blood culure

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blood culture with recent advances

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BLOOD CULTURE:

BLOOD CULTURE Dr. Ch. Preethi .

Learning objectives:

Learning objectives 1. Introduction 2. The blood culture 3. Principles of blood culture collection 4. Selection of the correct blood culture bottle 5. From collection to incubation 6. The choice of blood culture system 7. Interpretation of results 8. Blood culture contamination 9. New technologies used in conjunction with blood culture systems in the diagnosis of sepsis 10. Conclusion

1. Introduction:

1. Introduction Bacteraemia and sepsis is associated with a high mortality and an increased incidence of hospital stay and associated costs. Blood cultures are taken to establish microbial invasion of the vascular system. They are still considered to be the ‘gold standard’ for the detection of microbial pathogens related to bacteraemia and sepsis despite newer molecular techniques.

2. The blood culture:

2. The blood culture The term blood culture refers to a single venipuncture , either from a peripheral site or central or arterial line, with blood inoculated into one or more blood culture bottles. One bottle is considered a blood culture where two or more are considered a set. Multiple sets are from multiple venipunctures and are associated with different sites.

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Common mechanisms of sepsis include: Dissemination from a primary site after inadequate control by host defense mechanisms, intravascular device mediated or infection of the vascular system e.g. infective endocarditis . Transient bacteraemia occur due to translocation of bacteria e.g. during chewing with rapid clearance by immune mechanisms Intermittent bacteraemia where bacteria are periodically released into the blood from e.g. an abscess .

Indications for taking blood cultures:

Indications for taking blood cultures Acute bacterial sepsis Meningitis, Pneumonia, Osteomyelitis , Pyelonephritis Vascular Infective endocarditis Closed space infections Intra-abdominal abscesses Catheter related bacteraemia Follow up cultures Fungaemia , infective endocarditis

3. Principles of blood culture collection:

3. Principles of blood culture collection Collection of blood cultures is a critical component and can either positively affect the patient outcome by providing an accurate diagnosis or adversely affect the outcome by prolonging antimicrobial therapy and the length of hospital stay with the isolation of a contaminant. Timing, site, skin antisepsis, volume and number of cultures, training, clinical information and labeling, blood culture collection kits, etc should be considered carefully for accurate diagnosis.

Timing:

Timing 1.Blood cultures must be taken on suspicion of bacteraemia and not only around fever spikes. 2. Two sets must be taken at the onset of a 24 hour period within minutes apart. 3. Two more sets can be obtained during the 24 hour period if the clinical condition deteriorates. 4. Blood cultures must be obtained before administration of antimicrobial agents.

Site:

Site 1. Peripheral venipuncture is preferred. 2. If an invasive line is present, do both and correlate. 3. The culture from the CVC may be negative if antimicrobials are administered through the line, despite true bacteraemia .

Skin antisepsis:

Skin antisepsis Blood culture contamination can lead to significant increase in healthcare related costs. Skin antisepsis therefore plays a critical role in reducing these contaminants. The recommended agent for skin antisepsis when performing venipuncture is 2% chlorhexidine – gluconate in 70% isopropyl – alcohol. ChloraPrep ( Enturia Limited) is a commercial antisepsis system that uses a plastic applicator to release prepacked anti- septic into a sponge thereby reducing cross contamination from the care givers’ hands.

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Aseptic Technique

Volume and number of cultures:

Volume and number of cultures 1. The higher the volume, the higher the yield. 2. 4 blood culture bottles with upto 10ml of blood in each bottle in a 24 hour period may be necessary to detect > 99% of microorganisms. 3. As least 1 ml must be cultured in neonates. 4. Adequate blood – broth ratio of 1:10 must be achieved to dilute the effects of inhibitory substances and antimicrobials present in the blood.

Clinical information and labeling:

Clinical information and labeling 1. Label bottle with the date, time and site where the sample was obtained from. 2.Fill the requisition appropriately.

Rejection of unacceptable specimen :

Rejection of unacceptable specimen information on the label does not match the information on the requisition. transported in improper temperature , medium. Insufficient quantity Leaking If received in a fixative

Selection of the correct blood culture bottle:

Selection of the correct blood culture bottle Blood culture sets generally consist of aerobic and anaerobic bottle. Various different bottles are available depending on the monitoring system used. Specifically designed to optimize recovery of both aerobic and anaerobic organisms.

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Type of blood culture medium BHI broth Trypticase soy broth Supplemented peptone broth Thiol broth Columbia broth Columbia broth + 10% sucrose Columbia broth + 20% sucrose Dextrose phosphate broth Brucella broth Brucella broth + 10% sucrose

From collection to incubation:

From collection to incubation Blood cultures that have been collected must reach the laboratory as soon as possible and generally receive high attention for immediate incubation to allow optimal growth of organisms and rapid recovery without compromising . The duration of incubation is calculated from the time of insertion until the time of removal. Bottles will be removed, thus considered negative, when no positive signal is obtained after a certain amount of incubation time has elapsed. This time before removal will depend on the blood culture system used. For manual broth – based systems, 7 days are the recommended incubation time . It is 4 to 5 days with the continuous monitoring blood culture systems .

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Extended incubation (up to 14 to 21 days) is recommended to detect more fastidious organisms specifically the HACEK organisms ( Haemophilus , Actinobacillus , Cardiobacterium , Eikenella , and Kingella species) that are involved in endocarditis . T he method of detection and not the time of incubation is critical to detect these organisms.Bactec9240 system can detect the HACEK organisms within 5 days. Alternative methods e.g. a lysis centrifugation system for dimorphic fungi and molecular methods for Bartonella henselae may be of more value than prolonged incubation .

The choice of blood culture system:

The choice of blood culture system Factors that affect the choice of blood culture system: Sensitivity for organism recovery Time to positivity Workload capacity User friendly Costs

Time to positivity:

Time to positivity Time to positivity (TTP) is a parameter provided by the automated blood culture system and is calculated from the time of incubation until a positive signal is detected. TTP can be influenced by various factors e.g. the bacterial load, the growth rate of the micro-organism, the presence of antibacterial substances in the blood as well as source of infection and clinical features.

Various blood culture systems :

Various blood culture systems Manual blood culture systems Lysis centrifugation systems Continuous monitoring blood culture systems

Manual blood culture systems:

Manual blood culture systems The conventional manual method entails inoculating a commercially provided blood culture bottle, incubating the bottle at the required temperature and atmosphere with daily inspection of the bottle for macroscopic evidence of growth e.g. turbidity, haemolysis or colonies. Once growth is observed, a sample can be obtained for Gram staining and subculture for further identification. Bottles are incubated for 7 days and terminal subculture is mandatory.

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Variations to the conventional manual method is combining agar in the form of paddles to the broth (biphasic medium). These systems allow for more frequent subculturing by inverting the bottles to bring the broth into contact with the agar. These bottles can be inspected for growth and Gram staining with presumptive identification to be performed from the agar.

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Check for microbial growth, indicated by colonies growing on the agar slope, usually beginning at the agar-broth interface. Colonial appearances Colonies of staphylococci , S. typhi , Brucellae , and most coliforms can usually be seen easily, whereas colonies of S. pneumoniae , Neisseria species, S. pyogenes , and Y. pestis are less easily seen. Pseudomonas and Proteus species produce a film of growth on the agar. When growth is present: – Subculture on blood agar, chocolate agar, and MacConkey agar. – Incubate the blood agar and MacConkey agar plates aerobically and the chocolate agar plate in a carbon dioxide atmosphere (candle jar). – Examine a Gram stained smear of the colonies. Depending on the bacteria seen, test the colonies further (e.g. for coagulase , catalase , oxidase , urease , and motility).

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Large Gram positiverods -(C. perfringens ) Subculture on lactose egg yolk milk agar and incubate anaerobically motile, urease and oxidase negative Gram negative rods are isolated Subculture the colonies on Kligler iron agar catalase positive,Gram negative coccobacilli are isolated Suspect Brucella species and send for identification. Mark it as ‘ High Risk ’.

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The Septi-Chek (BD Diagnostics) blood culture system is a biphasic-agar slide system that uses a standard blood culture bottle containing brain heart infusion or trypticase soy broth connected to a second plastic chamber with a trisurface panel consisting of chocolate, Mackonkey and malt agar. The slide chamber is screwed onto the bottle after inoculation and incubated at 35°C for 4 – 6 hours. The bottle is then inverted for the first subculture and can be inverted at various intervals thereafter to optimize isolation.

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The Oxoid Signal System ( Oxoid Unipath , Basingstoke,England ) is unique in the sense that it is a one bottle system. After inoculation of a standard blood culture bottle a second chamber is attached with a long needle that extends below the surface of the blood – broth mixture. This closed space system uses CO2 production to detect growth. Any gas produced will increase the pressure in the headspace and allow some of the blood – broth mixture to enter into the chamber from where sampling, Gram staining and subculturing can be performed. This system thus signals the laboratory towards possible growth without using an automated system.

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Advantages Disadvantages Evaluated favourably in detecting growth More false positives Cost effective Lower yield of anaerobes Useful in small laboratories with small workload Labour intensive, need to visibly inspect for growth

Lysis centrifugation systems:

Lysis centrifugation systems The principle of this test is explained in its name. The Wampole Isostat / Isolater Microbial System (Inverness Medical) is a single tube test that uses saponin for lyses of erythrocytes and neutrophils , followed by centrifugation and subsequent inoculation of solid agar media for isolation. The system is useful for the recovery of slow growing and fastidious organisms including filamentous moulds, dimorphic fungi and Bartonella henselae . This method also allows quantification to be performed, however limitations include a higher rate of contamination, excessive hands on time and toxic effects of the saponin that can inhibit growth.

Continuous monitoring blood culture systems:

Continuous monitoring blood culture systems These systems are considered an advance in clinical microbiology and are the current preferred platform for blood culture testing worldwide. Introduced in 1970s. e.g. the Bactec series started with radiometric systems which was later replaced with non – radiometric systems. Today we face automated and computerized continuous monitoring blood culture systems. The three main commercially available systems are the BacT /ALERT blood culture system ( bioMérieux , Durham, N.C), Bactec 9000 series (BD Microbiology, Cockeysville,MD ) and VersaTREK system (Trek Diagnostic Systems, Cleveland, Ohio). All three systems have expandable detection units with self-contained incubation chambers and minimal bottle manipulation as agitation is achieved via rocking or vortexing . The principle of detection of these systems is based release of CO2 in the presence of microorganism metabolism.

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The Bact /ALERT and Bactec systems both depend on a pH change due to the production of CO2 to detect growth. The Bactec9240 systems’ bottles have a sensor at the bottom that emits a fluorescent light as the CO2 concentration increases, that will pass via an emission filter to a light sensitive diode. The system measures the voltage every 10 minutes and compares the new value with the previous value and emits a positive signal as soon as the threshold value is reached.

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The BacT /ALERT 3D system uses CO2 sensitive chemical sensor that is separated from the blood – broth mixture via a unidirectional membrane. Once the CO2 concentration increases, the colour will change from green to yellow, this is measured with a photosensitive detector.

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The VersaTREK system monitors changes in the bottle headspace every 24 minutes. Both gas consumption and production are monitored. As a result other gasses e.g. O2 and H2 are also detected. The system differs from the other systems in that the aerobic bottles are vortexed with a magnetic stir bar to increase oxygenation.

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Advantages Disadvantages Higher sensitivity for organism recovery High implementation cost Faster TTP Equipment must be maintained Fully automated and computerized Need continuous power supply Easy loading and unloading of bottles Expandable to accommodate larger or smaller volumes

Interpretation of results:

Interpretation of results Interpretation of positive blood culture results are challenging to both clinicians and microbiologists. contamination rates are high upto 50%. need for tools to assist in distinguishing contaminants from pathogens.

Organism identity to indicate significance or contamination:

Organism identity to indicate significance or contamination True pathogen Probable contaminant Staphylococcus aureus Coagulase negative staphylococci ( CoNS )* Streptococcus pneumoniae Bacillus species Escherichia coli Propionibacterium acnes Other Enterobacteriaceae Corynebacterium spp Pseudomonas aeruginosa Candida albicans

Parameters as tools to distinguish contaminants from pathogens :

Parameters as tools to distinguish contaminants from pathogens Clinical Laboratory Fever Identity of the microorganism Leucocytosis Number of positive sets Positive imaging Number of positive bottles (within set) Time to positivity

Blood culture contamination:

Blood culture contamination Various strategies must be implemented to decrease blood culture contamination rates: training staff with regard to aseptic collection technique feedback with regard to contamination rates and implementation of blood culture collection kits. Although skin antisepsis can reduce the burden of contamination, 20% of skin organisms are located deep within the dermis and are unaffected by antisepsis . The practice of changing needles before bottle inoculation should be abandoned as it increases the risk to acquire needle stick injuries without decreasing contamination rates . Also discarding the initial aliquot of blood taken from CVCs does not reduce contamination .

New technologies used in conjunction with blood culture systems in the diagnosis of sepsis:

New technologies used in conjunction with blood culture systems in the diagnosis of sepsis

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In hospital settings where resistance profiles of circulating micro-organisms are known, the use of rapid identifying methods to guide empiric antimicrobial usage is critical to improve patient outcomes. Research efforts are focused on developing molecular tests that can be performed without prior culturing with continuous monitoring systems, however these assays are limited to date. Molecular assays performed on positive blood culture bottles has improved sensitivity compared to conventional culturing methods, and has decreased turnaround times compared to routine culture .

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The Lightcycler ® SeptiFast (Roche Diagnostics, Manheim, Germany) is a multiplex realtime PCR system that can detect up to 25 common pathogens involved in sepsis from one single blood sample within 6 hours. Significantly higher yields were observed from patients already on antimicrobial therapy. Although this method is considered culture – independent but must be further evaluated.

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A new strategy for the detection of blood stream pathogens include PCR/ Electrospray ionization and mass spectrometry (PCR/ESI-MS). This technique in short amplifies broadly conserved regions of bacterial and fungal genomes followed by mass spectrometric analysis by weighing the PCR amplicons and comparing the product with known standards. The commercial assay is the Bac Spectrum Assay that runs on the PLEX-ID (Abbott Molecular).

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Matrix-assisted Laser Desorption/Ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is currently widely applied on post culture isolates for rapid identification. The system use MS signals created and compare them to standard signal patterns within a database. The use directly from positive blood culture bottles needs further evaluation but the advantage of this technology shows promise for the future.

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The Prove-it sepsis assay ( Mobidiag , Helsinki, Finland) is a DNA-based microarray platform can identify more than 50 Gram positive and Gram negative bacteria that can cause sepsis as well as detect the presence of the mecA gene that codes for methicillin resistance in S. aureus from positive blood culture bottles. Sensitivity and specificity compared to conventional culture are reported to be 94.7% and 98.8% respectively .Due to the multiplexing capabilities this assay can also be expanded to detect pathogens involved in fungaemia .

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The Xpert MRSA/SA Blood culture assay (Cepheid) was evaluated favourably for the detection and discrimination between methicillin resistant staphylococcus aureus (MRSA) and methicillin susceptible staphylococcus aureus (MSSA) . Although this method is limited with regard to the range of pathogens it will guide initial empiric therapy towards a better clinical outcome.

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Another new approach to enhance earlier specie identification from positive blood culture bottles following Gram staining include the Peptide Nucleic Acid Fluorescence In situ Hybridization assay (PNA-FISH). This assay uses probes that target specific conserved bacterial and fungal genomic regions and can distinguish between e.g. S. aureus and non – S. aureus as well as different Candida species.

conclusion:

conclusion The various new technologies appears attractive, however implementation will come at great cost and are not cost effective for routine laboratories at present. Certainly the rapidity of results being generated and the ability to detect pathogens unlikely to grew on conventional media comes as a great advantage. The clinical significance of enhanced detection of circulating microbial DNA must be established.

Thank you:

Thank you

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