Bloodstream infections (BSIs) 200,000 to 300,000 cases of BSI (US) mortality - 20% to 50%; timely detection and identification - most important functions of the microbiology laboratory. It helps to establish an infectious etiology and provide susceptibility testing for optimization of Antimicrobial therapy
Blood cultures are done to Detect the presence of bacteria or yeasts, which may have spread from a specific site in the body into the bloodstream.
BSI Rate of multiplication of microorganisms > the rate of removal by RES of the body = Blood Stream infection occur Failure of body’s defense systems to clear or failure to attempts to remove /drain the localized infection – persistent BSI occur
Routes Organisms in Circulation Bactermia Organisms enter the bloodthrough the lymphatic system Focus lung, Meninges, Direct entry to blood stream - in Joints skin GUT,or Bone infective endocarditis, infected AV fistula, mycotic aneurisms, suppurative thrombophlebitis and etc colonized IV catheters
Sudden rush of organisms generally cleared by the defense system in minutes to hours Fixed macrophage in spleen and liver play major role in clearance- Bacterial virulent factors hinders the clearance ex capsule etc When body response to the infective agents in blood – Systemic signs of symptoms of illness occur (SIRS) – the condition is known as sepsis
Common sources of BSI 19% 27% Intra Vascular Devises Genito Urinary Tract Respiratory Tract 17% Bowel and peritoneum 8% Skin Billiary Tract 5% 12% Intra Abdominal abcesses 3% 5% Other Known Sites 4% Unknown Sites
Clinical Pattern of Blood Stream InfectionsTransient Intermittent ContinuousManipulation of infected Undrained intra Cardinal feature of endosites ex- abdominal abscessese, vascular infections mostabscesses, furuncles, and perinephric abcess, notably acute, sub acute IEcellulitis Prostatic abscesses et –Instrumentation of these are common causesmucosal surfaces colonized of PUOwith regionalflora(Dental, urologicprocedures)Surgery involving prostate, First few weeks of Typhoidhysotectomies, suction fever and Brucellosiscurattage, debridement ofinfected wounds and burns
Breakthrough bacteremia Bacteremia which occur while a patient is receiving systemic therapy with antibacterial agents to which organism is susceptible Can occur early in the infection due to inadequate antibiotic concentration Later due to inadequate drainage of a focus or impairment host defenses
Bacteremia also occur in Early in the course of many systemic and localized infections 50 to 80% patients with meningitis 5 to 30% patients with pneumonia 20 to 70% Septic arthritis 30 to 50% osteomyelitis 5 to 90% patients with gonococcal and meningococcal infections
Principles Procedure appear very simple key elements often are ignored by health-care workers, thereby yielding false-positive tests (i.e., contaminated cultures).
Key steps1. best available site for culture,2. aseptic technique,3. adequate volume of blood,4. sufficient number of blood culture sets.5. Timing of blood culture
1.Selection of the Best AvailableSite Easily accessible, readily available & minimal discomfort to the pt low contamination rate As a rule ,vessels of lower extremes> Upper limb vessels antecubital veins- preferred Arterial blood = venous blood Avoid blood drawing through lines
Blood obtain through intravascular devices Often discouraged False positive results due to colonizers of the line (twice than the normal procedure) Often carried out to when suspecting Line infections. When blood drawn through lines, paired with a second culture obtained by peripheral venipuncture aid in interpreting
Blood through the CVC hematology-oncology patients, blood cultures drawn through either the central catheter or peripheral vein show excellent negative predictive value. Blood cultures drawn through an indwelling central venous catheter have low positive predictive value Therefore, a positive result from a catheter needs clinical interpretation and may require confirmation.
Blood for culture using a existing line is used in especially for patients on hemodialysis or for patients with hematologic and other malignancies, because of convenience and reduction of trauma to the veins
2. Skin Antisepsis avoid false positives – due to skin flora First Second RemarksStandard 70% isoproyl alcohol • 1 to 2 % iodine tincture (30S) Contaminatios ansPractice (30 s) • or iodophore (aquaous iodine Allergic reactions solution (1.5 to 2 min) more with iodophhoresOther l 0.2% chlorine peroxide Less contaminationsagents 10% povidone iodine than 1 (two studies)Other 70% alcohol 2% chlohexidine gluconate Found to be lessagents contamination with these 0.5% chlorhexidine Safe in neonates < 7d in 70% alcohol
2002 recommendation 2% chlorhexidine considered as the agent of choice But, not in children < 2yrs
Method cleansed with 70% isopropyl or ethyl alcohol and allowed to air dry. A second cleansing should be performed using 1% to 2% tincture of iodine or 10% povidone-iodine solution applied concentrically;(this should be allowed to air dry before the vein is punctured.)
Contamination of Blood cultures could be Initial Contaminants Prevent contamination during collection of Blood for culture – from skin flora – skin flora –CoNS, Diptheroids etc Latent Contamination- usually bacterial and is often observed long after cultures are initiated. Apparently the bacteria are present endogenously in the initial plant material and are not obviously pathogenic in situ –from broths or hands of the operator - they increase in titer and overrun the cultures. Latent contamination is particularly dangerous because it can easily be transferred among cultures Introduced Contamination – Contamination can also occur as a result of poor sterile technique or dirty lab conditions.- Fungi and Bacillus sp
3.Blood Volume Direct relationship between the volume and detection of bacteremia or fungemia Blood volume of each culture (Culture set) is the single most important variable in recovering microorganisms
Several studies using conventional and early generations auto mated blood culture systems demonstrated direct relationship between diagnostic yield and volume of blood When volume of blood increased from 2ml to 20ml, the yield increased 30% to 50% Cocerill et al showed that for adults optimum volume is 20ml for a culture
Effect of Volume of Blood Cultured on the Diagnostic Yield of Blood Cultures Mermel, L. A. et. al. Ann Intern Med 1993;119:270-272
bacteremia in adults In infants and young Overwhelming infections childrenlow order of magnitude magnitude of bacteremia Irrespective of age categoryoften <1 to 10 CFU/mL). tends to be greater (often bacteriamia would be > 10000 >100 CFU/mL) CFU/mlHigher volume of blood A comparatively low Generally gives a positiverequired from adults, amount of blood is results even with a leastspecially in instances where a sufficient sensitive culture systemsprior antibiotics givenCultures containing lower blood volumes still should be processed for patient care,with the notation that suboptimal blood volume might give false-negative results.
pediatric patients Optimum volume of blood culture not defined certinity A linear relationship exists with these patients too. Generally pediatric patients have relatively high degree of bacteremia But reported low level of around ≤ 10 CFU/ml bacteremia among 68% of infants(Up to 2 months)
Pediatric patientsAge group Recommended volumeneonates 1 to 2 mL of blood per cultureages 1 month to 2 years 2 to 3 mLbloo d per cultureolder children 3 to 5 mL for blood per culturefor adolescents.[4 10 to 20 mL blood per culture
Kellogg described a total volume of blood for culture depending on total blood volume ( 4 to 4.5 % of patients total blood volume) Weight of the Total blood Recommended V per Total V per % of total patient V(ml) culture culture blood V Kg lb Culture I Culture II ≤1 ≤1 50 - 99 2 - 2 4% 1.1-2 2.2-4.4 100-200 2 2 4 4%2.1-12.7 4.5-27 >200 4 2 6 3%12.8-36.3 28-80 >800 10 10 20 2.5% >36.2 >80 >2200 20 20 40 1.8-2.7%
Kellogg et al recommended blood culture volumes for infants and children based on weight and estimated total blood volume of the child. The recommendations are based on the premise that low level bacteremia (1–10 CFU/mL of blood) can occur in pediatric populations and that its detection can be optimized by culturing up to 4.5% of a childs total blood volume.
Blood cultures containing volumes of >30 mL May contribute to nosocomial anemia Blood may clot in the syringe
4.Number of Blood Culture Sets Two or three blood cultures are adequate for detecting episodes - caused by common microbial pathogens. Using conventional, non-automated blood culture found that total of 20ml per set gives proper result
Culture Positive Rate using manual/ or early generation automated systems Study Two with patients with IE Mayo Clinic 91% 99% 99% Study Two Uni of 88% Colarado 80% 65% 1 st Culture 2nd Culture 3rd Culture
No of blood culture sets are useful to interpreting clinical significance of positive blood cultures One positive test is sufficient for primary pathogens Opportunistic pathogens and Regional flora need more than one culture result for interpretation
Based on the data available Rarely necessary to collect more than two blood culture sets per 24 hours. It is not appropriate to collect only a single blood specimen for culture. A single blood culture will not have sufficient volume for optimal detection of bacteremias and fungemias, and the significance of a positive result with CONS and Diptheroids
Recommendations for the timing of blood culturesCondition or syndrome RecommendationSuspected acute primary bacterimia or Obtain two or three blood cultures, one rightfungemia – Meningitis, after the other, from different sitesOsteomyelitis,arthritis, or pneumoniaFever of uncertain origin (ex occult abscess. Obtain two or three blood cultures. One rightTyphoid fever, brucellosis or other after the other, from different sites initiallyundiagnosed febrile syndrome If they are negative after 24 to 48 hrs incubation. Obtain two or more cultures, one right after other, from different sitesSuspected bactermia or fungema or with Consider alternative blood culture methodspersistantly negative blood cultures designed to entrance recovery of mycobacteria, fungi, and rare fastidious micro organisms
Common patterns of culture positivity Type of bacterimia Example when one blood culture was positive Continuous infective endocarditis subsequent cultures positive in 95% to 100% casesBacterimia or fungaemia Other causes subsequent cultures positivenot due to endocarditis 75% to 80% casesBlood culture positivity Chances for subsequent culture due to contaminants to be positive is 5% highly likely to be negative
Timing of blood cultures Chills(and rigors) – occur 1 hr of lag period Fever follows chills and rigors Therefore, some recommend collection of blood culture soon before chills/rigors or fever spike
Timing of blood cultures Fever Bacterimia Fever spike Chills / RigorBacteremia –symptomsappear after ≈ I hr of lag period
Timing of Blood Cultures The optimal time for collection - just before the onset of a shaking chill not possible to anticipate the precise timing. - common practice to draw blood cultures when fever is detected As a general rule, it is reasonable to obtain two blood culture sets simultaneously, especially if antibiotic therapy is going to be initiated; in less urgent situations - blood cultures may be spaced at intervals. Li et al found no significant difference in detection of BSI from blood cultures obtained simultaneously - versus at intervals during a 24-hour period. timing of blood cultures should be a clinical decision - acuity of the patients illness and whether immediate antimicrobial therapy will be administered.
Variables which govern the culture results1. Culture Medium2. Ratio of Blood to Broth3. Inactivation of Antimicrobial Agents4. Duration of Incubation of Blood Cultures
1. Culture Medium No medium optimally supports the growth of all potential bloodstream pathogens. Even same basal medium (e.g., soybean casein digested broth) - not perform equally Manufacturers supplement the basal media with proprietary supplements
2.Ratio of Blood to Broth Inhibitory substances in blood impairs bacterial growth – False negatives Different methods are used to nullify the effects
Factors in blood which inhibit Mechanisms available to Systemsmicrobial growth counteract the mechanismsComplement, lysozyme, and Dilution with culture broth below Manual/phagocytic leukocytes the critical threshhold or action automatedAntimicrobial agents –Inhibition of Dilution with broth and addition of Automatedsusceptible agents resins (adsorption), Activated charcoal or enzymes- β lactamases **optimal dilution of blood in broth is 5- to 10-fold (i.e., a blood : broth ratio of 1:5 to 1:10).
3.Inactivation of Antimicrobial Agents System Method available AdvantageBACTEC, BD Biosciences, media containing resins that Media containing Sparks, Md bind antimicrobial agents, resins/charcoal detect more BacT/Alert, Organon adding activated charcoal to episodes of bacteremia thanTeknika Corp., Durham, some of its medium others media which doesn’t N.C.) formulations. containing additives specially gram positives* this is a one reason for shifting of major pathogens of bacteremia from BNB to GPC ** Resin containing media are very costly than ordinary media
4. Duration of Incubation of Blood Cultures Variable As most clinical microbiology laboratories use Continuous Monitoring systems – for majority of pathogens 5 d of incubation is enough Some recommend – 4 d Longer incubation - for fastidious pathogens Bartonella, Legionella, Brucella, and certain fungi.
prolonged incubation for culture neg IE (e.g., the HACEK group of fastidious gram-negative bacteria) is controversial Blood cultures for the detection of mycobacteria should be incubated for four weeks.(HIV/AIDS and Immunocompromised
Manual system Transfer of organisms from culture bottle to media Blind subculture Subculture when changes occur in medium – turbidity, froth, deposits, pellicles and hemolysis High rates of false positivty and false negativity Labour intensive
Lysis Centrifugation The Isolator blood culture system (Wampole Laboratories, Cranbury, N.J Principle of lysis-centrifugation- Blood is inoculated to culture tubes that contain a lysis solution. After lysis of the blood cells and tube is centrifuged Is unique as the only non-broth-based blood culture system After removing the supernatant , pellet is inoculated directly to agar media that support the growth of potential blood pathogens including bacteria, fungi, and mycobacteria.
Advantages Best system available to detect dimorphic fungi and Bartonella species ; Disadvantages labor-intensive, more processing needed More manipulations may place technologists at an increased risk of - blood-borne pathogens & category III and IV organisms Reduced detection of anaerobes, Haemophilus species, and pneumococci – due to delayed processing
Automated systems Early systems New systemThe BACTEC radiometric system – Continuous-monitoring blood cultureReplaced in most countries due to safety systems (CMBCS). labor reduction and more rapid detection of microbial growth 1. BacT/Alert (Organon Teknika Corp., Durham, N.C.), 2. BACTEC 9000 (BD Biosciences, Sparks, Md.), 3. and ESP (Trek Diagnostic Systems, Westlake, Ohio). ms
CMBCS Has modular incubation and agitation units, a central computer, monitoring of each bottle in the system for microbial growth at 10- to 24-minute intervals, culture bottles that accept up to 10 mL of blood per bottle. Results of individual bottle readings from the incubator modules are transmitted to the systems computer for data storage and analysis, and growth curves are calculated according to sophisticated algorithms. Testing and data accumulation occur around the clock. these systems have been able to detect evidence of microbial growth 1 to 1.5 days sooner result in fewer instrument false-positive signals than the earlier systems.
Specific Pathogen groups Some microorganisms are isolated infrequently- Nutritionally fastidious These organism groups may not be detected using routine blood culture methods and systems. 1. Anerobes 2. Nutritionally Variant Streptococci 3. Fungi 4. Bartonells sp 5. Mycobacteria
Anaerobic Bacteria decrease in the incidence of anaerobic bacteremia for only about 3% of all BSIs, Currently, no consensus about the routine use of anaerobic bottles some authorities recommend their selective use only - in patients at risk for anaerobic infections.
Fungi Increasing Trend as the etiologic agents of BSIs. Incubation temperature varies filamentous fungi grow better at 27° to 30° C, while yeasts may show better growth at 37° C. The duration of incubation most yeasts grow in two to three days, the dimorphic fungi may take as long as three to six weeks. Variety of media, tryptic soy, columbia, throglycollate and brain heart infusion (BHI) broths all have been successful for growing yeasts and molds from blood. Biphasic media (e.g., Castañeda bottles) traditionally have been considered best for growth of fungi from blood. Lysis Cenrifugation is better for intracellular fungi
Bartonella Species Bartonella henselae and Bartonella quintana are isolated best by using the Isolator system anecdotal reports of isolation using the BACTEC blood culture system as well.
Mycobacteria Blood cultures for mycobacteria are usually restricted to immunocompromised patients, particularly those with AIDS Either the Isolator system, or the BACTEC radiometric system using 13A blood culture medium can be used for mycobacterial blood cultures. From the Isolator tube the sediment is inoculated onto Middlebrook 7H11 media.
Nutritionally Variant Streptococci (NVS) Similar to viridans group Require pyridoxal for growth Separate species – Abiotrophia considered whenever broth-based cultures appear positive with gram-positive cocci, but usual subcultures are negative. 1. a subculture to blood agar supplemented with 0.001% pyridoxal o 2. demonstration of satelliting on a blood agar plate, disc 0.001% pyridoxal or along a staphylococcal streak,
Interpretation of positive blood culture1. Organisms – considered as true pathogens2. Organisms of questionable significance
Organisms – considered as true pathogens Common blood isolates that always or nearly always (>90%) represent true infection include Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli and other members of the Enterobacteriaceae family, Pseudomonas aeruginosa, and Candida albicans.
Other microorganisms Organisms rarely (<5%) represent true bacteremia Corynebacterium species, Propionibacterium acnes, More problematic are group viridans streptococci, enterococci, and coagulase-negative staphylococci (CoNS), ***represent true bacteremia in 38%, 78%, and 15% of cases
CoNS Common blood culture contaminants, and important pathogens in patients with intravascular devices and implanted prosthetic materials. Some authorities suggest that the number of positive blood culture bottles - predicts the clinical significance of CoNS Systematic evaluations find this criterion unreliable
RAPID METHODS FOR IDENTIFICATION OF ORGANISMS IN BLOOD CULTURES Future ?