Blood culture
Blood culture | |
---|---|
MeSH | D000071997 |
LOINC | 600-7 |
MedlinePlus | 003744 |
A blood culture is a
To perform the test, blood is drawn into bottles containing a liquid formula that enhances microbial growth, called a
The containers are placed in an
Procedures for culturing the blood were published as early as the mid-19th century, but these techniques were labour-intensive and bore little resemblance to contemporary methods. Detection of microbial growth involved visual examination of the culture bottles until automated blood culture systems, which monitor gases produced by microbial metabolism, were introduced in the 1970s. In developed countries, manual blood culture methods have largely been made obsolete by automated systems.
Medical uses
Blood is normally
When sepsis is suspected, it is necessary to draw blood cultures to identify the causative agent and provide targeted
The pathogens most frequently identified in blood cultures include
Procedure
Collection
Blood cultures are typically drawn through
A typical blood culture collection involves drawing blood into two bottles, which together form one "culture" or "set". One bottle is designed to enhance the growth of
Blood culture bottles contain a
Many commercially manufactured bottles contain a
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 the ratio of growth medium to blood is comparatively lower. A 1:10 to 1:5 ratio of blood to culture medium is suggested to optimize microbial growth.[28][39] For routine blood cultures in adults, the Clinical and Laboratory Standards Institute (CLSI) recommends the collection of two sets of bottles from two different draws, with 20–30 mL of blood drawn in each set.[11] In children, the amount of blood to be drawn is often based on the child's age or weight.[36][40] If endocarditis is suspected, a total of six bottles may be collected.[41]
Culturing
After the blood is collected, the bottles are incubated at body temperature to encourage the growth of microorganisms. Bottles are usually incubated for up to five days in automated systems,[43] although most common bloodstream pathogens are detected within 48 hours.[44] The incubation time may be extended further if manual blood culture methods are used or if slower-growing organisms, such as certain bacteria that cause endocarditis, are suspected.[43][45] In manual systems, the bottles are visually examined for indicators of microbial growth, which might include cloudiness, the production of gas, the presence of visible microbial colonies, or a change in colour from the digestion of blood, which is called hemolysis. Some manual blood culture systems indicate growth using a compartment that fills with fluid when gases are produced, or a miniature agar plate which is periodically inoculated by tipping the bottle.[46] To ensure that positive blood cultures are not missed, a sample from the bottle is often inoculated onto an agar plate (subcultured) at the end of the incubation period regardless of whether or not indicators of growth are observed.[47]
In developed countries, manual culture methods have largely been replaced by automated systems that provide continuous computerized monitoring of the culture bottles.[48] 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 levels of gases inside the bottle—most commonly carbon dioxide—which serve as an indicator of microbial metabolism.[46] An alarm or a visual indicator alerts the microbiologist to the presence of a positive blood culture bottle.[49] If the bottle remains negative at the end of the incubation period, it is generally discarded without being subcultured.[47]
A technique called the lysis-centrifugation method can be used for improved isolation of slow-growing or
Identification
If growth is detected, a microbiologist will perform a
In traditional methods, the blood is then subcultured onto
It typically takes 24 to 48 hours for sufficient growth to occur on the subculture plates for definitive identification to be possible.
Microorganisms may also be identified using automated systems, such as instruments that perform panels of biochemical tests,[65] or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in which microbial proteins are ionized and characterized on the basis of their mass-to-charge ratios; each microbial species exhibits a characteristic pattern of proteins when analyzed through mass spectrometry.[66]
Because bloodstream infections can be life-threatening, timely diagnosis and treatment is critical,
Even faster diagnosis could be achieved through bypassing culture entirely and detecting pathogens directly from blood samples. A few direct testing systems are commercially available as of 2018, but the technology is still in its infancy. Most panels detect only a limited number of pathogens, and the sensitivity can be poor compared to conventional blood culture methods. Culturing remains necessary in order to carry out full antimicrobial sensitivity testing.[73]
Antibiotic susceptibility testing
Antimicrobial treatment of bloodstream infections is initially
Rapid administration of effective antimicrobial drugs is crucial in the treatment of sepsis,[8] so several methods have been developed to provide faster antibiotic sensitivity results. Conventional AST methods can be carried out on young growth from the subculture plate,[76] pellets of microorganisms obtained from concentration and purification of the positive blood culture, or directly from the culture bottle.[77][78] Because direct testing methods do not isolate the organisms, they do not provide accurate results if more than one microorganism is present, although this is an infrequent occurrence in blood cultures.[76] Another source of error is the difficulty in standardizing the amount of bacteria in the sample (the inoculum), which has a profound effect on the test results.[79]
Limitations
Blood cultures are subject to both false positive and false negative errors. In automated culture systems, identification of positive bottles is based on the detection of gases produced by cellular metabolism, so samples with high numbers of white blood cells may be reported as positive when no bacteria are present. Inspection of the growth curve produced by the instrument can help to distinguish between true and false positive cultures, but Gram staining and subculturing are still necessary for any sample that is flagged as positive.[61]
Blood cultures can become contaminated with microorganisms from the skin or the environment, which multiply inside the culture bottle, giving the false impression that those organisms are present in the blood.[11] Contamination of blood cultures can lead to unnecessary antibiotic treatment and longer hospital stays.[29] The frequency of contamination can be reduced by following established protocols for blood culture collection, but it cannot be eliminated;[83] for instance, bacteria can survive in deeper layers of the skin even after meticulous disinfection of the blood draw site.[29] The CLSI defines an acceptable contamination rate as no greater than 3% of all blood cultures.[11] The frequency of contamination varies widely between institutions and between different departments in the same hospital;[83] studies have found rates ranging from 0.8 to 12.5 percent.[29]
When faced with a positive blood culture result, clinicians must decide whether the finding represents contamination or genuine infection. Some organisms, such as S. aureus or Streptococcus pneumoniae, are usually considered to be pathogenic when detected in a blood culture, while others are more likely to represent contamination with skin flora; but even common skin organisms such as coagulase-negative staphylococci can cause bloodstream infections under certain conditions. When such organisms are present, interpretation of the culture result involves taking into account the person's clinical condition and whether or not multiple cultures are positive for the same organism.[29]
False negatives may be caused by drawing blood cultures after the person has received antibiotics or collecting an insufficient amount of blood. 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.[11] 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.[28]
Not all pathogens are easily detected by conventional blood culture methods. Particularly
History
Early blood culture methods were labour-intensive.[86] One of the first known procedures, published in 1869, recommended that leeches be used to collect blood from the patient.[87] A microbiology textbook from 1911 noted that decontamination of the draw site and equipment could take over an hour, and that due to a lack of effective methods for preserving blood, the cultures would sometimes have to be prepared at the patient's bedside. In addition to subculturing the broth, some protocols specified that the blood be mixed with melted agar and the mixture poured into a petri dish.[86] In 1915, a blood culture collection system consisting of glass vacuum tubes containing glucose broth and an anticoagulant was described. Robert James Valentine Pulvertaft published a seminal work on blood cultures in 1930,[88] specifying—among other insights—an optimal blood-to-broth ratio of 1:5, which is still accepted today.[87] The use of SPS as an anticoagulant and preservative was introduced in the 1930s and 40s and resolved some of the logistical issues with earlier methods.[86] From the 1940s through the 1980s, a great deal of research was carried out on broth formulations and additives, with the goal of creating a growth medium that could accommodate all common bloodstream pathogens.[87]
In 1947, M.R. Castañeda invented a "biphasic" culture bottle for the identification of Brucella species, which contained both broth and an agar slant, allowing the agar to be easily subcultured from the broth;[42] this was a precursor of some contemporary systems for manual blood cultures.[43] E.G. Scott in 1951 published a protocol described as "the advent of the modern blood culture set".[86] Scott's method involved inoculating blood into two rubber-sealed glass bottles; one for aerobes and one for anaerobes. The aerobic bottle contained trypticase soy broth and an agar slant, and the anaerobic bottle contained thioglycollate broth. The lysis-centrifugation method was introduced in 1917 by Mildred Clough, but it was rarely used in clinical practice until commercial systems were developed in the mid-1970s.[86][89]
Automated blood culture systems first became available in the 1970s.
A major issue with the early BACTEC systems was that they produced radioactive waste, which required special disposal procedures,[50] so in 1984 a new generation of BACTEC instruments was released that used spectrophotometry to detect CO2.[91] The BacT/ALERT system, which indirectly detects production of CO2 by measuring the decrease in the medium's pH, was approved for use in the US in 1991. Unlike the BACTEC systems available at the time, the BacT/ALERT did not require a needle to be introduced into the bottle for sampling; this reduced the frequency of contamination[91] and made it the first system to provide truly continuous monitoring of blood cultures.[92] This non-invasive measurement method was adopted in 1992 by the BACTEC 9000 series, which used fluorescent indicators to detect pH changes.[93] The Difco ESP, a direct predecessor of the contemporary VersaTREK system[86] which detects gas production by measuring pressure changes, was also first approved in 1992.[91] By 1996, an international study found that 55% of 466 laboratories surveyed were using the BACTEC or BacT/ALERT systems, with other automated systems accounting for 10% of the total.[94]
Notes
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