Practice Of Clinical Microbiology Research Articles

Evaluation of different methods for in vitro susceptibility testing of colistin in carbapenem resistant Gram-negative bacilli.

The increasing antibiotic resistance like the advent of carbapenem resistant Enterobactarales (CRE), Carbapenem Resistant Acinetobacter baumanii (CRAB), and Carbapenem Resistant Pseudomonas aeruginosa (CRPA) has led to to the use of toxic and older drugs like colistin for these organisms. But worldwide there is an increase in resistance even to colistin mediated both by chromosomes and plasmids. This necessitates accurate detection of resistance. This is impeded by the unavailability of a user-friendly phenotypic methods for use in routine clinical microbiology practice. The present study attempts to evaluate two different methods - colistin broth disc elution and MIC detection by Vitek two in comparison to CLSI approved broth microdilution (BMD) for colistin for Enterobactarales, Pseudomonas aeruginosa , and Acinetobacter baumanii clinical isolates. Colistin susceptibility of 6013 carbapenem resistant isolates was determined by BMD, Colistin Broth Disc Elution (CBDE), and Vitek two methods and was interpreted as per CLSI guidelines. The MIC results of CBDE, Vitek two were compared with that of BMD and essential agreement (EA), categorical agreement (CA), sensitivity, specificity, very major error (VME), major error (ME) and Cohen's kappa (CK) was calculated. The presence of any plasmid-mediated colistin resistance (mcr-1, 2, 3, 4 and 5) was evaluated in all colistin-resistant isolates by conventional polymerase chain reaction. Colistin resistance was found in 778 (12.9 %) strains among the carbapenem resistant isolates. Klebsiella pneumoniae had the highest (18.9 %) colistin resistance by the BMD method. MIC of Vitek two had sensitivity ranging from 78.2-84.8% and specificity of >92 %. There were 171 VMEs and 323 MEs by Vitek two method, much more than CLSI acceptable range. The highest percentage of errors was committed for Acinetobacter baumanii (27.8 % of VME and 7.9 % ME). On the other hand, the CBDE method performed well with EA, CA, VME and ME within acceptable range for all the organisms. The sensitivity of the CBDE method compared to gold standard BMD varied from 97.5-98.8 % for different strains with a specificity of more than 97.6 %. None of the isolated colistin resistant organisms harboured mcr plasmids. As BMD has many technical complexities, CBDE is the best viable alternative available for countries like India. A sensitive MIC reported by Vitek two needs to be carefully considered due high propensity for VMEs particularly for Klebsiella spp.

The Use of Machine Learning for Image Analysis Artificial Intelligence in Clinical Microbiology.

The growing transition to digital microbiology in clinical laboratories creates the opportunity to interpret images using software. Software analysis tools can be designed to use human-curated knowledge and expert rules, but more novel artificial intelligence (AI) approaches such as machine learning (ML) are being integrated into clinical microbiology practice. These image analysis AI (IAAI) tools are beginning to penetrate routine clinical microbiology practice, and their scope and impact on routine clinical microbiology practice will continue to grow. This review separates the IAAI applications into 2 broad classification categories: (i) rare event detection/classification or (ii) score-based/categorical classification. Rare event detection can be used for screening purposes or for final identification of a microbe including microscopic detection of mycobacteria in a primary specimen, detection of bacterial colonies growing on nutrient agar, or detection of parasites in a stool preparation or blood smear. Score-based image analysis can be applied to a scoring system that classifies images in toto as its output interpretation and examples include application of the Nugent score for diagnosing bacterial vaginosis and interpretation of urine cultures. The benefits, challenges, development, and implementation strategies of IAAI tools are explored. In conclusion, IAAI is beginning to impact the routine practice of clinical microbiology, and its use can enhance the efficiency and quality of clinical microbiology practice. Although the future of IAAI is promising, currently IAAI only augments human effort and is not a replacement for human expertise.

Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study.

Background: Recent advances in nucleic acid amplification technique (NAAT)-based identification of pathogens in blood stream infections (BSI) have revolutionized molecular diagnostics in comparison to traditional clinical microbiology practice of blood culture. Rapid pathogen detection with point-of-care diagnostic-applicable platform is prerequisite for efficient patient management. The aim of the study is to evaluate an in-house developed, lyophilized OmiX-AMP pathogen test for the detection of top six BSI-causing bacteria along with two major antimicrobial resistance (AMR) markers of carbapenem and compare it to the traditional blood culture-based detection.Materials and methods: One hundred forty-three patients admitted to the Medical Intensive Care Unit, Narayana Hrudayalaya, Bangalore, with either suspected or proven sepsis, of either gender, of age ≥18 years were enrolled for the study. Pathogen DNA extracted from blood culture sample using OmiX pReP method was amplified at isothermal conditions and analyzed in real time using OmiX Analysis software.Results: Among the processed 143 samples, 54 were true negative, 83 were true positive, 3 were false negative, and 2 were false positive as analyzed by OmiX READ software. Gram-negative bacteria (91.3%) and gram-positive bacteria (75%) were detected with 100% specificity and 95.6% sensitivity along with the AMR marker pattern with a turnaround time of 4 hours from sample collection to results.Conclusion: OmiX-AMP pathogen test detected pathogens with 96.5% concordance in comparison to traditional blood culture. Henceforth, OmiX-AMP pathogen test could be used as a readily deployable diagnostic kit even in low-resource settings.How to cite this article: Maheshwarappa HM, Guru P, Mundre RS, Lawrence N, Majumder S, Sigamani A, et al. Validation of an Isothermal Amplification Platform for Microbial Identification and Antimicrobial Resistance Detection in Blood: A Prospective Study. Indian J Crit Care Med 2021;25(3):299–304.

A Day in the Life of a Consultant Microbiologist

The practice of Clinical Microbiology has many different forms and takes place in many different environments. From reference laboratories to rural community hospitals to public health laboratories, there are a diverse set of needs for microbiology expertise that extend far beyond the traditional model. In a larger clinical laboratory setting, such as an academic medical center or a reference laboratory, there are often personnel dedicated exclusively to the oversight and practice of microbiology. However, in many other environments and situations, there is a void of microbiology expertise which must be filled. These unmet needs can be addressed by a non-traditional microbiologist, the consultant. Like the practice of microbiology itself, serving as a microbiology consultant can take many forms and can be an appealing alternative for some who are looking to create a more flexible life-style yet continue to utilize their microbiology expertise. The following discussion will outline some key considerations for those looking to start their own microbiology consulting business. In addition, the manuscript will describe the various consulting opportunities that exist and what the “day in a life” of a consultant microbiologist might look like.

Applications of Artificial Intelligence in Clinical Microbiology Diagnostic Testing

The use of artificial intelligence (AI) computer software to interpret data has become part of our everyday lives, and these AI algorithms are becoming part of our everyday laboratory practices. Many AI tools are beginning to demonstrate their real or potential utility in clinical microbiology laboratory practice. In this introduction to applications of AI in clinical microbiology diagnostic testing, the authors introduce AI and machine learning to those familiar with routine clinical microbiology practice. The discussion explores the role of AI for image analysis including Gram stains, ova and parasite exam, and digital plate reading of bacterial cultures. AI's role in advanced analysis of matrix-assisted laser desorption-ionization/time of flight mass spectrometry (MALDI-TOF) mass spectral data and whole genome sequence data of microbes is also discussed. In the future, computers and clinical laboratory scientists will work more closely together to provide optimal efficiency and quality in clinical microbiology laboratory practice, and this close collaboration between humans and machines is expected to improve patient care.

Digital PCR-An Emerging Technology with Broad Applications in Microbiology.

The PCR and its variant, quantitative PCR (qPCR), have revolutionized the practice of clinical microbiology. Continued advancements in PCR have led to a new derivative, digital PCR (dPCR), which promises to address certain limitations inherent to qPCR. Here we highlight the important technical differences between qPCR and dPCR, and the potential advantages and disadvantages of each. We then review specific situations in which dPCR has been implemented in clinical microbiology and the results of such applications. Finally, we attempt to place dPCR in the context of other emerging technologies relevant to the clinical laboratory, including next-generation sequencing. dPCR offers certain clear advantages over traditional qPCR, but these are to some degree offset by limitations of the technology, at least as currently practiced. Laboratories considering implementation of dPCR should carefully weigh the potential advantages and disadvantages of this powerful technique for each specific application planned.

Laboratory automation impact on antimicrobial resistance

Antibiotic resistance in common bacterial pathogens, such as Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, has significantly limited the therapeutic options available for management of infectious diseases. While the indiscriminant use of broad spectrum antibiotics is a significant contributing factor, a more fundamental problem exists. Diagnostic microbiology test results have historically been available too late to be useful. This is, in part, due to the nature of the test methods and in part due to workflow practices in the laboratory. Thus, patients remain on empiric treatments that are frequently ineffective or unnecessarily too broad spectrum1,2. Microscopy and bacterial cultures are mainstays in the microbiology lab, using techniques developed more than 100 years ago. Although microbiologists speak with pride about the ‘art' of their science, the clinical value of the diagnostic tests is frequently lost because of the delays in reporting results with these ‘traditional' approaches. Fortunately, the practice of clinical microbiology is undergoing a dramatic transformation with the introduction of molecular diagnostics, primarily for rapid diagnosis of infections caused by viruses and difficult to grow bacteria, MALDI-TOF mass spectrometry for identification of bacteria, mycobacteria and fungi, and automation of all practices in bacteriology.

Automated Extraction of Microbial DNA From Formalin-Fixed, Paraffin-Embedded Tissue Using the Siemens Tissue Preparation System

We describe the adoption of an automated nucleic acid extraction platform, Siemens Tissue Preparation System (TPS), for use with formalin-fixed, paraffin-embedded (FFPE) tissues intended for PCR microbial testing. The TPS uses a liquid handling robot to automate deparaffinization, followed by extraction and purification of nucleic acids using magnetic particle-based isolation. While it has been used previously for human DNA extraction, this is the first incorporation of the TPS into clinical microbiology practice for microorganism detection to our knowledge. Our laboratory transitioned to use of the TPS from manual deparaffinization of FFPE tissue with subsequent DNA extraction using Roche MagNA Pure (MP) instruments (manual/MP method). Unpublished data from our group demonstrated that the TPS performed better than the manual/MP method for FFPE tissue extraction (27% higher positivity in 86 FFPE specimens tested by both methods). We tracked total testing volumes, laboratory efficiency, TPS errors, and workflow. Additionally, we added a microtome instrument to our clinical microbiology laboratory to section FFPE blocks in order to increase efficiency by avoiding reliance on the histopathology laboratory. Since implementation, 710 FFPE specimens were processed on the TPS, with 724 downstream PCR tests run on extracts for eight laboratory-developed assays: Mycobacterium tuberculosis (MTB), Bartonella spp, Tropheryma whipplei, Coxiella burnetii, Kingella kingae, Coccidioides spp, Acanthamoeba spp, and Balamuthia mandrillaris/Naegleria fowleri. The most common tests run after TPS extraction included 468 (64.6%) MTB, 156 (21.5%) Bartonella, and 70 (9.7%) T whipplei; positivity rates were 4.9%, 6.4%, and 4.3%, respectively. Regarding workflow, use of the TPS decreased total processing time by approximately 70%. The TPS workflow from tissue block sectioning to DNA extraction averaged a total of 3.2 hours, of which 0.7 hours (21.8%) were hands-on time (microtome sectioning and TPS specimen loading/unloading). Comparatively, our manual/MP method averaged 10.6 hours, of which 1.1 hours (10.5%) involved hands-on time (microtome sectioning, manual deparaffinization using xylene and 95% ethanol, and MP loading/unloading). Approximately 7 hours of the manual/MP method were devoted to proteinase K lysis. The TPS accommodates 48 specimens per run, with each run lasting 2.5 hours. Our laboratory currently performs one run daily with an average of four specimens per run. Eleven (6.25%) failure events were noted in 176 runs over the past 9 months. Nine (82% of the errors) were attributed to robotic mechanical problems and two (18%) to human errors (reagent misplacement). The system software alerts the user when errors occur so the problem can be fixed and/or the run repeated. In conclusion, the automated TPS extraction system for FFPE is rapid and limits exposure to hazardous chemicals with minimal run failures. Transition of FFPE sectioning to the microbiology laboratory from the histology laboratory also increased efficiency.

Lowering the Barriers to Routine Whole-Genome Sequencing of Bacteria in the Clinical Microbiology Laboratory.

Whole-genome sequencing of bacterial isolates is increasingly being used to predict antibacterial susceptibility and resistance. Mason and coauthors describe the phenotypic susceptibility interpretations of more than 1,300 Staphylococcus aureus isolates tested against a dozen antistaphylococcal agents, and they compared these findings to susceptibility predictions made by analyzing whole-genome sequence data (J Clin Microbiol 56:e01815-17, 2018, https://doi.org/10.1128/JCM.01815-17). The genotype-phenotype susceptibility interpretations correlated in 96.3% (2,720/2,825) of resistant findings and 98.8% (11,504/11,639) of susceptible findings. This work by Mason and colleagues is helping to lower the barriers to using whole-genome sequencing of S. aureus in clinical microbiology practice.

Reproducing Clinically Significant Multi-Organism Cultures to Improve Clinical Microbiology Education and Practice.

Medical microbiology courses focus on clinically relevant organisms, but designing laboratory experiments that mimic clinical specimens can be challenging. Many clinical specimens produce multi-organism cultures, which are difficult to reproducibly create in the academic laboratory. With experience with only single organism cultures, students may find it challenging to transition to the clinical laboratory where they must identify pathogens and normal flora from mixed cultures. Here, we present protocols for the creation of multi-organism cultures for mock wound, stool, urine, and throat cultures with medically relevant bacteria that allow these principles to be taught in an academic laboratory prior to clinical experiences.

The mass-spectrometric evaluation of сarbapenemase activity of Pseudomonas aeruginosa.

The Pseudomonas aeruginosa is among number of leading opportunistic pathogens. The evaluation of sensitivity of hospital isolates of P. aeruginosa to antibiotics is an important stage in the struggle with Pseudomonas sepsis pathology. The purpose of study is to confirm diagnostic efficiency of mass spectrometry approach in evaluation of сarbapenemase activity in clinical isolates of P. aeruginosa. The study was targeted to detection of сarbapenemases in 50 clinical isolates of P. aeruginosa, non-sensitive to сarbapenemas (control group - 9 isolates of P. aeruginosa sensitive to сarbapenemas). The comparative analysis was implemented concerning the results obtained using laser desorption ionization time-of-flight mass spectrometry and using such common techniques as phenotype detection of presence of metallo-beta-lactamase using E-tests and detection of presence of genes of carbapenemases (VIM, IMP, NDM) using polymerase chain reaction in real-time. The metallo-beta-lactamase activity was established in 14 (28%) out of 50 non-sensitive to сarbapenemas strains. All of them had genes of carbapenemases VIM-type. No IMP and NDM genes were detected in any strain. The VIM genes were detected only in metallo-beta-lactamase positive strains and metallo-beta-lactamase activity was registered only in carriers of VIM genes. According data of MALDI-TOF, all metallo-beta-lactamase and VIM positive strains demonstrated increased capacity of hydrolyzing meropenem. The percentage of hydrolysis under testing of the given strains made up to from 7.6 to 59.3. The absence of carbapenemase activity was demonstrated by 36 (72%) out of 50 strains non-sensitive to сarbapenemases with percentage of hydrolysis from 0 to 4. None of 9 control isolates sensitive to сarbapenemases had metallo-beta-lactamase activity, carried analyzed genes of сarbapenemas and hydrolyzed meropenem. The MALDI-TOF mass spectrometry is a perspective technique to be applied in practice of clinical microbiology for detect isolates of P. aeruginosa, producing сarbapenemases.

Uncommon non-fermenting Gram-negative rods as pathogens of lower respiratory tract infection

Glucose non-fermenting Gram-negative bacteria are ubiquitous environmental organisms. Most of them are identified as opportunistic, nosocomial pathogens in patients. Uncommon species are identified accurately, mainly due to the introduction of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology practice. Most of these uncommon non-fermenting rods are isolated from lower respiratory tract samples. Their significance in lower respiratory tract infections, such as rules of their testing are not clarified yet. The aim of this study was to review the clinical microbiological features of these bacteria, especially their roles in lower respiratory tract infections and antibiotic treatment options. Lower respiratory tract samples of 3589 patients collected in a four-year period (2013-2016) were analyzed retrospectively at Semmelweis University (Budapest, Hungary). Identification of bacteria was performed by MALDI-TOF MS, the antibiotic susceptibility was tested by disk diffusion method. Stenotrophomonas maltophilia was revealed to be the second, whereas Acinetobacter baumannii the third most common non-fermenting rod in lower respiratory tract samples, behind the most common Pseudomonas aeruginosa. The total number of uncommon non-fermenting Gram-negative isolates was 742. Twenty-three percent of isolates were Achromobacter xylosoxidans. Beside Chryseobacterium, Rhizobium, Delftia, Elizabethkingia, Ralstonia and Ochrobactrum species, and few other uncommon species were identified among our isolates. The accurate identification of this species is obligatory, while most of them show intrinsic resistance to aminoglycosides. Resistance to ceftazidime, cefepime, piperacillin-tazobactam and carbapenems was frequently observed also. Ciprofloxacin, levofloxacin and trimethoprim-sulfamethoxazole were found to be the most effective antibiotic agents. Orv Hetil. 2018; 159(1): 23-30.

Syndromic Panel-Based Testing in Clinical Microbiology.

The recent development of commercial panel-based molecular diagnostics for the rapid detection of pathogens in positive blood culture bottles, respiratory specimens, stool, and cerebrospinal fluid has resulted in a paradigm shift in clinical microbiology and clinical practice. This review focuses on U.S. Food and Drug Administration (FDA)-approved/cleared multiplex molecular panels with more than five targets designed to assist in the diagnosis of bloodstream, respiratory tract, gastrointestinal, or central nervous system infections. While these panel-based assays have the clear advantages of a rapid turnaround time and the detection of a large number of microorganisms and promise to improve health care, they present certain challenges, including cost and the definition of ideal test utilization strategies (i.e., optimal ordering) and test interpretation.

Assessing Clinical Microbiology Practice Guidelines: American Society for Microbiology Ad Hoc Committee on Evidence-Based Laboratory Medicine Practice Guidelines Assessment.

As part of the American Society for Microbiology (ASM) Evidence-Based Laboratory Medicine Practice Guidelines Committee of the Professional Practice Committee, an ad hoc committee was formed in 2014 to assess guidelines published by the committee using an assessment tool, Appraisal of Guidelines for Research Evaluation II (AGREE II). The AGREE II assessment helps reviewers determine whether published guidelines are robust, transparent, and clear in presenting practice recommendations in a standardized manner. Identifying strengths and weaknesses of practice guidelines by ad hoc assessments helps with improving future guidelines through the participation of key stakeholders. This minireview describes the development of the ad hoc committee and results from their review of several ASM best practices guidelines and a non-ASM practice guideline from the Emergency Nurses Association.

Applications of MALDI Mass Spectrometry in Clinical Chemistry.

MALDI-TOF mass spectrometry (MS) is set to make inroads into clinical chemistry because it offers advantages over other analytical platforms. These advantages include low acquisition and operating costs, ease of use, ruggedness, and high throughput. When coupled with innovative front-end strategies and applied to important clinical problems, it can deliver rapid, sensitive, and cost-effective assays. This review describes the general principles of MALDI-TOF MS, highlights the unique features of the platform, and discusses some practical methods based upon it. There is substantial potential for MALDI-TOF MS to make further inroads into clinical chemistry because of the selectivity of mass detection and its ability to independently quantify proteoforms. MALDI-TOF MS has already transformed the practice of clinical microbiology and this review illustrates how and why it is now set to play an increasingly important role in in vitro diagnostics in particular, and clinical chemistry in general.

Proficiency testing for bacterial whole genome sequencing: an end-user survey of current capabilities, requirements and priorities

The advent of next-generation sequencing (NGS) has revolutionised public health microbiology. Given the potential impact of NGS, it is paramount to ensure standardisation of ‘wet’ laboratory and bioinformatic protocols and promote comparability of methods employed by different laboratories and their outputs. Therefore, one of the ambitious goals of the Global Microbial Identifier (GMI) initiative (http://www.globalmicrobialidentifier.org/) has been to establish a mechanism for inter-laboratory NGS proficiency testing (PT). This report presents findings from the survey recently conducted by Working Group 4 among GMI members in order to ascertain NGS end-use requirements and attitudes towards NGS PT. The survey identified the high professional diversity of laboratories engaged in NGS-based public health projects and the wide range of capabilities within institutions, at a notable range of costs. The priority pathogens reported by respondents reflected the key drivers for NGS use (high burden disease and ‘high profile’ pathogens). The performance of and participation in PT was perceived as important by most respondents. The wide range of sequencing and bioinformatics practices reported by end-users highlights the importance of standardisation and harmonisation of NGS in public health and underpins the use of PT as a means to assuring quality. The findings of this survey will guide the design of the GMI PT program in relation to the spectrum of pathogens included, testing frequency and volume as well as technical requirements. The PT program for external quality assurance will evolve and inform the introduction of NGS into clinical and public health microbiology practice in the post-genomic era.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0902-3) contains supplementary material, which is available to authorized users.

Classification, identification, and clinical significance of Haemophilus and Aggregatibacter species with host specificity for humans.

The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology practice. However, identification of some strains will still be problematic, necessitating DNA sequencing of multiple housekeeping gene fragments or full-length 16S rRNA genes.

Performance of Kiestra Total Laboratory Automation Combined with MS in Clinical Microbiology Practice

BackgroundMicrobiological laboratories seek technologically innovative solutions to cope with large numbers of samples and limited personnel and financial resources. One platform that has recently become available is the Kiestra Total Laboratory Automation (TLA) system (BD Kiestra B.V., the Netherlands). This fully automated sample processing system, equipped with digital imaging technology, allows superior detection of microbial growth. Combining this approach with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) (Bruker Daltonik, Germany) is expected to enable more rapid identification of pathogens.MethodsEarly growth detection by digital imaging using Kiestra TLA combined with MS was compared to conventional methods (CM) of detection. Accuracy and time taken for microbial identification were evaluated for the two methods in 219 clinical blood culture isolates. The possible clinical impact of earlier microbial identification was assessed according to antibiotic treatment prescription.ResultsPathogen identification using Kiestra TLA combined with MS resulted in a 30.6 hr time gain per isolate compared to CM. Pathogens were successfully identified in 98.4% (249/253) of all tested isolates. Early microbial identification without susceptibility testing led to an adjustment of antibiotic regimen in 12% (24/200) of patients.ConclusionsThe requisite 24 hr incubation time for microbial pathogens to reach sufficient growth for susceptibility testing and identification would be shortened by the implementation of Kiestra TLA in combination with MS, compared to the use of CM. Not only can this method optimize workflow and reduce costs, but it can allow potentially life-saving switches in antibiotic regimen to be initiated sooner.

Automatic identification of mixed bacterial species fingerprints in a MALDI-TOF mass-spectrum

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been broadly adopted by routine clinical microbiology laboratories for bacterial species identification. An isolated colony of the targeted microorganism is the single prerequisite. Currently, MS-based microbial identification directly from clinical specimens can not be routinely performed, as it raises two main challenges: (i) the nature of the sample itself may increase the level of technical variability and bring heterogeneity with respect to the reference database and (ii) the possibility of encountering polymicrobial samples that will yield a 'mixed' MS fingerprint. In this article, we introduce a new method to infer the composition of polymicrobial samples on the basis of a single mass spectrum. Our approach relies on a penalized non-negative linear regression framework making use of species-specific prototypes, which can be derived directly from the routine reference database of pure spectra. A large spectral dataset obtained from in vitro mono- and bi-microbial samples allowed us to evaluate the performance of the method in a comprehensive way. Provided that the reference matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints were sufficiently distinct for the individual species, the method automatically predicted which bacterial species were present in the sample. Only few samples (5.3%) were misidentified, and bi-microbial samples were correctly identified in up to 61.2% of the cases. This method could be used in routine clinical microbiology practice.

Performance of Kiestra Total Laboratory Automation Combined with MS in Clinical Microbiology Practice

Performance of Kiestra Total Laboratory Automation Combined with MS in Clinical Microbiology Practice