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 Table of Contents  
SHORT COMMUNICATION
Year : 2016  |  Volume : 18  |  Issue : 2  |  Page : 131-134

Evaluation of direct sensitivity testing as a method for early initiation of treatment in Gram-negative sepsis


Department of Microbiology, M. S. Ramaiah Medical College, Bengaluru, Karnataka, India

Date of Web Publication30-Nov-2016

Correspondence Address:
Latha Roy Shanthraju
Department of Microbiology, M. S. Ramaiah Medical College, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1282.194953

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  Abstract 

Purpose: For antibiotic susceptibility results, conventional culture and sensitivity methods take 48 h after a blood culture are flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. The objective of this study was to evaluate direct sensitivity test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 h earlier. Materials and Methods: Blood cultures that flagged positive from 1st January 2015 to 30th June 2015 by BacT/ALERT were stained by Gram stain. All blood cultures with only one kind of Gram-negative bacteria by Gram stain were simultaneously cultured and sensitivity tests put up directly (DST) from the broth using disk-diffusion method according to the British Society of Antimicrobial Chemotherapy guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST). Results of DST (test method) and AST (reference method) were compared for agreements or errors. Results: Of the fifty Gram-negative isolates tested, we observed 91.5% categorical agreement or no error (κ = 0.407, P < 0.001). Conclusions: DST using disk diffusion from positive blood culture broths helps initiate early targeted antibiotic therapy. There is a high concordance between DST and AST.

Keywords: Antibiotic resistance, bacteraemia, blood culture, direct sensitivity, inoculum size


How to cite this article:
Shanthraju LR, Devi G. Evaluation of direct sensitivity testing as a method for early initiation of treatment in Gram-negative sepsis. J Acad Clin Microbiol 2016;18:131-4

How to cite this URL:
Shanthraju LR, Devi G. Evaluation of direct sensitivity testing as a method for early initiation of treatment in Gram-negative sepsis. J Acad Clin Microbiol [serial online] 2016 [cited 2017 May 26];18:131-4. Available from: http://www.jacmjournal.org/text.asp?2016/18/2/131/194953


  Introduction Top


Sepsis is one of the major causes of mortality and morbidity in hospitals. Bloodstream infections affect approximately 2% of all hospitalised patients and 70% of patients admitted to the Intensive Care Units.[1] Detection of bloodstream infections is one of the most important tasks performed in the microbiology laboratory. Rapid identification of isolates/agents and their antimicrobial susceptibility is essential for guiding clinicians to select the most appropriate treatment for patients with bloodstream infections. Kumar et al. demonstrated an increase in mortality of 7.6% for every hour by which antimicrobials were delayed in septic shock.[2] International guidelines for the management of severe sepsis and septic shock also recommend that appropriate antimicrobial therapy should be administered within 1 h of recognition of severe sepsis or septic shock.[3] For antibiotic susceptibility results, the conventional culture and sensitivity method take 48 h if done as per Clinical and Laboratory Standards Institute (CLSI) guidelines.[4] In case of direct sensitivity test (DST), therapy can be initiated as early as 24 h after a blood culture is flagged positive by automated systems. Although not mentioned in CLSI, standardisation of broth for direct sensitivity is mentioned in the British Society for Antimicrobial Chemotherapy (BSAC) guidelines.[5]


  Materials and Methods Top


  • Period of study: 1st January 2015 to 30th June 2015
  • Place: M. S. Ramaiah Medical College and Hospital, Bengaluru, Karnataka, India
  • Objective: To evaluate DST as a potential tool to get reliable antibiotic susceptibility results 24 h earlier
  • Inclusion criteria: Blood cultures with only one kind of Gram-negative bacteria by Gram stain
  • Exclusion criteria: Blood cultures with more than one type of growth and Gram-positive cocci by Gram stain
  • Selection criteria: A total of 2500 blood cultures received from various departments with suspected bacteraemia were included in the study. Among which, 300 blood cultures which were positive are processed. After Gram staining, only those with only one kind of Gram-negative bacteria were taken for DST and antibiotic susceptibility test (AST). Out of 50 isolates, 7 isolates were paired and remaining 43 were single blood sample.


Blood cultures that flagged positive by BacT/ALERT were stained by Gram stain.

All positive broths were cultured on Blood agar and McConkey's agar followed by DST using disk-diffusion method as per the BSAC guidelines. All blood cultures with only one kind of Gram-negative bacteria by Gram stain were simultaneously cultured and sensitivity tests put up directly (DST) from the broth using disk-diffusion method according to the BSAC guidelines.

Direct susceptibility testing by disk-diffusion method

For DST of Gram-negative isolates, one drop of blood culture medium (BacT/ALERT) was added to 5 ml 0.45% saline. This dilution was shown to result in: M. S. Ramaiah Medical College and Hospital, Bengaluru, Karnataka, India, on the Mueller-Hinton agar plates used for disk-diffusion testing after incubation at 35°C-37°C in air for 18-20 h as described by the standards of the BSAC Methods for Antimicrobial Susceptibility Testing 2011 and 2012.

[Figure 1] shows DST inoculums as per BSAC criteria.
Figure 1: Direct sensitivity testing inoculums size as per the British Society for Antimicrobial Chemotherapy criteria

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Culture and antibiotic susceptibility testing (conventional method)

A drop of the positive blood culture broth was plated onto a MacConkey agar and Columbia agar with 5% sheep blood (bioMerieux, France) and incubated at 37°C overnight to obtain isolated colonies. These colonies were added to peptone water (bioMerieux, France) to make a suspension equivalent to a 0.5 McFarland standard, adjusted using a DensiCHEK Plus (bioMerieux, Inc.) based on colorimetric principle.

The following panels of antimicrobial disks (HiMedia) were applied: Amoxicillin/Clavulanate (20/10 μg), Cefuroxime (30 μg), Cefpodoxime (10 μg), Ceftazidime (30 μg), Ceftriaxone (30 μg), Aztreonam (30 μg), Piperacillin-Tazobactam (75/10 μg), Imipenem (10 μg), Meropenem (10 μg) and Tigecycline (15 μg).

The zone sizes of all antibiotics were recorded using measuring scale; and at the same time, susceptibility test was repeated from isolated colonies from subcultures, with inoculums prepared of McFarland 0.5 standard. Zone sizes were interpreted as 'sensitive' (S), 'resistant' (R) and 'intermediate' (I) according to CLSI recommendation.[4]

Data analysis

DST results of the isolates were compared with conventional AST. Results of DST (test method) and AST (conventional method) were compared for agreements or errors. The agreement was computed using kappa statistic. Percentage of agreement between the two methods was also calculated.

The errors were classified as:

  1. Minor discrepancy: Change from resistant or sensitive to intermediate category or vice versa
  2. Major discrepancy: If the organism was resistant by direct testing but susceptible by the standard method
  3. Very major discrepancy: When organisms were found to be susceptible by direct testing and resistant by the standardised method.



  Results Top


A total of 2500 blood cultures were received in our laboratory between January and June 2015. All 300 blood cultures that were flagged as positive by the BacT/ALERT system were followed. Positive blood culture bottles were first analysed by Gram staining. A total of fifty positive blood cultures showing monomicrobial Gram-negative bacilli were included in study.

A total of fifty Gram-negative bacterial isolates were compared for DST and AST as shown in [Figure 2]. DST results were available 18-24 h after a blood culture was signalled positive by BacT/ALERT compared to 36-48 h for AST results. The total numbers and percentages of various isolates from blood cultures are given in [Table 1]. Among fifty Gram-negative isolates, seven isolates ( Escherichia More Details coli and Klebsiella spp.) showed minor variations only in susceptibility testing with Imipenem and Meropenem antibiotic discs. No variations were observed in other isolates as shown in [Table 2]. For all fifty Gram-negative isolates, we observed 91.5% categorical agreement or no error (κ = 0.407, P < 0.001).
Figure 2: Images of antibiotic susceptibility test and direct sensitivity test

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Table 1: Species distribution among the positive blood cultures available for direct susceptibility test and antimicrobial susceptibility test


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Table 2: Comparison of interpretive results with direct and standard methods for antimicrobial susceptibility testing of Gram - negative rods cultured from blood


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  Discussion Top


DST inoculum as opposed to standard AST inoculum is a better representative of bacterial isolates in the sample. DST by disk-diffusion method selects some colony variants which may be potentially resistant to the particular antibiotic and are visible inside the sensitive zone or as double zones. The organisms giving rise to double zone (potentially resistant clones) can be confirmed by Gram stain and identification tests to reassure purity of lawn culture of DST plates. Then, they can be separately tested for antibiotic sensitivity. Alternatively, in the final report, we may advice not to use that particular antibiotic, which was reported sensitive based on AST. Otherwise, the potential resistant clones may get selected during treatment with that antibiotic and give rise to antibiotic resistance and treatment failure. This becomes even more important in dealing blood cultures of immunocompromised neutropenic patients. This study involved fifty Gram-negative isolates for comparison of direct and conventional AST methods. The number of Gram-negative isolates for DST comparison included in this study was comparable to previously published studies of direct AST with positive blood cultures.[6],[7] This study demonstrates that DST of Gram-negative isolates from BacT/ALERT bottles using disk-diffusion method performs well since 91.5%, categorical agreement was noted.

Johnson and Washington found direct susceptibility test as both feasible and accurate with only 2.4% minor and 1% major discrepancies as compared with standardised susceptibility testing.[8] Mirerett also found no 'very major' discrepancy and only 0.3% major discrepancy.[9] Doern et al. found 1.6% minor, 1.5% major and 0.1% 'very major' discrepancies when he compared the two methods.[10]


  Conclusions Top


DST of positive blood cultures can help clinicians to initiate appropriate antibiotic treatment 24 h earlier than conventional AST. Early initiation of treatment can lead to significant reductions in patient morbidity, mortality and costs. DST of Gram-negative isolates from BacT/ALERT bottles using disk-diffusion method showed 91.5% categorical agreement. There is a high concordance between DST and conventional AST.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Dombrovskiy VY, Martin AA, Sunderram J, Paz HL. Facing the challenge: Decreasing case fatality rates in severe sepsis despite increasing hospitalizations. Crit Care Med 2005;33:2555-62.  Back to cited text no. 1
    
2.
Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006;34:1589-96.  Back to cited text no. 2
    
3.
Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, et al. Surviving sepsis campaign: International guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 2008;36:296-327.  Back to cited text no. 3
    
4.
Clinical Laboratory Standards Institute. M100S Performance Standard for Antimicrobial Disk Susceptibility Tests. Approved Standard. 26 th ed. CLSI Document M02-A12, M07-A10, M11-A8. Wayne, PA: Clinical Laboratory Standards Institute; 2015.  Back to cited text no. 4
    
5.
Andrews JM; BASC Working Party on Susceptibility Testing. BSAC standardized disc susceptibility testing method (version 5). J Antimicrob Chemother 2006;58:511-29.  Back to cited text no. 5
    
6.
Bearman GM, Wenzel RP. Bacteremias: A leading cause of death. Arch Med Res 2005;36:646-59.  Back to cited text no. 6
    
7.
Fraser A, Paul M, Almanasreh N, Tacconelli E, Frank U, Cauda R, et al. Benefit of appropriate empirical antibiotic treatment: Thirty-day mortality and duration of hospital stay. Am J Med 2006;119:970-6.  Back to cited text no. 7
    
8.
Johnson JE, Washington JA 2 nd . Comparison of direct and standardized antimicrobial susceptibility testing of positive blood cultures. Antimicrob Agents Chemother 1976;10:211-4.  Back to cited text no. 8
    
9.
Stanley M. Comparison of direct and standard antimicrobial discsusceptibility method. J Clin Microbiol 1979;4:482-7.  Back to cited text no. 9
    
10.
Doern GV, Scott DR, Rashad AL, Kim KS. Evaluation of a direct blood culture disk diffusion antimicrobial susceptibility test. Antimicrob Agents Chemother 1981;20:696-8.  Back to cited text no. 10
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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