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Background: The critical factors for optimal recovery of microbial pathogens from blood are not only the basis for how we perform blood cultures on a daily basis, but are also important for development of all current blood-culture systems. Because much of this research was conducted and published between 1975 and 2010 there is a general sense that many physicians and scientists may not be familiar with it, but it is critical for performing and interpreting blood cultures., Objectives: To review the critical factors in the laboratory recovery and isolation of pathogenic microorganisms in blood., Sources: Literature review of published papers, select reviews and updated guidelines., Content: This review focuses on the critical factors that affect the recovery isolation of pathogenic microorganisms from individuals with bloodstream infections. Contemporary blood-culture systems, and current methods for blood-culture collection, have been designed to incorporate these critical factors so as to optimize recovery and isolation of pathogens while minimizing contamination., Implications: It is important for microbiologists and practitioners to understand how and why these critical factors affect current blood-culture practices to improve patient management. Future research should emphasize which of these critical factors will still play a role in the era of molecular diagnostic tests, which will no longer be relevant, and which new critical factors have yet to be defined., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

To determine the optimal anaerobic companion bottle to pair with BACTEC Plus Aerobic/F medium for recovery of pathogenic microorganisms from adult patients with bacteremia and fungemia, we compared Plus Anaerobic/F bottles with Standard Anaerobic/F bottles, each of which was filled with 4 to 6 ml of blood. The two bottles were paired with a Plus Aerobic/F bottle filled with 8 to 12 ml of blood. A total of 14,011 blood culture sets were obtained. Of these, 11,583 sets were received with all three bottles filled adequately and 12,257 were received with both anaerobic bottles filled adequately. Of 818 clinically important isolates detected in one or both adequately filled anaerobic bottles, significantly more staphylococci (P < 0.001), streptococci (P < 0.005), Escherichia coli isolates (P < 0.02), Klebsiella pneumoniae isolates (P < 0.005), and all microorganisms combined (P < 0.001) were detected in Plus Anaerobic/F bottles. In contrast, significantly more anaerobic gram-negative bacilli were detected in Standard Anaerobic/F bottles (P < 0.05). Of 397 unimicrobial episodes of septicemia, 354 were detected with both pairs, 30 were detected with Plus Aerobic/F-Plus Anaerobic/F pairs only, and 13 were detected with Plus Aerobic/F-Standard Anaerobic/F pairs only (P < 0.05). Significantly more episodes of bacteremia caused by members of the family Enterobacteriaceae (P < 0.05) and aerobic and facultative gram-positive bacteria (P < 0.025) were detected with Plus Anaerobic/F bottles only. In a paired-bottle analysis, 810 of 950 isolates were recovered from both pairs, 90 were recovered from Plus Aerobic/F-Plus Anaerobic/F pairs only, and 50 were recovered from Plus Aerobic/F-Standard Anaerobic/F pairs only (P < 0.001). Paired Plus Aerobic/F-Plus Anaerobic/F bottles yielded significantly more staphylococci (P < 0.001), streptococci (P < 0.05), and members of the family Enterobacteriaceae (P <0.001). We conclude that Plus Anaerobic/F bottles detect more microorganisms and episodes of bacteremia and fungemia than Standard Anaerobic/F bottles as companion bottles to Plus Aerobic/F bottles in the BACTEC 9240 blood culture system.

Iodophor and alcohol pledgets were compared with the Medi-Flex Prep Kit II for skin disinfection before venipuncture. Of 12,367 blood cultures collected, 6,362 were done with conventional pledgets and 6, 005 were done with Medi-Flex kits. Contamination occurred in 351 of 6,362 blood cultures (5.5%; range, 3.7 to 8.1%) with conventional pledgets versus 328 of 6,005 (5.5%; range, 3.5 to 7.5%) with Medi-Flex kits.

A total of 9,446 blood cultures were collected from adult patients at three university-affiliated hospitals. Of these, 8,943 cultures were received with both aerobic bottles filled adequately; 885 yielded 1,016 microorganisms, including 622 isolates (61%) that were the cause of sepsis, 337 isolates (33%) that were contaminants, and 57 isolates (6%) that were indeterminate as the cause of sepsis. With the exception of Staphylococcus aureus, which was recovered more often from VITAL aerobic bottles, more pathogenic microorganisms were recovered from BACTEC NR6 (aerobic) bottles than from VITAL aerobic bottles. Growth of pathogenic microorganisms was detected earlier in VITAL aerobic bottles. A total of 8,647 blood cultures were received with both anaerobic bottles filled adequately; 655 yielded 740 microorganisms, including 486 isolates (66%) that were the cause of sepsis, 215 isolates (29%) that were contaminants, and 39 isolates (6%) that were indeterminate as the cause of sepsis. More pathogenic microorganisms were recovered from VITAL anaerobic bottles than from BACTEC NR7 (anaerobic) bottles. Growth of pathogenic microorganisms was detected earlier in VITAL anaerobic bottles. In 8,500 sets all four bottles were received adequately filled. When paired aerobic and anaerobic bottle sets (systems) were compared, more pathogenic microorganisms (again with the exception of S. aureus) were recovered from the BACTEC system. For the 304 septic episodes (253 unimicrobial and 51 polymicrobial), significantly more were detected by the BACTEC system. We conclude that VITAL requires modification to improve recovery of pathogenic microorganisms to make it competitive with other commercially available blood culture systems.

The presence of microorganisms in a patient's blood is a critical determinant of the severity of the patient's illness. Equally important, the laboratory isolation and identification of a microorganism present in blood determine the etiologic agent of infection, especially when the site of infection is localized and difficult to access. This review addresses the pathophysiology and clinical characteristics of bacteremia, fungemia, and sepsis; diagnostic strategies and critical factors in the detection of positive blood cultures; characteristics of manual and instrument approaches to bacteremia detection; approaches for isolating specific microorganisms associated with positive blood cultures; and rapid methods for the identification of microorganisms in blood cultures.

FAN medium was formulated to improve microbial recovery, particularly for fastidious microorganisms and for microorganisms causing sepsis in patients receiving antimicrobial therapy. In a controlled clinical evaluation performed at four university-affiliated hospitals, FAN anaerobic bottles were compared with standard anaerobic bottles for yield, speed of detection of microbial growth, and detection of septic episodes. A total of 10,431 blood culture sets were received; both anaerobic bottles of 7,694 blood culture sets were adequately filled with blood. Altogether, 925 isolates were recovered: 557 that were the cause of sepsis, 99 that were indeterminate as the cause of sepsis, and 269 contaminants. More Staphylococcus aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Escherichia coli (P < 0.02), and all microorganisms combined (P < 0.005) were recovered from FAN bottles; more nonfermentative gram-negative bacilli (P < 0.05), Torulopsis glabrata (P < 0.001), and other yeasts (P < 0.01) were recovered from standard bottles. Growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Enterococcus faecalis (P < 0.025), streptococci other than Streptococcus pneumoniae (P < 0.01), and all microorganisms combined (P < 0.001) was detected earlier in standard bottles; growth of more isolates of E. coli (P < 0.05) and anaerobic bacteria (P < 0.01) was detected earlier in FAN bottles. The mean times to detection were 14.2 and 16.1 h for standard and FAN bottles, respectively. More septic episodes caused by S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.005), members of the family Enterobacteriaceae (P < 0.02), and all microorganisms combined (P < 0.02) were detected in FAN bottles; more septic episodes caused by nonfermentative gram-negative bacilli (P < 0.025) and yeasts (P < 0.005) were detected in standard bottles. In summary, more isolates (except for strict aerobes) were recovered from FAN bottles than from standard anaerobic bottles. Similarly, significant more septic episodes (except for those caused by strict aerobes) were detected with FAN bottles than with standard anaerobic bottles. With the exception of E. coli and anaerobic bacteria, growth of more isolates was detected earlier in standard anaerobic bottles.

A new medium, FAN, designed to enhance the recovery of microorganisms, has been developed for the BacT/Alert blood culture system (Organon Teknika Corp., Durham, N.C.). We compared the yield and speed of detection of microorganisms in 6,847 adequately filled paired aerobic standard and FAN bottles at four university hospitals. Of 499 clinically significant microorganisms isolated from one or both bottles, significantly more Staphylococcus aureus isolates (P < 0.001), coagulase-negative staphylococci (P < 0.001), yeasts (P < 0.01), and all microorganisms combined (P < 0.001) were recovered from the FAN bottles. Overall, the speeds of detection of positive cultures did not differ between the two medium formulations; mean times to detection in the standard and FAN bottles were 16.1 and 16.0 h, respectively. When a subset of patients on antimicrobial therapy was evaluated, significantly enhanced yield from the FAN bottle was evident for staphylococci. Overall, the FAN bottle outperformed the standard aerobic BacT/Alert bottle.

Helicobacter cinaedi (formerly Campylobacter cinaedi) was first detected in the fecal flora of homosexual men. Since 1984, 11 case reports of H. cinaedi bacteremia have been published; most cases have presented as a nonspecific febrile illness in homosexual men infected with the human immunodeficiency virus (HIV). We identified seven additional cases of H. cinaedi bacteremia in two Denver hospitals within a 5-year period, which suggests that this illness is not as rare as was previously thought. Six of these cases of H. cinaedi bacteremia occurred in homosexual men who were infected with HIV. Four patients presented with the distinctive cutaneous manifestation of multifocal cellulitis, and two patients had monoarticular arthritis. Microbiological diagnosis of this infection was delayed by the slow growth of the bacterium in nonradiometric blood culture bottles. Although the patients' bacteremia was prolonged, their response to treatment was excellent. In contrast to campylobacter infections in HIV-infected patients, H. cinaedi bacteremia did not relapse after a course of effective therapy. H. cinaedi bacteremia should be suspected in HIV-infected individuals who present with an indolent febrile illness, particularly in the presence of multifocal cellulitis and/or arthritis.

Bottles developed for use in the BacT/Alert automated blood culture system (Organon Teknika Corp., Durham, N.C.) can accept up to 10 ml of blood without falling below a 1:5 ratio of blood to broth. We compared the yield and speed of detection of microorganisms in 13,128 adequately filled, paired, aerobic bottles inoculated with 5 versus 10 ml of blood at three university hospitals. A total of 798 microorganisms causing sepsis grew in one or both bottles. The overall recovery of microorganisms from 10-ml samples exceeded that from 5-ml samples (P < 0.001); the increased yield attributed to the additional 5 ml in the samples was 7.2%. The increased yield from 10-ml inocula was most marked for Escherichia coli (P < 0.01) and other members of the family Enterobacteriaceae (P < 0.001). Ten-milliliter samples did not yield more gram-positive bacteria, nonfermentative gram-negative rods, or yeasts. When both bottles were positive, the bottles inoculated with 10 ml of blood showed growth sooner (P < 0.001). Earlier detection with 10-ml inocula was especially notable for coagulase-negative staphylococci (P < 0.001), streptococci (P < 0.001), E. coli (P < 0.025), and other members of the family Enterobacteriaceae (P < 0.025). We conclude that an increase in the volume of blood inoculated into BacT/Alert aerobic blood culture bottles from 5 to 10 ml will increase the overall yield and the speed of detection of clinically important blood pathogens.

Most, if not all, rare or fastidious microbial pathogens can be recovered from standard blood culture bottles and media using routine laboratory methods. Because they are only rarely recovered from blood cultures, relatively few data exist concerning the ability of different blood culture systems to recover these microorganisms. Thus, clinical microbiologists should take a conservative approach when processing blood cultures that may contain one of the microorganisms reviewed in this article, including the use of additional media, media supplements, blind and terminal subcultures, or prolonged incubation and testing.

For the first time, clinical microbiologists have the option of purchasing or leasing a variety of automated blood culture systems. Although the performance characteristics of the BACTEC radiometric and nonradiometric blood culture systems are well-documented, we are only beginning to learn the performance characteristics of CMBCS. Well-designed and executed controlled clinical trials are needed to establish performance characteristics and to address issues that are unique to CMBCS. In particular, controlled clinical comparisons of different CMBCS are needed. In terms of the impact on clinical microbiology laboratories, there are no published data to support claims that the use of these systems decreases laboratory workload, staffing, or costs. Although such an impact seems reasonable and even likely, unequivocal documentation of such savings would make it easier for many laboratorians to justify the cost of purchasing or leasing one of these systems. Similarly, there are no published data regarding the long-term reliability of these systems while in clinical use. A documented track record of mechanical and electrical reliability would be useful to clinical microbiologists contemplating the acquisition of a new blood culture system.

Various factors are important in the laboratory detection of bacteremia and fungemia. These include clinical factors, the type of blood culture system, and laboratory processing of blood culture bottles. Most commercial blood culture products have been designed to take these factors into account and, thus, are relatively similar. There are differences, however, and it should not be assumed that these products have identical performance characteristics. To optimize microbial recovery, clinicians should order and collect the proper number of blood cultures at the earliest possible time and before the administration of antimicrobial agents. Clinical microbiologists should select a blood culture system that optimizes the recovery of common microbial pathogens and should use blood culture systems according to the manufacturers' recommendations.

Clinical microbiologists must be able to evaluate critically the blood culture literature for patient care purposes, for sound laboratory management, and for decision making in regards to purchase or lease of a new system. This skill has never been more important due to the many new commercially available blood culture products and technologies. Critical assessment of the literature requires familiarity with blood culture terminology and an understanding of those factors critical in designing scientifically valid clinical evaluations.

For 273 patients evaluated for disseminated Mycobacterium avium complex infection, a total of 1,047 mycobacterial blood cultures (MBCs) were submitted; the M. avium complex was recovered from 140 (13%) of the specimens. Results for the paired MBCs were highly concordant: in 392 of 462 (85%) culture sets, both MBCs were negative, in 53 of 462 (11%) sets, both MBCs were positive, and in only 17 of 462 (4%) sets was one culture positive and the other negative. Acid-fast smears were done on sediments from 671 specimens; smears were positive for 4 of 98 (4%) cultures that grew the M. avium complex. A single MBC should be obtained and then repeated if negative and disseminated M. avium complex infection is still clinically suspected. Use of direct acid-fast smears of sediments is not a reliable means of detecting mycobacteremia.

Because of the declining frequency of anaerobic bacteremia, routinely using half the collected blood volume for anaerobic culture has been challenged. There is no data indicating whether more clinically relevant isolates would be recovered if all or most of the given blood sample were cultured aerobically. In this two-part study, we reviewed cases of anaerobic bacteremia to determine what proportion occurred in situations when anaerobes would be expected and then estimated the yield of different culture approaches by reanalyzing the data from a large prospective clinical blood culture study. The records of 61 patients who had an anaerobic isolate (excluding Propionibacterium species) recovered only from an anaerobic bottle were examined to define clinical settings in which such isolates occur. Fifty-six (92%) patients had clinically important isolates, and the source of infection was obvious at the time of culture in 47 of the 56 (84%). Of 56 patients, 36 (64%) had abdominal signs and symptoms, including 12 with recent abdominal surgery. Of nine patients without an obvious source of infection, six were on high-dose steroids. Relative yields were compared for (i) one aerobic bottle and one anaerobic bottle (5 ml to each) for all blood cultures, (ii) two aerobic bottles (5 ml to each), or (iii) two aerobic bottles plus an extra anaerobic bottle (only for clinically suspected anaerobic sepsis) (5 ml to each). The third approach had the highest yield (475 isolates), because the routine use of two aerobic bottles recovered more Candida spp., members of the family Enterobacteriaceae, and nonfermenters than did the first approach (448 isolates) (P < 0.02), and clinically directed culturing for anaerobes would recover anaerobes missed with the second approach (458 isolates). Our data suggest that the use of two aerobic bottles with selective culturing for anaerobes could increase the number of clinically relevant isolates by at least 6% compared with the current practice of inoculating an aerobic bottle and an anaerobic bottle with equal volumes of blood.

The BACTEC high-blood-volume fungal medium (HBV-FM) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) was compared with the Isolator (IS) tube and the BACTEC Plus 26 (BP26) blood culture bottle for the ability to recover fungi from the blood of adult patients suspected of having fungemia. A total of 6,836 blood culture sets that fulfilled criteria for inclusion in the study were received. Three separate comparisons were performed: 4,907 HBV-FM versus IS, 4,886 BP26 versus HBV-FM, and 4,949 BP26 versus IS. For the HBV-FM versus IS comparison, 218 isolates were recovered: 125 (57.3%) were bacteria and 93 (42.7%) were fungi. HBV-FM was comparable to IS for recovery of yeasts, but IS was superior for recovery of Histoplasma capsulatum (25 versus 0 isolates recovered [P < 0.001]). Growth of Torulopsis glabrata was detected earlier (P < 0.05) in HBV-FM bottles. For the BP26 versus HBV-FM comparison, 229 isolates were recovered: 161 (70.3%) were bacteria, and 68 (29.7%) were fungi. HBV-FM was superior for recovery of T. glabrata (P < 0.025) and all fungi combined (P < 0.025). There were no statistically significant differences in the speed of detection of microbial growth. For the BP26 versus IS comparison, 251 isolates were recovered: 165 (65.7%) were bacteria, and 86 (34.2%) were fungi. IS was superior for recovery of H. capsulatum (P < 0.001), T. glabrata (P < 0.05), and fungi other than H. capsulatum (P < 0.025). BP26 was superior for recovery of all bacteria combined (P < 0.001) and viridans group streptococci (P < 0.01). Growth of T. glabrata (P < 0.05) was detected earlier in IS tubes. Growth of Staphylococcus aureus (P < 0.01), viridans group streptococci (P < 0.01), Pseudomonas aeruginosa (P < 0.05), and all microorganisms combined (P < 0.05) was detected earlier in BP26 bottles. For yeast, 57 of 59 (96.6%), 79 of 80 (98.7%), and 64 of 67(95.5%) were recovered from BP26 bottles, HBV-FM bottles, and IS tubes, respectively, by day 14; for H. capsulatum, 14 of 36 (38%) isolates were recovered from IS tubes by day 14. Mean times of recovery were similar for BACTEC bottles and IS. We conclude that (i) for recovery of fungi from blood cultures, HBV-FM is equivalent to IS (with the exception of H. capsulatum); (ii) for recovery of bacteria, BP26 is superior to IS; (iii) BP26 bottles are inferior to both HBV-FM bottles and IS tubes for recovery of T. glabrata; and (iv) HBV-FM bottles must be paired with another blood culture bottle or system to optimize detection of bacteremia.

In a collaborative study at three university hospitals, the recovery of microorganisms and the speed of detection of microbial growth by the BacT/Alert (Organon Teknika Corporation, Durham, N.C.) and BACTEC 660/730 (Becton-Dickinson Diagnostic Instrument Systems, Sparks, Md.) nonradiometric blood culture systems were compared. A total of 5,918 comparisons were made between BacT/Alert aerobic and BACTEC NR 6A bottles and 5,992 comparisons were made between BacT/Alert anaerobic and BACTEC NR 7A bottles. Each bottle was inoculated with 5 ml of blood. The overall recoveries of microorganisms from the two aerobic bottles were comparable; members of the family Enterobacteriaceae were recovered more often from BacT/Alert aerobic bottles alone (P less than 0.001). The overall recoveries of microorganisms from the anaerobic bottles were not significantly different. Growth of Staphylococcus aureus (P less than 0.001), coagulase-negative staphylococci (P less than 0.01), streptococci (P less than 0.001), Escherichia coli (P less than 0.01), other members of the family Enterobacteriaceae (P less than 0.02), and Pseudomonas aeruginosa (P less than 0.05) was detected earlier in BacT/Alert aerobic bottles. Growth of S. aureus (P less than 0.001), coagulase-negative staphylococci (P less than 0.05), enterococci (P less than 0.01), Streptococcus pneumoniae (P less than 0.02), viridans group streptococci (P less than 0.05), E. coli (P less than 0.001), Klebsiella pneumoniae (P less than 0.01), and other members of the family Enterobacteriaceae (P less than 0.001) was detected earlier in BacT/Alert anaerobic bottles. In a system-versus-system comparison, more gram-positive cocci were recovered from the BACTEC system alone (P < 0.05), and more members or the family Enterobacteriaceae were recovered from the BacT/Alert system alone (P < 0.001). As a system, the BacT/Alert system detected growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.01), streptococci (P < 0.001), E. coli (P < 0.001), other members of the familyEnterobacteriaceae (P < 0.001), and P. aeruginosa (P < 0.05) earlier than the BACTEC system did. Significantly fewer (40 versus 1,183) false-positive results occurred with the BacT/Alert system. We conclude that the BacT/Alert and BACTEC 660/730 nonradiometric systems are comparable for recovering clinically significant microorganisms form adult patients with bacteremia or fungemia, but that the BacT/Alert system detects microbial growth earlier than the BACTEC system does, with significantly fewer false-positive results.