Impact of Reporting Gram Stain Results From Blood Culture Bottles on the Selection of Antimicrobial Agents

Abstract

Table 1

Clinical Data for 482 Episodes of Positive Blood Cultures^*

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Materials and Methods

Clinically significant bacteremia is an important cause of serious morbidity and mortality. In a previous report of 843 episodes of positive blood cultures from 707 patients, bacteremia-associated mortality was 17.5%.¹ In another report of 955 bacteremic episodes, mortality due to unidentified organisms was 44%, and even when the organisms were known, mortality was still as high as 25%.²

To reduce mortality and morbidity in bacteremic patients, it is necessary to initiate effective antimicrobial therapy as soon as possible. Many reports indicate a relationship between inadequate antimicrobial therapy and unsatisfactory outcome.^3–8 Bacteremia frequently causes sepsis and septic shock. Because prompt institution of therapy active against the causative pathogen is one of the most important predictors of outcome, physicians must establish a system for rapid administration of a rationally chosen drug or combination of drugs.⁹

The clinical microbiology laboratory has an important role in the management of bacteremia. Detecting pathogenic microorganisms in blood cultures and testing antimicrobial susceptibility always assists in selecting the appropriate antimicrobial agent. The most important and primary test to perform on any positive blood culture is Gram stain,¹⁰ which is the most rapid and simplest test to characterize microorganisms. It is therefore highly likely that the information provided by the Gram stain will help to assess the adequacy of antimicrobial therapy selected after collecting blood culture specimens and before final identification of the microorganism.¹¹ In recent reports, the impact of Gram stain results on patient mortality has been documented.^12–15 On the other hand, there remains the possibility that Gram stain results do not match with the final identification of microorganisms. This would carry a risk leading to inadequate antimicrobial therapy and potentially affecting patients’ clinical course and mortality.

The aim of our study was to assess the advantages and disadvantages of reporting Gram stain results for selecting antimicrobial agents and correlating these results with the patients’ hospital courses.

Results

Study Period and Patient Selection

For the 12-month period, April 1, 2005, through March 31, 2006, all inpatients with positive blood cultures clinically recognized as reflecting true pathogen presence were evaluated at Nihon University Itabashi Hospital, Tokyo, Japan. Microorganisms appearing in blood cultures were defined as true pathogens when it was necessary to start treating the patient with antimicrobials. If several blood culture sets were undertaken simultaneously, they were dealt with as 1 episode. Following the literature,¹ if patients had positive blood culture results repeatedly over 48 hours following the previous positive results, they were included as new positive episodes.

Processing of Samples in the Microbiology Laboratory

For blood culture, we used culture bottles (BACTEC bottles 92F and 93F as 1 set or 94F for small children, Becton Dickinson Diagnostic Systems, Franklin Lakes, NJ) and automated systems (BACTEC 9240 and 9120, Becton Dickinson Diagnostic Systems), with continuous agitation. Microorganisms were examined by Gram stain (neo-B and M Wako, Wako Pure Chemical Industries, Osaka, Japan) using an uncentrifuged drop of blood taken from the culture bottle. This result was compared with results after subculture on standard culture media, with identification and antimicrobial susceptibility tests performed as appropriate for suspected microorganisms using standard procedures recommended by the Clinical and Laboratory Standards Institute. To identify bacterial species, a number of methods were used, including assessment of morphologic criteria and the appearance of Gram-stained colonies, biochemical characteristics with manual procedures or commercial kits (API series, bioMérieux, l’Etoile, France; and N-ID test SP-18, Nissui Pharmaceutical, Tokyo, Japan), or an automated identification system (VITEK 2 system, bioMérieux). For susceptibility tests, disk-diffusion methods, microdilution methods, and the VITEK 2 system were used.

Table 2

Changes in Antimicrobial Therapy After Results of Gram Stain Reported^*

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For positive blood culture bottles, results of the Gram stain were reported immediately to the physicians by telephone using categories such as gram-positive cocci, gram-positive rods, gram-negative cocci, gram-negative rods, fungi, and multiple microorganisms. Written reports were sent to an online reference system after definitive identification had been made and susceptibilities were obtained.

Table 2

Changes in Antimicrobial Therapy After Results of Gram Stain Reported^*

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Reporting the Results of Microbiologic Examinations

Discussion

Clinical Data

For each microorganism, mortality and duration of hospital stay of episodes with incorrect Gram stain results were compared with the data for other episodes with correct results.

For univariate analyses, statistical significance was calculated by using the χ² test or Fisher exact probability test. For comparisons of distributions, the Mann-Whitney U test was used. All statistical analyses were performed with Statmate II (ATMS, Tokyo, Japan).

During the study period, 482 blood culture–positive episodes in 374 patients were clinically recognized as true bacteremia. The age range of patients was 0 to 96 years (median, 67 years). Of the patients, 291 were male and 191 female Table 1.

Clinical data pertaining to infectious episodes are also shown in Table 1. The Department of Gastroenterology and Hepatology, the Department of Hematology and Rheumatology, and the Department of Emergency and Critical Care Medicine were the 3 departments with the highest frequency of true bacteremia. The most common sites of infection were the bloodstream infection (catheter-related) followed by undetermined and gastrointestinal/biliary tract. Malignant diseases, ongoing chemotherapy, and diabetes mellitus were frequent predisposing conditions.

Suspected microorganisms as assessed by Gram stain of blood cultures are shown in Table 2. The most common was Staphylococcus spp, followed by the Enterobacteriaceae family and other streptococci. Multiple microorganisms were also frequent. Table 2 also shows changes in antimicrobial therapy after the results of the Gram stain were reported. “Adjust spectrum” means that antimicrobials selected for empiric therapy did not cover microorganisms detected by Gram stain; more effective antimicrobials were then substituted (eg, ceftazidime changed to vancomycin to cover Staphylococcus spp).

For Gram stains indicating Staphylococcus spp, some changes in antimicrobial therapy in 107 (60.1%) of 178 episodes were instituted. In particular, in 20 (11.2%) of these episodes, no antimicrobial therapy had been given before Gram stain results were reported, but antimicrobial therapy was started thereafter. In contrast, when the results of the Gram stain were Streptococcus pneumoniae or other streptococci, antimicrobial therapy was changed in 4 (33%) of 12 and 15 (43%) of 35 episodes, respectively. The frequency of changing antimicrobials in episodes of Staphylococcus spp was significantly greater than in episodes of other streptococci (P < .05), but there was no difference between Staphylococcus spp and S pneumoniae.

When the results of Gram stain were Enterobacteriaceae family, antimicrobial therapy was changed in 45 (30.6%) of 147 episodes. In particular, patients with 10 (6.8%) of these episodes had not received antimicrobial therapy before the staining results were reported. In contrast, when the results were Pseudomonas spp and related organisms, the episodes for which antimicrobial therapy was changed numbered 16 (59%) of 27. Changing antimicrobials in episodes of Pseudomonas spp and related organisms was thus significantly more frequent than in episodes of Enterobacteriaceae family (P < .05).

Impact of Reporting the Results of Gram Stains on Antimicrobial Therapy

Patients with all 21 episodes reported as yeasts had already received some antimicrobial therapy, but after Gram stain results were reported, additional or changed antifungal agents were instituted in 12 episodes. This frequency would probably have been even higher had 2 patients not already died by the time the Gram stain result became available.

Accuracy of Gram Stain Results and Incorrectly Identified Episodes

In 38 episodes, multiple microorganisms were recognized by Gram stain, and for 18 (47%), some changes of antimicrobial therapy were required. Here, choice of antimicrobials depended on the site of infection and other clinical considerations.

Table 4

Details for Episodes With Incorrect Identification by Gram Stain and Progress of Antimicrobial Therapy

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Table 3 shows the accuracy of Gram stain results. The results for the Staphylococcus spp, S pneumoniae, other streptococci, Corynebacterium spp, Neisseria/Moraxella spp, Pseudomonas spp and related organisms, and yeasts groups were in perfect agreement with culture identification results.

Table 5

Clinical Information for 12 Patients With Incorrect Gram Stain Results

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However, in 12 episodes, the results of Gram stain were not consistent with the results of culture identification. One episode in the undetermined gram-positive cocci group was finally identified as Acinetobacter baumannii. Of 9 episodes in the Enterobacteriaceae family group, 6 were finally identified as Acinetobacter spp, and 3 contained Pseudomonas aeruginosa. There were 2 episodes in the multiple microorganisms group. One episode recognized as Staphylococcus spp and Pseudomonas spp and related organisms was identified as only P aeruginosa, and another recognized as Bacillus spp and Pseudomonas spp and related organisms was identified as Clostridium perfringens and P aeruginosa. In 9 episodes (75%), antimicrobials were changed after culture identification and antimicrobial susceptibility data became available. Especially in 7 episodes, the choice of antimicrobials was inappropriate because of incorrect Gram stain results. Details of the microbiologic features and change of antimicrobials are shown in Table 4.

Conclusions

Outcome

Of 12 episodes with incorrectly identified species, 2 yielded 2 bacterial genera at the same time. Patients with 6 (50%) of these 12 episodes died despite antimicrobial therapy; duration of hospital stay was 23 to 418 days (median, 77 days). Table 6 also shows the outcome and duration of hospital stay for episodes with incorrect Gram stain results, summarized and compared with episodes with correct results for each microorganism. Even though no statistically significant differences in mortality and hospital days were observed between incorrectly and correctly identified groups, the comparison of each microorganism is described here in detail.

Statistical Analysis

In 7 episodes with incorrect identification by Gram stain, 2 patients died (29%). However, only 1 patient with a correctly identified episode also died (100%).

In 3 episodes with incorrect Gram stain results, 2 patients died (67%), whereas among 24 correctly identified episodes, 10 died (42%).

Forty patients had infections with multiple microorganisms according to culture identification. Of 4 patients with incorrectly identified episodes (cases 9–12 in Tables 4 and 5), 2 died (50%). Among 36 correctly identified episodes, 11 patients died (31%).

Clinical Data

Clinical microbiology laboratories have a key role in the management of bacteremia because they are the first to be in a position to register infection from the positive blood culture results. Appropriate early antimicrobial therapy improves the clinical course and reduces mortality in patients with bacteremia.^3–8

Gram stain is the simplest and most rapid test to assess microorganisms directly from positive blood culture bottles and is important for starting adequate antimicrobial agents as soon as possible.¹¹ Recent studies have examined the impact of reporting Gram stain results on patient mortality.^2,12–15 The American Society for Microbiology (ASM) indicates that the single most important first test to be performed on any positive blood culture is Gram stain and states that the terminology used to report such results should be as descriptive as possible.¹⁰ Thus, we reported the Gram stain results as shown in Table 2 in a manner intended to be as useful and easily understandable as possible for clinicians.

However, the assessment of Gram stain results and microscopic examination is quite subjective, being influenced by medical technologists’ skill and experience. Thus, Gram stain results are occasionally inconsistent with the final identification of microorganisms, which represents a risk leading to inadequate antimicrobial therapy and can affect the clinical course and mortality. In our clinical laboratory, Gram stain morphologic examination of direct smears follows the recommendations of the ASM,¹⁶ and results are reviewed by supervisory personnel for quality control. So in our study, as shown in Table 3, except for 12 mismatched episodes, Gram stain results completely matched the corresponding culture identification.

References

We found that when the Gram stain result was Staphylococcus spp, physicians changed or started antimicrobial therapy suitable for Staphylococcus spp significantly more frequently than for the other streptococci group. Staphylococcus spp were most frequently isolated from blood cultures, and all episodes were identified as Staphylococcus spp correctly. In the present study, Staphylococcus spp were the main causative microorganisms of catheter-related systemic infections (137 episodes [79.2%]). Among 107 episodes with a change of antimicrobials after Gram stain results became available, antimicrobials active against methicillin-resistant Staphylococcus aureus had been started in 100 (93.5%). Furthermore, in 89 (89.0%) of 100 episodes, the same antimicrobials were continued after culture identification and susceptibility tests. In only 11 cases could the use of anti–methicillin-resistant S aureus agents be discontinued because the causative microorganisms were not resistant to methicillin. Thus, it is clearly safe and advantageous to report definitive Staphylococcus spp to initiate treatment effective for one of the most important health care–associated infections.

In the gram-negative rods series, when Pseudomonas spp and related organisms were identified, physicians changed or started antimicrobial therapy significantly more frequently than when the Enterobacteriaceae family was reported. This is important for selecting antipseudomonal agents because the administration of appropriate antimicrobial therapy is essential for a good outcome in P aeruginosa bacteremia.¹⁷ In our study, in 16 episodes (59%) of Pseudomonas spp and related organisms, it was necessary to change antimicrobials started for empiric therapy, although the infected sites were not identified in 6 episodes. Only 2 episodes were in patients from the Department of Hematology and Rheumatology, and none showed leukocytopenia. These results might cause difficulties in anticipating infection with Pseudomonas spp and related organisms at the time of selecting empiric therapy. All episodes in the Pseudomonas spp and related organisms group were correctly identified as nonfermentative gram-negative rods, and continued administration of antipseudomonal agents after culture identification and susceptibility tests was required. However, bacteria in 4 episodes were resistant to antimicrobials selected after the Gram stain result was reported. Therefore, one solution for appropriate therapy would be to use multiple antipseudomonal agents after Gram stain by referring to the antibiogram of each hospital. In our clinical laboratory, Pseudomonas spp and related organisms are recognized as gram-negative thin bacilli, medium length to long with rounded to pointy ends, following the criteria in the ASM manual.¹⁶ As a result, the sensitivity and specificity values for the Gram stain for distinguishing nonfermentative gram-negative rods from all gram-negative rods defined by culture were 71.1% and 98.9%, respectively, and the positive and negative predictive values were 94.1% and 93.0%, respectively (unpublished data). Even though the recognition of Pseudomonas spp and related organisms by Gram stain has not been standardized, we conclude that it is helpful and safe to report results definitively to treat patients with bacteremia caused by nonfermentative gram-negative rods appropriately and rapidly. The problem of prediction of Acinetobacter spp will be discussed later.

In the gram-negative rods series, when Pseudomonas spp and related organisms were identified, physicians changed or started antimicrobial therapy significantly more frequently than when the Enterobacteriaceae family was reported. This is important for selecting antipseudomonal agents because the administration of appropriate antimicrobial therapy is essential for a good outcome in P aeruginosa bacteremia.¹⁷ In our study, in 16 episodes (59%) of Pseudomonas spp and related organisms, it was necessary to change antimicrobials started for empiric therapy, although the infected sites were not identified in 6 episodes. Only 2 episodes were in patients from the Department of Hematology and Rheumatology, and none showed leukocytopenia. These results might cause difficulties in anticipating infection with Pseudomonas spp and related organisms at the time of selecting empiric therapy. All episodes in the Pseudomonas spp and related organisms group were correctly identified as nonfermentative gram-negative rods, and continued administration of antipseudomonal agents after culture identification and susceptibility tests was required. However, bacteria in 4 episodes were resistant to antimicrobials selected after the Gram stain result was reported. Therefore, one solution for appropriate therapy would be to use multiple antipseudomonal agents after Gram stain by referring to the antibiogram of each hospital. In our clinical laboratory, Pseudomonas spp and related organisms are recognized as gram-negative thin bacilli, medium length to long with rounded to pointy ends, following the criteria in the ASM manual.¹⁶ As a result, the sensitivity and specificity values for the Gram stain for distinguishing nonfermentative gram-negative rods from all gram-negative rods defined by culture were 71.1% and 98.9%, respectively, and the positive and negative predictive values were 94.1% and 93.0%, respectively (unpublished data). Even though the recognition of Pseudomonas spp and related organisms by Gram stain has not been standardized, we conclude that it is helpful and safe to report results definitively to treat patients with bacteremia caused by nonfermentative gram-negative rods appropriately and rapidly. The problem of prediction of Acinetobacter spp will be discussed later.