Multilaboratory comparison of proficiencies in susceptibility testing of Helicobacter pylori and correlation between agar dilution and E test methods.

Susceptibility testing was performed at seven Canadian microbiology laboratories and the Helicobacter Reference Laboratory, Halifax, Nova Scotia, Canada, to assess susceptibility testing proficiency and the reproducibility of the results for clarithromycin and metronidazole and to compare the Epsilometer test (E test) method to the agar dilution reference method. Control strain Helicobacter pylori ATCC 43504 (American Type Culture Collection) and 13 clinical isolates (plus duplicates of four of these strains including ATCC 43504) were tested blindly. The National Committee for Clinical Laboratory Standards (NCCLS) guidelines for agar dilution testing were followed, and the same suspension of organisms was used for agar dilution and E test. Antimicrobials and E test strips were provided to the investigators. Methods were provided on a website (www.Helicobactercanada.org). Each center reported MICs within the stated range for strain ATCC 43504. Compared to the average MICs, interlaboratory agreements within 2 log(2) dilutions were 90% (range, 69 to 100%) for clarithromycin by agar dilution, with seven very major errors [VMEs], and 85% (range, 65 to 100%) by E test, with three VMEs. Interlaboratory agreements within 2 log(2) dilutions were 83% (range, 50 to 100%) for metronidazole by agar dilution, with six VMEs and eight major errors (MEs), and 75% (range, 50 to 94%) by E test, with four VMEs and four MEs. At lower and higher concentrations of antibiotic, E test MICs were slightly different from agar dilution MICs, but these differences did not result in errors. When a standardized protocol based on NCCLS guidelines was used, most participants in this study correctly identified clarithromycin- and metronidazole-susceptible and -resistant strains of H. pylori 93% of the time by either the agar dilution or E test method, and the numbers of errors were relatively equivalent by both methods.

Four-day incubation for detection of bacteremia using the BACTEC 9240.

Records of 29,356 blood cultures performed between April 1994 and April 1997, using the BACTEC 9240 continuous monitoring blood culture system, were reviewed retrospectively. From these, 3,127 blood culture vials became positive. Of 95 blood culture isolates detected after three days of incubation, 63 were recovered on day four and 32 on day five. Twenty-six contaminants were recovered on day four, and 21 on day five. Chart review was performed for all day four and five isolates that did not meet our definition of a contaminant. Of the 40 isolates that were clinically insignificant, 31 were recovered on day four, and nine on day five. Of eight clinically significant isolates, six were recovered on day four, and two on day five. Our data support a four-day incubation protocol for the recovery of all clinically significant bacteria with overall sensitivity reduced by only 0.06% when compared with a five-day protocol.

Helicobacter pylori: primary susceptibility to clarithromycin in vitro in Nova Scotia.

Resistance to antimicrobial agents is a major determinant of the efficacy of regimens to eradicate Helicobacter pylori. Clarithromycin (CLA) has become one of the most commonly used antibiotics for treatment of H pylori infection. In this study, the rate of primary resistance to CLA in H pylori isolated from patients was determined. One hundred sixty-two strains were recovered from patients before treatment. Strains were grown and inoculated onto Mueller-Hinton agar with 7% sheep blood. CLA epsilometer gradient agar diffusion test (E test) strips were used to test for susceptibility. Appropriate control organisms were tested to validate the assay. Plates were incubated at 37 degrees C in a microaerophilic atmosphere for up to five days. E test results were easy to interpret. Strains were considered resistant if the minimum inhibitory concentration (MIC) was 2 micrograms/mL or greater. Three strains were resistant (two strains with MIC 8 micrograms/mL and one strain with MIC 12 micrograms/mL) and 159 strains were sensitive (MICs ranged from less than 0.016 to 0.38 micrograms/mL). Ninety per cent of the strains had MICs of 0.023 micrograms/mL. Primary resistance was 1.8%. These susceptibility data support the use of CLA for the treatment of H pylori in the Nova Scotia population.

Subcutaneous dirofilariasis in Nova Scotia.

A case of dirofilariasis in a 78-year-old woman from Nova Scotia is described along with the histological findings and the basis of identification of the parasite. The patient developed a subcutaneous nodule, which was excised. Dirofilaria ursi and ursi-like nematodes are a rare cause of subcutaneous nodules. This is the first time dirofilariasis has been diagnosed in an adult in Atlantic Canada.

Direct immunofluorescence for the diagnosis of legionellosis.

Culture and direct immunofluorescent microscopy (DFA) results for Legionella pneumophila were reviewed over a two-year period. In the first year, a positive result was defined as having at least one morphologically typical fluorescing organism. In the second year, a positive was defined as at least five typical fluorescing organisms. Despite these stricter criteria and other measures to reduce the possibility of reagent contamination, there was no statistically significant difference in the sensitivity or specificity of the DFA in the two years for sputa, deep specimens or overall. Of 37 sputum specimens from infected patients, 16 were positive on DFA. Thirty-two of 38 positive patients were detected by sputum culture. DFA can provide rapid diagnostic information but cannot be used to rule out the diagnosis. Sputum is a useful specimen for the initial laboratory investigation of patients with legionellosis.

Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori.

Colonisation of the respiratory tract with Legionella pneumophila for 63 days before the onset of pneumonia.

We report the case of a 70-year-old man who was admitted to hospital A 66 days before developing Legionella pneumophila pneumonia 6 days after open heart surgery at hospital C. The strain of L. pneumophila recovered from the patient's sputum was of the same subtype (monoclonal antibody type, enzyme type, plasmid profile, and restriction endonuclease pattern) as a strain of L. pneumophila in the potable water supplied to the room where he stayed in hospital A. We conclude that the patient's respiratory tract became colonised by L. pneumophila while he was in hospital A and persisted for at least 63 days until he developed pneumonia requiring antibiotic treatment while in hospital C.

Pathogenic marine vibrio species in selected Nova Scotian recreational coastal waters.

Seven heavily frequented coastal recreation sites serving Metropolitan Halifax and Dartmouth were investigated to determine the numbers and species of pathogenic marine vibrios (PMV) present. Seawater, mussels and sea gull feces were cultured using quantitative methods and the effects of temperature and fecal pollution noted. Emergency rooms serving the sites under surveillance were monitored for PMV-related infections. All 11 recognized species of pathogenic marine vibrios were recovered from the 7 sites. Estuarine sites yielded a greater variety of species and greater numbers of PMV than non-estuarine sites. Culture of hand washings after immersion in seawater did not demonstrate contamination of skin by PMV. We did not demonstrate any cases of PMV infection associated with the 7 surveillance sites. PMV contamination of marine recreational waters does not frequently result in superficial infections.

A comparison of calcofluor white, potassium hydroxide, and culture for the laboratory diagnosis of superficial fungal infection.

A total of 207 skin scrapings were prospectively studied using potassium hydroxide (KOH), calcofluor white (CW), and culture to determine the clinical usefulness of each microscopic method. For dermatophytes (prevalence 13.2%), CW had a sensitivity of 92% and a specificity 95%, giving a positive predictive value of 74% and negative predictive value of 99%. KOH had a sensitivity of 88% and a specificity of 95%, giving a positive predictive value of 73% and a negative predictive value of 98%. CW was simple, rapid, and easy to read. For dermatophyte infection, CW results are as useful as KOH results.

Rapid identification of Candida albicans using 4-methylumbelliferyl N-acetyl-beta-galactosaminide.

Rapid identification of Candida albicans is performed mainly by the germ-tube test. However, recent reports have suggested that up to 5% of C. albicans species can give false negative results. We describe the use of 4-Methylumbelliferyl N-acetyl-beta-D-galactosaminide (4-MAG) conjugate as an alternative to the germ-tube test. Our results indicate that, in comparison to the germ-tube test, the 4-MAG test has a sensitivity of 100% and a specificity of 92%. Candida tropicalis can give false-positive results, and that a further screening test is required to identify this species. Problems reading end-points were not encountered.

Improved biochemical screening procedure for small clinical laboratories for vero (Shiga-like)-toxin-producing strains of Escherichia coli O157:H7.

Of 327 sorbitol-negative fecal isolates of Escherichia coli, 37 O157:H7 Vero (Shiga-like)-toxin-producing strains were identified. The specificity of the basic sorbitol screen was improved from 11.3 to 33.6% by the exclusion of organisms with negative ornithine and lysine decarboxylase reactions.

Direct antimicrobial susceptibility testing of urines positive in the MS-2 urine screening program.

A study was performed using the Abbott MS-2 system, in which positive urine screening specimens were set up directly to an antimicrobial susceptibility test. These results were compared with standard techniques. The overall correlation for urines containing single pathogens was 95.4%. Results were available 5-8 hr after receipt by the laboratory.

In vitro susceptibility of Pseudomonas species to carbenicillin and trimethoprim-sulfamethoxazole.

We compared susceptibility tests of 47 Pseudomonas aeruginosa isolates and 40 Pseudomonas species to carbenicillin and trimethoprim-sulfamethoxazole by the MS-2 and Sceptor systems and agar dilution. The major and very major errors encountered in these tests in the MS-2 and Sceptor systems raise doubts about the accuracy of these methods for testing P. aeruginosa and confirm that they should not be used for testing the susceptibility of Pseudomonas species to the two drugs tested.