Multicenter Assessment of Gram Stain Error Rates

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories.     

Direct identification of bacteria from charcoal-containing blood culture bottles using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Several protocols for direct matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) from positive blood cultures are currently used to speed up the diagnostic process of bacteraemia. Identification rates are high and results are accurate for the BACTEC? system and for charcoal-free bottles. Only a few studies have evaluated protocols for charcoal-containing BacT/ALERT bottles reaching substantially lower identification rates. We established a new protocol for sample preparation from aerobic and anaerobic positive charcoal-containing BacT/ALERT blood culture bottles and measured the protein profiles (n?=?167). Then, we integrated this protocol in the routine workflow of our laboratory (n?=?212). During the establishment of our protocol, 74.3?% of bacteria were correctly identified to the species level, in 23.4?%, no result and in 2.4?%, a false identification were obtained. Reliable criteria for correct species identification were a score value?≥1.400 and a best match on rank 1-3 of the same species. Identification rates during routine workflow were 77.8?% for correct identification, 20.8?% for not identified samples and 1.4?% for discordant identification. In conclusion, our results indicate that MALDI-TOF MS is possible, even from charcoal-containing blood cultures. Reliable criteria for correct species identification are a score value?≥1.400 and a best match on rank 1-3 of a single species.     

Rapid method for direct identification of bacteria in urine and blood culture samples by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: intact cell vs. extraction method

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a fast and reliable technology for the identification of microorganisms with proteomics approaches. Here, we compare an intact cell method and a protein extraction method before application on the MALDI plate for the direct identification of microorganisms in both urine and blood culture samples from clinical microbiology laboratories. The results show that the intact cell method provides excellent results for urine and is a good initial method for blood cultures. The extraction method complements the intact cell method, improving microorganism identification from blood culture. Thus, we consider that MALDI-TOF MS performed directly on urine and blood culture samples, with the protocols that we propose, is a suitable technique for microorganism identification, as compared with the routine methods used in the clinical microbiology laboratory.     

Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert^? SA (bioMérieux), and charcoal-containing BacT/Alert^? FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert^? SA, and BacT/Alert^? FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for Gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for Gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.     

Evaluation of Matrix-Assisted Laser Desorption Ionization?Time of Flight Mass Spectrometry for Identification of Mycobacterium species,Nocardia species,and Other Aerobic Actinomycetes

The value of matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) for the identification of bacteria and yeasts is well documented in the literature. Its utility for the identification of mycobacteria and Nocardia spp. has also been reported in a limited scope. In this work, we report the specificity of MALDI-TOF MS for the identification of 162 Mycobacterium species and subspecies, 53 Nocardia species, and 13 genera (totaling 43 species) of other aerobic actinomycetes using both the MALDI-TOF MS manufacturer''s supplied database(s) and a custom database generated in our laboratory. The performance of a simplified processing and extraction procedure was also evaluated, and, similar to the results in an earlier literature report, our viability studies confirmed the ability of this process to inactivate Mycobacterium tuberculosis prior to analysis. Following library construction and the specificity study, the performance of MALDI-TOF MS was directly compared with that of 16S rRNA gene sequencing for the evaluation of 297 mycobacteria isolates, 148 Nocardia species isolates, and 61 other aerobic actinomycetes isolates under routine clinical laboratory working conditions over a 6-month period. MALDI-TOF MS is a valuable tool for the identification of these groups of organisms. Limitations in the databases and in the ability of MALDI-TOF MS to rapidly identify slowly growing mycobacteria are discussed.     

Identification of Anaerobic Bacteria by Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry with On-Plate Formic Acid Preparation

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria.     

Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics

This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.     

Direct identification of Staphylococcus aureus in blood culture fluid with a commercial latex agglutination test.

A commercial latex agglutination slide test (SeroSTAT Staph, Scott Laboratories, Inc., Fiskeville, R.I.) accurately identified Staphylococcus aureus when applied directly to blood culture fluid containing staphylococci. This latex agglutination test exhibited 100% accuracy when 30 seeded aerobic and anaerobic radiometric blood cultures (15 strains of S. aureus, 15 strains of other staphylococcal species) were tested blindly. In 36 actual clinical specimens yielding 16 isolates of S. aureus and 20 isolates of Staphylococcus epidermidis, 94.4% accuracy was achieved. The latex agglutination test provided positive test results before objective criteria of blood culture positivity such as radiometric growth indices and Gram stains became positive.     

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

SUMMARY

Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the “nuts and bolts” of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.     

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory.     

Isolation of a strictly anaerobic strain of Staphylococcus epidermidis

Staphylococcus epidermidis is a well-characterized, nonfastidious, aerobic gram-positive coccus commonly isolated in the clinical microbiology laboratory. Although coagulase-negative staphylococci, including Staphylococcus epidermidis, are often considered a contaminant in the clinical laboratory, an increasing number of reports describe their pathogenesis, in particular in infections of prosthetic devices. This article describes the isolation of a strictly anaerobic strain of Staphylococcus epidermidis in pure culture from the site of an infected prosthetic hip. This isolate was unique in that it grew only under strictly anaerobic conditions. Initially, the isolate was thought to be a known anaerobic gram-positive coccus. However, certain key biochemical and antimicrobial tests performed as part of the standard laboratory identification procedure were not consistent with results expected for any known anaerobic gram-positive coccus; the isolate was catalase positive and metronidazole and penicillin resistant. This isolate was characterized by further biochemical analysis, antimicrobial testing, and nucleic acid sequencing. This paper presents the first documented isolation of a strictly anaerobic Staphylococcus epidermidis strain, confirmed by rpoB gene sequencing.     

Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium

Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.     

High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.Identification of bacteria and yeasts is generally based on conventional phenotypic methods, encompassing culture and growth patterns on specific media, Gram staining, and morphological and biochemical characteristics. Although results of Gram staining can be achieved within minutes, complete identification usually takes 1 or more days. In addition, tests may be difficult to interpret or inconclusive and require specialized staff. Recent molecular methods for microbial identification, such as real-time PCR, sequence analysis, or microarray analysis, have found some application in bacteriology. However, these methods do not provide the complete solution in routine bacterial identifications. To optimize care of patients with infectious diseases, there still is an urgent need for rapid and simple techniques for microbial identification.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used to analyze many different biological molecules. The application of microbial identification based on species-specific spectra of peptides and protein masses by mass spectrometry was first reported about 30 years ago (1). By further improvement of the technique, a rapid, accurate, easy-to-use, and inexpensive method has become available for identification of microorganisms (4, 14, 27). MALDI-TOF MS can be used for accurate and rapid identification of various microorganisms, such as Gram-positive bacteria (2, 3, 9, 10, 22, 26), Enterobacteriaceae (5), nonfermenting bacteria (6, 19-21), mycobacteria (12, 16, 24), anaerobes (10, 23), and yeasts (18, 25). Most studies have reported on MALDI-TOF MS identification of a single strain or family of microorganisms in a research setting. Only one study applied MALDI-TOF MS for identification of bacteria—but not yeasts—in conventional microbiology settings but did not evaluate the results for individual bacteria at the species level (27). In the present study, identification of bacteria by MALDI-TOF MS was extensively evaluated for both bacterial and yeast species identification in an academic medical microbiologic laboratory.     

Improved clinical laboratory identification of human pathogenic yeasts by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The key to therapeutic success with yeast infections is an early onset of antifungal treatment with an appropriate drug regimen. To do this, yeast species identification is necessary, but conventional biochemical and morphological approaches are time-consuming. The recent arrival of biophysical methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), in routine diagnostic laboratories holds the promise of significantly speeding up this process. In this study, two commercially available MALDI-TOF MS species identification systems were evaluated for application in clinical diagnostics, using a geographically diverse collection of 1192 clinical yeast and yeast-like isolates. The results were compared with those of the classical differentiation scheme based on microscopic and biochemical characteristics. For 95.1% of the isolates, all three procedures consistently gave the correct species identification, but the rate of misclassification was greatly reduced in both MALDI-TOF MS systems. Furthermore, several closely related species (e.g. Candida orthopsilosis/metapsilosis/parapsilosis or Candida glabrata/bracarensis) could be resolved by both MALDI-TOF MS systems, but not by the biochemical approach. A significant advantage of MALDI-TOF MS over biochemistry in the recognition of isolates novel to the system was observed. Although both MALDI-TOF MS systems employed different approaches in the database structure and showed different susceptibilities to errors in database entries, these were negligible in terms of clinical usefulness. The time-saving benefit of MALDI-TOF MS over biochemical identification will substantially improve fungal diagnostics and patient treatment.     

Rapid identification of bacterial and fungal pathogens from positive blood cultures by MALDI-TOF MS

Sepsis is a syndrome characterized by a systemic inflammatory response due to severe infection. Early detection of causal agents and appropriate antimicrobial treatment reduce mortality. Conventional microbiological methods often do not provide time critical results for an optimal early management. We used an in-house protocol based on Tween 80 to process 109 positive blood cultures for bacteria and yeast identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and results were compared to standard reference or automated methods. MALDI-TOF MS correctly identified 91.7% of the isolates. Correct identification was obtained for 57/62 (91.9%) aerobic/facultative anaerobic Gram-positive isolates, 53 (85.5%) at species level, and 4 (6.4%) at the genus level; 32/32 (100%) aerobic/facultative anaerobic Gram-negative isolates, 31 (96.9%) at species level, and 1 (3.1%) at the genus level; 7/7 (100%) obligate anaerobes, all at the genus level; 3/7 (42.8%) fungi, all at genus level. Overall, the median identification time of MALDI-TOF MS vs reference standard methods was significantly shorter: median (interquartile range) 7.1 h (4.7–10.2) vs 48.1 h (32.5–50.0), p < 0.0001. MALDI-TOF MS is a valuable tool for rapid identification of pathogens in septic patients. An in-house protocol based on Tween 80 can be used to process positive blood cultures.     

Evaluation of the Bruker MALDI Biotyper for Identification of Gram-Positive Rods: Development of a Diagnostic Algorithm for the Clinical Laboratory

Reported matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing.     

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry: revolutionizing clinical laboratory diagnosis of mould infections

The clinical diagnosis of mould infections currently involves complex species identification based on morphological criteria, which is often prone to error. Employing an extensive mould species reference spectral library (up to 2832 reference spectra, corresponding to 708 strains from 347 species), we assessed the extent to which matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) enhanced the accuracy of species identification. MALDI-TOF MS data were validated against morphology-based and DNA sequence-based results with 262 clinical isolates collected over a 4-month period in 2013. The implementation of MALDI-TOF MS resulted in a dramatic improvement in mould identification at the species level (from 78.2% to 98.1%) and a marked reduction in the misidentification rate (from 9.8% to 1.2%). We then compared the mould identification results obtained before (i.e. 2011) and after (i.e. 2013) the implementation of MALDI-TOF MS in routine identification procedures, which showed an improvement from 64.57% to 100%. Reassessment of a set of isolates from 2011 with this procedure, including MALDI-TOF MS, yielded an increase in species diversity from 16 to 42 species. Finally, application of this procedure during a 16-month period (2012–2013) enabled the identification of 1094 of 1107 (98.8%) clinical mould isolates corresponding to 107 distinct species. MALDI-TOF MS-based mould species identification may soon challenge traditional techniques in the clinical laboratory, as patient prognosis is largely contingent on rapid and accurate diagnosis.     

Use of MALDI-TOF MS technique for rapid identification of bacteria from positive blood cultures

We evaluated the feasibility of same-day routine aerobic bacterial identification using the following procedures: Picking colonies from 4 and 6 h incubated subculture from positive blood culture bottle and analyzing them by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The matched identification rate of this procedure at the species level was 80.6% (141/175) for the 4-h cultures compared with overnight cultures and 90.9% (159/175) for the 6-h cultures. Thus, our technique provides an easy and rapid method for identification of aerobic bacteria in routine clinical microbiology laboratories.     

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not.In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment.     

Direct identification of clinical pathogens from liquid culture media by MALDI-TOF MS analysis

ObjectivesWe propose using MALDI-TOF MS as a tool for identifying microorganisms directly from liquid cultures after enrichment of the clinical sample in the media, to obtain a rapid microbiological diagnosis and an adequate administration of the antibiotic therapy in a clinical setting.MethodsTo evaluate this approach, a series of quality control isolates were grown in thioglycollate (TG) broth and brain heart infusion (BHI) broth and extracted under four different protocols before finally being identified by MALDI-TOF MS. After establishing the best extraction protocol, we validated the method in a total of 300 liquid cultures (150 in TG broth and 150 in BHI broth) of different types of clinical samples obtained from two tertiary Spanish hospitals.ResultsThe initial evaluation showed that the extraction protocol including a 5 minute sonication step yielded 100% valid identifications, with an average score value of 2.305. In the clinical validation of the procedure, 98% of the microorganisms identified from the TG broth were correctly identified relative to 97% of those identified from the BHI broth. In 24% of the samples analysed, growth by direct sowing was only successful in the liquid medium, and no growth was observed in the direct solid agar cultures.ConclusionsUse of MALDI-TOF MS plus the sonication-based extraction method enabled direct and accurate identification of microorganisms in liquid culture media in 15 minutes, in contrast to the 24 hours of subculture required for conventional identification, allowing the administration of a targeted antimicrobial therapy.