|Year : 2010 | Volume
| Issue : 2 | Page : 138-142
Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma
B Behera1, P Mathur2, B Gupta3
1 Department of Microbiology, Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi - 110 029, India
2 Department of Laboratory Medicine, Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi - 110 029, India
3 Department of Anaesthesiology and Critical Care, Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi - 110 029, India
|Date of Submission||27-Mar-2009|
|Date of Acceptance||26-Nov-2009|
|Date of Web Publication||16-Apr-2010|
Department of Laboratory Medicine, Jai Prakash Narayan Apex Trauma Centre, All India Institute of Medical Sciences, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
Purpose: The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. Materials and Methods: A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. Results: No 'very major' discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in β lactam - β lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Conclusions: Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.
Keywords: Acridine orange stain, bloodstream infection, blood culture, direct sensitivity, Gram stain, trauma
|How to cite this article:|
Behera B, Mathur P, Gupta B. Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma. Indian J Med Microbiol 2010;28:138-42
|How to cite this URL:|
Behera B, Mathur P, Gupta B. Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma. Indian J Med Microbiol [serial online] 2010 [cited 2020 Aug 11];28:138-42. Available from: http://www.ijmm.org/text.asp?2010/28/2/138/62491
| ~ Introduction|| |
Despite substantial advances in trauma care, critical traumatic injuries remain one of the leading causes of death.  Increased mortality in the critically ill adult trauma population is not solely due to the direct effects of trauma . Patients who survive the initial injury may experience an increased risk of mortality resulting from nosocomial infection. , Nosocomial blood stream infection (BSI) was the second most common type of nosocomial infection among trauma patients in one study.  In another study, the rate of nosocomial BSIs among trauma patients was reported to be higher than that among patients in surgical intensive care units (ICUs).  Rapid detection, identification and antimicrobial susceptibility testing of bacteria isolated from blood are crucial in trauma patient management. Several studies have shown that prompt appropriate initial antimicrobial therapy in sepsis improves the prognosis. ,, Continuously monitoring automated blood culture systems have emerged as the new standard in blood culture technology, enabling detection of a positive blood culture within 6-24 h of initial incubation.  An overnight agar medium subculture from positive blood bottles is the initial step in the microbial identification of pathogens causing bacteraemia. This conventional culture method is time-consuming, and several days are usually required for microbial recovery, biochemical identification of the bacterial isolate and determination of antimicrobial susceptibility.  Gram stain and direct sensitivity testing from positive blood culture bottle can be used to guide appropriate treatment in critical patients.  Recently, notification of AO stain results for Gram stain negative blood cultures was reported to have significant impact on the clinical management of BSI.  Early communication between the clinical microbiologist and the clinicians responsible for the care of each patient may increase the proportion of patients receiving appropriate treatment in sepsis. ,
Therefore, the present study was designed to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early appropriate treatment in trauma patients with clinical suspicion of sepsis. Another aim of this study was to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies.
| ~ Materials and Methods|| |
The study was conducted over a period of one year (1 st January 2008 to 31 st December 2008) at the clinical microbiology laboratory of a level 1 Trauma Centre, which is a 190 bedded, trauma care hospital of India, with neurosurgery, polytrauma, general surgery and orthopaedics wards and ICUs. The centre has 36 ICU beds and 30 polytrauma beds.
All blood culture samples (5-10 ml in BacT/ALERT FA aerobic blood culture bottle, Bio Merieux Inc., Durham, NC) received from patients with suspected septicaemia during one-year period was included in the study. The blood culture bottles were subjected to automated screening for at least five days for bacteria or two weeks for yeast growth, by BacT/ALERT continuous monitoring blood culture system (Bio Merieux Inc., Durham, NC).  From each bottle reported positive by the system, an aliquot of broth was Gram stained and the result was directly reported to the clinician. Acridine orange (AO) stain (Difco Becton Dickinson, Sparks, Mass.) was performed on all BacT/ALERT-positive, Gram stain negative blood culture bottles as well as on blood culture broths showing polymicrobial growth in Gram staining according to the instructions of the manufacturer.  The blood cultures were sub-cultured on blood agar, MacConkey agar, and direct susceptibility testing was performed on MHA (Mueller-Hinton agar) with antibiotics panel according to Gram stain result without any attempt to standardize the turbidity of broth. The zone diameters of inhibition were recorded using measuring scale after overnight incubation at 37◦ C. Zone sizes were interpreted as 'sensitive' (S), 'resistant' (R) and 'intermediate' (I) according to CLSI recommendation.  The growth obtained on the media was identified using conventional bacteriological and mycological methods and by the Vitek 2 system. ,,, The accuracy of initial Gram stain was compared with culture findings. Antibiotics sensitivity was performed from the culture plate by disc diffusion according to CLSI guidelines and the Vitek 2 system. Staphylococcus aureus (ATCC 29213); E. coli (ATCC 25922) and Pseudomonas aeruginosa i>(ATCC 27853) were included as quality control strain. The results of direct susceptibility from blood culture broth and pure growth using McFarland 0.5 standard turbidity were compared for errors.
Minor discrepancy was interpreted if there was a change from resistant or sensitive to intermediate category or vice versa. A discrepancy was considered to be 'major' if the organism was resistant by direct testing but susceptible by the standard method. Discrepancies were considered to be 'very major' when organisms were found to be susceptible by direct testing and resistant by the standardized method. It has been reported that an overall category error rate of <10% is acceptable for antimicrobial susceptibility testing, including <1.5% very major and <3% major errors. 
| ~ Results|| |
During the study period (January 2008 to 31 st December 2008), total of 2219 blood culture samples were processed in the clinical microbiology laboratory. Of these, 509 (509/2219, 22.9%) gave a positive signal and were subjected to Gram stain. The Gram stain and subsequent culture findings of blood culture bottles are shown in [Table 1]. A total of 19 blood culture bottles were reported as positive by the system but were Gram-stain negative. Upon sub-culturing, five of them showed no growth. Thus, the overall rate of false-positive blood culture bottles was 0.9%. In our study, 14 (73.6%) of the 19 Gram-stain negative bottles grew organisms upon sub-culturing. AO staining detected micro-organisms in 12 of these 14 cultures [Figure 1]. Only two positive cultures growing coagulase-negative Staphylococci were missed by AO staining. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining.
Comparison of interpretive results with direct and standard methods for antimicrobial susceptibility testing of gram-negative rods and gram-positive cocci is shown in [Table 2] and [Table 3]. In this study no 'very major' discrepancy was found. Out of 3221 organisms on which the antibiotic combinations were tested in gram-negative bacteria, 1.8% (61/3221) minor discrepancy was found [Table 2]. The discrepancies were more pronounced in Enterobacteriaceae as compared to nil-fermenters, which comprised majority of non-Enterobacteriaceae isolates. Out of 2064 organisms on which the antibiotic combinations were tested in gram-positive bacteria, 5.2% (109/2064) minor discrepancy was found [Table 3]. Direct sensitivity testing was not reliable for reporting of methicillin resistance and vancomycin resistance in Staphylococcus aureus and coagulase-negative Staphylococci [Table 3]. At our centre, in view of high prevalence of multi-drug resistance in Gram-negative isolates (Unpublished data), it is a routine practice to put tigecycline (except in Pseudomonas and Proteus spp.) and polymyxin B in Gram-negative isolates. In our experience, a high degree of error occurs in direct sensitivity testing of these two precious antibiotics and should be reported with caution.
| ~ Discussion|| |
Many studies have documented the importance of prompt appropriate antimicrobial therapy in the clinical outcome of BSI. ,, Recently, notification of Gram stain results of blood cultures was reported to have a significantly higher impact on antimicrobial management of bloodstream infections than the release of antimicrobial susceptibility data.  In our study, Gram staining proved particularly helpful in the early detection of candidaemia [Figure 2]. Two-third of the microorganisms missed by Gram stain was Gram-positive cocci. Since BacT/ALERT FA bottles contain absorbent charcoal particles, Gram-positive cocci are camouflaged, particularly if the load is low. AO stain was particularly helpful in detecting Gram-positive cocci as well as budding yeast cells, which were otherwise missed by Gram stain.
Empirical therapy is usually administered early in the clinical course of suspected BSI at our tertiary trauma care facility, prior to reporting of positive blood culture results. With respect to antimicrobial management, the most important information provided by the clinical microbiology laboratory appeared to be the release of AST data based on direct sensitivity. Since1970s, multiple studies have compared the direct versus standard method. Direct susceptibility method is found 94-97% in agreement; yet repeating of sensitivity with standard method has been recommended. No 'very major' discrepancy was found in this study. About 5.2 and 1.8% of minor error rates were noted in Gram-positive bacteria and gram-negative bacteria, respectively, while comparing the two methods. Johnson et al, found direct susceptibility test as both feasible and accurate with only 2.4% minor and 1% major discrepancies as compared with standardized susceptibility testing.  Mirerett also found no 'very major' discrepancy and only 0.3% major discrepancy.  Doern found 1.6% minor, 1.5% major and 0.1% 'very major' discrepancies when he compared the two methods. 
Our study shows that the Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. According to our results, this is especially important when managing infections caused by Gram-negative rods. It is difficult to explain why more discrepancies were noted in β-lactams-β-lactamase inhibitor combinations in Gram-negative rods. Reporting of methicillin and vancomycin resistance in Staphylococcus aureus and coagulase-negative Staphylococci requires stringent conditions like 0.5 Mc Farland turbidity standard, exact 24-h incubation etc.  In this study, we did not attempt to standardize turbidity of broth before susceptibility testing; this may have accounted for high degree of error in these two antibiotics. Similarly, CLSI breakpoints for tigecycline sensitivity testing are not available till date. Hence, one should be cautious in direct sensitivity testing of this important antimicrobial.
Timely communication between the microbiological laboratory and the clinician responsible for each patient, when the Gram stain and direct sensitivity result is available, significantly improves targeting of early appropriate antimicrobial therapy. Further studies of different patient populations are needed to address the impact of provision of AST results based on direct sensitivity on antimicrobial drug administration to bacteraemia patients in other care settings. However, while reporting broad-spectrum antibiotics with direct sensitivity test, one should be cautious. These are the major antibiotic used for life-threatening infections. In case of being heavy/lighter standard inoculums or marginal zones, repeating with standard method should be preferred to minimize the chances of error.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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