Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures

A new radiometric method for the automatic detection of bacterial growth in blood cultures has been compared with conventional methods. A total of 2,967 cultures from 1,280 patients suspected of having bacteremia were studied. A 2-ml amount of blood was inoculated into culture media in which the glucose was labeled with carbon-14. The release of (14)CO(2) by bacterial metabolism was checked hourly for 18 to 24 hr, daily for the next 2 days, and, on the 12th day, with an automated instrument. A 10-ml amount of blood was studied by conventional bacteriological techniques. In 125 cultures from 50 patients, there was bacterial growth in at least one of the routine media. Of these, the radiometric method detected 102 cultures from 40 patients. In 111 cultures from 48 patients, there was radiometric detection of bacterial growth. In all of these cultures, there was detection of bacterial growth in subcultures from the radioactive medium. Of these, the routine laboratory detected 98 cultures from 40 patients. Neither method detected all patients with bacteremia. Among the 57 patients positive by one or both methods, routine techniques detected bacteria in 87% and the radiometric method detected bacteria in 85%. Seventy per cent of the cultures were detected first by the radiometric method, 65% on the day of inoculation. Our results suggest that the radiometric method is faster than conventional techniques and comparable in accuracy. Its great advantage is that it is simple, automatic, and can be extended to automatic detection of bacterial sensitivity to antibiotics.     

Usefulness of the BACTEC MYCO/F lytic system for detection of mycobacteremia in a clinical microbiology laboratory

278 BACTEC MYCO/F lytic system blood cultures for mycobacteria were evaluated between 1997 and 1999. Sixty of them were read as positive by the system, being considered 15 of them as false positives. Twenty-seven yielded mycobacterial growth (13 Mycobacterium avium from 3 patients and 14 Mycobacterium tuberculosis from 8 patients). Other bacteria isolated were coagulase-negative Staphylococcus (13 samples), Corynebacterium sp. (5 samples), Salmonella enteritidis (2 samples) and Klebsiella pneumoniae (1 sample). Five of these isolates were considered as true episodes of bacteremia. The average time for detection of mycobacteria was 12.6 days for M. avium and 26.4 days for M. tuberculosis. BACTEC MYCO/F lytic system is useful for detection of mycobacteremia in clinical microbiology laboratories.     

Bacteremia After Genitourinary Tract Manipulation: Bacteriological Aspects and Evaluation of Various Blood Culture Systems

A total of 300 patients undergoing various types of urological procedures was studied for incidence of bacteremia. An osmotically stabilized anaerobic broth with sodium polyanethol sulfonate (Liquoid) yielded more positive blood cultures than any other culture system and was also the best system by far for recovery of anaerobes. The membrane filter showed faster growth and, therefore, facilitated faster identification of the infecting organism. There was a 31% incidence of bacteremia in the patients having transurethral resection of the prostate, 17% in the cystoscopy group, 24% in the urethral dilation group, and 8% in the urethral catheterization group. The organisms found most frequently isolated in blood cultures were enterococci and Klebsiella pneumoniae. Notable were a relatively large number of anaerobes and two protoplasts. The major source of the bacteremia was previous urinary tract infection, but evidence is presented which indicates that the prostate gland and the normal urethral flora are other significant sources.     

Measurement of Microbial DNA Polymerase Activity Enables Detection and Growth Monitoring of Microbes from Clinical Blood Cultures

Surveillance of bloodstream infections (BSI) is a high priority within the hospital setting. Broth-based blood cultures are the current gold standard for detecting BSI, however they can require lengthy incubation periods prior to detection of positive samples. We set out to demonstrate the feasibility of using enzymatic template generation and amplification (ETGA)-mediated measurement of DNA polymerase activity to detect microbes from clinical blood cultures. In addition to routine-collected hospital blood cultures, one parallel aerobic blood culture was collected and immediately refrigerated until being transported for ETGA analysis. After refrigeration holding and transport, parallel-collected cultures were placed into a BACTEC incubator and ETGA time-course analysis was performed. Of the 308 clinical blood cultures received, 22 were BACTEC positive, and thus were initially selected for ETGA time course analysis. The ETGA assay detected microbial growth in all 22 parallel-positive blood cultures in less time than a BACTEC incubator and also yielded genomic DNA for qPCR-based organism identification. In summary, feasibility of detecting microbes from clinical blood culture samples using the ETGA blood culture assay was demonstrated. Additional studies are being considered towards development of clinically beneficial versions of this methodology.     

Radiometric Method for Detection of Bacteremia

A study was performed with simulated blood cultures to evaluate the production of (14)CO(2) as an index of bacterial growth. With a range of inoculum sizes from 4 to 4,250 colony-forming units, it was not possible to detect (14)CO(2) within 6 hr after inoculation in 59 separate experiments. In a limited trial with patients' blood cultures, the radiometric method failed to provide any earlier evidence of bacteremia than did routine broth cultures.     

Radiometric Detection of Bacteremia in Neonates

The predicted prevalence of false positive blood cultures due to hyperactive neonatal blood cells in a radiometric detection system was confirmed. Suppression of this blood background radioactivity in the system was achieved by using a hypertonic medium containing 10% sucrose. The radiometric system produced accurate results as fast as the conventional blood culturing method, saved labor and minimized the recovery of extraneous contaminants.     

Comparison of the conventional culture,the manual fluorescent MGIT system and the automated fluorescent MGIT 960 culture system for the detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> ssp. <Emphasis Type="Italic">avium</Emphasis> in tissues of naturally infected hens

Different methods for the detection of Mycobacterium avium ssp. avium (MAA) in naturally infected hens were compared. They included the conventional culture method (solid Herrold’s and Stonebrink media and liquid Sula medium) and newly developed liquid culture systems, the manual mycobacteria growth indicator tube (M-MGIT) and the fully automated BACTEC MGIT 960 system (A-MGIT). 152 tissues originating from 15 naturally infected hens have been processed. The overall detection rates (percentage of positive cultures from the number of positive cultures determined by all the methods together) were 60, 70 and 76 % for the conventional media, M-MGIT and A-MGIT systems, respectively, the mean time of mycobacteria detection being 32.6, 17.6 and 14.6 d, respectively. The lowest contamination rate (2.0 %) was found in A-MGIT compared with M-MGIT (4.6 %) and conventional media (10.4 %).     

Comparison of the BACTEC System with Blind Subculture for the Detection of Bacteremia

One thousand blood specimens were cultured in BACTEC vials containing modified Columbia broth in aerobic, anaerobic, and hypertonic formulations. Radiometric readings and subcultures were performed on aerobic and hypertonic vials at 24 h and 7 days, and on anaerobic vials at 48 h and 7 days. Significant numbers of false-positive BACTEC readings were obtained. Although all positive cultures were eventually detected by the BACTEC, approximately 20% of blood specimens yielding positive subcultures at 24 h did not give positive BACTEC readings until 48 h.     

Epidemiology of bloodstream infections and time to detection of positive blood cultures: an evaluation of the automated BacT/Alert and BACTEC 9240 systems

Data of 3,097 blood culture sets processed with the BacT/Alert system in 1997 were compared to those of 3,158 blood culture sets processed with BACTEC 9240 in 1999. Agents responsible for bloodstream infections (BSI) were detected in 15.9% and 20.0% of blood cultures in 1997 and 1999, respectively. The incidence of BSI was 9.3 (1997) vs. 11.3 (1999) per 1,000 admissions. In both years, S. aureus was the most frequent isolate, followed by E. coli. Overall, the mean detection time (MDT) obtained with the BACTEC 9240 was significantly shorter than that of the BacT/Alert. Significant MDT differences were found for all organisms, except for Enterobacteriaceae (12.7 vs. 10.6 h). With both systems, over 95% positive samples were detected within 3 days, indicating that a 4-day incubation protocol may disclose most BSI agents. Thus, the added speed of the BACTEC 9240 allowed a particularly fast clinical management of septic patients.     

Comparison of Macroscopic, Microscopic, and Radiometric Examinations of Clinical Blood Cultures in Hypertonic Media

Clinical blood cultures were collected in the Bactec 8A flask (Johnston Laboratories, Cockeysville, Md.) and examined macrosopically, microscopically, and radiometrically in an effort to determine which approach produced the fastest detection time. Of 360 blood cultures found to contain organisms by subculture, 334 were first detected by Bactec, 98 by macroscopic examination, and 68 by microscopic examination. Examination times were at 4, 8, 16, 24, 36, and 48 h after collection of the specimen. Sixteen hours after specimen collection, microscopic examination had detected 31 positive cultures, macroscopic examination had detected two positive cultures, and Bactec had detected 160 positive cultures. By the end of the first 24 h of incubation, Bactec had detected 313 (93%) of those cultures eventually found to be positive. Although Bactec produced the fastest detection time in an overwhelming majority of the cultures, it failed to detect three of three Candida spp. cultures, three of five Bacteroides spp. cultures, and six of 32 Enterococcus spp. cultures during the first 48 h of incubation.     

The detection of positive blood cultures in children using the acridine orange, gram, and methylene blue stains

The efficacy of the acridine orange (AO), gram (G), and methylene blue (MB) microscopic procedures was analyzed using positive blood cultures monitored by a radiometric procedure (Bactec) in children. Sixty-eight positive cultures included the following isolates: Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, Enterobacteriaceae, Staphylococcus aureus, Candida spp., and seven other pathogens. The MB stain yielded the highest detection rate, 99%, in comparison with 94 and 93% for the AO and G stains, respectively. Since the MB stain yielded comparable results to the AO procedure with no requirement for a fluorescent microscope, the former method is recommended for confirming the presence and initial characterization of microorganisms in blood cultures monitored by Bactec from children.     

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.     

手工和仪器两种血培养结果差异性分析

目的对手工法双相血培养瓶和BACTEC9120全自动血培养仪的阳性率作回顾性分析。方法将血液标本同时接种双相血培养基和BACTEC9120全自动血培养仪配套血瓶中,将阳性结果移种血平板,如为阴性再移种巧克力平板。结果370例血培养,双相血培养瓶阳性25例,阳性率为6．76％（25／370）,树脂需氧（儿童）瓶BACTEC9120报警显示阳性59例,阳性率为15．9％（59／370）,阳性标本移种到血平板及巧克力平板阳性54例,阳性率为14．6％（54／370）,假阳性5例,假阳性率为1．4％（5／370）,共有29例树脂需氧（儿童）瓶阳性,而双相血培养瓶为阴性,P〈0．001。结论BACTEC9120全自动血培养仪提高阳性率,缩短阳性的报告时间优于传统的双相血培养基。     

Isolated intracranial hypertension: a rare presentation of neurobrucellosis

Brucella melitensis infection is endemic in the eastern and south-eastern Anatolia regions of Turkey. We report an unusual case of brucella meningitis presenting with bilateral papilla stasis, diplopia and absence of other neurological involvement. Diagnosis was made by positive culture of Brucella spp. with a BACTEC 9120 system with inoculation of the patient's cerebrospinal fluid (CSF). This is the first report of isolation of Brucella spp. from CSF on a BACTEC 9120 system for diagnosis of meningitis. This case demonstrated that brucella meningitis may present with very slight symptoms, and inoculation of CSF into BACTEC bottle besides conventional cultures improves the detection of Brucella in endemic areas such as Turkey.     

Transient Bacteremia Due to Suction Abortion: Implications for SBE Antibiotic Prophylaxis

The incidence and character of the bacteremia associated with elective suction abortion was investigated in volunteer subjects aged 19 to 35 years who were to undergo first trimester abortion by suction curettage. One hundred and forty-four blood cultures were obtained from thirteen pregnant and four non-pregnant (control) subjects matched for age. Transient bacteremia occurred during or soon after suction abortion in 11 of 13 (84.7%) study subjects. Four of these patients were bacteremic after bimanual pelvic examination, just prior to initiation of the abortion procedure. Seven others developed bacteremia temporally related to cervical dilatation and suction abortion. The bacteremia was intermittent in some, persistent in others, existed as long as one hour after the procedure, and was transient in all patients. Microorganisms isolated from the blood were all normal genital tract flora and were predominantly anaerobes, although alpha hemolytic streptococci were also recovered. Mixed bacteremia occurred in six patients. In contrast, blood cultures from four non-pregnant women were sterile. This study indicates that the systemic circulation-uterine cavity barrier is significantly disrupted during abortion by suction curettage permitting endogenous genital tract microorganisms to gain access into the bloodstream. These observations also suggest that there may be some risk of developing endocarditis during suction abortion in patients with cardiac deformities, and lend some support to the current practice of giving antibiotic prophylaxis to abortion patients with cardiac lesions which predispose them to endocarditis.     

A new MSPQC system for rapid detection of pathogens in clinical samples

A new multi-channel series piezoelectric quartz crystal (MSPQC) system for detection of pathogens in clinical sample was proposed. Some factors, which affect the detection of pathogens by using MSPQC, were all investigated. A total of 650 clinical samples were detected by MSPQC and compared with licensed BACTEC 9120 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA) simultaneously in the Third Xiangya Hospital of Central South University, China. When the incubation period was 5 days, two systems had similar detected results: the MSPQC system detected 123 growth of 650 (18.92%) bottles while the BACTEC 9120 detected 125 growth of 650 (19.23%) bottles. The MSPQC had 2 false-positive signals and 2 false-negative signals. However, BACTEC 9120 had 3 false-positive signals and 0 false-negative signals. Further identifications of bacteria were run by VITEK-2 (bioMérieux China Ltd.), 5% sheep blood trypticase soy agar (SBA) and chocolate agar (CA). Comparing with BACTEC 9120, MSPQC system possesses following advantages: shorter average detection time, less blood volume needed, less false-positive results and low cost. It can also provide information in real time. So MSPQC has a wonderful perspective in clinical application.     

Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BackgroundAutomated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell–based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed.MethodsMicroorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry.ResultsThe BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h).ConclusionsWhen a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).     

A Note on Positive BACTEC Readings Produced by Mycoplasma

Five of nine mycoplasma strains tested were associated with a positive radiometric reading, suggesting that Mycoplasma spp. should be considered during the subculturing of clinical materials found positive upon screening by the BACTEC system     

Comparative evaluation of the MGIT and BACTEC culture systems for the recovery of Mycobacterium avium subsp. paratuberculosis from milk

AIMS: To compare the detection capabilities of the non-radiometric MGIT (Mycobacteria Growth Indicator Tubes) and radiometric BACTEC 460TB culture systems (Becton Dickinson, Cowley, Oxford, UK) for recovering Mycobacterium avium subsp. paratuberculosis from milk. METHODS AND RESULTS: Ultra heat treated (UHT) milk samples spiked with different levels of M. paratuberculosis (10-107 cells ml-1) were inoculated into MGIT and BACTEC media (containing recommended supplements) with and without prior chemical decontamination of the milk samples with 0.75% (w/v) cetylpyridinium chloride for 5 h. Time for the detection of growth in days was recorded for each culture system, and a M. paratuberculosis count for each milk sample was calculated from BACTEC readings using a published formula. Correlation between MGIT and BACTEC detection times was 0.6983. Both culture systems were capable of detecting 10-100 M. paratuberculosis cells ml-1 in milk within 30-40 days when no decontamination treatment was applied, but only 102-103 cells ml-1 or greater when chemical decontamination was applied before culture. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The non-radiometric MGIT system could be substituted for the radiometric BACTEC system for the culture of M. paratuberculosis from milk without loss of detection sensitivity. Chemical decontamination before culture caused a significant reduction in numbers of viable M. paratuberculosis in all spiked milk samples resulting in decreased detection capability for both culture systems.     

Microbiologic analysis of results from blood cultures

The aim of our investigations was the microbiological analysis together with the evaluation of sensitivity of bacteria frequently isolated from blood cultures. Blood samples were taken from patients with symptoms suggesting bacteremia in Rydygier's Hospital in Cracow. A total of 11,170 blood samples taken from 1997 to 2000 were tested. Automatic VITAL system (bioMerieux) was applied to culture and detect microorganisms. Bacteria were identified by ATB system (bioMerieux). Susceptibility was detected by ATB and disc diffusion method. Percentage of positive results relating to detection of microorganisms of clinical significance was 16.9% (1891 cultures). Staphylococcus spp. (Staph. epidermidis in range 22.8% to 21.9%), Enterococcus spp. and Streptococcus spp. were most frequently isolated species among aerobic Gram-positive bacteria. In 2000, compared to 1997 the number of isolates of MRSA increased considerably (from 1.8% to 6.8%). In blood infections the increase of frequency of E. coli bacteria was also noted: 6.1% and 11.4% in 1997 and 2000, respectively. Among non-fermentant bacilli the percentage of occurrence of P. aeruginosa in the period of 4 years was comparable in the range 7.3% in 1997 to 7.2% in 2000. The increase in the frequency of blood infections of A. baumanii was also noticed (respectively from 4.8% to 9.9%). Susceptibility of P. aeruginosa strains to selected beta-lactame antibiotics and aminoglycosydes increased in 2000 in comparison to 1999. A. baumanii strains were 100% sensitive only to imipenem.