Laboratory diagnosis of bloodstream infection
المؤلف:
APURBA S. SASTRY , SANDHYA BHAT
المصدر:
Essentials Of Medical Microbiology 2021
الجزء والصفحة:
3rd edition , p297-299
2025-10-23
38
Diagnosis of bloodstream infection depends on the isolation of the causative agent from blood by performing blood culture.
specimen collection for Blood culture
Extreme care should be taken while collection of blood for culture, as there is a high-risk of contamination with skin flora.
- Site: Blood for culture should always be collected in pairs; from two separate venipuncture and 2 separate skin decontamination process. If a central line is present, then one sample from the central line and one from the venipuncture should be collected
- Preparation of the site: To avoid contamination with skin flora, blood should be collected under strict aseptic conditions using sterile disposable syringe (Fig. 1)
- Skin decontamination: Skin should be disinfected by two-step procedure—first, treated with 70% isopropyl alcohol and then a second antiseptic solution such as povidone iodine or chlorhexidine should be applied
* The disinfectants should be applied in a circular motion (5 cm in diameter), starting from the centre to the periphery
* The area should be allowed to air dry before venipuncture.
- Timing of collection: Blood should be collected before starting antimicrobial therapy. If the antimicrobial agent is already started, then the best time of collection is just before the next dose of the antimicrobial agent
- Blood volume: Blood specimen is drawn using a sterile syringe and needle. Higher the volume of blood, greater is the chance of isolation (yield increases by 3.2% per mL of blood cultured). At least 8–10 mL of blood per bottle for an adult and 1–3 mL per pediatric bottle is recommended
- Number of blood cultures: At least 2–3 blood culture sets (each set consists of two bottles: 1 aerobic and 1 anaerobic) are required to have good isolation rate (around 65%, 80% and 95% with one, two and three sets respectively). Multiple blood cultures should be collected for endocarditis cases
- Dispensing: Collected blood is then directly dispensed into either blood culture bottle at the bedside; either a conventional or automated blood culture. Change of needle between collection and dispensing, an old practice is no longer recommended
- Transport of blood specimen: The collected blood is gently mixed with the broth and then transported immediately to the Microbiology laboratory. In case of delay, blood culture bottle should never be refrigerated. It can be kept at 35°C in an incubator (if available) or left at room temperature.
Fig1. Steps of collection of blood for culture.
conventional culture medium
The method used for the conventional blood culture is as follows
- Types of media: There are two types of conventional blood culture media (Figs 1A and B)
* Monophasic medium: It contains 50–100 mL of brain heart infusion (BHI) broth
* Castaneda’s biphasic medium: It consists of BHI agar slope and BHI broth (50 mL).
- Dilution: The blood is inoculated in the medium at a dilution of 1:5 so that the antibacterial components in the blood, if any, will get diluted
- SPS (sodium polyanethol sulfonate) is added to the medium as an anticoagulant. It also counteracts the bactericidal action of blood
- Incubation: Upon receipt, the bottles should be directly incubated in the upright position at 37° C for up to 7 days
- Repeat subcultures are made from the BHI broth onto blood agar and MacConkey agar
* From monophasic medium: Subcultures are made when the broth becomes turbid or periodically (blind subcultures) for one week. There is a risk of contamination due to opening of the cap of the bottle every time when subcultures are done
* In biphasic medium, the subcultures can be made just by tilting the bottles so that the broth runs over the agar slope. There is lower risk of contamination as it obviates the opening of the cap of the bottle. The colonies appear over the agar slant, which is further used for identification.
Fig2. a to c: Blood culture bottles: a. Monophasic medium (BHI broth); B. Biphasic medium (Castaneda’s), containing BHI broth and BHI agar slant; c. BacT/ALERT bottle. Source: A to C. Department of Microbiology, JIPMER, Puducherry (with permission).
Automated culture media
BACTEC and BacT/ALERT are the automated blood culture systems. The most advanced system is Bact/ALERT Virtuo. In these systems, the growth is continuously monitored and reading is recorded every 15–20 min (Fig. 1C).
When the growth is detected, the system gives a positive signal. Then the bottle is removed and processed similarly as done for conventional bottles. Automated systems are much superior to conventional media in terms of faster isolation and increased sensitivity. More so, they also help in diagnosing catheter related bloodstream infection (CRBSI) by determining the differential time to positivity.
Identification
The isolated organism is identified by colony morphology, Gram staining, followed by either conventional biochemical reactions or automated identification system such as MALDI-TOF or VITEK.
Antimicrobial susceptibility Test (AST)
Antimicrobial susceptibility test is carried out for guiding the institution of appropriate therapy. Minimum inhibitory concentration (MIC) based method (e.g. VITEK) is preferred over disk diffusion method when testing for blood isolates. It is ideal for endocarditis isolates, especially while reporting AST result of penicillin.
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