1%) of the patients infected with campylobacters. The results suggest a bimodal age distribution with highest rates in young adults aged 20 to 29 years and children below 10 years of age. A majority of the campylobacters were isolated from travellers returning from abroad, and, to a lesser extent, from immigrants, particularly from Asia. Immigrants accounted for 45.2% of the patients below 10 years of age. The number of cases increased during the warmer months of the year. Travelling habits could, at least in part, explain the observed seasonality, age distribution, and geographical origin of infection. Eight outbreaks of Campylobacter enteritis were detected, five of which were family outbreaks, whereas three involved people from different families.     

Observations on primary health care in Ontario, Canada.

The Canadian health care system has developed very differently from that of its neighbour, the United States of America. It has a publicly financed and administered universal insurance plan which provides good access to high quality medicine, free at the point of delivery. Increasing costs, however, mean that painful political decisions on health will have to be made. Experiments with alternative means of financing primary health care provision and the Canadian approach to postgraduate education may offer useful ideas for general practice in the United Kingdom.     

Cadmium chloride susceptibility, a characteristic of Campylobacter spp.

We report a simple diagnostic characteristic useful in the presumptive identification of Campylobacter jejuni and Campylobacter coli. Filter paper disks impregnated with cadmium chloride were placed on streaked agar medium. Zones of growth inhibition for Campylobacter spp. occurred at 1.25 micrograms per disk. Other enteropathogens (Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, Escherichia coli, and Yersinia enterocolitica) were resistant to at least 40 micrograms per disk, with the exception of a strain of Shigella flexneri, which showed first susceptibility at 10 micrograms per disk. Most of the 52 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 2.5 micrograms of cadmium chloride per disk. At 20 micrograms per disk, Campylobacter isolates from clinical sources were significantly (P less than 0.01) more susceptible to cadmium chloride inhibition than were those from meat samples.     

Residents' duty hours in the province of Ontario, Canada.

The author explains the history of the Professional Association of Internes and Residents of Ontario (PAIRO), Canada, founded in 1968-69 to represent postgraduate medical trainees in negotiations with the Ontario Hospital Association over issues of trainees' stipends. Over the years, the negotiations evolved to cover a number of other issues, including duty hours, and established the principle that binding arbitration would be used to resolve any disputes between the two parties that could not be resolved through negotiation. At present, PAIRO negotiates a biannual collective agreement with the Ontario Council of Teaching Hospitals (OCOTH), whose features the author describes. The most important provisions of the 2000-2002 PAIRO-OCOTH agreement on the limits of duty hours are described. The author then comments that while such limits have benefited programs and residents, there is concern that the limits decrease the opportunities for trainees to be involved in the care of patients with a wide variety of medical conditions. Also, the duty-hours limits have required some services to use attending physicians or outside health professionals to perform duties previously carried out by trainees, creating problems that the author describes.     

Incidence of Smith-Lemli-Opitz syndrome in Ontario, Canada

Smith-Lemli-Opitz syndrome (OMIM 270400) (SLOS) is caused by inherited enzymatic deficiency of 3beta-hydroxysterol-Delta7-reductase (7-dehydrocholesterol-Delta7-reductase, DHCR7). SLOS is diagnosed clinically by the demonstration of elevated levels of 7-dehydrocholesterol (7DHC) in body fluids or tissues. SLOS is associated with mental retardation of variable degree and severe behavior abnormalities. The physical abnormalities range from minor facial anomalies to lethal malformations of the central nervous system, heart, kidneys, and other organs. The exact incidence of SLOS is not known. Although there exist estimates of the incidence of SLOS ranging from 1 in 20,000 to 1 in 60,000, no prospective studies of the incidence of SLOS, based on the clinical data and biochemical diagnosis of SLOS, have been performed. Five unrelated cases of SLOS were diagnosed in Ontario during a 12-month period. The diagnoses were made based on the demonstration of elevated 7DHC in plasma or amniotic fluid. The birth rate for Ontario for that period was 132,000 births. The incidence of SLOS in Ontario was at least 1 in 26,500 pregnancies in 1999-2000. Given that 86% of the population of Ontario is of European origin, the incidence of SLOS in the Ontario population of European origin was at least 1 in 22,700. As infants with mild forms of SLOS born during this period may remain undiagnosed, these numbers likely are underestimates. This observation has implications for prenatal and newborn screening for this potentially treatable inherited disorder.