Automated blood culture system part 1

Diagnostic Microbiology

1. AUTOMATED BLOOD CULTURE SYSTEMS Mrs. SARANYA DEVI K

2. Automated blood culture systems are the primary choice for the detection of pathogens from blood specimens. Instead of manually read broth-based conventional methods, laboratories use continuously monitored, automated blood culture systems for the processing of blood culture specimens nowadays. These systems have increased the yield of blood cultures, reduced the time for organism recovery, and diminished the laboratory technologist’s hands-on time. Several automated blood culture systems are available commercially. Some of the most popular continuous-monitoring blood culture systems are: 

3. BacT /ALERT ( bioMérieux ), BACTEC ( Becton Dickinson and Company) Vital system ESP(EXTRA SENSING POWER) system Versa TREK system  (Thermo Scientific) Commonly used instrument based blood culture techniques Available from 1970-radiometric method From mid 1980-Non radiometric type.

4. The BacT /ALERT Microbial Detection System utilizes a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO2) that is dissolved in the culture medium. If microorganisms are present in the test sample, carbon dioxide is produced as the organisms metabolize the substrates in the culture medium . When growth of the microorganisms produces CO2 , the color of the gas-permeable sensor installed in the bottom of each culture bottle changes from green to yellow. 1. BacT /ALERT

5. The BacT /ALERT System and culture bottles provide both a microbial detection system and a culture media with suitable nutritional and environmental conditions for organisms which might be present in the test sample. Inoculated bottles are placed into the instrument where they are incubated and continuously monitored for the presence of microorganisms that will grow in the BacT /ALERT bottles.

6. Each bottle is placed bottom down into a receiving well, directed by a barcode on the bottle, which is integrated in the computer to match patient information. The wells containing rack that gently rocks back and forth when door is closed. At 10 min interval, a light beam from emitting diodes is projected through an excitation filter to reflect off the CO2 sensitive sensor in the bottom of each bottle. The reflecting light is directed through an emission filter to photosensitive detection that in turn is connected to a computer compiler. As soon as the accumulation of CO2 is sufficient in the bottle to alert the sensor, an audible or visible alert is generated and the position of the bottle is immediately flagged, which is in the form of graph. Procedure:

7. BacT /ALERT 1.BacT/ALERT 3D- (240 POSITION) BacT/ALERT ® Culture Media

8. Principle Measures CO2 (carbon dioxide) produced by metabolism of organisms same as BacT /alert e xcept that it uses fluorescent rather then spectral light to detect changes in conc of Co2. 1 st BACTEC Semi automated Detect radiolabelled BACTEC NR-860 Fully automated Incubator, shaker and detector in one instrument Measures CO2- Infrared photometry BACTEC9240 and BACTEC9120 (commonly used) - Continous monitoring Measures CO2- by FLUORESCENCE BACTEC

9. The BD BACTEC™ Automated Blood Culture System utilizes fluorescent technology in detecting the growth of organisms in the blood culture bottles. When microorganisms are present in the cultured vials, they metabolize nutrients in the culture medium, releasing carbon dioxide into the medium. A dye in the sensor at the bottom of the culture bottles will react with CO2. This modulates the amount of light that is absorbed by a fluorescent material in the sensor . The instrument’s photo detectors measure the level of fluorescence, which corresponds to the amount of CO2 released by the organisms. Then the measurement is interpreted by the system according to preprogramed positivity algorithms. BACTEC CULTURE BOTTLES Depending on the type of suspected pathogen, several broth formulas are available, including media for the recovery of aerobic and anaerobic bacteria, mycobacteria, and fungi.

10. Name of the medium Use BD BACTEC™ Plus Aerobic medium All-purpose medium capable of supporting the growth of aerobic and facultative organisms. BD BACTEC™ Plus Anaerobic medium All-purpose medium for common obligate anaerobic and facultative organisms. BD BACTEC™ Peds Plus media Accommodate small volume and optimally detect common pediatric and non-pediatric organisms BD BACTEC™ MYCO/F LYTIC Growth medium for Mycobacterium sp

11. The uniqueness of BACTEC™ fluorescence sensor technology allows for fully-automated, walkaway testing using a continuous-monitoring instrument that agitates and incubates BACTEC blood culture bottles, resulting in earlier detection of positives blood culture . The system also provides advance algorithms for each media types, for special circumstances such as low blood volume, pediatric specimens or to detect slow growing organisms such as Haemophilus and Neisseria . The algorithms provide rapid detection of pathogens in blood culture . The combination of media-specific algorithms with growth phase-specific algorithms enhances the sensitivity and time to detection, even in the case of delayed vial entry (≤48 hours if are not incubated or held at room temperature) and for bacteria that only generate limited amounts of carbon dioxide (e.g. Pseudomonas aeruginosa ) Advantage

12. Culture for AFB is the most specific test for TB and allows direct identification and determination of susceptibility of the causative organism. Access to the organisms, however, may require lymph node/sputum analysis, bronchoalveolar lavage, or aspirate of cavity fluid or bone marrow. In addition, obtaining the test results is slow (3-8 wk ), and they have a very low positivity in some forms of disease . Routine culture uses a nonselective egg medium (Lowenstein-Jensen or Middlebrook 7H10) and often requires more than 3-4 weeks because of the 22-hour doubling time of M tuberculosis. BACTEC FOR TB DETECTION

13. However, mycobacterial bacteremia ( bacillemia ) is detectable using blood cultures only if specialized systems are used; these bacilli have specific nutrient growth requirements not met by routine culture systems . Such blood cultures should be used for all patients with HIV infection who are suspected of having TB, because bacillemia is particularly prevalent in this population. If available, in fact, these cultures should be used for any patient highly suspected of having active TB. NOW a days Radiometric broth culture (BACTEC radiometric system) of clinical specimens was found to significantly reduce the time (10-14 days) for mycobacterial recovery.

14. Procedure Remove the flip-off cap from the BD BACTEC vial top and inspect the vial for cracks, contamination, excessive cloudiness in medium, and bulging or indented septum. DO NOT USE if any defect is noted. Before inoculating, swab the septum with alcohol (iodine is NOT recommended). Aseptically inject or draw directly 8–10 mL of specimen per vial. If the sample volume is decreased recovery will not be as great as with larger volumes. Place inoculated vials in the BD BACTEC fluorescent series instrument as soon as possible for incubation and monitoring.

15. 4.Vials entered into the instrument will be automatically tested every ten minutes for the duration of the testing protocol period. 5.Positive vials will be determined by the BD BACTEC fluorescent series instrument and identified as such. 6 .Remove the positive vials, subculture in an appropriate solid culture medium (Chocolate Agar, Blood Agar and MacConeky Agar) and Gram-stain.  7. Gram stain  will provide a necessary preliminary report which can help physician to take appropriate decision while waiting for the culture report. 8.If the growth is observed in the solid culture medium, note the colony morphology, do the necessary testing to identify the isolate as well as perform antimicrobial susceptibility test.