Rapid detection of microorganisms in blood cultures of newborn infants utilizing an automated blood culture system.

BACKGROUND: Neonatal sepsis is a low incidence, high-risk disease with many sepsis work-ups performed to detect a single case. Seventy-two hours of antibiotic therapy have been traditionally recommended pending negative culture results. Improved culture media and new technology integrated into blood culture systems could shorten incubation time required to detect positive culture results. This would then change the length of antibiotic therapy in the management of the newborn infant with suspected sepsis. In addition, previous data supporting the 72-hour recommendation were retrospectively acquired, utilized nonautomated systems, and reported in an era with a different population of microorganisms cultured in special care nurseries. OBJECTIVE: Evaluate the time of incubation to detect positive blood cultures from newborn infants with suspected sepsis using a computer-assisted, automated blood culture system, ESP (Trek Diagnostic Systems, Inc, Westlake, OH). DESIGN: Prospective, observational study. PATIENTS AND SETTING: All positive blood culture results that were obtained from term and preterm newborn infants born from November 1993 through June 1997 at a publicly funded hospital with over 6000 live births per year. METHODS: As positive blood culture results were identified, data were prospectively obtained from the patient's medical record. The computer algorithm in the automated blood culture system determined the time to positivity. Time to positivity was determined for blood cultures obtained before the initiation antimicrobial therapy and compared with those cultures obtained after beginning therapy. Time to positivity was also evaluated for clinically important Gram-positive and Gram-negative bacteria and yeast. RESULTS: Four hundred fifty-five positive blood culture results were obtained from 222 patients. Gram-positive organisms accounted for 80% (366/455) of the positive culture results, Gram-negative organisms accounted for 11% (48/455), and yeast for 9% (41/455). Virtually all cultures growing clinically significant Gram-positive and Gram-negative organisms were positive by 24 to 36 hours of incubation. Cultures growing Staphylococcus epidermidis were virtually all positive after 36 to 48 hours of incubation. Of cultures growing yeast, 88% (36/41) were positive by 48 hours of incubation. There was no difference in time to positivity in pretherapy or posttherapy obtained positive blood cultures. Prenatally administered antibiotics did not affect time to positivity in positive cultures drawn on the first day of life. In a selected group of microorganisms that are the frequent cause of bacteremia in term infants, 97% and 99% of cultures were positive by 24 to 36 hours of incubation when only pretherapy cultures are evaluated. CONCLUSIONS: The ESP blood culture system identified 77%, 89% and 94% of all microorganisms at 24, 36, and 48 hours of incubation in aerobic cultures obtained from both term and preterm infants. Introduction of antimicrobial therapy did not affect time to positivity. Reducing duration of antibiotic therapy to 24 to 36 hours should be considered in term, asymptomatic newborn infants undergoing evaluation for suspected sepsis for maternal indications. Confirmation of similar rapidity of detection using other blood culture systems should be undertaken.

Clinical laboratory evaluation of a bacteriuria detection device for urine screening.

The BAC-T-SCREEN (BTS) (Marion Laboratories, Inc., Kansas City, MO) is a 2 1/2-minute urine screen designed to detect culture-negative specimens. A total of 1,609 urine specimens were tested by the BTS, and results were compared with quantitative culture methods. One hundred and forty-eight (9.2%) specimens were not screened successfully by the BTS because they contained interfering pigments or clogged the test filters. A total of 1,461 specimens were tested successfully. The sensitivity, specificity, positive predictive value, and negative predictive value for specimens containing greater than or equal to 10(5) CFU/mL were 98.0, 72.2, 57.3, and 99.0%, respectively. These values for specimens containing greater than or equal to 10(4) CFU/mL were 93.2, 77.2, 69.2, and 95.5%, respectively.

A rapid and inexpensive method for processing induced sputum for detection of Pneumocystis carinii.

The authors describe a method to process induced sputum specimens for detection of Pneumocystis carinii which is simple, rapid and inexpensive. Induced sputum and bronchoalveolar lavage (BAL) were obtained within a 24-hour period from 41 patients who were HIV-positive and had pulmonary symptoms suspicious for P carinii pneumonia. Induced sputum or BAL fluid was placed into Saccomanno's fixative, blended, and centrifuged. The sediment was stained for P carinii cysts by a modified method with Fungi-Fluor Solution A (Polysciences, Warington, PA) and the Genetic Systems Pneumocystis carinii Immunofluorescence Antibody (Genetic Systems, Seattle, WA). The Genetic Systems stain on the BAL specimen was positive in 35 patients and was the standard for comparison. With a single induced sputum, the Genetic Systems stain detected 31 (89%) positive patients, whereas the Fungi-Fluor stain detected 21 (60%). The sensitivity for detecting P carinii cysts in induced sputum was significantly greater (P < 0.05) for the Genetic Systems stain.

Lactobacillemia of amniotic fluid origin.

Four cases of amnionitis due to Lactobacillus sp and three cases of bacteremia are reported. Postpartum endometritis developed in all four cases. Lactobacillus bacteremia occurred in two infants. A review of the literature on serious infections due to Lactobacillus sp is also presented. The importance of considering the clinical role of any microorganism isolated in pure culture from symptomatic patients is discussed.

Evaluation of the FiltraCheck-UTI for detection of bacteriuria.

The FiltraCheck-UT1 (FC) is a disposable colorimetric urine screen for bacteriuria that requires less than 1 min to perform. The results of the FC and two other urine screens, the Bac-T-Screen (BTS) and the Chemstrip LN test strip (LN), were compared with quantitative culture method. A total of 551 urine specimens were tested. The sensitivity of FC, BTS, and LN for probable pathogens at greater than or equal to 10(5) CFU/ml was 94.8%, 97.4%, and 77.9% respectively. These values for specimens with probable pathogens at greater than or equal to 10(4) CFU/ml were 91.1%, 92.1%, and 74.3%, respectively. When the LN was combined with the FC or BTS as a urine screen, the sensitivity for probable pathogens was improved.

Identification of Mycobacterium tuberculosis in sputum by direct inoculation of the BACTEC NAP vial.

The Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, and M. africanum) can be differentiated from mycobacteria other than M. tuberculosis (MOTT bacilli) with the BACTEC NAP test (Johnston Laboratories, Becton, Dickinson & Co., Towson, MD), by selectively inhibiting their growth with p-nitro-alpha-acetyl-amino-beta-hydroxypropriophenone (NAP). The BACTEC NAP test is recommended for isolated mycobacterial cultures. In this report, a direct NAP test is performed by immediate inoculation of BACTEC NAP vials with processed sputum specimens that stain acid-fast positive. Seventy-six of 80 M. tuberculosis were correctly identified and all of the MOTT bacilli (nine M. avium complex and one M. kanasii) were correctly classified. The average time required for identification of M. tuberculosis with the direct BACTEC NAP test described here is more convenient than the recommended indirect test, and it is an accurate and rapid method to differentiate the M. tuberculosis complex from MOTT bacilli.

Pulmonary coccidioidal pseudomycetoma.

Coccidiomycosis is rarely associated with a pulmonary mycetoma. We report a patient with progressive cavitary coccidiomycosis, whose initial radiographic and clinical appearance simulated a mycetoma. Examination of the surgically resected lung showed necrotizing Coccidioides immitis granulomas with spherules and arthroconidialike structures, but no evidence of a mycetoma. We propose the term pulmonary coccidioidal pseudomycetoma as the best descriptor for this patient's clinical, radiographic, pathologic, and microbiologic presentation.

Direct antimicrobial drug susceptibility testing of Mycobacterium tuberculosis by the radiometric method.

Direct-drug-susceptibility tests were performed on clinical specimens positive for acid-fast bacilli by either Ziehl-Neelsen or fluorochrome staining. The results of conventional agar dilution (Vestal, 1975) and a modified radiometric (BACTEC) method were compared. A total of 580 smear-positive specimens were tested by the BACTEC method at three separate sites. Three hundred and seventy-seven of these were culture positive for M. tuberculosis, and 343 (91%) yielded acceptable direct-susceptibility-test results. We used the conventional method to determine that 343 of 519 smear-positive specimens were culture positive for M. tuberculosis, and 212 (62%) produced acceptable results within 3 wks. Conventional results were reported in 3-4 wks, while the time required to obtain results with the BACTEC method ranged from 5 to 21 days (average 11.5 days). Results indicate that the radiometric method provides reportable results more frequently with time savings as compared to the conventional method.

Comparison of several culture media used for studies on mycobacteriophages.

The value of RVA, N-1, 7H10, 7H11 and Sauton's media for studies on mycobacteriophage infeciton and lysis of mycobacteria was assessed. Experiments were made with mycobacteriophages BGI, BKI, CRI-3, G37, and LG, all of which lyse Mycobacterium smegmatis strain 607B, and with mycobacteriophage DS6A which lyses Mycobacterium tuberculosis strain H37Rv. The methods involved "direct lysis", the measurement of "routine test dilutions" and counts of plaque-forming units. It was found that N-1, 7H10 and 7H11 media gave better overall results than RVA medium for M. smegmatis strain 607B and its phages, and that RVA medium was generally the most useful for M. tuberculositems employed.

Modification of the fungi-fluor and the genetic systems fluorescent antibody methods for detection of Pneumocystis carinii in bronchoalveolar lavage specimens.

Four hundred forty-five bronchoalveolar lavage specimens from patients with the human immunodeficiency virus were preserved in Saccomano's fixative and stained for Pneumocystis carinii cysts by a modified method with Fungi-Fluor Solution A (Polysciences, Warrington, Pa) and the Genetic Systems Pneumocystis carinii Immunofluorescence Antibody (Genetic Systems Corp, Seattle, Wash). The majority of patients had been treated for suspected P carinii pneumonia for a few days prior to collection of bronchoalveolar lavage specimens. P carinii cysts were detected in 194 (43.6%) specimens. Both stains identified P carinii in 166 (37.3%) specimens and were negative in 251 (56.4%), yielding a concordance rate of 93.7%. P carinii cysts were detected in 25 (5.6%) specimens by the Genetic Systems stain only, and in 3 (0.7%) specimens by the Fungi-Fluor stain only. The sensitivity for detecting cysts of P carinii was significantly greater with the Genetic Systems stain (P < .01).

Automated systems for identification of microorganisms.

Automated instruments for the identification of microorganisms were introduced into clinical microbiology laboratories in the 1970s. During the past two decades, the capabilities and performance characteristics of automated identification systems have steadily progressed and improved. This article explores the development of the various automated identification systems available in the United States and reviews their performance for identification of microorganisms. Observations regarding deficiencies and suggested improvements for these systems are provided.

Acid-fastness of Mycobacteri,m tuberculosis H37Rv following infection by mycobacteriophage DS6A.

Mycobacterium tuberculosis H37Rv demonstrates a loss of acid-fastness following exposure to specific mycobacteriophage DS6A. No effect was seen with another mycobacteriophage GS7 which does not lyse this organism.

Detection of asymptomatic bacteriuria in obstetric patients with a semiautomated urine screen.

Urine specimens (1215) from obstetric patients were evaluated for the presence of significant bacteriuria by the Bac-T-Screen (Marion Scientific Division, Marion Laboratories, Inc., Kansas City, Missouri) and a conventional semiquantitative culture method. Seventy (5.8%) specimens were not processed successfully by the Bac-T-Screen because pigments interfered with interpretation or the test filters clogged. The remaining 1145 specimens screened by the Bac-T-Screen were compared with a conventional culture method. The sensitivity, specificity, positive predictive value, and negative predictive value for specimens containing greater than or equal to 10(5) colony-forming units per milliliter were 97.4%, 70.4%, 19.0%, and 99.7%, respectively. These values for specimens containing greater than or equal to 10(4) colony-forming units per milliliter were 92.2%, 71.6%, 24.1%, and 98.9%, respectively.

Rapid method for identification and susceptibility testing of Escherichia coli bacteriuria.

This report describes a rapid method for the identification and susceptibility testing of Escherichia coli bacteriuria by use of the Autobac urine screen (AUS), a 5-h indole test, and the AutoMicrobic system E. coli Susceptibility Card (AMS-ECSC). All specimens that were AUS negative at 3 and 5 h were reported as negative. All specimens that were AUS positive at 3 or 5 h were removed from the refrigerator and streaked to a MacConkey-blood agar biplate with a 0.001-ml calibrated loop and incubated at 35 degrees C. The standard method for identification and susceptibility testing consisted of inoculating isolated colonies into the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card and the AutoMicrobic system General Susceptibility Card. Of 915 specimens, 212 (23.2%) were AUS positive at 3 h, of which 112 (52.8%) were indole positive when tryptophan broth was tested at 5 h. The sensitivity and specificity of this screening method for E. coli bacteriuria were 78.8 and 72.2%, respectively. If contaminated cultures were excluded, specificity was 94.4%. When the indole test was positive, 10 microliters of growth from tryptophan broth was used as inoculum for the AMS-ECSC. AMS-ECSC results were final at 12 h. The sensitivity and specificity of the AMS-ECSC for identification of E. coli were 90.4 and 55.0%, respectively. If contaminated cultures were excluded, specificity was 100%. AMS-ECSC susceptibility results demonstrated an overall agreement of 94.3% with the standard method, with 0.5% very major, 3.4% major, and 1.8% minor discrepancies. The biplate was examined after overnight incubation, and when colonies compatible in morphology with E. coli were present in significant numbers, AMS-ECSC results were reported. When discrepancies were found between biplate and AMS-ECSC results, the biplate was processed in the conventional manner. A rapid method for identifying and performing susceptibility tests for approximately 70% of the specimens with E. coli bacteriuria is described.

Rapid method for detection, identification, and susceptibility testing of enteric pathogens.

Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35 degrees C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion results at 99.2%. One Salmonella enteritidis and four Shigella spp. showed minor discrepancies with tetracycline. No very major or major discrepancies were observed.

4-h Identification of Pseudomonas aeruginosa with 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan.

A total of 361 gram-negative bacilli were evaluated for their ability to grow in the presence of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan. The minimum time required for the production of visual turbidity in brain heart infusion broth was determined to be 4 h in shake cultures at 35 degrees C. The minimal inhibitory concentration (MIC) of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan in brain heart infusion broth was determined for 174 isolates. The MICs for all 40 Pseudomonas aeruginosa isolates tested were greater than 50 micrograms/ml. The MICs for the other 53 pseudomonads tested were less than or equal to 5 micrograms/ml. Among 81 other gram-negative bacilli tested, the MICs for 4 were 15 micrograms/ml, the MICs for 15 were 10 micrograms/ml, the MICs for 21 were 5 micrograms/ml, and the MICs for 41 were less than or equal to 1 micrograms/ml. Based on these data, 361 gram-negative bacilli were inoculated into brain heart infusion broth containing 15 micrograms of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan per ml and incubated on a shaker for 4 h. The only bacteria that produced visual turbidity were identified as P. aeruginosa (170 of 170 isolates).

Differential medium for Vibrio cholerae.

A differential medium designed for rapid presumptive identification of Vibrio cholerae was described and shown to be useful for enumeration of viable cholera vibrios in the presence of other intestinal bacteria.

Phage typing of mycobacteria using paper discs.

The suitability of phage-impregnated paper discs for the phage typing of mycobacteria was studied. The relevance of the routine test dilution, the volume of the phage used, the mode of incubation, and the effect of prolonged storage of phage-impregnated paper discs were considered. By using paper discs, each impregnated with one of 5 different mycobacteriophages (BG1, BK1, G37, CRI-3 and LG) that lyse Mycobacterium smegmatis 607B, it was determined that 100 x the routine test dilution in a volume of at least 20 microliter was required for phage lysis. Soaked and dried paper discs produced larger areas of lysis than those with 20-microliter volumes. Soaked discs were found to be stable even after storage for 8 weeks at 4 degrees C.

Rapid presumptive identification of gram-negative rods directly from blood cultures by simple enzymatic tests.

Gram-negative rods were presumptively identified directly from blood cultures within 15 min as Escherichia coli, a member of the Klebsiella-Enterobacter group, or oxidase positive. Samples of artificially seeded blood cultures (193 cultures) and patient blood cultures (78 cultures) were filtered into a Dynadepth test card with the Bac-T-Screen instrument (Vitek, Inc., Hazelwood, Mo.). Triton X-100 was then filtered into the test card to lyse the blood cells but not the entrapped bacteria, and either methylumbelliferone-labeled substrates or oxidase reagent was applied to the filter surface. The oxidase test was read within 30 s, and the methylumbelliferone and indole tests were read after a 10-min incubation at room temperature. Positive beta-galactosidase, beta-glucuronidase, and indole test results predicted the identification of E. coli with a 96 to 100% sensitivity and a 99 to 100% specificity. Positive beta-xylosidase and beta-galactosidase test results and negative oxidase and beta-glucuronidase test results were 85 to 93% sensitive and 100% specific for a Klebsiella-Enterobacter organism. A positive oxidase test result and negative beta-glucuronidase, beta-xylosidase, and indole test results were highly predictive of Pseudomonas aeruginosa (sensitivity, 100%; specificity, 99%). The procedures described are rapid and simple and provide a direct presumptive identification of the gram-negative rods most commonly found in blood cultures.

Evaluation of a modified dye pour-plate auxanographic method for the rapid identification of clinically significant yeasts. Comparison with two commercial systems.

A modified dye pour-plate auxanographic (DPPA) method for the presumptive identification of medically important yeasts was evaluated, in a comparative study with a conventional procedure, the API 20C clinical yeast system (Analytab Products Inc.), and the Uni-Yeast-Tek (UYT) system. The 174 coded clinical isolates were members of the genera Candida, Cryptococcus, Rhodotorula, Saccharomyces, Torulopsis, and Trichosporon. The identification accuracies with DPPA, API, and UYT were 95%, 93%, and 99% respectively. DPPA and API required more time to inoculate but gave rapid identification profiles. UYT was simple to inoculate and both UYT and DPPA were easy to read. Cost analysis of the three rapid methods demonstrated DPPA to be the most economical making it a feasible alternative for small clinical laboratories as well as large laboratories possessing the facilities to make their own media.