Evaluation of the Performance of Direct Susceptibility Test by VITEK-2 from Positively Flagged Blood Culture Broth for Gram-Negative Bacilli

Introduction

Bloodstream infections (BSIs) are medical emergencies, which can cause serious morbidities as well as mortalities. BSIs are termed as the third leading cause of health care-related infections.^[1] The gold standard diagnostic method for BSI is culturing the causative organism from blood and providing the antimicrobial susceptibility testing (AST) result for the same.^[2] This will help in timely initiation of antimicrobial therapy. But, prolonged laboratory turnaround time (TAT) can increase mortality, time of hospital stay as well as cost of therapy. In the currently used conventional methods, empirical therapy can be started based on the Gram staining done from positive blood culture bottles.^[3,4] And final confirmatory results are based on biochemical identification and ASTs done from colonies grown on subculture and targeted therapy is given accordingly. As the whole process takes 48 hours or even longer, the need for a robust diagnostic method arises. With every hour of delay in initiating appropriate antimicrobial agents for sepsis, there is an increase in mortality by 7.6%.^[5]

Novel molecular methods and rapid phenotypic methods can help in faster identification, but lack the ability to identify broad range of pathogens and providing antibiotic susceptibility. Moreover, the hands-on processing time as well as monetary investments are high. So, still blood culture remains as the gold standard. Doing direct susceptibility testing (DST) on positive blood culture broth can expedite AST, at least by 24 hours. Since 1980s there have been a lot of studies aiming to standardize DST from blood culture using disc diffusion, automated and molecular testing.^[6-9] But the categorical error varies in different methods. DST methods are shown to be feasible from previous studies as there is good correlation with reference methods. But disc diffusion done directly from broth showed mixed results.^[9] With the advent of automation, laboratory work flows have been redesigned. Rapid susceptibility testing can be done from positive blood broth by automated Vitek system of Biomerieux. Automations help in standardizing the laboratory techniques with accurate results at a short TAT and also it is not labor intensive.^[10]

Our study aims to assess the various errors associated with DST done with automated blood culture system compared with the reference method of AST that is done from bacterial colonies.

Methodology

Results

One twenty positively flagged blood cultures during the study period were included in the study. For analysis to be valid, a minimum of 30 isolates should be included.^[12] So, 60 isolates in Enterobacteriaceae family were included, which comprise 30 Escherichia coli and 30 Klebsiella pneumoniae. Also, 60 nonfermenters were included, with 30 Pseudomonas aeruginosa and 30 Acinetobacter baumannii.

As explained in ►Table 1, nonfermenters showed a better categorical agreement of 97.6% compared with Enterobacteriaceae, which showed 97%. Among Enterobacteriaceae, both E. coli and K. pneumoniae showed categorical agreement of 97% each. Nonfermenters behaved in a different way. P. aeruginosa showed an excellent categorical agreement of 99.4%, whereas A. baumannii showed 95.8% which was least among all the four.

Assessment of categorical disagreement was also done. mE was highest in A. baumannii (3.8%), ME and VME were highest in K. pneumoniae (1.2 and 0.5%, respectively). Essential agreement was 97.1%. P. aeruginosa showed an excellent essential agreement of 99.4%. A. baumannii showed maximum essential disagreement (4.2%) among the isolates tested.

For selective reporting of antibiotics, analysis of DST and AST of individual antibiotics should be done. Among Enterobacteriaceae, most of the antibiotics in the Vitek panel showed an excellent categorical agreement > 95% except piperacillin/tazobactam and nitrofurantoin that showed only 93.3 and 85.0%, respectively. Categorical disagreement was more than acceptable level (> 3%) in mE for amoxicillin/clavulanic acid (3.3%), piperacillin/tazobactam (5.0%), imipenem (3.3%), and nitrofurantoin (13.3%). ME and VME were showing acceptable levels of categorical disagreement (< 3%). Essential disagreement of > 3% was seen in piperacillin/tazobactam (6.7%), imipenem (5.0%), meropenem (3.4%), amikacin (3.3%), gentamicin (3.3%), ciprofloxacin (3.4%), and nitrofurantoin (15.0%) as shown in ►Table 2.

Nonfermenters also showed excellent categorical agreement > 95% except levofloxacin (91.7%) and minocycline (90.0%). ME and VME showed excellent categorical agreement > 97%, but mE showed categorical disagreement > 3% for ticarcillin/clavulanic acid (95.0%), levofloxacin (8.3%), minocycline (10.0%), and tigecycline (3.3%) as shown in ►Table 3.

Discussion

Rapid AST for invasive infections like BSI is the need of the hour. Even though molecular mechanisms of resistance to different antibiotics can be detected by nucleic acid amplification tests, the practical utility is less. On a clinical perspective, rapid identification of antibiotics to which organism is susceptible is more important to modify the empirical antibiotics.^[13] Most of the studies were done based on disk diffusion method directly from blood culture broth and other clinical samples such as urine, bile, and respiratory samples.^[6,14] Here comes the importance of an automated AST system with software and disposable reagent cards like Vitek that has shorter TAT and gives MIC values also. There are only few studies, conducted on this idea, which implemented laborious workflow involving bacterial pellets as extra step.^[15-19] We have used supernatant after centrifugation as bacterial inoculums. And also most of the similar studies have used AST-N020, AST-202, AST-NO09 Vitek cards. We have used card no. N280 for fermenter organism and card no. N281 for nonfermenter organism that were easily available and routinely used card in most of the laboratories.

Barnini et al^[15] conducted similar study in Italy in 2014, where they used MALDI-TOF MS for identification and AST by Vitek-2. But their accordance of direct AST result with culture AST was less than our study. Usage of serum separator tubes or short-term subculture in liquid medium may be the reason for that. In Belgium, Maelegheer and Nulens^[16] did similar study in 2016, where they got a better concordance of 98.9% than our study. They microcentrifuged positive blood sample and MALDI-TOF identification and AST by BD Phoenix was performed. Study by Mauri et al^[17] showed an excellent categorical agreement of 98.1% and essential agreement of 97.7%. They used MALDI-TOF mass spectrometry identification and automated AST.

Among the isolates tested, A. baumannii showed maximum essential disagreement (4.2%). mE was highest in A. baumannii, whereas ME and VME were highest in K. pneumoniae, but they were within the acceptable range (< 3%). But in study by Mauri et al, ME and VME were not reported in K. pneumoniae. Analysis of drug–bug combination showed essential agreement less than allowed level (< 95%) in Enterobacteriaceae—piperacillin, tazobactam, and nitrofurantoin, with no ME and VME. Amoxiclav, Piptaz, and nitrofurantoin were showing mE for Enterobacteriaceae, which was observed in study done by Mauri et al also.^[17] In nonfermenters, levofloxacin and minocycline showed essential agreement less than 95%. MEs and VMEs were not reported. Pan et al showed less essential agreement (< 95%) for meropenem, imipenem, cefepime, ceftriaxone, and ceftazidime, which was not reflected in our study.^[18]

In this study we demonstrated that Vitek-2 can be used to provide rapid, highly accurate susceptibility reports directly from positive blood cultures with Enterobacteriaceae and nonfermenters. The advantages of using Vitek include widespread availability in majority of laboratories around the globe, so that the need for additional equipment and facilities can be circumvented. Less hands-on time and low additional cost are the added advantages. Moreover, there is no need of dedicated manpower to perform the test.^[19] In a study conducted by Höring et al, comparing Vitek-2 and BD Phoenix for direct blood culture inoculation, Vitek-2 demonstrated higher test accuracy.^[20]

Finally, in addition to the high performance for rapid AST, our method reduced the TAT also considerably.

Conclusion

The new procedure of doing direct AST from blood culture represents a simple, effective technique that can reduce the TAT by 24 hours, which in turn benefits the patient outcome by reducing the mortality, time to de-escalation, length of stay, and antibiotic-related side effects like Clostridium difficile infection.