Blood Culture Incubation Research Articles

A differential centrifugation protocol and validation criterion for enhancing mass spectrometry (MALDI-TOF) results in microbial identification using blood culture growth bottles

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) is a widely used tool in clinical microbiology for rapidly identifying microorganisms. This technique can be applied directly on positive blood cultures without the need for its culturing, thereby, reducing the time required for microbiological diagnosis. The present study proposes an innovative identification protocol applied to positive blood culture bottles using MALDI-TOF. We have processed 100 positive blood culture bottles, of which 36 of 37 Gram-negative bacteria (97.3 %) were correctly identified directly with 100 % of Enterobacteriaceae and other Gram-negative rods and 87.5 % of non-fermenting Gram-negative rods. We also correctly identified directly 62 of 63 of Gram-positive bacteria (98.4 %) with 100 % of Streptococcus, Enterococcus, and Gram-positive bacilli and 98 % of Staphylococcus. Applying the differential centrifugation protocol at the moment the automatic blood culture incubation system gives a positive reading together with the proposed validation criterion offers 98 % sensitivity (95 % confidence interval: 95.2-100 %). The MALDI-TOF system, thus, provides a rapid and reliable system for identifying microorganisms from blood culture growth bottles.

A Critical Appraisal of the Role of the Clinical Microbiology Laboratory in the Diagnosis of Bloodstream Infections

ABSTRACT The detection of bloodstream infections is one of the most important functions of clinical microbiology laboratories. Despite advances in blood culture technology and clinical studies that have focused on the detection of bacteremia and fungemia, perfection has not been achieved and uncertainties persist. This review provides perspectives on a number of areas, including the recommended number of blood cultures, duration of incubation of blood cultures, use of anaerobic, in addition to aerobic, blood culture media, value of the lysis-centrifugation method, processing and reporting of probable blood culture contaminants, and limitations of current blood culture methods and systems. We also address the handling of blood cultures in point-of-care locations that lack full microbiology capabilities.

Clinical Impact of Preincubation of Blood Cultures at 37°C

The effect of immediate incubation of blood cultures at 37°C on the turnaround time and the impact of Gram stain results on antimicrobial management were investigated. During a 6-month period, blood cultures collected at the emergency department outside laboratory operating hours were preincubated at 37°C until transportation to the laboratory. Upon the arrival of blood cultures at the laboratory, Gram stains and subcultures were made from all bottles prior to further incubation in the automated system (Bactec 9240). Data from 1 year earlier, when all blood cultures were stored at room temperature, were used for comparison. In the study period, 79 episodes of bacteremia were detected for 75 patients, compared to 70 episodes for 67 patients in the control period. Preincubation of blood cultures at 37°C resulted in a 15-h reduction in the median time to reporting of Gram stain results, from 34 to 19 h (P, <0.001). With preincubation, 3 episodes (4%) of bacteremia were not detected by the Bactec 9240 system. Based on the reporting of the Gram stain results, appropriate antimicrobial therapy was initiated for 12% of all patients with positive blood cultures, while for 24% the therapy was streamlined. Thus, immediate incubation of blood cultures reduced the time to reporting of Gram stain results. However, not all episodes of bacteremia were detected by the Bactec 9240 system after preincubation at 37°C. Blood culture results contributed importantly to appropriate antimicrobial management.

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.

Immediate Incubation of Blood Cultures Outside Routine Laboratory Hours of Operation Accelerates Antibiotic Switching

The aim of this prospective randomized controlled clinical trial was to assess the impact of immediate incubation of blood cultures delivered to the laboratory outside its hours of operation on turnaround times, antibiotic prescription practices, and patient outcomes. A continuously monitoring blood culture incubator was placed outside the laboratory, which was switched on (intervention arm) and off (control arm) in a randomized manner. Included were new bacteremia episodes of patients older than 18 years. During the 30-week study period, the first positive blood culture specimen of an episode had to be brought to the laboratory outside its hours of operation. The median time from specimen collection until growth detection was reduced by 10.1 h in the intervention arm (P < 0.001). For 46 of 66 (70%) episodes in the intervention arm and for 51 of 85 (60%) episodes in the control arm, the antibiotic regimen was changed (not significant). The median time until the first change in the antibiotic regimen was 42.8 h in the intervention arm and 64.0 h in the control arm (P, 0.024). There was no difference in length of stay or hospital mortality. Immediate incubation of blood cultures outside laboratory hours reduces turnaround times and accelerates antibiotic switching.

Not the usual cardiac rhythm device infection: A fastidious pathogen with several teaching points

Because cardiac device infections may include fastidious pathogens, extended incubation of blood cultures is suggested. A patient with an infection of a right ventricular lead implantable cardioverter defibrillator (ICD) system is described. The device was implanted 6 months earlier. The pathogen was identified as Haemophilus parainfluenzae, which was cultured within 72 hours and was presumably from a respiratory tract infection. Extended incubation was not necessary to culture this fastidious pathogen. Two large retrospective studies suggest that prolonged incubation for fastidious organisms is generally not necessary because of advances in culture media and automated blood culture systems.

Unexplained choroidal embolisation: remember the antiphospholipid syndrome

In January, 2005, a 58-year-old woman presented complaining of bilateral sudden painless loss of vision, followed by perception of fl oaters. She had a history of colon carcinoma 2 years previously, and had been diseasefree since her treatment. Her visual acuity was 20/100 in the right eye and 20/40 in the left. Fundus examination showed scattered multiple choroidal infarcts bilaterally in the posterior pole surrounding the macular area (fi gure A). A 2+ vitreal fl are was also seen bilaterally. Fluorescein angiography showed hypofl uoresence at the infarcted sites in early stages and hyperfl uoresence at the late stages. Physical examination showed only a grade 2/6 systolic murmur of the heart. Two-dimensional transoesophageal echocardiography showed mobile vegetations of the mitral valve. Extensive laboratory blood tests, including tumour markers, C-reactive protein, fi brinogen, rheumatoid factor, antinuclear antibodies, anti-double-stranded DNA antibodies, antineutrophil cytoplasmic antibodies, and C3 and C4 complement components, showed only a mild prolongation of activated partial thromboplastin time (37 s). Serological tests for bacterial and viral infections were negative. Long-duration incubation of blood and aqueoushumour cultures were negative. Abdominal ultrasonography, CT of the chest and abdomen, brain MRI, carotid doppler tests, and colonoscopy showed no abnormalities. Further testing gave a positive result for IgG anticardiolipin antibodies (IgG-aCL), with a binding index of 370 (index cut-off =100, which corresponds to the cut-off of 15–20 GPL units of the commercially available assays) on a highly sensitive, specifi c, and inter nationally accepted ELISA. She was also positive for IgM-aCL and lupus anticoagulant (ratio: 1·65; normal <1·2) by the diluted Russell’s viper venom test (HemosIL, Instrumentation Laboratory Company, Lexington, MA, USA). The IgG-aCL titre remained positive 6 weeks and 12 weeks after the fi rst test (binding index 456 and 379, respectively, which correspond to more than 80 GPL units with the conventional assays). Antiphospholipid syndrome (APS) accom panied by non-bacterial thrombotic endocarditis was considered, and anticoagulant treatment with acenocoumarol was started with target INR 3·0–3·5. After 4 weeks, her visual acuity had improved (right eye 20/50; left eye 20/25). Vitreal fl are disappeared 6 weeks after presentation. 8 weeks after her intitial presentation, transoesophageal echocardiography and cardiac MRI showed no abnormalities. At a 5-month follow-up appoint ment, her visual acuity had improved further (right eye 20/25; left eye 20/20). 1 year after her initial presentation, the patient’s fundus (fi gure B), visual acuity, and general health have remained stable. When last seen in May, 2006, she remained well. APS is an autoimmune disorder characterised by arterial or venous thrombosis, recurrent fetal loss, and circulating antiphospholipid antibodies. It has been implicated in a range of ocular disorders. The clinical features of ocular involvement seem to be diff erent in primary and secondary APS—vasculopathic eye disease involving retinal and choroidal vessels seems to be typical in the primary form, whereas retinal thrombosis is more common in APS associated with systemic lupus erythematosus. In our patient, APS was the main mechanism leading to choroidal infarction. Non-bacterial thrombotic endocarditis can accompany malignant disorders whether or not they are associated with APS. In our case, we were not able to detect a relapse of cancer, which is in accordance with a prospective study showing high aCL titres in some cancer patients even during clinical remission of the disease. These associations suggest the need for thorough followup for thrombotic events in aCL-positive cancer. Additionally, unexplained ocular manifestations should alert physicians for APS presence because the ophthalmological symptoms can be the fi rst clinically evident manifestations of this disorder, and anticoagulant treatment should be started as soon as possible.

Bartonella quintana prosthetic valve endocarditis detected by blood culture incubation beyond 10 days

We describe the case of a 45-year-old man with mitral and aortic prosthetic valve replacement who presented with symptoms of subacute bacterial endocarditis. Bartonella quintana was grown from blood after prolonged culture. The course of the disease was complicated by splenic infarction, glomerulonephritis resulting in progressive renal insufficiency, and cerebroventricular hemorrhage. Notably, cardiac ultrasonography showed no extensive vegetations but a strand-like lesion. Culture-positive B. quintana prosthetic valve endocarditis in a formerly healthy subject represents a newly observed entity. It should be added to the differential diagnosis of prosthetic valve endocarditis, especially when it presents with features suggesting subacute bacterial endocarditis.

Cardiobacterium hominis endocarditis: two cases and a review of the literature

Cardiobacterium hominis, a member of the HACEK group (Haemophilus parainfluenzae, Haemophilus aphrophilus, and Haemophilus paraphrophilus, Actinobacillus actinomycetemcomitans, C. hominis, Eikenella corrodens, and Kingella species), is a rare cause of endocarditis. There are 61 reported cases of C. hominis infective endocarditis in the English-language literature, 15 of which involved prosthetic valve endocarditis. There is one reported case of C. hominis after upper endoscopy and none reported after colonoscopy. Presented here are two cases of C. hominis prosthetic valve endocarditis following colonoscopy and a review of the microbiological and clinical features of C. hominis endocarditis. Patients with C. hominis infection have a long duration of symptoms preceding diagnosis (138+/-128 days). The most common symptoms were fever (74%), fatigue/malaise (53%), weight loss/anorexia (40%), night sweats (24%), and arthralgia/myalgia (21%). The most common risk factors were pre-existing cardiac disease (61%), the presence of a prosthetic valve (28%), and history of rheumatic fever (20%). Of the 61 cases reviewed here, the aortic valve was infected in 24 (39%) and the mitral valve in 19 (31%) patients. The average duration of blood culture incubation before growth was detected was 6.3 days (range, 2-21 days). Complications were congestive heart failure (40%), central nervous system (CNS) emboli (21%), arrhythmia (16%), and mycotic aneurysm (9%). C. hominis is almost always susceptible to beta-lactam antibiotics. Ceftriaxone is recommended by the recently published American Heart Association guidelines. The prognosis of C. hominis native valve and prosthetic valve endocarditis is favorable. The cure rate among 60 patients reviewed was 93% (56/60). For prosthetic valve endocarditis, the cure rate was 16/17 (94%). Valve replacement was required in 27 (45%) cases.

Emerging Data Indicating that Extended Incubation of Blood Cultures Has Little Clinical Value

terium avium complex isolates recovered from a patient with AIDS and recovery of 1 Legionella species, all of which were detected using the lysis-centrifugation method) among 215 patients for whom the very extensive special protocol was used. During the same period, 24 HACEK bacteria were detected in ∼14,000 routine blood cultures, all of which were processed in accordance with the laboratory’s standard 5-day incubation protocol. Potential limitations of this study are that the data were generated from a single institution and that the observations may apply to only one of the commercially-available CMBCSs (Bactec; BD Diagnostic Systems). Accordingly, it could be argued that the findings cannot be generalized to other health care centers or other blood culture systems. However, there are emerging data from

Time to Positivity in Staphylococcus aureus Bacteremia: Possible Correlation with the Source and Outcome of Infection

Staphylococcus aureus bacteremia often persists and causes metastatic infections. It is unknown whether the time between blood culture incubation and growth detection (i.e., the time to positivity) in a continuously monitored system--a probable surrogate marker of bacteremia severity--correlates with outcome. We performed a prospective, observational study involving adult inpatients who had S. aureus bacteremia between 1 January 2002 and 30 June 2003 at a 600-bed teaching hospital. Measurements included time to positivity in initial blood culture series, duration of bacteremia, rate of metastatic infection, and outcome. A total of 376 S. aureus bacteremias (> or = 1 positive blood culture result) were reported for 357 patients aged 18-103 years (median age, 59 years); 64 bacteremias were excluded because blood was drawn after antibiotic therapy was started (n = 59) or through an intravascular catheter (n = 5). The source of infection was identified in 244 series (78.2%). Metastatic infection was detected in 25 bacteremias (8.0%). The mortality rate was 25.6%. The duration of bacteremia (determined in 251 series) was 1-59 days (median duration, 1 day; 70th percentile, 3 days). The time to positivity ranged from 4.2 to 98.2 h (median time to positivity, 15.5 h) and was significantly shorter for patients with an endovascular source of infection (14.9+/-5.4 vs. 19.5+/-10.6 h; P < .0005), extended duration (i.e., > or = 3 days) of bacteremia (14.1+/-4.2 vs. 18.6+/-9.2 h; P < .0005), and metastatic infection (12.9+/-5.9 vs 18.0+/-9.3 h; P = .007). Analysis of a range of cutoff values demonstrated that a time to positivity of < or = 14 h yielded the best sensitivity and specificity for predicting the source and outcome of infection. Logistic regression analyses revealed that a time to positivity of < or = 14 h was an independent predictor of an endovascular source of infection (P < .0005), extended bacteremia (P < .0005), metastatic infection (P < .0005), and attributable mortality (P = .017). Time to positivity in S. aureus bacteremia may provide useful diagnostic and prognostic information. Growth of S. aureus within 14 h after the initiation of incubation may identify patients with a high likelihood of endovascular infection sources, delayed clearance, and complications.

Brucellosis in San Diego

Although aggressive public health measures have greatly reduced the number of brucellosis cases in the United States, there is a resurgence of interest in this worldwide zoonosis because of its potential as a bioweapon and its 8-fold higher incidence in California, Texas, and the other borderlands between the United States and Mexico compared with the national rate. Accordingly, we reviewed the clinical records of 28 patients diagnosed at a university hospital in San Diego, CA, between 1979 and 2002 to look for new epidemiologic trends and to test the hypothesis that there are species-specific differences in clinical presentations. In contrast to the latest California-wide study completed in 1992, Brucella abortus infections were more common (73%) than Brucella melitensis after 1992, and women were more commonly infected (77% compared with 39%) than men. Major risk factors remained Hispanic ethnicity, travel to Mexico, and ingestion of nonpasteurized dairy products. Analysis of diagnostic procedures suggested that the traditional practice of prolonged incubation of blood cultures increased their sensitivity for Brucella, even in automated radiometric systems. Direct comparison of the clinical manifestations of infections with B. abortus and B. melitensis strongly supported differences in acute presentations. B. melitensis presented more acutely as fevers of unknown origin with statistically significant higher rates of abdominal tenderness, hepatomegaly, splenomegaly, thrombocytopenia, pancytopenia, and hepatic dysfunction. These results suggest that the epidemiology of brucellosis in California may be evolving, and they show, to our knowledge for the first time in a single series, that species-specific differences in presentations may account for some of the protean manifestations of brucellosis. Familiarity with manifestations of brucellosis and the optimal laboratory techniques for its diagnosis could help physicians protect the public against this reemerging, under-recognized zoonosis.

Reassessment of the routine anaerobic culture and incubation time in the BacT/Alert FAN blood culture bottles

A total of 9,130 blood cultures were collected from adult patients with suspected bloodstream infections. The recommended 20 mL sample of blood was divided equally between the aerobic and anaerobic FAN bottles and monitored in the BacT/Alert Microbial Detection System for a total of 5 days. There were 757 clinically significant positive culture pairs from 291 patients. Significant differences were found with greater recovery of Pseudomonas aeruginosa (p<0.001), Acinetobacter spp. ( p = 0.002), coagulase-negative staphylococci other than Staphylococcus epidermidis ( p = 0.002), and Candida spp. ( p<0.001) from the aerobic bottle and greater recovery of anaerobic bacteria ( p<0.001) from the anaerobic bottle. Significantly more episodes of P. aeruginosa bacteremia ( p<0.003) and candidemia ( p<0.001) were detected by the aerobic FAN bottle and significantly more episodes of anaerobic bacteremia ( p<0.001) were detected by the anaerobic FAN bottle (Table 2). No other significant differences between systems in their detection of bacteremias were noted. Anaerobic bacteremias were encountered in diverse and often unpredictable clinical settings. All clinically significant episodes of bloodstream infection were detected within 4 days of incubation of their cultures. We conclude routine, rather than selective, use of the anaerobic FAN bottle in the blood culture set and a 4-day incubation of blood cultures in the BacT/Alert aerobic and anaerobic FAN bottles is an appropriate routine procedure.

The detection of positive blood cultures by the AccuMed ESP-384 system: the clinical significance of three-day testing

A total of 805 positive blood cultures obtained over a 4-month period during 1997 by the AccuMed ESP-384 system were evaluated and compared with 471 positive blood cultures obtained during 1989. Of the 805 microorganisms isolated in 5-day culture and testing by the ESP-384, 671 (83.4%) were detected within the first 2 days and 758 (94.2%) within 3 days. There were 47 (5.7%) microorganisms detected in culture Days 4 and 5 from 42 patients and review of the medical records from these patients demonstrated that no significant changes in clinical management were instituted as a result of detection and identification of these isolates. These 47 organisms were considered to be zero to equivocal clinical relevance. These data suggest that a 3-day routine blood culture incubation protocol with the ESP-384 system may be sufficient when considering detection rate, clinical relevance, and cost-effectiveness. The microbial spectrum and relative frequencies in this study were found to be similar to those of positive blood cultures obtained during 1989.

Prolonged incubation of blood and bone marrow cultures in 12B bottles processed on the BACTEC 460 TB system does not increase microbial recovery

Standard references continue to recommend testing specimens processed on the BACTEC TB System for 8 weeks, despite evidence that mycobacteria are rarely recovered beyond 5–6 weeks. To clarify this issue, we retrospectively reviewed all positive blood/bone marrow cultures processed during a 17-month period when specimens were tested for 6 weeks. We then prolonged the incubation period to 8 weeks during the subsequent 5 months. Excluding Mycobacterium genavense, only 1 of 159 mycobacterial isolates was recovered during and none were recovered beyond the 5th week of incubation and testing on the BACTEC TB System.

Cytological effects of 50 Hz electromagnetic fields on human lymphocytes in vitro

Incubation of human peripheral blood cultures in the presence of an electromagnetic field (EMF) of 50 Hz and 5 mT leads to stimulation of the cell cycle of dividing lymphocytes but has no influence on the frequencies of sister-chromatid exchanges. Comparative studies with two different exposure systems and with different culture temperatures indicate that the effect on the cell cycle results from the EMF and is not a thermal effect. These data support the assumption that with respect to their suspected carcinogenic effects EMFs have no initiating but probably promoting effects.

Should aerobic blood cultures be shaken intermittently or continuously?

Stationary incubation of aerobic blood cultures was compared with intermittent shaking in aerobic Vacutainer 2630 bottles with agar slants during the first 24 h, and was simultaneously compared with the continuously shaken aerobic Bactec 6A bottles as a reference system. Intermittent shaking did not significantly increase or decrease the seven days yield of the Vacutainer bottles as compared with the continuously agitated Bactec 6A bottles. When one of 604 paired Vacutainer bottles was agitated for eight h and the other incubated stationary, the speed of growth detection was significantly greater in the agitated bottle (p less than 0.001), but significantly less than with continuously agitated Bactec 6A bottles (p less than 0.001). Agitation for 14 h showed the same pattern of detection speed. These results suggest that it is desirable initially to agitate aerobic blood cultures either intermittently or continuously.

Extended incubation of lysis-centrifugation blood cultures yields a newly recognized pathogen

Extended incubation of lysis-centrifugation blood cultures yields a newly recognized pathogen

Recovery of a newly recognized pathogen through extended incubation of lysis-centrifugation blood cultures

Recovery of a newly recognized pathogen through extended incubation of lysis-centrifugation blood cultures

Controlled evaluation of the effect of atmosphere of incubation on detection of bacteremia and fungemia in supplemented peptone broth.

To evaluate the role of atmosphere of incubation in the detection of clinically important bacteremia and fungemia in adults, we compared the yield of microorganisms from 10,541 paired 5-ml samples of blood incubated aerobically and anaerobically. The medium, supplemented peptone broth (SPB) with 0.03% sodium polyanetholesulfonate, and the ratio of blood to broth (1:10) were the same for all cultures. Only cultures with adequate blood samples (greater than or equal to 80% of stated volume) were compared statistically. More fungi (P less than 10(-7) ) grew in continuously vented bottles of SPB. Aerobic incubation also favored (P less than 0.01) isolation of Neisseria gonorrhoeae and Eubacterium; more than 80% of these bacterial organisms were detected only in vented bottles. Anaerobic incubation (plugged venting units) did not significantly favor the isolation of any genus of microorganisms, although an estimated 11% more Bacteroidaceae grew in the unvented bottle of SPB. By comparison of our data with published results for other media, we conclude that the need for both aerobic and anaerobic incubation of blood cultures is dependent upon the medium used and the microorganisms likely to be encountered. Vented incubation of blood cultured in SPB is crucial for detection of fungi and some bacteria. Routine use of an unvented bottle of SPB may not be worthwhile for patients in whom Bacteroidaceae cause bacteremia infrequently. However, when Bacteroidaceae are suspected as the cause of sepsis, use of an unvented bottle of SPB is prudent.