Lysis-centrifugation Blood Culture Research Articles

Direct blood culturing on solid medium outperforms an automated continuously monitored broth-based blood culture system in terms of time to identification and susceptibility testing

Pathogen identification and antimicrobial susceptibility testing (AST) should be available as soon as possible for patients with bloodstream infections. We investigated whether a lysis-centrifugation (LC) blood culture (BC) method, combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and Vitek 2 AST, provides a time advantage in comparison with the currently used automated broth-based BC system. Seven bacterial reference strains were added each to 10 mL human blood in final concentrations of 100, 10 and 1 CFU/mL. Inoculated blood was added to the Isolator 10 tube and centrifuged at 3000 g for 30 min, then 1.5 mL sediment was distributed onto five 150-mm agar plates. Growth was observed hourly and microcolonies were subjected to MALDI-TOF MS and Vitek 2 as soon as possible. For comparison, seeded blood was introduced into an aerobic BC bottle and incubated in the BACTEC 9240 automated BC system. For all species/concentration combinations except one, successful identification and Vitek 2 inoculation were achieved even before growth detection by BACTEC. The fastest identification and inoculation for AST were achieved with Escherichia coli in concentrations of 100 CFU/mL and 10 CFU/mL (after 7 h each, while BACTEC flagged respective samples positive after 9.5 h and 10 h). Use of the LC-BC method allows skipping of incubation in automated BC systems and, used in combination with rapid diagnostics from microcolonies, provides a considerable advantage in time to result. This suggests that the usefulness of direct BC on solid medium should be re-evaluated in the era of rapid microbiology.

Detection of fungal DNA in lysis–centrifugation blood culture for the diagnosis of invasive candidiasis in neonatal patients

We report data concerning the detection of fungal DNA directly from lysis–centrifugation blood culture to assess its value in the detection of fungaemia in 86 of the 347 patients admitted to the neonatal intensive-care unit between January 2009 and December 2010. The sensitivity and specificity of the PCR were 87.5% and 98.5%, respectively, with a positive predictive value of 93.3% and a negative predictive value of 97.1%. Detection of fungal DNA directly from blood culture Isolator 1.5 microbial tubes, without prior cultivation, is a promising approach for the rapid detection of Candida spp. in neonates with suspected candidaemia.

Whipple's disease diagnosis following the use of TNF- blockade

A 46-year-old man with inflammatory back pain and large and small joint arthritis was diagnosed with AS 10 years following symptom onset. Physical examination showed decreased vertebral range of motion. Laboratories revealed ESR of 40 mm/h, RF negative, ANA negative with positive HLA-B27. Plain radiographs demonstrated sacroiliac pseudo-widening. Anti-inflammatories, MTX (25 mg s.c. weekly) and prednisone (7.5–10 mg daily) were initiated. Five months later, infliximab (5 mg/kg increased to 10 mg/kg every 6 weeks) was added for inflammatory back pain with improvement. After 2 years of infliximab, the patient developed fevers and migratory arthritis precipitating hospitalization. Abnormal laboratories included white blood cell (WBC) count 36 k/mm3, haemoglobin (Hgb) 11.6 g/dl and ESR 58 mm/h. Evaluation including WBC scan, histoplasma antigen, brucella serology, routine and lysis centrifugation blood cultures, hepatitis and HIV serology, and bone marrow biopsy were negative. Symptoms resolved with cessation of infliximab and MTX. Three months later, re-challenge with adalimumab and MTX for recurrent inflammatory symptoms resulted in fevers, migratory arthritis and new weight loss with diarrhoea. Endoscopy with duodenal biopsy showed altered architecture and intracellular bacilli on periodic acid-Schiff stain (Fig. 1). Trophyrema whipplei was detected from blood and duodenal tissue by PCR. Intravenous ceftriaxone (2 g daily for 2 weeks) was commenced followed by trimethoprim-sulphamethoxazole with improved symptoms. Fig. 1 Detection of Whipple's disease. (A) Duodenal mucosa showing loss of normal villous architecture with foamy macrophages filling and expanding the lamina propria (haematoxylin and eosin stain, objective magnification 10×). (B) Intense periodic acid–schiff ... Trophyrema whipplei is a ubiquitous organism, rarely associated with multisystemic and relapsing disease. The organism may be detectable by PAS staining of involved organ tissue or with 16S rRNA gene identification [1]. Arthropathies associated with T. whipplei may precede diagnosis and exhibit axial and peripheral involvement, with symptom exacerbation following TNF-α inhibitors [1, 2]. Our case illustrates the consideration of alternative aetiologies in patients with articular symptoms, most importantly following clinical deterioration on immunomodulatory agents like TNF-α inhibitors. Disclosure statement: The authors have declared no conflicts of interest.

Quantitation of Candida CFU in Initial Positive Blood Cultures

One potential limitation of DNA-based molecular diagnostic tests for Candida bloodstream infection (BSI) is organism burden, which is not sufficiently characterized. We hypothesized that the number of CFU per milliliter (CFU/ml) present in an episode of Candida BSI is too low for reliable DNA-based diagnostics. In this study, we determined Candida burden in the first positive blood culture and explored factors that affect organism numbers and patient outcomes. We reviewed records of consecutive patients with a positive blood culture for Candida in the lysis-centrifugation blood culture system (Isolator, Wampole Laboratories, Cranbury, NJ) from 1987 to 1991. Descriptive statistics and logistic regression analyses were performed. One hundred fifty-two episodes of Candida BSI were analyzed. Patient characteristics included adult age (72%), indwelling central venous catheters (83%), recent surgery (29%), neutropenia (24%), transplant (14%), and other immune suppression (21%). Rates of treatment success and 30-day mortality for candidemia were each 51%. The median CFU/ml was 1 (mode 0.1, range 0.1 to >1,000). In the multivariate analysis, pediatric patients were more likely than adults to have high organism burdens (odds ratio [OR], 10.7; 95% confidence interval [95% CI], 4.3 to 26.5). Initial organism density did not affect patient outcome. Candida CFU/ml in the first positive blood culture of a BSI episode varies greatly; >50% of cultures had ≤1 CFU/ml, a concentration below the experimental yeast cell threshold for reliable DNA-based diagnostics. DNA-based diagnostics for Candida BSI will be challenged by low organism density and the need for sufficient specimen volume; future research on alternate targets is warranted.

Histoplasma capsulatum fungemia in patients with acquired immunodeficiency syndrome: detection by lysis-centrifugation blood-culturing technique

Progressive disseminated histoplasmosis (PDH) is an increasingly common cause of infection in patients with acquired immune deficiency syndrome (AIDS). We report 21 cases of PDH associated with AIDS diagnosed by lysis-centrifugation blood culture method. The most prevalent clinical findings were fever, weight loss, respiratory symptoms, and mucocutaneous lesions. Chest roentgenogram showed diffuse pulmonary infiltrates in 13 of 21 patients (62%). Bronchoalveolar fluid has yielded positive culture in four patients only in medium with cycloheximide.

What is the clinical significance of positive blood cultures with Aspergillus sp in hematopoietic stem cell transplant recipients? A 23 year experience

Hematopoietic stem cell (HSC) transplantation is the most frequent underlying predisposing condition to invasive aspergillosis. However, the significance of positive blood culture with Aspergillus sp in this particular population remains uncertain. We retrospectively reviewed all blood cultures performed in 1453 patients who received HSC transplant at our institution between 1980 and 2002. We identified 19 patients with positive blood cultures with Aspergillus sp. Only one of these patients had clinical, histologic or microbiologic evidence of invasive aspergillosis. Thus, even in a population at highest risk for invasive aspergillosis, positive blood cultures with Aspergillus sp remain unusual, and cannot be readily associated with invasive aspergillosis. A case by case assessment by treating physicians of the clinical and radiologic parameters should be systematically made to establish the significance of aspergillemia. Single bottle positivity, obtained with the lysis-centrifugation blood culture system, is a common indicator of pseudoaspergillemia.

Bacteremia due to Mycobacterium tuberculosis or M. bovis, Bacille Calmette-Guérin (BCG) among HIV- positive children and adults in Zambia.

Among adults with advanced HIV infection in developing countries, bacteremia due to Mycobacterium tuberculosis (MTB) is common and bacteremia due to M. bovis (bacille Calmette-Guérin; BCG) is rare. Comparable data are not available for children with HIV. To compare the prevalence of bacteremia due to M. tuberculosis or M. bovis BCG in hospitalized children and adults with HIV infection in a developing country with a high prevalence of tuberculosis and HIV and > 95% BCG immunization coverage. Descriptive cross-sectional study. Prospectively hospitalized patients in Lusaka, Zambia who were suspected to have HIV infection underwent phlebotomy for HIV ELISA, HIV viral load, and lysis-centrifugation blood culture for mycobacteria. Histories were obtained and patients were examined for BCG scars. Mycobacterial isolates were identified using DNA probes for MTB complex (MTBC), multiplex PCR and IS6110 typing. The median age of 387 HIV-positive children was 15 months; 98% were BCG immunized. The median age of 344 HIV-positive adults was 32 years; 44% were BCG immunized. Blood cultures were positive for mycobacteria in six children (2%) and 38 adults(11%) (P < 0.001). The six pediatric isolates included five MTBC (40% clustered) and one BCG. The 38 adult isolates included 36 MTBC (16% clustered) and two M. avium complex. Bacteremia due to MTB is less common among children than adults with advanced HIV infection in Zambia. Bacteremia due to M. bovis BCG is rare even among children with recent BCG immunization and symptomatic HIV infection.

Granulomatous Disease Associated withBartonellaInfection in 2 Dogs

Shortly after removal of an engorged tick from the left ear, a 4‐year‐old Greyhound was referred for evaluation of fever and a rapidly enlarging mass in the region of the left submandibular lymph node. Histopathologic evaluation of the lymph node resulted in a diagnosis of severe granulomatous lymphadenitis. An 11‐year‐old mixed‐breed dog was referred for evaluation of a 6‐week history of serous nasal discharge. Histologic examination of a surgical biopsy from a nasal mass indicated multifocal granulomatous inflammation with fibrosis. Serum samples obtained from both dogs were reactive by immunofluorescent assay toBartonella vinsoniisubsp.berkhoffiiantigens (reciprocal titers of 128). AlthoughBartonellaorganisms were not isolated by lysis centrifugation blood culture,BartonellaDNA was amplified from tissue samples obtained from each dog (lymph node biopsy from dog 1 and nasal biopsy from dog 2) using primers that amplify a portion of the 16S rRNA gene followed by Southern blot hybridization using a genus‐specific probe. Additionally, restriction fragment length polymorphism (RFLP) analysis of aBartonella‐specific citrate synthase gene product obtained from dog 2 resulted in a restriction pattern identical toB. vinsoniisubsp.berkhoffii.This is the 1st report of granulomatous disease in dogs associated withBartonellainfection.

Bartonella vinsonii subsp. berkhoffii and related members of the alpha subdivision of the Proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis.

Cardiac arrhythmias, endocarditis, or myocarditis was identified in 12 dogs, of which 11 were seroreactive to Bartonella vinsonii subspecies berkhoffii antigens. Historical abnormalities were highly variable but frequently included substantial weight loss, syncope, collapse, or sudden death. Fever was an infrequently detected abnormality. Cardiac disease was diagnosed following an illness of short duration in most dogs, but a protracted illness of at least 6 months' duration was reported for four dogs. Valvular endocarditis was diagnosed echocardiographically or histologically in eight dogs, two of which also had moderate to severe multifocal myocarditis. Four dogs lacking definitive evidence of endocarditis were included because of seroreactivity to B. vinsonii antigens and uncharacterized heart murmurs and/or arrhythmias. Alpha proteobacteria were not isolated from the blood by either conventional or lysis centrifugation blood culture techniques. Using PCR amplification and DNA sequencing of a portion of the 16S rRNA gene, B. vinsonii was identified in the blood or heart valves of three dogs. DNA sequence alignment of PCR amplicons derived from blood or tissue samples from seven dogs clustered among members of the alpha subdivision of the Proteobacteria and suggested the possibility of involvement of one or more alpha proteobacteria; however, because of the limited quantity of sequence, the genus could not be identified. Serologic or molecular evidence of coinfection with tick-transmitted pathogens, including Ehrlichia canis, Babesia canis, Babesia gibsonii, or spotted fever group rickettsiae, was obtained for seven dogs. We conclude that B. vinsonii subsp. berkhoffii and closely related species of alpha proteobacteria are an important, previously unrecognized cause of arrhythmias, endocarditis, myocarditis, syncope, and sudden death in dogs.

Detection of Candida albicans in blood by PCR in a rabbit animal model of disseminated candidiasis

A model of acute disseminated Candida albicans infection in New Zealand rabbits was developed to determine the sensitivity and accuracy of polymerase chain reaction (PCR) assay compared with the lysis-centrifugation blood culture method. Primers used amplify a DNA fragment from the multicopy gene coding for the small subunit rRNA, highly conserved in fungi. The sensitivity of PCR achieved in rabbit blood samples spiked with Candida albicans was 10–50 CFU/100 μL. A nested-PCR increased the limit of detection 10-fold. The sensitivity achieved exclusively with the lysis-centrifugation method (37.5%) was higher than that obtained with PCR (25%), but lower than nested PCR (52.5%). The combination of both techniques, lysis-centrifugation and nested PCR, increased the overall sensitivity rate to 62.5%. These results have demonstrated that, globally, the nested PCR was more sensitive than both single PCR and lysis-centrifugation culture in detecting C. albicans in blood from immunecompetent rabbits with acute disseminated candidosis. PCR could be a useful complementary technique to traditional methods in the early diagnosis of candidemia.

The Yield of Bone Marrow Biopsy and Culture Compared with Blood Culture in the Evaluation of HIV-infected Patients for Mycobacterial and Fungal Infections

Purpose: To compare the clinical utility of bone marrow biopsy and culture specimens with blood cultures for mycobacterial and fungal infections among human immunodeficiency virus (HIV)-infected patients. Patients and Methods: All bone marrow biopsies obtained from HIV-infected patients at the University of Alabama at Birmingham (UAB) Medical Center during 1993 to 1995 were blindly reviewed in a standardized format. Bone marrow culture results and blood culture results obtained within 6 weeks of each bone marrow study were compiled. Medical records were reviewed to determine indications for performing bone marrow biopsies, empiric or prophylactic antimicrobial therapies preceding the biopsy, and CD4 counts. Results: Eighty-two bone marrow studies were obtained from 76 patients. Most were performed during the evaluation of fever, cytopenia, or weight loss. Of 55 bone marrow mycobacterial cultures, 13 yielded Mycobacterium avium complex (MAC) and 2 yielded M tuberculosis (MTB). Of 51 bone marrow fungal cultures performed, 2 yielded Cryptococcus neoformans and 1 Histoplasma capsulatum. All patients with a bone marrow culture positive for MAC had a CD4 count of 20 cells/mm 3 or less. The mean CD4 count in this group (±95% confidence interval) (8 ± 3 cells/mm 3) was lower than that of culture-negative cases (41 ± 25 cells/mm 3); P <0.015). When bone marrow cultures and mycobacterial blood cultures were concurrently obtained, results were usually in agreement between the two sites. The mean time until the report of positive mycobacterial bone marrow cultures (22 ± 5 days) was similar to that for blood cultures (24 ± 3 days). Most (84%) patients with multiple mycobacterial cultures had completely concordant results (all positive or all negative). When blood or bone marrow culture yielded mycobacteria, only 29% of the corresponding bone marrow examinations revealed stainable acid-fast bacilli (AFB). In contrast, all 3 cases with positive fungal bone marrow cultures also had stainable organisms on histologic examination. Conclusions: The combined use of bone marrow biopsy and culture as well as blood cultures provide the maximum diagnostic yield when evaluating patients with AIDS for mycobacterial or fungal infections. However, when mycobacterial infections were diagnosed, bone marrow results seldom provided more immediate or specific information than lysis centrifugation blood cultures. A single lysis centrifugation blood culture should be the first step in the routine evaluation of HIV-infected patients when disseminated MAC infection is suspected.

Fungal Blood Cultures Not Useful in AIDS

Lysis centrifugation blood cultures (e.g., Dupont isolator) are commonly used because they may increase the recovery of fungi compared with conventional blood culture systems such as BACTEC. The high incidence of disseminated fungal infections in febrile AIDS patients prompts many …

Lack of utility of the lysis-centrifugation blood culture method for detection of fungemia in immunocompromised cancer patients.

We retrospectively compared the utility of a fungal isolation device (Isolator) versus conventional techniques for recovering fungal organisms from blood cultures obtained from neutropenic cancer patients. Positive cultures were deemed true pathogens, possible pathogens, or contaminants according to laboratory and clinical criteria. Fifty-three patients had 66 positive blood cultures for fungi, nine on multiple occasions. In 20 episodes true pathogens were recovered, 6 from broth medium alone, 4 from the Isolator system alone, and 10 from both systems. False-negative cultures were noted in 4 of 20 (20%) cases in which broth medium was used and in 6 of 20 (30%) cases in which the Isolator system was used. Possible pathogens were detected in 4 of 66 blood culture-positive cases. Forty-two positive cultures were considered contaminants, 1 collected from standard medium and 41 of 42 (98%) which grew only in Isolators. Eleven of 18 patients with true fungal infections expired as a result of infection, while 4 of 33 patients with a contaminant expired, none from a fungal cause. We do not advocate the routine use of Isolator tubes in the evaluation of the febrile, neutropenic patient due to the high rates of false positives and of contamination.

Clinical Comparative Study of Quantitative Lysis Filtration and Lysis Centrifugation Blood Culture Techniques for Streptococcus Bacteremia

It has been suggested that transient bacteremia following oral procedures may cause infective endocarditis in susceptible patients and streptococci remain the most common pathogenic bacteria for infective endocarditis. Blood samples of 20 patients after tooth extraction were cultured by two different quantitative blood culture techniques; lysis filtration and lysis centrifugation (Isolater 10). Both techniques are performed by first lysing the blood cells, then followed by filtration or centrifugation. Isolated rates were compared by recovery of oral streptococci. The incidence of streptococcis bacteremia were 60% by lysis filtration and 5% by isolater 10. The concentration of oral streptococci in positive blood samples by lysis filtration ranged from 0.12 to 3.72 CFU/ml (mean, 0.62).Streptococcus constellatus andGemella morbillorum were isolated by both techniques in patient 1, the concentration was 3.72 CFU/ml by lysis filtration but only 0.2 CFU/ml by isolater 10.s. constellatus was the most common isolate. Also isolated wereS. intermedius, S. sanguis, S. mutans andG. morbillorum. Polymicrobial streptococcus bacteremia was found in 4 patients. A combination ofS. constellatus andG. morbillorum was isolated from three patients. In conclusion, lysis filtration was an excellent quantitative blood culture technique for the isolation of streptococci. Lysis filtration should provide a better comparison of the efficacy of different antibiotics used in the prevention of transient dental bacteremia.

Lysis—centrifugation blood cultures in the detection of tissue-proven invasive candidiasis disseminated versus single-organ infection

Several studies have demonstrated significantly higher frequency and more rapid detection of candidemia with blood culture methods performed by lysis-centrifugation (LC) in comparison with other techniques. Little is known, however, about the ability of LC blood culture methods to detect tissue-proven invasive candidiasis. We therefore investigated the sensitivity of LC blood cultures in the detection of tissue-proven invasive candidiasis. Between 1985 and 1991, invasive candidiasis was detected in 41 (5.1%) of 803 autopsies at the Clinical Center of the National Institutes of Health (Bethesda, MD, USA). Cases were classified as single-organ (SO) candidiasis (n = 20) and as disseminated candidiasis (DI) (n = 21). Patients with DI were more likely than those with SO to have a hematologic malignancy (71% vs 15%, P < 0.001) and to have gastrointestinal mucosal candidiasis (76% vs 25%, P = 0.003). LC detected fungemia in 16 (43%) of all 37 cases with blood cultures. When analyzed by classification, Candida spp. were isolated from blood in 11 (58%) of 19 patients with DI and in five (28%) of 18 patients with SO (P = 0.13). When analyzed by number of organs infected, blood cultures were positive in seven (78%) of nine patients with >3 organs infected by Candida in comparison to five (28%) of 18 patients with one organ infected (P = 0.024). The mean recovery time for Candida in blood cultures was 2.6 days in DI and 3.2 days in SO (P = 0.017). There was no difference in colonies of organisms per LC tube between patients with DI and those with SO. These findings indicate that LC blood cultures are more likely to detect deeply invasive candidiasis in direct relation to the number of tissue sites infected and that there is a critical need for development of more sensitive methods for detection of invasive candidiasis.

Incidence of Mycobacterium avium-intracellulare complex bacteremia in human immunodeficiency virus-positive patients.

The product-limit incidence of Mycobacterium avium-intracellulare complex (MAC) bacteremia in 1006 human immunodeficiency virus (HIV)-positive patients followed at one institution over a 3-year period from the day of AIDS diagnosis with monthly lysis-centrifugation blood cultures was 21% +/- 2% SE at 1 year and 43% +/- 3% at 2 years. The product-limit incidence of MAC bacteremia at 1 year after the patients' first CD4 cell count was related to both the CD4 cell count and to whether they had an AIDS diagnosis (both P less than .0001) but not to age, sex, or race. This incidence was 39% +/- 6% for CD4 cell counts of less than 10/mm3, 30% +/- 5% for 10-19/mm3, 20% +/- 4% for 20-39/mm3, 15% +/- 4% for 40-59/mm3, 8% +/- 3% for 60-99/mm3, and 3% +/- 1% for 100-199/mm3. MAC may eventually infect most if not all HIV-positive patients who do not die from another HIV-related event.

Late Recurrent Candida Endocarditis

Late recurrent Candida endocarditis (LRCE) developed on a prosthetic mitral valve 22 months after treatment for primary native mitral valve endocarditis. The LRCE was difficult to diagnose; results of two dimensional echocardiography and repeated blood cultures were negative. Only transesophageal echocardiography revealed a vegetation and only lysis centrifugation blood cultures demonstrated candidemia. Postmortem examination revealed a large Candida vegetation on the prosthetic valve and Candida in the mitral valve ring. This case and a review of the literature indicate that Candida endocarditis treated with amphotericin B and prosthetic valve replacement may recur months after treatment, and that LRCE, which is difficult to diagnose and treat, may be best prevented by lifelong antifungal suppressive therapy.

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

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

Concurrent bloodstream infection withHistoplasma capsulatumandMycobacterium tuberculosis

An immunosuppressed patient is the first to be reported with both histoplasmal fungemia and tuberculous bacteremia, emphasizing the need to consider polymicrobial infection in such patients and the utility of lysis-centrifugation and radiometric blood culture techniques.

Lysis centrifugatiomこよる血液培養法の真菌検出における基礎的ならびに臨床的検討

We compared the lysis centrifugation blood culture system using Isolator (Du Pont) to the conventional blood culture bottle using BCB system (Roch), and the following results were obtained. In the investigation of experimental fungemia made by human blood and Candida albicans, the organism was detected 9/12 after 1 day and 11/12 after 3 days by Isolator, although the organism was detected 3/12 after 3 days and 11/12 after 6 days by the culture bottle system. In the experimental fungemia of Candida tropicalis, the organism was detected 12/12 after 2 days by Isolator, although it was detected 1/12 after 6 days and 4/12 after 10 days with the culture bottle system. Isolator showed faster and a more sensitive detection rate for fungemia. Clinical investigation of 94 cases (180 cultures) showed a 11.1% isolation rate with Isolator system and a 7.1% isolation rate with the culture bottle system. In 43 cases which were examined by the both systems, 7 cases were positive with Isolator, 3 positive with culture bottle. The culture bottle system isolated only Candida albicans and Candida parapsilosis, where as the Isolator isolated Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida spp. This investigation suggested that the Isolator system was an excellent method for the detection of fungemia.