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In traditional methods, the blood is then subcultured onto [[agar plate]]s to [[Isolation (microbiology)|isolate]] the organism for culture and identification. The Gram stain results inform microbiologists about what [[growth media|types of agar plates]] should be used and what tests might be appropriate to provide a definitive identification of the organism.<ref>Ford, M (2019). pp. 91–2.</ref> Antibiotic susceptibilities are assessed on the isolate to inform clinicians with respect to appropriate antibiotics for treatment.<ref>Mahon, CR ''et al''. (2018). pp. 874–6.</ref> As of 2011, the pathogens most commonly isolated from blood cultures were [[coagulase-negative staphylococci]], ''[[Staphylococcus aureus]]'', ''[[Enterococcus]]'' species, and ''[[Candida albicans]]''.<ref>Mahon, CR ''et al''. (2018). p. 866.</ref>
In traditional methods, the blood is then subcultured onto [[agar plate]]s to [[Isolation (microbiology)|isolate]] the organism for culture and identification. The Gram stain results inform microbiologists about what [[growth media|types of agar plates]] should be used and what tests might be appropriate to provide a definitive identification of the organism.<ref>Ford, M (2019). pp. 91–2.</ref> Antibiotic susceptibilities are assessed on the isolate to inform clinicians with respect to appropriate antibiotics for treatment.<ref>Mahon, CR ''et al''. (2018). pp. 874–6.</ref> As of 2011, the pathogens most commonly isolated from blood cultures were [[coagulase-negative staphylococci]], ''[[Staphylococcus aureus]]'', ''[[Enterococcus]]'' species, and ''[[Candida albicans]]''.<ref>Mahon, CR ''et al''. (2018). p. 866.</ref>


It typically takes 24 to 48 hours for sufficient growth to occur on the subculture plates to definitively identify the organism and carry out antibiotic susceptibility testing.<ref name="mahon-874"/> Because bloodstream infections can be life-threatening, timely diagnosis and treatment is critical,<ref>Carroll, KC ''et al''. (2016). p. 756.</ref> and to this end a number of rapid identification methods have been developed for blood cultures.<ref name="mahon-874">Mahon, CR ''et al''. (2018). p. 874.</ref> One approach uses [[Matrix-assisted laser desorption/ionization|matrix-assisted laser desorption/ionization time-of-flight mass spectrometry]] (MALDI-TOF MS) to identify organisms directly from the blood culture bottle after separation and concentration procedures,<ref name="OpotaCroxatto2015">{{cite journal|last1=Opota|first1=O|last2=Croxatto|first2=A|last3=Prod'hom|first3=G|last4=Greub|first4=G|title=Blood culture-based diagnosis of bacteraemia: state of the art|journal=Clinical Microbiology and Infection|volume=21|issue=4|year=2015|pages=313–322|issn=1198743X|doi=10.1016/j.cmi.2015.01.003}}</ref> or from preliminary growth on the agar plate within a few hours of subculturing.<ref>Pitt, SJ (2018) p. 35.</ref> [[Polymerase chain reaction]] (PCR) can be used to identify microorganisms by detection of [[DNA sequence]]s specific to certain species in samples from the blood culture bottle; several PCR systems designed for the identification of common blood culture pathogens are commercially available.<ref>Gonzalez, MD & Jerris, RC. Chapter 7 in Dunne, WM & Burnham, CAD ''eds.'' (2018). sec. "Introduction"; "Summary".</ref>
It typically takes 24 to 48 hours for sufficient growth to occur on the subculture plates to definitively identify the organism and carry out antibiotic susceptibility testing.<ref name="mahon-874"/> Because bloodstream infections can be life-threatening, timely diagnosis and treatment is critical,<ref>Carroll, KC ''et al''. (2016). p. 756.</ref> and to this end a number of rapid identification methods have been developed for blood cultures.<ref name="mahon-874">Mahon, CR ''et al''. (2018). p. 874.</ref> One approach uses [[Matrix-assisted laser desorption/ionization|matrix-assisted laser desorption/ionization time-of-flight mass spectrometry]] (MALDI-TOF MS) to identify organisms directly from blood culture bottles after separation and concentration procedures,<ref name="OpotaCroxatto2015">{{cite journal|last1=Opota|first1=O|last2=Croxatto|first2=A|last3=Prod'hom|first3=G|last4=Greub|first4=G|title=Blood culture-based diagnosis of bacteraemia: state of the art|journal=Clinical Microbiology and Infection|volume=21|issue=4|year=2015|pages=313–322|issn=1198743X|doi=10.1016/j.cmi.2015.01.003}}</ref> or from preliminary growth on the agar plate within a few hours of subculturing.<ref>Pitt, SJ (2018) p. 35.</ref> [[Polymerase chain reaction]] (PCR) can be used to identify microorganisms by detection of [[DNA sequence]]s specific to certain species in samples from positive blood cultures; several PCR systems designed for the identification of common blood culture pathogens are commercially available.<ref>Gonzalez, MD & Jerris, RC. Chapter 7 in Dunne, WM & Burnham, CAD ''eds.'' (2018). sec. "Introduction"; "Summary".</ref> Some biochemical and immunologic tests can be performed directly on positive blood cultures, such as the [[Coagulase|tube coagulase]] test for identification of ''S. aureus''<ref>Mahon, CR ''et al''. (2018). p. 874.</ref> or [[latex agglutination]] tests for ''S. pneumoniae'';<ref>Ford, M (2019). p. 93.</ref> unlike PCR and MALDI-TOF, these methods may be practical for laboratories in low and middle income countries.<ref name="OmbeletBarbé2019">{{cite journal|last1=Ombelet|first1=S|last2=Barbé|first2=B|last3=Affolabi|first3=D|last4=Ronat|first4=JB|last5=Lompo|first5=P|last6=Lunguya|first6=O|last7=Jacobs|first7=J|last8=Hardy|first8=L|displayauthors=6|title=Best Practices of Blood Cultures in Low- and Middle-Income Countries|journal=Frontiers in Medicine|volume=6|year=2019|issn=2296-858X|doi=10.3389/fmed.2019.00131}}</ref>


==Limitations==
==Limitations==

Revision as of 01:55, 27 September 2020

Blood culture
See caption.
A microbiologist unloads blood culture bottles from a Bact-Alert machine, an automated system used to incubate blood cultures and detect microbial growth
ICD-990.52
MedlinePlus003744

A blood culture is a medical laboratory test used to detect bacteria or fungi in a person's blood.[1] Blood is normally sterile, and the presence of microbes in the blood indicates a bloodstream infection such as bacteremia or fungemia, which can result in sepsis.[2]

The test involves drawing the blood into bottles containing chemicals that encourage microbial growth, which are placed in an incubator for several days to allow the organisms to multiply.[3] If microbial growth is detected, a Gram stain is made from the blood culture bottle to confirm that organisms are present and to provide a preliminary identification. The blood is then inoculated onto an agar plate to isolate the organisms for further testing.[4] A positive Gram stain from a blood culture is considered a critical result and must immediately be reported to the clinician.[5]

To ensure accurate results, blood cultures are drawn using sterile technique. If the sample is contaminated with skin flora, the person will appear to have those organisms in their blood.[6] When a blood culture is performed, it is usually drawn in at least two different sets (one set of bottles from two different blood draw sites) so that contamination is easier to detect. If an organism only appears in one of the two sets, it is more likely to be a contaminant.[1]

Medical uses

When a patient shows signs or symptoms of a systemic infection, results from a blood culture can verify that an infection is present, and they can identify the type (or types) of microorganism that is responsible for the infection. For example, blood tests can identify the causative organisms in severe pneumonia, puerperal fever, pelvic inflammatory disease, neonatal epiglottitis, sepsis, and fever of unknown origin (FUO). However, negative growths do not exclude infection.

Procedure

Three clear bottles with differently coloured caps and labels.
Aerobic, anaerobic, and pediatric blood culture bottles

Collection

Blood culture samples are typically drawn through venipuncture. Collecting the sample from an intravenous line is not recommended, as this is associated with higher contamination rates, although blood cultures may be collected from both venipuncture and an intravenous line to diagnose catheter-associated infections.[7][8] Prior to drawing the blood, the tops of the blood culture bottles are disinfected using an alcohol swab,[7] and the venipuncture site is cleaned with alcohol followed by 2% chlorhexidine or tincture of iodine, then left to dry.[note 1] This is done to avoid contamination with organisms from the skin or the environment.[9] If other blood tests need to be drawn at the same time as a blood culture, the blood culture bottles are drawn first to minimize the risk of contamination.[10]

A typical blood culture collection involves drawing blood into a set of two bottles: one designed to enhance the growth of aerobic organisms, and one designed to grow anaerobic organisms. In children, infection with anaerobic bacteria is uncommon, so a single aerobic bottle may be collected to minimize blood loss,[11] although this may impede the growth of facultative anaerobes like Streptococcus pneumoniae.[12] It is recommended that at least two sets are collected from two separate venipunctures. This helps to distinguish true infection from contamination, as contaminants are less likely to appear in more than one set. Additionally, the collection of larger volumes of blood increases the likelihood that pathogens will be detected if present.[13]

It is important that the bottles are neither underfilled nor overfilled: underfilling can lead to false negative results as fewer organisms are present in the sample, while overfilling can inhibit microbial growth because there is a comparatively lower ratio of growth medium to blood. A 1:10 to 1:5 ratio of blood to culture medium is suggested to optimize microbial growth.[9][14] For routine blood cultures in adults, the Clinical and Laboratory Standards Institute recommends the collection of two sets of bottles from two different blood draws, with 20–30 mL of blood drawn in each set, half of which is distributed to each bottle.[7][14] In children, the amount of blood to be drawn is often based on the child's age or weight;[15][16] age-based protocols are discouraged because age correlates less well with blood volume than weight does.[9] If endocarditis is suspected, a total of six bottles may be collected in either two or three sets.[17]

Blood culture bottles contain a growth medium, which enhances the growth of microorganisms, and an anticoagulant that prevents the blood from clotting.[18] Sodium polyanethol sulfonate (SPS) is the most commonly used anticoagulant,[18] because unlike the anticoagulants used for other blood tests, it does not interfere with the growth of most organisms.[9] The exact composition of the growth medium varies, but aerobic bottles use a medium that is enriched with nutrients, such as brain-heart infusion or trypticase soy broth,[19] and anaerobic bottles typically add a reducing agent such as thioglycollate to the growth medium. The empty space in an anaerobic bottle is filled with a gas mixture that does not contain oxygen.[18][20] Many commercially available bottles contain a resin that absorbs antibiotics to reduce their action on the microorganisms in the sample.[7] Bottles designed for pediatric use contain a lower amount of growth medium to accommodate lower blood volumes and have additives that enhance the growth of pathogens more commonly found in children;[16] other specialized culture bottles may be used for fungi and mycobacteria.[20] In low and middle income countries, pre-formulated culture bottles can be prohibitively expensive, and it may be necessary to prepare the bottles manually; even this can present problems because access to the proper supplies and reagents is limited.[21]

Culturing

A few large, spherical, purple bacteria in small clusters on a faded pink background
Off-white colonies of bacteria growing on a blood agar plate
Left: Direct Gram stain from a positive blood culture bottle, showing spherical bacteria (cocci) that stain purple (Gram-positive). Right: Growth of Staphylococcus aureus, a Gram-positive coccus and a pathogen commonly isolated from blood cultures, on an agar plate.

After the blood culture bottles have been collected, they are incubated at body temperature to encourage the growth of microorganisms. Bottles are usually incubated for five days in automated systems, but the incubation time may be longer if manual blood culture methods are used or if slower-growing organisms, such as those that cause endocarditis, are suspected.[22][23] Throughout the incubation period, the bottles are examined for indicators of microbial growth. This can be done by manually inspecting the bottles for the presence of visible microbial colonies; cloudiness, which might indicate microbial growth; gas production; or digestion of blood (hemolysis). Some manual blood culture systems indicate microbial growth using a compartment that fills with fluid when gases are produced, or a miniature agar plate which is inoculated by tipping the bottle.[24]

In developed countries, these manual methods have largely become obsolete with the introduction of automated systems that continuously monitor the culture bottles.[25] These systems, such as the BACTEC, BacT/ALERT and VersaTrek, consist of an incubator in which the culture bottles are continuously mixed. Growth is detected by sensors that measure the production or consumption of certain gases inside the bottle, which serves as a indicator of microbial metabolism; production of carbon dioxide is a common detection principle.[26] An alarm or a visual indicator alerts the microbiologist to the presence of a positive blood culture bottle.[27]

Identification

If growth is detected, a microbiologist will perform a Gram stain on a sample of blood from the bottle for a rapid preliminary identification of the organism.[28] The Gram stain classifies bacteria as Gram-positive or Gram-negative and provides information about their shape—whether they are rod-shaped (referred to as bacilli), spherical (referred to as cocci), or spiral-shaped (spirochetes)—as well as their arrangement.[29] This information helps clinicians make decisions about the possible identity of the organism, which can allow them to select a more appropriate antibiotic treatment before the full culture and sensitivity results are complete.[28] Gram-positive cocci in clusters, for example, are typical of Staphylococcus species.[30]

A gloved hand holds a metal plate onto which microbial samples have been placed, ready to load it into the sampling area of the MALDI-TOF instrument
Loading a target plate containing microbial samples into a Bruker Biotyper, an instrument used for MALDI-TOF analysis in microbiology

In traditional methods, the blood is then subcultured onto agar plates to isolate the organism for culture and identification. The Gram stain results inform microbiologists about what types of agar plates should be used and what tests might be appropriate to provide a definitive identification of the organism.[31] Antibiotic susceptibilities are assessed on the isolate to inform clinicians with respect to appropriate antibiotics for treatment.[32] As of 2011, the pathogens most commonly isolated from blood cultures were coagulase-negative staphylococci, Staphylococcus aureus, Enterococcus species, and Candida albicans.[33]

It typically takes 24 to 48 hours for sufficient growth to occur on the subculture plates to definitively identify the organism and carry out antibiotic susceptibility testing.[34] Because bloodstream infections can be life-threatening, timely diagnosis and treatment is critical,[35] and to this end a number of rapid identification methods have been developed for blood cultures.[34] One approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify organisms directly from blood culture bottles after separation and concentration procedures,[36] or from preliminary growth on the agar plate within a few hours of subculturing.[37] Polymerase chain reaction (PCR) can be used to identify microorganisms by detection of DNA sequences specific to certain species in samples from positive blood cultures; several PCR systems designed for the identification of common blood culture pathogens are commercially available.[38] Some biochemical and immunologic tests can be performed directly on positive blood cultures, such as the tube coagulase test for identification of S. aureus[39] or latex agglutination tests for S. pneumoniae;[40] unlike PCR and MALDI-TOF, these methods may be practical for laboratories in low and middle income countries.[41]

Limitations

Blood cultures are subject to both false positive and false negative errors. False positive results occur 1–10% of the time.[42] Samples with high numbers of white blood cells may generate false positives in automated culture systems, as positive flagging is based on the detection of gases produced by cellular metabolism. Inspection of the growth curve produced by the instrument can help to distinguish between a true positive culture and a false positive, but Gram staining and culture are still necessary for any sample that is flagged as positive.[43] False negatives may be caused by collecting an insufficient amount of blood or drawing blood cultures after the person has received antibiotics. Additionally, certain organisms are difficult to culture and may not be detected by conventional methods.[7] The volume of blood drawn is considered the most important variable in ensuring that pathogens are detected: the more blood that is collected, the more pathogens are recovered.[7] However, if the amount of blood collected far exceeds the recommended volume, bacterial growth may be inhibited by natural inhibitors present in the blood and an inadequate amount of growth medium in the bottle. Over-filling of blood culture bottles may also contribute to iatrogenic anemia.[9]

History

Blood cultures were pioneered in the early 20th century.

Notes

  1. ^ Chlorhexidine is not used in infants under two months old, and iodine-based antiseptics are contraindicated in low-birth-weight infants.[9]

References

  1. ^ a b American Association for Clinical Chemistry (15 November 2019). "Blood Culture". Lab Tests Online. Retrieved 12 May 2020.
  2. ^ Mahon, CR et al. (2018). p. 863.
  3. ^ Mahon, CR et al. (2018). p. 871.
  4. ^ "Blood culture". MedlinePlus Medical Encyclopedia. 16 September 2019. Retrieved 12 May 2020.
  5. ^ Mahon, CR et al. (2018). pp. 868–71
  6. ^ Mahon, CR et al. (2018). pp. 868–9.
  7. ^ a b c d e f Garcia, RA; Spitzer, ED; Beaudry, J; Beck, C; Diblasi, R; Gilleeny-Blabac, M; Haugaard, C; Heuschneider, S; Kranz, BP; McLean, K; Morales, KL; Owens, S; Paciella, ME; Torregrosa, E (2015). "Multidisciplinary team review of best practices for collection and handling of blood cultures to determine effective interventions for increasing the yield of true-positive bacteremias, reducing contamination, and eliminating false-positive central line–associated bloodstream infections". American Journal of Infection Control. 43 (11): 1222–1237. doi:10.1016/j.ajic.2015.06.030. ISSN 0196-6553. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)
  8. ^ Septimus, E (1 August 2019). "Collecting Cultures: a Clinician Guide". Centers for Disease Control and Prevention. Archived from the original on 24 September 2020. {{cite web}}: |archive-date= / |archive-url= timestamp mismatch; 25 September 2020 suggested (help)
  9. ^ a b c d e f Mahon, CR et al. (2018). p. 869.
  10. ^ Pagana, KD et al. (2014). p. xiii.
  11. ^ Pitt, SJ (2018) p. 34.
  12. ^ Revell, P & Doern, C. Chapter 8 in Dunne, WM & Burnham, CAD eds. (2018). sec. "Anaerobic Blood Culture Media".
  13. ^ Mahon, CR et al. (2018). p. 870.
  14. ^ a b Tibbetts, RJ & Robinson-Dunn, B. Chapter 10 in Dunne, WM & Burnham, CAD eds. (2018). sec. "Introduction".
  15. ^ Revell, P & Doern, C. Chapter 8 in Dunne, WM & Burnham, CAD eds. (2018). sec. "Specimen Collection".
  16. ^ a b Dien Bard, J; McElvania TeKippe, E; Kraft, CS (2016). "Diagnosis of Bloodstream Infections in Children". Journal of Clinical Microbiology. 54 (6): 1418–1424. doi:10.1128/JCM.02919-15. ISSN 0095-1137.
  17. ^ Bennett, JE et al. (2019). p. 202.
  18. ^ a b c Atkinson-Dunn, R. & Dunne, WM. Chapter 2 in Dunne, WM & Burnham, CAD eds. (2018). sec. "Introduction".
  19. ^ Procop, GW et al. (2017). p. 194.
  20. ^ a b Ford, M (2019). p. 85.
  21. ^ Baron, EJ (2019). "Clinical Microbiology in Underresourced Settings". Clinics in Laboratory Medicine. 39 (3): 359–369. doi:10.1016/j.cll.2019.05.001. ISSN 0272-2712.
  22. ^ Mahon, CR et al. (2018). p. 871.
  23. ^ Procop, GW & Koneman, EW (2017). p. 199.
  24. ^ Mahon, CR et al. (2018). pp. 871–2.
  25. ^ Carroll, KC et al. (2015). p. 756.
  26. ^ Mahon, CR et al. (2018). pp. 871–2.
  27. ^ Procop, GW & Koneman, EW (2017). pp. 197–8.
  28. ^ a b Ford, M (2019). p. 89.
  29. ^ Turgeon, ML (2016). pp. 492–3.
  30. ^ Carroll, KC et al. (2016). p. 203.
  31. ^ Ford, M (2019). pp. 91–2.
  32. ^ Mahon, CR et al. (2018). pp. 874–6.
  33. ^ Mahon, CR et al. (2018). p. 866.
  34. ^ a b Mahon, CR et al. (2018). p. 874.
  35. ^ Carroll, KC et al. (2016). p. 756.
  36. ^ Opota, O; Croxatto, A; Prod'hom, G; Greub, G (2015). "Blood culture-based diagnosis of bacteraemia: state of the art". Clinical Microbiology and Infection. 21 (4): 313–322. doi:10.1016/j.cmi.2015.01.003. ISSN 1198-743X.
  37. ^ Pitt, SJ (2018) p. 35.
  38. ^ Gonzalez, MD & Jerris, RC. Chapter 7 in Dunne, WM & Burnham, CAD eds. (2018). sec. "Introduction"; "Summary".
  39. ^ Mahon, CR et al. (2018). p. 874.
  40. ^ Ford, M (2019). p. 93.
  41. ^ Ombelet, S; Barbé, B; Affolabi, D; Ronat, JB; Lompo, P; Lunguya, O; Jacobs, J; Hardy, L (2019). "Best Practices of Blood Cultures in Low- and Middle-Income Countries". Frontiers in Medicine. 6. doi:10.3389/fmed.2019.00131. ISSN 2296-858X. {{cite journal}}: Unknown parameter |displayauthors= ignored (|display-authors= suggested) (help)CS1 maint: unflagged free DOI (link)
  42. ^ Farron, ML & Ledeboer, NA. Chapter 11 in Dunne, WM & Burnham, CAD eds. (2018). sec. "Staining Techniques and Subculture".
  43. ^ Ford, M (2019). p. 90.

Bibliography

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