Blood culture examinations at a community hospital without a microbiology laboratory: using an automated blood culture system and performing a Gram stain on positive culture bottles in the institution

To elucidate the existence of microorganisms from blood culture bottles in hospitals without a microbiology laboratory, we changed the system of blood culture examinations. The Oxoid signal blood culture system and submission of all blood cultures to the clinical testing industry was used from July 2002 to December 2002 (first period). Use of the BacT/Alert system and performing of Gram stain for positive culture bottles in our institutions was conducted from January 2003 to June 2003 (latter period). A total of 210 and 193 blood cultures were processed during the first and latter periods, respectively. There were 40 (19.0%) positive cultures in the first period and 32 (16.6%) positive cultures in the latter period. The times from the specimen collection to the Gram stain result that were required were 3.8 and 1.0 days in the first period and the latter period, respectively. The times required for the final report of the blood cultures in the first period and in the latter period were 5.8 and 4.9 days, respectively. We conclude that using a continuous monitoring, automated blood culture system and performing Gram stain for positive culture bottles in institutions without microbiology laboratories may be useful for medical doctors to rapidly determine the existence of microorganisms and to begin adequate antiinfective therapy.     

BaCT/Alert全自动血培养系统在儿科中的应用研究

目的评价BacT/A1ert连续监测血培养系统检出儿科常见血培养分离菌的孵育时间.方法分析BacT/Alert全自动血培养系统检出1 426份新生儿和小儿阳性血培养瓶败血症病原菌的检出时间特点.结果1 426份新生儿和小儿血培养标本被系统检出阳性培养瓶133份,分离阳性率9.33%,其中革兰阳性菌占75.8%、革兰阴性菌占21.9%、真菌占2.3%;133份阳性培养瓶被系统于12,24,36,48,72 h及96 h的检出率分别为10.4%,67.5%,85.4%,93.7%,96.7%和99.7%.结论BacT/AlerT全自动血培养系统可在48 h以内检出93.7%的儿科败血症重要病原菌,实验室可在系统监测2 d后发出血培养的一级报告,48 h系统监测结果为阴性的培养瓶可能培养出真菌、四联球菌、溶血巴斯德菌及某些认为是污染的革兰阳性杆菌,可将系统监测时间确定为5 d.     

Early adjustment of empirical antibiotic therapy of bloodstream infections on the basis of direct identification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and Gram staining results

IntroductionTo assess the potential added value of rapid MALDI-TOF MS-based identification of bacteria in positive blood cultures to the information provided by Gram staining for adequate empirical antibiotic treatment adjustments in patients with bloodstream infections (BSI).MethodsWe conducted a retrospective, single-center, pre-post quasi-experimental study. In the pre-MALDI-TOF MS phase of the study antibiotic adjustments were made on the basis of Gram stain results, whereas in the MALDI-TOF MS phase they were based on information provided by Gram staining and MALDI-TOF MS results. No antimicrobial stewardship program for BSI was in place within the study period. Antibiotic regimens were categorized as correct, improvable or incorrect.ResultsCohorts were matched for demographics, clinical characteristics of patients and bacterial species involved. Enterobacteriales were the most represented in both study periods (67%), followed by Non-fermenting Gram-negative bacilli and Gram-positive cocci. The number of patients receiving correct, improvable and incorrect empirical antibiotic treatments was comparable for both study periods (P = 0.45, P = 0.57, P = 0.87, respectively). The percentage of patients who ended up receiving correct treatment following modified empirical antibiotic regimens was significantly higher (P = 0.008) in the MALDI-TOF MS phase (27 patients/38.6%) than in the pre-MALDI-TOF MS phase of the study (11 patients/15.7%), although overall adequate coverage of the bacteria causing the infection was comparable across the study periods (90%).ConclusionGram stain results offer valuable information for early adjustment of empirical antibiotic therapies for BSI. Nevertheless, rapid identification of bacteria involved in BSI by MALDI-TOF MS provides added value to achieve this aim.     

Demonstration and utility of clustered pseudohyphae on Gram-stained smears from Candida albicans-positive blood cultures

The presence of clustered and branched pseudohyphae was investigated on Gram-stained smears of 78 consecutive yeast-positive blood cultures. The accuracy of the method was 96.1%, with a positive predictive value of 96.6% for Candida albicans. These findings demonstrate that the presence of clustered and branched pseudohyphae on Gram stain may be used for the rapid and presumptive identification of C. albicans from yeast-positive blood culture bottles.     

BIOFIRE® Blood Culture IDentification 2 (BCID2) panel for early adaptation of antimicrobial therapy in adult patients with bloodstream infections: a real-life experience

Our objective was to assess the effectiveness of a multiplex PCR panel for blood culture identification (BCID2) on the implementation of appropriate antimicrobial therapy. We conducted a monocentric pre/post study comparing the time to result from direct microscopic examination (DE) to bacterial identification (BI) in positive blood cultures between 2 different periods: P1 without BCID2 and P2 with BCID2. Appropriate treatments prescribed before DE and after DE / BCID2 and after BI / BCID2 were compared using direct proportion comparison and survival analysis. For mono-microbial bloodstream infections, the proportion of appropriate antimicrobial treatment after DE was 50% in P1 vs. 87.5% after BCID2 in P2 (P < 0.001) for Gram-negative bacteria and 33.0% in P1 vs. 64.4% in P2 (P < 0.01) for Gram-positive bacteria. A significant difference (P = 0.04) was recorded with survival curves for Gram positive bacteria. BCID2 seems effective in reducing the time for prescribing appropriate antimicrobials.     

The ability of Procalcitonin,lactate, white blood cell count and neutrophil-lymphocyte count ratio to predict blood stream infection. Analysis of a large database

BackgroundThe global burden of death due to sepsis is considerable. Early diagnosis is essential to improve the outcome of this deadly syndrome. Yet, the diagnosis of sepsis is fraught with difficulties. Patients with blood stream infection (BSI) are at an increased risk of complications and death. The aim of this study was to determine the diagnostic accuracy of four readily available biomarkers to diagnose BSI in patients with suspected sepsis.MethodsIn this retrospective, observational, Electronic Medical Record based study we compared the accuracy of procalcitonin (PCT), serum lactate concentration, total white blood cell (WBC) count and the neutrophil-lymphocyte count ratio (NLCR) to diagnose BSI in adult patients presenting to hospital with suspected sepsis. Based on the blood culture results patients were classified into 1 of the following 5 groups: i) negative blood cultures, ii) positive for a bacterial pathogen, iii) positive for a potential pathogen, iv) fungal pathogen and v) potential contaminant. Group 2 was further divided into Gram –ve and Gram +ve pathogens. Receiver operating characteristic (ROC) curves were constructed to compare the diagnostic performance of the biomarkers.ResultsThere were 1767 discreet patient admissions. The median PCT concentration differed significantly across blood culture groups (p < 0.0001). The highest median PCT concentration was observed in patients with a Gram-negative pathogen (17.1 ng/mL; IQR 3.6–49.7) and the lowest PCT in patients with negative blood cultures (0.6 ng/mL; IQR 0.2–2.8). The AUROC was 0.83 (0.79–0.86) for PCT, 0.68 (0.64–0.72) for the NLCR, 0.55 (0.51–0.60) for lactate concentration and 0.52 (0.48–0.57) for the WBC count. The AUROC for PCT was significantly greater than that of the NLCR (p < 0.0001). A PCT less than 0.5 ng/mL had a negative predictive value of 95% for excluding BSI. The best cut-off value of PCT for predicting BSI was 1.5 ng/ml.ConclusionOur results suggest that PCT of less than 0.5 ng/mL may be an effective screening tool to exclude BSI as the cause of sepsis, while the diagnosis of BSI should be considered in patients with a PCT above this threshold. The total WBC count and blood lactate concentration may not be reliable biomarkers for the diagnosis of BSI. The NLCR may be a useful screening test for BSI when PCT assays are not available.     

Retrospective analysis of clinical data associated with patients enrolled in a molecular diagnostic feasibility study highlights the potential utility for rapid detection of bloodstream infection

Background

Measurement of pathogen DNA polymerase activity by enzymatic template generation and amplification (ETGA) has shown promise in detecting pathogens in bloodstream infection (BSI). We perform an in-depth analysis of patients with clinical BSI enrolled in ETGA feasibility experiments.

Methods

In addition to hospital blood cultures, 1 study aerobic culture bottle was drawn from patients with suspected BSI. The study bottle was split into 2 bottles and was additionally subjected to ETGA analysis. Enzymatic template generation and amplification sensitivity/specificity for BSI detection was determined against the Centers for Disease Control BSI definition. When split cultures were both positive, time course analysis was performed to determine time to detection. The records of patients with BSI were reviewed for presence of systemic inflammatory response syndrome, antibiotic timing and appropriateness, and organism identification.

Results

Of 307 enrollees, 38 met the Centers for Disease Control BSI definition. Seventy-four percent met systemic inflammatory response syndrome criteria on admission. Antibiotic coverage was adequate in 76% of patients. Antibiotics were more often delayed in afebrile patients (odds ratio, 5).Twenty-seven of the split study culture bottles were positive in at least 1 sample, and ETGA detected microbes within all samples (sensitivity/specificity, 70.3%/99.3%). Of these, 22 were culture positive in both split study bottles and underwent ETGA time course analysis. Enzymatic template generation and amplification detected microbes within these 3-fold faster than culture.

Conclusions

Patients with BSI often have diagnostic and treatment delays. Enzymatic template generation and amplification provides clinically meaningful data more rapidly than cultures. Future development should focus on real-time application of assays that detect microbes at the molecular level.     

Development of subsequent bloodstream infection in patients with positive Hickman catheter blood cultures and negative peripheral blood cultures

There are limited data on the incidence of subsequent bloodstream infection (BSI) and the effect of systemic antibiotics in patients who had positive catheter-drawn blood cultures (CBC) and negative peripheral blood cultures (PBC). We retrospectively reviewed all paired blood cultures from patients with Hickman catheter in the hematology-oncology ward between January 1997 and December 2008. There were 112 episodes with positive CBC and negative PBC. Nine episodes (8.0%; 95% CI, 3.0-13.1%) led to subsequent BSI within 28 days. Subsequent BSI developed in 6 of 31 episodes (19%) where empiric antibiotics were inappropriate but in 3 of 81 episodes (4%) where empiric antibiotics were appropriate (P = 0.01). Subsequent candidemia (50%, 2 of 4) was more common than subsequent bacteremia (6%, 7 of 108) (P = 0.03). In conclusion, for patients with positive CBC and negative PBC, the overall incidence of subsequent BSI was 8.0%, and inappropriate empiric antibiotics was associated with subsequent BSI.     

Predominance of Enterobacteriaceae Isolates in Early Positive Anaerobic Blood Culture Bottles in BacT/Alert System

We collected and analyzed the time to detection (TTD) of blood cultures in the BacT/Alert automated system from 2002 to 2007. Among the 10,893 monomicrobial isolates from a total of 133,735 blood culture sets, the recoveries of aerobic bottles were compared with those of anaerobic bottles in this study. Significantly more Gram‐positive cocci (except Staphylococcus aureus and enterococci), glucose nonfermentative Gram‐negative bacteria, and yeast were recovered from aerobic bottles than from anaerobic bottles. The average TTD was 19.0 hr and 20.1 hr for the aerobic and anaerobic bottles, respectively, and 96.8% of the microorganisms were detected within the first 72 hr. Of the 5,489 microorganisms recovered from both of the blood culture bottle pair, microbial growth was significantly more often detected first in the anaerobic bottles than the aerobic bottles for Enterobacteriaceae except Serratia marcescens, while S. aureus, coagulase‐negative staphylococci and Pseudomonas aeruginosa were more often detected first in the aerobic bottles. According to these data, we conclude that the earlier positivity of anaerobic bottles is a useful marker for rapid presumptive identification of Enterobacteriaceae infection. J. Clin. Lab. Anal. 27:113–120, 2013. © 2013 Wiley Periodicals, Inc.     

Recovery of Coccidioides immitis from blood and abscess fluid using the BacT/Alert system

We report the recovery of Coccidioides immitis from the blood and abscess fluid of two separate patients by using two automated blood culture systems. In the first case, an aspirate from a neck abscess containing C. immitis spherules was serially diluted and inoculated into liquid media used by the BacT/Alert and the Bactec NR660 blood culture systems. BacT/Alert bottles inoculated with 10⁵, 10⁴, 10³, 10², 10, and two spherules produced a positive signal at 19, 24, 35, 42, 57, and 62 h postinoculation, respectively. Bactec NR660 bottles containing >10² spherules and 10 spherules produced a positive signal after approximately 72 and 96 h of incubation, respectively. In the second case, a blood specimen incubated in BacT/Alert blood culture broth was signaled positive after 82 h of incubation. No organisms were detected by Gram stain of the broth, but C. immitis grew after blind subculture. Our observations demonstrate that these rapid blood culture systems are capable of supporting growth of C. immitis. To our knowledge, this report is the first to detect C. immitis by these blood culture systems.     

What happens when automated blood culture instrument detect growth but there are no technologists in the microbiology laboratory?

We investigated blood cultures that revealed growth at night when no technologists were in the microbiology laboratory and at other times when the laboratory was staffed. On average, it took 7 h and 26 min and 2 h and 12 min for laboratory personnel to report positive blood culture Gram stain results to physicians when growth was detected by automated instruments during the former and latter time periods, respectively. When positive blood culture results led to a change in therapy, it took an average of 8 h and 21 min and 5 h and 26 min in the former and latter groups, respectively, from detection to when the order was written.     

阳性血培养中阴性杆菌快速鉴定方法探讨

目的：结合荧光原位杂交法和直接VITEK法，快速鉴定阳性血培养中的阴性杆菌。方法：当BacT／ALERT3D120报警后，涂片革兰氏染色，若发现阴性杆菌，首先与大肠杆菌特异性的探针杂交，结果阳性者可直接报告为大肠杆菌；结果阴性者，将阳性血培养标本中的细菌离心后制成混悬液，采用VITEK直接法鉴定。所有菌株同时按标准法分纯后，采用VITEK标准法鉴定，比较两法的鉴定结果。结果：分离到的86株阴性杆菌中，在6h内可鉴定94％的细菌，90％为正确鉴定。结论：结合荧光原位杂交法和直接VITEK法，可在一天时间内报告鉴定阴性杆菌结果，并可将其鉴定至种的水平，有助于病人的早期选用抗生素。     

Microbiology of liver abscesses and the predictive value of abscess gram stain and associated blood cultures

Although rare, pyogenic liver abscesses are potentially fatal. We evaluated the predictive value of Gram stain of liver abscess aspirates and temporally associated blood cultures. Gram stains detected bacteria in 79% of the liver abscesses tested. The sensitivity and specificity of Gram stain of the liver abscesses were 90% and 100% for Gram-positive cocci (GPC) and 52% and 94% for Gram-negative bacilli (GNB). The sensitivities of the blood cultures for any GPC and GNB present in the liver abscess were 30% and 39%, respectively. Although, Gram stains and blood cultures offer incomplete detection of the microbial contents of pyogenic liver abscesses, both tests should always accompany liver abscess cultures.     

The use of a differential fluorescent staining method to detect bacteriuria

BACKGROUND: This report describes a differential staining method which distinguishes gram-positive from gram-negative bacteria in fluorescence. Gram-positive bacteria appear yellow and gram-negative bacteria appear green. MATERIAL AND METHODS: The method is based on two fluorochromes, one acting in the wavelength of red, i.e. the acridine orange, and another acting in the wavelength of green, i.e. the fluorescein, which together form a red/ green system. In this report we compared the accuracy of the differential fluorescent staining method and the Gram stain in screening for bacteriuria, as detected by conventional cultures. RESULTS: A total of 1487 urine samples were tested. 289 cultures were positive. 237 specimens grew a single organism at 10(5) and 10(4) CFU/ml. 224 smears were detected by the differential fluorescent staining method and 162 were detected by Gram stain. 1198 samples failed to grow organisms at 10(5) and 10(4) CFU/ml. 107 smears were falsely positive by the fluorescent staining procedure and 289 were falsely positive by the Gram stain. On the basis of the culture results, the sensitivity of the differential fluorescent staining method was 94.5% and that of the Gram stain 68.3%. The specificity of the fluorescent staining procedure was 91.6% and that of the Gram stain 75.8%. The positive predictive value and the negative predictive value of the fluorescent staining method were 67.6% and 98.8%, respectively. Those of the Gram stain were 35.9% and 92.3%, respectively. CONCLUSIONS: A wide range of microbiological and chemical techniques are available to identify bacteria in urine. This fluorescent staining method represents a simple, rapid, reliable method with low-running costs. The main advantage of this technique is that it enables the microbiologist to exclude the presence of bacteria in the urine within a short time after specimen receipt and to eliminate a large number of specimens for culture with significant cost saving. Another advantage of the method is that it allows to distinguish gram-positive from gram-negative bacteria in positive slides on the same day the sample is obtained. The stained smears were easily interpreted, even when the bacterial counts in the specimen were low.     

Bloodstream infection caused by Campylobacter lari

We describe a case of bloodstream infection (BSI) caused by Campylobacter lari in a 58-year-old man diagnosed with lumbar pyogenic spondylitis. Anaerobic blood cultures, taken on the day of admission and on hospital day 4, were positive after 30 h of incubation, although no bacteria were detected by Gram staining. After subculture on 5 % sheep blood agar for 2 days at 35 °C in a 5 % CO₂ environment, capnophilic, curved, gram-negative bacteria were recovered. The bacteria were identified as C. lari using a combination of phenotypic identification methods and partial 16S rRNA gene sequencing. The BSI was eradicated following combination therapy with intravenous tazobactam/piperacillin, oral erythromycin, and sulfamethoxazole/trimethoprim. These results suggest that accurate identification, to the species level, is important to determine effective treatment of BSI caused by Campylobacter spp. and can help us to understand the epidemiology.     

Direct gram stain and urease test to detect Helicobacter pylori.

Antral biopsies were obtained by gastrointestinal endoscopy on 143 adult patients with dyspeptic symptoms of gastritis or peptic ulcer disease. A direct Gram stain and a direct urease test were performed on each biopsy in addition to culture. Forty-three biopsies (30%) were considered positive for Helicobacter pylori based on culture or histologic examination, or both. Thirty-one biopsies (72% sensitivity) were positive for both direct tests, whereas 95 of 100 negative cultures were negative for both tests. Thirty-eight of the 43 positive biopsies were Gram stain positive (sensitivity, 88%; specificity, 100%). The direct urease test alone was positive at 4 hr for 29 biopsies (sensitivity, 67%; specificity, 100%) and at 24 hr for 38 biopsies (sensitivity, 74%; specificity, 95%). Rapid presumptive diagnosis of H. pylori in antral biopsies was obtained when at least one direct test, Gram stain or urease, was positive.     

Improvement of positive blood culture detection by agitation

To evaluate the advantages of agitation in reducing the detection time and increasing the recovery rate of positive blood cultures, 1,000 three-bottle sets of tryptic soy broth on adult inpatients were analyzed. Two bottles were transiently vented, one of which was agitated (250 rpm) for 7-19 hr at 35 degrees C. The other vented bottle and the anaerobic bottle were incubated stationary at 35 degrees C. Smears and subcultures were performed 7-19 hr after collection on both agitated and nonagitated vented bottles. Subcultures were done on all bottles at 72 hr and smears were performed on the anaerobic bottle. There were 137 of 1000 (13.7%) positive cultures from 90 patients. The agitated bottle detected 112 of 137 (81.8%) positive cultures, was the first or only means of detection in 57 of 137 cultures (41.6%), and was the only positive bottle in 30 of 137 (21.9%) cultures. The nonagitated vented bottle detected 89 of 137 (65.0%) of positive cultures and was the only means of detection in 13 of 137 (9.5%), but was never the first means of detection. The anaerobic bottle detected 76 of 137 (55.5%) of positive cultures, was the first or only means of detection in 11 of 137 (8.0%), and was the first means of detection in one of 137 (0.7%) cultures. When both the agitated and nonagitated bottle were positive, the agitated bottle was positive on the average 35 hr earlier. We conclude that agitation of the vented bottle in a conventional blood culture system significantly decreases the detection time of positive blood cultures and increases the number of positive blood cultures detected.     

Impact of postgraduate education on physician practice for community-acquired pneumonia

Background Clinical practice guidelines on community‐acquired pneumonia (CAP) are widely recognized by hospitals in Japan; however, little is known about the effect of postgraduate education on physicians' adherence to the guidelines or on patient outcomes. Method We conducted a chart review of inpatient CAP cases at a single teaching hospital in Japan from 2003 to 2005, during which the educational programme for residents was gradually reinforced by the introduction of multifaceted education and training in the management of infectious diseases. To assess the effects of this educational programme, we measured process indicators such as usage of diagnostic tests, choice of antibiotics, and clinical outcomes, including length of antibiotic treatment, length of stay, and mortality. Results Several improvements were observed after educational intervention: (1) more frequent blood, sputum cultures, and Gram stain tests; (2) less frequent use of broad‐spectrum antibiotics as the initial empiric therapy (from 50% to 12%) and on hospital day 5 (from 66.7% to 10%); and (3) median length of stay was shorter after intervention (16.5 days to 13 days). Conclusions Our findings suggest that multifaceted educational intervention for residents focused on diagnostic efforts, including Gram stain and cultures, choice of antibiotics with the appropriate spectrum, and de‐escalation of antibiotics, can increase adherence to CAP guidelines as well as improve clinical outcomes.     

Rapid Testing Using the Verigene Gram-Negative Blood Culture Nucleic Acid Test in Combination with Antimicrobial Stewardship Intervention against Gram-Negative Bacteremia

Rapid identification of microorganisms and antimicrobial resistance is paramount for targeted treatment in serious bloodstream infections (BSI). The Verigene Gram-negative blood culture nucleic acid test (BC-GN) is a multiplex, automated molecular diagnostic test for identification of eight Gram-negative (GN) organisms and resistance markers from blood culture with a turnaround time of approximately 2 h. Clinical isolates from adult patients at the University Maryland Medical Center with GN bacteremia from 1 January 2012 to 30 June 2012 were included in this study. Blood culture bottles were spiked with clinical isolates, allowed to incubate, and processed by BC-GN. A diagnostic evaluation was performed. In addition, a theoretical evaluation of time to effective and optimal antibiotic was performed, comparing actual antibiotic administration times from chart review (“control”) to theoretical administration times based on BC-GN reporting and antimicrobial stewardship team (AST) review (“intervention”). For organisms detected by the assay, BC-GN correctly identified 95.6% (131/137), with a sensitivity of 97.1% (95% confidence interval [CI], 90.7 to 98.4%) and a specificity of 99.5% (95% CI, 98.8 to 99.8%). CTX-M and OXA resistance determinants were both detected. Allowing 12 h from Gram stain for antibiotic implementation, the intervention group had a significantly shorter duration to both effective (3.3 versus 7.0 h; P < 0.01) and optimal (23.5 versus 41.8 h; P < 0.01) antibiotic therapy. BC-GN with AST intervention can potentially decrease time to both effective and optimal antibiotic therapy in GN BSI.     

Diagnostic utility of the genital Gram stain in ED patients

Objective

The study aimed to determine the diagnostic usefulness of the genital Gram stain in an emergency department (ED) population.

Methods

A linked-query of an urban, tertiary-care, university- affiliated hospital laboratory database was conducted for all completed Chlamydia trachomatis and Neisseria gonorrhoeae DNA probes, Trichomonas vaginalis wet preps, and genital Gram stains performed on ED patient visits between January and December 2004. Positive criteria for a Gram stain included greater than 10 white blood cells per high-power field, gram-negative intracellular/extracellular diplococci (suggesting N gonorrhoeae), clue cells (suggesting T vaginalis), or direct visualization of T vaginalis organisms. DNA probes were used as the gold standard definition for N gonorrhoeae and C trachomatis infection.

Results

Of 1511 initially eligible ED visits, 941 were analyzed (genital Gram stain and DNA probe results both present), with a prevalence of either C trachomatis or N gonorrhoeae of 11.4%. A positive genital Gram stain was 75.7% sensitive and 43.3% specific in diagnosing either C trachomatis and/or N gonorrhoeae infection, and 80.4% sensitive and 32.2% specific when the positive cutoff was lowered to more than 5 white blood cells/high-power field. No Gram stains were positive for T vaginalis (with 47 positive wet mounts), and clue cells were noted on 117 Gram stains (11.6%).

Conclusion

Gram stains in isolation lack sufficient diagnostic ability to detect either C trachomatis or N gonorrhoeae infection in the ED.