Recovery of Histoplasma capsulatum from BACTEC TB media.

From 1990 through 1994, we fortuitously isolated Histoplasma capsulatum from six patients with AIDS whose specimens of blood were processed by the BACTEC system using Middlebrook broth selective for acid-fast bacilli (13A medium). Growth indices became positive after an average of 17 days of incubation (range, 11 to 20 days). No acid-fast bacilli were seen, but small budding yeasts characteristic of H. capsulatum were present.     

Evaluation of BACTEC MYCO/F Lytic medium for recovery of mycobacteria, fungi, and bacteria from blood

MYCO/F Lytic medium (MFL), a liquid medium developed for use with the BACTEC 9240 blood culture system, was compared to the Isolator system (IS) for the recovery of fungi and to the BACTEC 13A medium for the recovery of mycobacteria. Recovery of bacteria was compared to routine BACTEC Plus Aerobic/F (AF) blood cultures. Microbial growth was detected in 203 (17%) of 1,166 blood cultures. Fifty-seven specimens were positive for fungi: 35 were positive with both IS and MFL; six were positive with IS only (three Candida albicans, one Histoplasma capsulatum, one Candida glabrata, and one Fusarium species isolate); three were positive with AF only (two C. albicans and one Candida parapsilosis isolate); and 13 were positive with MFL only (five C. glabrata, three C. albicans, two Candida krusei, two Candida tropicalis, and one C. parapsilosis isolate; P > 0.05 versus IS). Eighteen of 19 blood cultures positive for H. capsulatum grew in both IS and MFL, although the time to detection for MFL was greater. The mean time to detection for all fungi was 8.15 days for IS and 12.07 days for MFL. Seven hundred forty specimens were also cultured for mycobacteria with MFL and 13A. Forty-four grew mycobacteria; 38 were positive with both 13A and MFL; and 16 were positive with MFL only. Mycobacterium avium was recovered from 41 specimens; 36 were positive for both systems and 5 were positive for MFL alone. MFL was also compared to the AF bottle for the same 740 specimens. MFL and AF both detected 34 of the 40 clinically significant bacteria, while IS detected only 15 of 40. In summary, MFL is an excellent medium for the recovery of fungi, mycobacteria, and bacteria; however, the time to detection of H. capsulatum is increased.     

Radiometric detection of yeasts in blood cultures of cancer patients.

During a 12-month period, 19,457 blood cultures were collected. Yeasts were isolated from 193 cultures derived from 76 cancer patients. Candida albicans or Candida tropicalis accounted for 79% of isolates. Of the three methods compared, the radiometric method required 2.9 days to become positive, "blind" subculture required 2.6 days, and Gram stains required 1 day. However, the radiometric method was clearly superior in detecting positive cultures, since 73% of all cultures were first detected radiometrically, 22% were detected by subculture, and only 5% were detected by Gram stain. Although 93% of the isolates were detected by aerobic culture, five (7%) isolates were obtained only from anaerobic cultures. Seven days of incubation appear to be sufficient for the radiometric detection of yeasts.     

Acridine orange as a screen for organisms in clinical specimens and comparison with gram's stain

We compared the result of acridine orange and Gram's stains with the results of culture for 202 wound swabs and 188 fluid specimens. Cerebrospinal fluid was excluded from the study. Acridine orange was more sensitive and less specific than Gram's stain compared with findings that have been previously reported. A difference in the sensitivity was observed between the two stains and between the types of specimens examined themselves. The sensitivity of acridine orange and Gram's stains was 83% and 49% for swabs and 66% and 45% for fluids, respectively. The negative predictive values for acridine orange and Gram's stains were 60% and 40% for swabs and 84% and 81% for fluids, respectively. Overall, the sensitivity for acridine orange and Gram's stains was 75% and 64% with negative predictive values of 75% and 63%, respectively; specificity was 75% (acridine orange) and 97% (Gram's stain) and did not differ significantly between the two specimen types. Acridine orange was cleaner, faster, easier to perform and read, and less costly than Gram's stain for screening purposes. Slides that were positive by acridine orange staining should be stained with Gram's stain for specificity and for the Gram's-stain reaction report. Acridine orange is recommended for screening smears, with positive results confirmed by Gram's stain.     

Evaluation of acridine orange stain for detection of microorganisms in blood cultures.

A pH 4.0 buffered solution of the fluorochrome acridine orange was used to stain samples of 2,704 blood cultures that failed to yield visible evidence of growth after 1 day of incubation. Results obtained by the staining method were compared with those obtained by aerobic and anaerobic subcultures simultaneously performed upon the same cultures. Of the 109 culture-positive blood specimens initially detected by the acridine orange and the subculture methods, 85 (78%) were detected by both acridine orange and subcultures techniques, 14 (12.8%) were detected by subculture alone, and 10 (9.2%) were detected by acridine orange alone. The differences between the subculture and acridine orange methods were not found to be statistically significant (P less than 0.1). The acridine orange method represents a rapid and inexpensive alternative to conventional subculture techniques for the detection of bacteria in blood cultures that fail to yield visible evidence of growth after 1 day of incubation.     

Early detection of positive blood cultures by the acridine orange staining technique.

Staining 2,205 macroscopically negative blood cultures with acridine orange after 6 to 17 h of inoculation and incubation was as sensitive as an early subculture in detecting positive blood cultures. Of the 179 positive blood cultures, 30 (16.8%) were detected by acridine orange alone, 19 (10.6%) were detected by early subculture alone, 84 (46.9%) were detected by both techniques, and 46 (25.7%) were not detected by either method. The latter group includes cultures that became positive after 48 h of incubation. Acridine orange staining of smears prepared from macroscopically negative blood cultures after 6 to 17 h is a rapid, reliable method to detect positive blood cultures.     

Evaluation of the BACTEC radiometric method for recovery of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis from acid-fast smear-positive specimens.

A total of 463 respiratory specimens, all smear positive for acid-fast bacteria, were inoculated onto routine solid media and into BACTEC 7H12 Middlebrook medium for detection of mycobacterial growth. Conventional drug susceptibility testing (1% proportion method) was performed on Middlebrook 7H10/7H11 medium, and radiometric susceptibility testing was performed on BACTEC 7H12 medium. The average detection times for BACTEC-positive cultures were 8.3 days for Mycobacterium tuberculosis and 5.2 days for mycobacteria other than tuberculosis; by conventional methods, they were 19.4 and 17.8 days, respectively. These detection times do not include time required for identification, which was done by the conventional method only. There was an excellent correlation in the recovery rates of mycobacteria by the two methods. Drug susceptibility test results of M. tuberculosis isolates by the two methods showed 95.1 to 100% overall agreement. The average reporting time for drug susceptibility results ranged from 4.2 to 6.9 days for the BACTEC method and 13.7 to 21 days for the conventional methods. An average of 18 days was required by the BACTEC method for complete recovery and drug susceptibility testing of M. tuberculosis, as compared with 38.5 days for the conventional methods.     

Acridine orange staining of smears for demonstration ofMycobacterium tuberculosis

Acridine orange staining for the detection of mycobacteria was compared with staining by auramine 0 and with mycobacterial culture in a series of 1071 clinical specimens. A total of 78 (7 %) specimens were positive by staining. No false positive or negative findings were recorded by the acridine orange method. The two fluorochromes proved equal in their ability to detect mycobacteria in specimens from culture positive cases of tuberculosis. In the rapid bacteriological diagnosis of tuberculosis, acridine orange offers a good alternative to auramine 0 which is considered carcinogenic.     

Comparison of a radiometric method (BACTEC) and conventional culture media for recovery of mycobacteria from smear-negative specimens.

The BACTEC system and three conventional media (Middlebrook 7H10, selective Middlebrook 7H11 [S7H11], and Lowenstein-Jensen [LJ] were compared for their mean recovery times and recovery rates of mycobacteria from acid-fast, smear-negative clinical specimens. Of the 71 smear-negative, culture-positive specimens recovered from 2,165 submitted smear-negative cultures, the BACTEC system detected 71.8%, compared with 88.7% for the conventional three-medium system. When media were individually compared, BACTEC medium (Middlebrook 7H12) was more successful in recovering mycobacteria (71.8%) than was LJ (62%), Middlebrook medium 7H10 (55.9%), or Middlebrook S7H11 medium (52.1%). Middlebrook 7H11 medium containing sodium selenate was also evaluated and did not increase the recovery rate or decrease the recovery time of mycobacterial species when compared with LJ, Middlebrook 7H10, S7H11, and 7H12 media. The mean detection time for the BACTEC system was less than that by conventional methods for the seven species of mycobacteria recovered. Detection times for Mycobacterium tuberculosis on the BACTEC system and conventional cultural systems were 13.7 and 26.3 days, respectively.     

Increased sensitivity of the BACTEC 460 mycobacterial radiometric broth culture system does not decrease the number of respiratory specimens required for a definitive diagnosis of pulmonary tuberculosis

The BACTEC 460 radiometric mycobacterial broth culture system has consistently demonstrated faster and increased recovery of Mycobacterium tuberculosis from respiratory specimens of patients with pulmonary tuberculosis than conventional culture methods. We thus questioned whether three sputa were still necessary to definitively diagnose pulmonary tuberculosis if the BACTEC radiometric culture system were in use. We performed a retrospective analysis of 430 sequential respiratory specimens submitted from 143 patients and from which M. tuberculosis had been recovered by in vitro culture and simultaneously assessed the diagnostic yield of acid-fast smear in this same cohort. M. tuberculosis was recovered from the first specimen for 117 (82%) of the 143 patients, from the second for 14 patients (10%; cumulative rate, 92%), and from the third for 12 patients (8%; cumulative rate, 100%). With the exception of those for bronchial brushings, recovery rates of M. tuberculosis were comparable for all respiratory specimen types (expectorated sputum, induced sputum, tracheal aspirates, bronchoalveolar lavage fluids). Only 46 (32%) of these 143 patients had acid-fast bacilli detected in smears; acid-fast bacilli were detected in the first submitted specimen for 44 patients (96%) and in the second for the remaining 2 patients (4%; cumulative rate, 100%). Culture- or smear-positive rates for sequential specimens obtained from AIDS patients were comparable to those for non-AIDS patients. Overall, the diagnostic culture yield of sequentially submitted specimens was not different from previously published studies in which the BACTEC radiometric culture system had not been used. Despite the documented enhanced ability of the BACTEC 460 radiometric mycobacterial culture system to recover M. tuberculosis more often and faster than conventional methods, three sequential respiratory specimens (regardless of type) were still necessary to definitively diagnose pulmonary tuberculosis.     

Acridine orange stain in the early detection of bacteria in blood cultures

A total of 1,592 blood cultures without macroscopic signs of bacterial growth in the first 12–24 h of incubation were processed for both acridine orange stain and blind subculture. One hundred and twenty-one (7.6 %) blood cultures were positive by either method; of these, 105(8.68 %) were positive by both methods, 11 (9.1 %) positive by acridine orange and negative by subculture, and 5 (4.1 %) negative by acridine orange and positive by subculture. The difference between the 116 blood cultures positive by acridine orange and the 110 blood cultures positive by subculture was not statistically significant (p>0.1). Gram stain performed on all acridine orange positive cultures failed to reveal bacteria in 14 cases. Acridine orange staining is a sensitive, rapid and reliable method for detecting bacteria in blood cultures early during incubation. The method is inexpensive and easy to perform and can be substituted for blind subcultures.     

Bone marrow examination for the diagnosis of mycobacterial and fungal infections in the acquired immunodeficiency syndrome.

In a series of 342 bone marrow examinations from 314 patients with human immunodeficiency virus infection, 70 examinations (20%) detected opportunistic mycobacterial or fungal infections. One hundred eleven of the 314 patients had such infections, and, hence, 63% (70/111) were detected by bone marrow examination. Special stains for microorganisms detected 16 (32%) of 50 Mycobacterium avium complex infections, 10 (22%) of 45 Mycobacterium tuberculosis infections, eight (73%) of 11 Histoplasma capsulatum infections, and five (83%) of six Cryptococcus neoformans infections. Bone marrow cultures detected 36 (72%) of the 50 M avium complex infections, 13 (29%) of the 45 M tuberculosis infections, and 63% of the fungal infections. Marrow examination revealed infection in only one of the 70 specimens (1%) collected to evaluate thrombocytopenia alone or hematologic malignancy, but in 69 (25%) of 274 with fever, neutropenia, anemia, or miscellaneous other indications for marrow examination. Granulomas were detected in 102 (30%) of the biopsy specimens, including 71 (64%) of those in cases with mycobacterial or fungal infection. The granulomas showed caseous necrosis in nine cases, all in patients with tuberculosis, and the 27 cases with tuberculosis-associated granulomas tended to show large, tightly cohesive granulomas. The presence of granulomas correlated with opportunistic infection in 82 (80%) of 102 cases. Without granulomas, special stains were positive in only eight (3%) of 240 specimens. These results suggest that (1) bone marrow granulomas are a common and valuable histologic clue to opportunistic infection; (2) without them, special stains may not be a cost-efficient way to diagnose such infection; and (3) bone marrow examination can be a useful method of diagnosing opportunistic mycobacterial and fungal infections in patients with fever, anemia or neutropenia, and underlying human immunodeficiency virus infection.     

Use of lysis-centrifugation (isolator) and radiometric (BACTEC) blood culture systems for the detection of mycobacteremia.

Patients with acquired immune deficiency syndrome may develop infection with mycobacteria, particularly Mycobacterium avium-M. intracellulare (MAI). These infections can frequently be associated with demonstrable mycobacteremia with the organism. In this study, we compared the sensitivity of a radiometric (BACTEC system; Johnston Laboratories, Inc., Towson, Md.) liquid medium culture system with that of conventional solid mycobacterial culture media for cultures of blood from these patients. Both systems were inoculated with blood concentrate prepared by lysis-centrifugation (Isolator; Du Pont Co., Wilmington, Del.). Of 46 acquired immune deficiency syndrome patients whose blood was cultured, 28% had cultures positive for MAI. Patients had from less than 1 to more than 100 MAI colonies per ml of blood. Lowenstein-Jensen and Middlebrook 7H11 agars were comparable in recovery of MAI. BACTEC 12A vials containing double the standard volume of medium (4 ml) were more sensitive and were positive slightly earlier than vials containing the standard volume (2 ml). Conventional media detected 98% of positive cultures; BACTEC vials containing double volumes of medium detected 94% of positive cultures, whereas single-volume vials detected 77%. BACTEC vials were positive approximately 5 to 6 days sooner than slants or plates containing conventional media. For a few cultures, the use of unconcentrated blood was compared with the use of Isolator-concentrated blood by using each of these as inocula for BACTEC vials. Results for these cultures suggested that, although the use of Isolator-concentrated blood resulted in greater sensitivity than the use of unconcentrated blood would, the use of unconcentrated blood would still result in the detection of at least 78% of positive cultures.     

Direct identification of Staphylococcus aureus in blood culture fluid with a commercial latex agglutination test.

A commercial latex agglutination slide test (SeroSTAT Staph, Scott Laboratories, Inc., Fiskeville, R.I.) accurately identified Staphylococcus aureus when applied directly to blood culture fluid containing staphylococci. This latex agglutination test exhibited 100% accuracy when 30 seeded aerobic and anaerobic radiometric blood cultures (15 strains of S. aureus, 15 strains of other staphylococcal species) were tested blindly. In 36 actual clinical specimens yielding 16 isolates of S. aureus and 20 isolates of Staphylococcus epidermidis, 94.4% accuracy was achieved. The latex agglutination test provided positive test results before objective criteria of blood culture positivity such as radiometric growth indices and Gram stains became positive.     

Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of broth blood cultures.

Bartonella quintana was isolated from 34 BACTEC nonradiometric aerobic resin blood cultures for 10 adults. Nine patients were initially diagnosed by routine acridine orange staining of routine cultures that had been incubated for 8 days. All subcultures grew on chocolate agar within 3 to 12 days (median, 6 days). The PLUS 26 high-volume aerobic resin medium, combined with acridine orange stain and subculture, is an effective system for detection and isolation of B. quintana from blood.     

Acridine orange staining and radiometric detection of microorganisms in blood cultures.

To determine whether acridine orange (AO) staining of blood cultures could be used as a substitute for blind subculture when used in conjunction with the BACTEC system (Johnston Laboratories, Inc., Towson, Md.), the two methods were compared on all BACTEC-negative specimens. Since blind subcultures were routinely performed in our laboratory on days 2 and 6 of incubation, AO staining was also performed on these days. Cultures which were BACTEC positive on day 1 of incubation were not included in the study. Of the 2,395 bottles tested after 2 days of incubation, 106 were subculture positive. Of these, 96 (90.6%) were also AO positive and BACTEC positive, 3 (2.8%) were AO positive and BACTEC negative, and 7 (6.6%) were AO negative and BACTEC positive. Of the 3,487 bottles tested on day 6 of incubation, 14 were subculture positive; 7 (50%) of these were AO positive and BACTEC positive, and seven were AO positive and BACTEC negative. Of the total of 10 culture-positive bottles missed by BACTEC, all were positive, and all 10 companion aerobic bottles were BACTEC positive. In both phases of the experiment, there was a total of only four false-positive AO stains. As a result of this investigation, we have substituted AO staining for blind subculturing of BACTEC-negative bottles.     

Evaluation of a commercial radiometric system for primary isolation of mycobacteria over a fifteen-year period

The rate of recovery and time to detection of mycobacteria from clinical specimens were analysed for specimens inoculated into both radiometric Middlebrook 7H12 (Bactec 12B, originally 12A) medium and Lowenstein-Jensen egg-based medium (with and without sodium pyruvate) over the 15-year period from 1980 to 1994. A total of 19,679 Bactec vials were inoculated together with Lowenstein-Jensen slopes, with 2198 mycobacterial isolates detected. The mean times to detection for Bactec and Lowenstein-Jensen were 11.7 days and 23.9 days, respectively. In 195 cases mycobacteria were isolated from Bactec and not from Lowenstein-Jensen, whereas the reverse was true in 42 cases. The number of contaminated Bactec vials was 488 and the number of contaminated Lowenstein-Jensen slopes 448.     

Recovery and susceptibility testing of Mycobacterium tuberculosis from extrapulmonary specimens by the BACTEC radiometric method

This study was carried out to evaluate the sensitivity and rapidity of the BACTEC radiometric techniques for isolation and susceptibility testing of mycobacteria from extrapulmonary specimens. Concentrated specimens of urine, pleural fluid, and blood as well as other extrapulmonary specimens were processed for the recovery of mycobacteria and for drug susceptibility testing, employing conventional and BACTEC radiometric methods. Out of 483 specimens processed, 20 were found to be positive for Mycobacterium tuberculosis on the conventional Lowenstein -Jensen medium, and 19 were found to be positive in the BACTEC 7H12 medium. Average recovery times were 22.5 days for the conventional method and 10.9 days for the BACTEC method. When isolated cultures were tested for susceptibility to streptomycin, isoniazid, rifampin, and ethambutol, results were reported at an average time of 22 and 5.4 days for the conventional and BACTEC methods, respectively, with good correlation.     

Comparison of acridine orange, methylene blue, and Gram stains for blood cultures.

Direct microscopic screening of blood cultures by Gram stain or methylene blue stain is time consuming and frequently insensitive. Therefore, we evaluated a fluorescent-staining procedure that uses acridine orange (AO) at pH 3.5 and compared it with the methylene blue and Gram stain procedures. All smears were prepared within 24 h of receiving the culture, fixed with methanol, and examined without the results of the companion smears being known. AO-stained smears were examined with incident-light fluorescence at 600 x magnification and confirmed at 1,500x magnification. All bottles macroscopically positive within 24 h were excluded from the study. Of 2,946 cultures entered into the study, 204 (6.9%) were positive within 3 days. The sensitivity and specificity of AO based on these culture results were 52 and 98%, respectively, compared with 38% sensitivity and 99% specificity by methylene blue and Gram stains. The AO staining procedure is a simple, sensitive, screening technique for the early detection of positive blood cultures.     

Multicenter Comparison of ESP Culture System II with BACTEC 460TB and with Lowenstein-Jensen Medium for Recovery of Mycobacteria from Different Clinical Specimens, Including Blood

The recently developed ESP Culture System II (AccuMed, Chicago, Ill.) was compared with radiometric BACTEC 460TB (Becton Dickinson, Towson, Md.) and with Lowenstein-Jensen medium for recovery of mycobacteria from over 2,500 clinical specimens both of respiratory and nonrespiratory origin, including blood. The majority of the 219 mycobacterial isolates (129) belonged to the Mycobacterium tuberculosis complex, followed by 37 isolates of the Mycobacterium avium complex (MAC) and 53 isolates of eight other mycobacterial species. Rates of recovery obtained with BACTEC, ESP, and Lowenstein-Jensen medium were 89, 79, and 64%, respectively, with such differences being statistically significant. Different media and systems appeared to behave differently when the more frequently detected organisms were considered: M. tuberculosis complex isolates grew better with BACTEC, and MAC isolates grew better with ESP. An analysis of the combinations of Lowenstein-Jensen medium with BACTEC and with ESP did not reveal significant differences in recovery rates. With regard to the times needed for the detection of positive cultures, they were significantly longer on Lowenstein-Jensen medium (average, 28 days) than with the remaining two systems, between which there was no difference (average, 18 days). We conclude, therefore, that the ESP system, when used in combination with a solid medium, performs as well as the thoroughly validated radiometric BACTEC system and offers the advantages of full automation and absence of radioisotopes.