Martha J. Bale

An experimental model for study ofCandida survival and transmission in human volunteers

In order to determine the potential for cross-transmission ofCandida spp. between health-care workers and patients, the survival of clinical isolates of five species ofCandida on the palms of human volunteers was tested. One hundred µl of a McFarland 1.0 density suspension (5×105 cfu) from an overnight culture ofCandida albicans, Candida krusei, Candida parapsilosis, Candida tropicalis andCandida glabrata was used as inoculum. The degree of hydrophobicity of the differentCandida species was also tested and did not influence the survival. The half-lives were brief, being 9.5, 12.4, 7.4, 12.8, 9.6 min forCandida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, andCandida tropicalis, respectively, but at 45 min 2.6 × 103 to 3 × 104 organisms remained on the hands. Survival ofCandida albicans for as long as 24 h on inanimate surfaces was observed. Transmission from one hand to a second hand occurred in 69 % of the experiments and from the first to a third hand in 38 %. Transmission to and from inanimate surfaces was successful in most of the experiments (90 %). This experimental model aids in the biological study ofCandida spp. and suggests some of the potential mechanisms of transmission.

Multicenter comparison of a colorimetric microdilution broth method with the reference macrodilution method for in vitro susceptibility testing of yeast isolates

This multicenter study was performed to compare a colorimetric microdilution method with the NCCLS M27-P reference macrodilution method for the testing of yeast isolates against amphotericin B, fluconazole, and 5-fluorocytosine (5FC). Testing was performed on ten yeast isolates in five independent laboratories. All sites tested each isolate a total of 20 times with both methods. MICs were read after 48 h incubation. The macrodilution MIC reference range was defined as the modal MIC +/- 1-log2 dilution for each organism-antifungal agent combination. Agreement between the M27-P reference range results and the microdilution MICs was 86% with amphotericin B, 90% with fluconazole, and 93% with 5FC. Based on these data, it is apparent that new approaches, such as the colorimetric microdilution method, will provide MIC values comparable to the M27-P macrodilution method in a format that is more practical for use in a busy clinical laboratory.

Comparison of identification systems for Staphylococcus epidermidis and other coagulase-negative Staphylococcus species

Three commercially available systems (API Staph-Trac, API 20GP, and Vitek GPI), used to identify coagulase-negative staphylococci, were evaluated against 277 bloodstream isolates, including 94 isolates of Staphylococcus epidermidis and 183 isolates of other coagulase-negative Staphylococcus species. The conventional method of Kloos and Schleifer served as the reference method. Controls included 14 ATCC type culture strains of coagulase-negative staphylococci. The API Staph-Trac system showed the highest rate of agreement with reference method, correctly identifying 73% of the isolates. The Vitek GPI System had an overall rate of agreement of 67% and the API 20GP system correctly identified 61%. The API Staph-Trac system correctly identified 94% of the isolates of S. epidermidis compared with 64% by both Vitek GPI and API 20GP. The most common error for both Vitek GPI and API 20GP systems was the failure to identify organisms contained within the database of the systems. Because none of the tested commercial identification systems identified non-epidermidis coagulase-negative Staphylococcus species with a high degree of accuracy, the systems need to be markedly improved or new systems developed.

Antifungal activity of a new triazole, D0870, compared with four other antifungal agents tested against clinical isolates of Candida and Torulopsis glabrata

D0870 is a new triazole agent with potent, broad-spectrum antifungal activity. We investigated the in vitro activity of D0870, fluconazole, itraconazole, amphotericin B, and 5-fluorocytosine (5FC) against 319 clinical isolates of Candida spp. and Torulopsis glabrata. In vitro susceptibility testing was performed using a microdilution broth method performed according to NCCLS guidelines. D0870 was very active (MIC90 of 0.12 microgram/ml and 0.5 microgram/ml at 24 and 48 h incubation, respectively) against all of the yeast isolates. D0870 was 2- to 32-fold more active than amphotericin B and 2- to 8500-fold more active than 5FC. By comparison with the other triazoles, D0870 was generally 2- to 16-fold more active than itraconazole and > or = 16-fold more active than fluconazole. More than half (53%) of C. albicans isolates with elevated fluconazole and itraconazole MICs (> or = 128 micrograms/ml and > 8.0 micrograms/ml, respectively) were inhibited by < or = 1.0 microgram/ml of D0870. Based on these studies, D0870 has promising antifungal activity and warrants further in vitro and in vivo investigation.

Value of the hybritech ICON Candida assay in the diagnosis of invasive candidiasis in high-risk patients

A total of 314 sera from 114 patients at risk for invasive candidiasis were analyzed for the presence of antigenemia using the Hybritech enzyme immunoassay (EIA) for detection of Candida mannan in serum (ICON Candida Assay, Hybritech Inc., San Diego, CA). Fourteen patients (12%) had invasive candidiasis documented by positive blood cultures, deep biopsy culture, and histopathology or autopsy, and five patients had probable invasive candidiasis based on a single positive blood culture and no additional signs of candidiasis. Nine patients had candiduria, 43 patients had mucous membrane colonization, 25 patients were not colonized but received empiric amphotericin B, and 18 patients were not colonized and not treated with amphotericin B. All sera were enzymatically extracted, heat treated, and reacted in a solid-phase sandwich EIA. Test results were read visually and with the ICON reader. The sensitivity and specificity of the mannan EIA in detection of documented invasive candidiasis was 86% and 92%, respectively. The positive predictive value was 60% and the negative predictive value was 98%. Among all patients with invasive candidiasis (documented plus probable), the sensitivity was 68%, the positive predictive value 62%, and the negative predictive value 94%. Specimens were positive within 3 days of the first positive culture in 11 (79%) of 14 patients with documented invasive candidiasis.

Application of the Etest to antimicrobial susceptibility testing of Legionella spp.

Development of susceptibility tests for Legionella spp. has been difficult because of the specific growth requirements of this organism. The most commonly used media, buffered charcoal-yeast extract (BCYE) agar contains charcoal, which is known to inactivate some antimicrobial agents. This study compared five antimicrobial (erythromycin, clindamycin, ofloxacin, doxycycline, and rifampin) minimum inhibitory concentration (MIC) values as determined by agar dilution using BCYE, agar dilution using this same media without the charcoal [buffered yeast extract (BYE) agar], and the Etest on BCYE media. The MIC50 and MIC90 for this group of Legionella spp. were greater with BCYE than with BYE. Erythromycin and ofloxacin (fourfold change) were the most affected by the charcoal in the medium. The Etest MIC and agar dilution MIC with BYE were comparable. The Etest rifampin results demonstrated that the Legionella spp. strains were very susceptible (< 0.016 micrograms/ml, producing very large zones) requiring use of one half of the Etest strip, a whole test strip on an individual 100-mm plate of BCYE, or use of a new low-MIC-range Etest strip. The Etest on BCYE provides a simple, readily available, and accurate method unaffected by medium components for susceptibility testing of Legionella spp.

Prospective evaluation of the Gen-Probe assay for detection of Legionellae in respiratory specimens

A prospective evaluation of a DNA probe assay for detection ofLegionella species was performed on 427 consecutive respiratory specimens submitted over an 18-month period. The Gen-Probe assay utilizing both low (⩾4.0) and high (>7.0) ratio threshold values was compared to direct fluorescent antibody staining (DFA) as a predictor of isolation ofLegionella on culture. The highest sensitivity (63 %) was obtained with the lower threshold ratio, but was not significantly different from the result obtained with a threshold ratio of >7.0 (50 %, p=0.722) or DFA results (44 %, p=0.479). The specificity of the DNA probe assay was improved with the high threshold (99 %) compared either to the low threshold ratio (95 %, p=0.0002) or DFA (97 %, p=0.055). When the DNA probe was compared to DFA and/orLegionella isolation on culture, a significantly lower specificity (97 % versus 99 %, p=0.0006) and higher sensitivity (74 % versus 37 %, p=0.013) was obtained with a threshold value of ⩾4.0 than >7.0. Ten of 20 specimens with a DNA probe ratio between 4.0 and 7.0 were DFA positive, although only two were isolated on culture. The DFA assay and both probe threshold ratios have a high negative predictive value when compared to culture. However, only the threshold ratio of >7.0 has a sufficiently high positive predictive value to be useful alone. Although the DNA probe appears to be a practical alternative to DFA testing for the rapid diagnosis ofLegionella infections, false-negative results emphasize the importance of obtaining several specimens for testing, and confirm the fundamental role of culture in the diagnosis ofLegionella infections.

Evaluation of growth characteristics on blood agar and eosin methylene blue agar for the identification of Candida (Torulopsis) glabrata

Candida albicans and Candida (Torulopsis) glabrata are the most common species of yeast encountered in the clinical laboratory. In this study, we sought to evaluate simple means of screening cultures for the presence or absence of C. glabrata. Twelve thousand five hundred (12,500) consecutive cultures were evaluated for sufficient yeast growth to warrant identification. When detected (369 isolates), the amount of growth on eosin methylene blue agar (EMB) versus sheep blood agar (BAP) (both incubated in 5% CO2), wet mount morphology, and germ tube production were evaluated. All germ tube-negative yeasts were definitively identified using the Vitek YBC card. Of the 369 yeast isolates included in this study, 225 were C. albicans, 102 C. glabrata, and 42 other Candida species. Growth on EMB was greater than BAP for 92 isolates; all identified as C. glabrata. When EMB growth was equal to or less than BAP, 10 isolates were C. glabrata and 267 were other Candida ssp. An accurate presumptive identification of C. glabrata may be made using the observation of greater growth on EMB versus BAP. When coupled with the germ tube test, the majority of yeast isolates could be identified by these simple methods in our laboratory.

Vitek GPS card susceptibility testing accuracy using direct inoculation from Bactec 9240 blood culture bottles

The emergent need for antimicrobial susceptibility testing (AST) data for the therapy of bacteremic patients has led to the development of rapid methods and local procedure modification of some commercial AST products such as the direct inoculation from blood culture systems. We compared the Vitek GPS card results using direct and standardized inoculation with a reference broth microdilution method for 112 consecutive staphylococcal bloodstream infections (seven drugs). Among the 28 Staphylococcus aureus strains, 0%-3.6% total error/drug was observed with both Vitek inoculation procedures. However, the only oxacillin-resistant strain was not detected (100% true very-major error). For 84 coagulase-negative staphylococci (CNS), the direct inoculation procedure had an 11.9% very-major error rate for oxacillin, ampicillin-sulbactam, and cephalothin, plus 4.8% very-major error rate for ciprofloxacin and trimethoprim-sulfamethoxazole (total error rate 4.8%-16.7% for five of seven drugs compared). The Vitek direct inoculation procedure routinely missed 20.4% of oxacillin-resistant CNS strains. The use of Vitek direct inoculation procedures for staphylococcal bloodstream infection isolates (from BACTEC 9240 cultures) produced serious false-susceptible results; this procedure should be avoided in favor of routine package insert-recommended Vitek procedures or other reference-quality overnight incubation susceptibility tests.

Application of a biotyping system and DNA restriction fragment analysis to the study of Serrati marcescens from hospitalized patients

We applied a nine-test biotyping system to the study of 138 Serratia marcescens isolates from 93 patients hospitalized in 33 different hospital bed units. Test reproducibility for a panel of 10 isolates tested in triplicate on 3 separate days was 100%. Overall, the biotyping system delineated 25 strains. In examining strain variation among isolates obtained from multiple anatomic sites over time we found that the same biotype was recovered from tracheal aspirates, urine, wounds, and blood in a given patient and that these strains were carried over time. In general, patients were infected or colonized with their own distinct biotype of S. marcescens. Temporally related isolates from seven surgical intensive care unit (SICU) patients and six unrelated control isolates were typed by biotyping and DNA restriction fragment analysis (RFA). The distribution of biotypes was similar among SICU outbreak and control isolates, with five distinct biotypes among the seven SICU isolates. Each isolate had a different DNA subtype by RFA, confirming the lack of nosocomial transmission of a single strain. These results will be useful in studying the epidemiology of S. marcescens.