Identification of Streptococcus pneumoniae revisited.

The sensitivities and specificities of several different diagnostic assays for Streptococcus pneumoniae were assessed using 99 clinical isolates of S. pneumoniae and 101 viridans streptococci and were as follows: Pneumoslide, 99 and 87%, respectively; Directigen, 100 and 85%, respectively; Phadebact, 100 and 98%, respectively; deoxycholate drop test, 99 and 98%, respectively; deoxycholate tube test, 100 and 99%, respectively; optochin, 99 and 98%, respectively; and Gram Positive Identification Card, 90 and 96%, respectively. Identification of clinical isolates of S. pneumoniae should be confirmed using one or more diagnostic assays with well-documented high (e.g., > or =95%) sensitivities and specificities.

Application of the Sherlock Mycobacteria Identification System using high-performance liquid chromatography in a clinical laboratory.

There is a growing need for a more accurate, rapid, and cost-effective alternative to conventional tests for identification of clinical isolates of Mycobacterium species. Therefore, the ability of the Sherlock Mycobacteria Identification System (SMIS; MIDI, Inc.) using computerized software and a Hewlett-Packard series 1100 high-performance liquid chromatograph to identify mycobacteria was compared to identification using phenotypic characteristics, biochemical tests, probes (Gen-Probe, Inc.), gas-liquid chromatography, and, when necessary, PCR-restriction enzyme analysis of the 65-kDa heat shock protein gene and 16S rRNA gene sequencing. Culture, harvesting, saponification, extraction, derivatization, and chromatography were performed following MIDI's instructions. Of 370 isolates and stock cultures tested, 327 (88%) were given species names by the SMIS. SMIS software correctly identified 279 of the isolates (75% of the total number of isolates and 85% of the named isolates). The overall predictive value of accuracy (correct calls divided by total calls of a species) for SMIS species identification was 85%, ranging from only 27% (3 of 11) for M. asiaticum to 100% for species or groups including M. malmoense (8 of 8), M. nonchromogenicum (11 of 11), and the M. chelonae-abscessus complex (21 of 21). By determining relative peak height ratios (RPHRs) and relative retention times (RRTs) of selected mycolic acid peaks, as well as phenotypic properties, all 48 SMIS-misidentified isolates and 39 (91%) of the 43 unidentified isolates could be correctly identified. Material and labor costs per isolate were $10.94 for SMIS, $26.58 for probes, and $42.31 for biochemical identification. The SMIS, combined with knowledge of RPHRs, RRTs, and phenotypic characteristics, offers a rapid, reasonably accurate, cost-effective alternative to more traditional methods of mycobacterial species identification.

Frequency of low-level bacteremia in children from birth to fifteen years of age.

A single blood culture inoculated with a small volume of blood is still frequently being used for the diagnosis of bacteremia in children because of the continued belief by many that bacteria are usually found in high concentrations in the blood of pediatric patients with sepsis. To determine the importance of both blood volume cultured and the number of culture devices required for the reliable detection of pathogens in our pediatric population, blood from children from birth to 15 years of age and with suspected bacteremia at York Hospital (a 500-bed community hospital) was inoculated into at least a Pediatric Isolator (Wampole Laboratories; 1.5 ml of blood) or a standard Isolator (10 ml of blood) and a bottle of ESP anaerobic broth (Trek Diagnostic Systems; 0.5 to 10 ml of blood). The use of a second Isolator and additional aerobic and anaerobic bottles and the total blood volume recommended for cultures (2 to 60 ml) depended on the weight and total blood volume of each patient. One hundred forty-seven pathogens were recovered from the blood of 137 (3.6%) of 3,829 children for whom culturing was done. Of 121 septic episodes for which the concentration of pathogens in the blood could be determined using Isolators, 73 (60. 3%) represented low-level bacteremia (

Variation in Microbial Identification System accuracy for yeast identification depending on commercial source of Sabouraud dextrose agar.

The accuracy of the Microbial Identification System (MIS; MIDI, Inc. ) for identification of yeasts to the species level was compared by using 438 isolates grown on prepoured BBL Sabouraud dextrose agar (SDA) and prepoured Remel SDA. Correct identification was observed for 326 (74%) of the yeasts cultured on BBL SDA versus only 214 (49%) of yeasts grown on Remel SDA (P < 0.001). The commercial source of the SDA used in the MIS procedure significantly influences the system's accuracy.

Limitations of the current microbial identification system for identification of clinical yeast isolates.

The ability of the rapid, computerized Microbial Identification System (MIS; Microbial ID, Inc.) to identify a variety of clinical isolates of yeast species was compared to the abilities of a combination of tests including the Yeast Biochemical Card (bioMerieux Vitek), determination of microscopic morphology on cornmeal agar with Tween 80, and when necessary, conventional biochemical tests and/or the API 20C Aux system (bioMerieux Vitek) to identify the same yeast isolates. The MIS chromatographically analyzes cellular fatty acids and compares the results with the fatty acid profiles in its database. Yeast isolates were subcultured onto Sabouraud dextrose agar and were incubated at 28 degrees C for 24 h. The resulting colonies were saponified, methylated, extracted, and chromatographically analyzed (by version 3.8 of the MIS YSTCLN database) according to the manufacturer's instructions. Of 477 isolates of 23 species tested, 448 (94%) were given species names by the MIS and 29 (6%) were unidentified (specified as "no match" by the MIS). Of the 448 isolates given names by the MIS, only 335 (75%) of the identifications were correct to the species level. While the MIS correctly identified only 102 (82%) of 124 isolates of Candida glabrata, the predictive value of an MIS identification of unknown isolates as C. glabrata was 100% (102 of 102) because no isolates of other species were misidentified as C. glabrata. In contrast, while the MIS correctly identified 100% (15 of 15) of the isolates of Saccharomyces cerevisiae, the predictive value of an MIS identification of unknown isolates as S. cerevisiae was only 47% (15 of 32), because 17 isolates of C. glabrata were misidentified as S. cerevisiae. The low predictive values for accuracy associated with MIS identifications for most of the remaining yeast species indicate that the procedure and/or database for the system need to be improved.

Frequency of low level bacteremia in infants from birth to two months of age.

BACKGROUND: The frequency of low level bacteremia (< or = 10 colony-forming units/ml) in infants from birth to 2 months of age and the optimal volume of blood and number of blood cultures to be collected have not been well-documented. During 1991 guidelines at this hospital for collection of blood for culture from these infants were revised. METHODS: Blood from each infant with suspected bacteremia was usually inoculated into an Isolator 1.5 Microbial Tube (1.5 ml of blood) and a bottle of anaerobic broth (0.5 to 3.0 ml of blood). The use of a second Isolator tube and the total blood volume recommended for culture (2 to 6 ml) depended on the weight and total blood volume of each infant. RESULTS: Forty-four bacterial pathogens were recovered from the blood of 40 (2.5%) of 1589 infants. Of 34 infants from whose blood the concentration of pathogens could be determined, 23 (68%) had low level bacteremia. Of 50 isolates of pathogens recovered from Isolator cultures, 32 (64%) were detected in counts of < or = 10 colony-forming units/ml. When 2 or 3 blood culture devices were inoculated with a total of 2 to 6 ml of blood from each infant, significantly more cases of bacteremia were detected (34 (3.0%) of 1126 infants had positive blood cultures) than when only one culture device containing < or = 1.5 ml of blood was used (2 (0.5%) of 398 infants had positive blood cultures; P = 0.008). However, when 4 or more culture devices were inoculated with a total of > 6 ml of blood from each infant (5 (7.7%) of 65 infants had positive blood cultures), the difference in recovery of pathogens compared with the culturing of from 2 to 6 ml of blood per infant was not significant (P = 0.089). CONCLUSIONS: Low level bacteremia was common in our infants' patient population. The culturing of up to 6 ml of blood which represented up to 4.5% of an infant's total blood volume was required for detection of the pathogens.

Occurrence and documentation of low-level bacteremia in a community hospital's patient population.

To document the incidence of low-level bacteremia in the patient population of this study, two blood culture sets were collected from symptomatic patients weighing more than 80 pounds. Each blood culture set consisted of a lysis-centrifugation tube and three bottles containing different culture broths, each inoculated with 10 mL blood. Pathogens from 63 (26.4%) and 48 (20.1%) of the 239 culture-positive patients were recovered from only one and two of the eight culture devices, respectively, representing low-level bacteremia. Isolates from another 60 (25.1%) of the 239 patients were recovered from all eight of the culture devices, representing high-level bacteremia. Whether patients had low-level or high-level bacteremia, there were mostly insignificant differences in the types of species recovered, in the percentages of patients for whom therapy was initiated or changed following the laboratory's reports, and in the clinical signs, symptoms, and characteristics of the patients. Clinically documented, low-level bacteremia is relatively common in this community hospital's patient population. Culturing of up to 80 mL of blood was required for detection of all pathogens from patients weighing more than 80 pounds.

Clinical comparison of isolator and thiol broth with ESP aerobic and anaerobic bottles for recovery of pathogens from blood.

The recovery of pathogens and the speed of their detection were determined for our conventional blood culture system (an Isolator [Wampole] and a 100-ml Thiol bottle [Difco]) compared with automated ESP aerobic and anaerobic bottles (80 ml each; Difco). Each of the four culture devices was inoculated with approximately 10 ml of blood from symptomatic patients weighing more than 80 lb (ca. 36 kg). From 7,070 sets of cultures for 2,841 patients, 607 clinically significant isolates were recovered: 456 (75.1%) from the Isolator, 353 (58.2%) from Thiol, 377 (62.1%) from ESP aerobic bottles, and 346 (57.0%) from ESP anaerobic bottles. Of the 607 isolates, 149 (24.5%) were detected only with the conventional system (Isolator and/or Thiol), and 65 (10.7%) were detected only with the ESP two-bottle system (P < 0.001). Our conventional system allowed for detection of significantly more isolates of members of the family Enterobacteriaceae (P < 0.001), Staphylococcus aureus (P < 0.01), Staphylococcus spp. (coagulase-negative) (P < 0.01), and Enterococcus spp. (P < 0.05), and ESP facilitated detection of significantly more isolates of S. pneumoniae (P < 0.01). When all four devices in a culture set were positive for the same isolate, no microbial species or group was detected significantly earlier ( > or = 24 h) by either blood culture system. The Isolator contamination rate (4.8%) was > or = 6 times the rate for any of the bottles. Of pathogens detected by the Isolator, 50% were recovered in counts of < or = 1.0 CFU/ml and 18% were recovered only as a single colony. The ESP system offered an automated, less labor-intensive blood culture system for which routine subcultures were not required, but the important considerations of culturing large volumes of blood and of obtaining at least two sets from each patient in our population were reemphasized.

Detection of group A streptococci by aerobic culture and a new simplified immunoassay in three pediatric practices and a hospital laboratory.

Duplicate throat swabs for detection of group A streptococci were collected in three pediatric offices from 1,035 patients with symptoms of pharyngitis. In the collecting office and in the hospital laboratory, one swab from each patient was first inoculated to sheep blood agar (incubated at 35 degrees C aerobically for 2 days) and then tested for group A streptococcal antigen by using the SMART enzyme immunoassay technique (New Horizons Diagnostics Corp) incubated for up to 24 hours. Group A streptococci were recovered in culture (from one or both swabs) and serologically identified from 444 (42.9%) of the patients. Pediatric offices numbers 1, 2, and 3 detected 84.4%, 84.6%, and 82.2%, respectively, of their patients who had positive cultures (in the office and/or laboratory) by using their own culture system and 82.6%, 71.1%, and 84.9%, respectively, of these same patients by using the SMART technique. In the laboratory, SMART test sensitivity and specificity were 71.4% and 98.7%, respectively, after 5 minutes of test incubation. However, SMART test sensitivity improved to 86.5% after overnight incubation of the immunoassay and to 91.3% if the data from one defective lot of seven SMART production lots studied were excluded. SMART test results which are negative after 5 minutes of incubation should therefore be confirmed both by reincubation of the antigen test up to 24 hours and by culture.

Comparison of the TestPack Strep A enzyme immunoassay system with anaerobically incubated cultures for detection of group A streptococci from oropharyngeal swabs.

To find a rapid and sensitive test for detection of Group A streptococci (GABS), the performance of the TestPack Strep A (TPSA; Abbott) was compared with culture with the use of rayon-tipped throat swabs from symptomatic patients six months to 90 years of age. Each swab was first inoculated to a 5% sheep blood agar plate and then tested for GABS antigen with the use of the TPSA and the manufacturer's instructions. Cultures were incubated anaerobically at 35 degrees C for 36-48 hours unless positive results were obtained after one night. GABS were identified with a fluorescent antibody method or a latex antibody test. From 1,616 throat swabs, 296 (18.3%) of the cultures contained GABS. The sensitivity and specificity of the TPSA were 73.3% and 94.8%, respectively, whereas the predictive values of positive and negative results were 75.9% and 94.1%, respectively. Results varied significantly, however, with different production lots of TPSA. The TPSA does not appear to provide a sensitive alternative to an anaerobic culture for detection of GABS.

Performance of an enzyme immunoassay test and anaerobic culture for detection of group A streptococci in a pediatric practice versus a hospital laboratory.

The ability of pediatricians and hospital laboratory personnel to detect group A streptococci in patients with suspected streptococcal pharyngitis was evaluated using the TestPack Strep A and anaerobic culture. Duplicate throat specimens (for similar processing by both the pediatricians and laboratory technologists) were simultaneously collected on rayon-tipped swabs from patients with symptoms of pharyngitis. Each swab was first inoculated to a 5% sheep blood agar plate, then tested for group A streptococcus antigen using the TestPack Strep A according to the manufacturer's instructions. Cultures were incubated anaerobically at 35 degrees C for 2 nights unless positive after 1 night. Group A streptococci were identified using specific antisera. Pediatric office or laboratory cultures from 112 (31.3%) of the 358 patients contained group A streptococci. Of the patients with positive cultures, 96 (85.7%) and 107 (95.5%) were detected by the pediatricians and laboratory, respectively. Respective findings with the TestPack Strep A by the pediatricians and laboratory were sensitivity 68.8% and 74.8%, specificity 94.3% and 95.6%, predictive value of a positive result 81.5% and 87.9%, and predictive value of a negative result 89.2% and 89.9%. Anaerobic culture was significantly more sensitive than the TestPack Strep A for detection of group A streptococci by both the pediatricians (P less than 0.005) and laboratory personnel (P less than 0.05).

Suitability of a throat culture method for evaluation of group A streptococcal antigen detection kits.

Previous reports have indicated a wide variation in observed sensitivity of antigen-detection kits for group A streptococci. Before undertaking an evaluation of these new kits, the sensitivity of the throat culture technic routinely used by this laboratory was reexamined. Each throat swab was directly inoculated to sheep blood agar containing trimethoprim-sulfamethoxazole (SXT X BA) and drug-free sheep blood agar (SBA) plates. Swabs were then washed in saline and the saline used to inoculate one more of each type of medium. SXT X BA cultures were incubated aerobically (5 to 10% CO2), and SBA cultures were incubated anaerobically, both for two days at 35 degrees C. From 726 patients, 164 (22.6%) of the specimens contained group A streptococci, 99% detected on directly inoculated cultures and 100% on cultures inoculated with the saline wash. Either an aerobically (CO2) incubated SXT X BA or an anaerobically incubated SBA, directly inoculated and held for two days, appears to offer a satisfactory reference culture method for the recovery of group A streptococci.