Rapid Identification Of Microorganisms Research Articles

Flash MS/MS proteotyping allows identifying microbial isolates in 36 s of mass spectrometry signal.

Rapid identification of microorganisms is essential for medical diagnostics, sanitary controls, and food safety. High-throughput analytical platforms currently rely on whole-cell MALDI-TOF mass spectrometry to process hundreds of samples per day. Although this technology has become a reference method, it is unable to process most environmental isolates and opportunistic pathogens due to an incomplete experimental spectrum database. In most cases, its discriminating power is limited to the species taxonomical rank. By recording much more sequence information at the peptide level, proteotyping by tandem mass spectrometry is able to identify the taxonomic position of any microorganism in the tree of life and can be highly discriminating at the subspecies level. We propose here a methodology for ultra-fast identification of microorganisms by tandem mass spectrometry based on direct sample infusion and a highly sensitive procedure for data processing and taxonomic identification. Results obtained on reference strains and hitherto uncharacterized bacterial isolates show identification to species level in 36 s of tandem mass spectrometry signal, 102 s when including the injection procedure. Flash proteotyping is highly discriminating, as it can provide information down to strain level. The methodology enables high throughput identification of isolates, opening up new prospects, particularly in culturomics, and diagnostics.

A-290 Evaluation of a Commercial Turbidity Monitoring System Followed by Mass Spectrometry to Rapid Identification of Microorganisms from Urine Samples

Abstract Background Urinary tract infections (UTIs) are one of the most common bacterial infections in worldwide and represent a worried public health problem, especially due to a significant rise in global antibiotic use in recent years. The purpose of the study was to evaluate the performance of Uro-Quick (UQ) system (Alifax) and the Vitek-MS system (bioMérieux) to identify bacteria isolates directly from urine samples. Methods We analyzed 352 clinical samples of urine. From automated screening by UQ system was used 500 µL of homogenized urine sample manually pipetted into a UQ vial containing 4 mL of broth. The inoculated vials were manually introduced into the Uro-Quick reading unit and incubated for 3 h. The signals are processed by software which monitors the growth curves and calculates the microbial count as colony forming units (CFU/mL). Sample preparation to the mass spectrometry (Vitek-MS) was performed using an in-house protocol based on a centrifugation-wash (CW) method. In parallel with the Vitek-MS direct identification, all the urine samples were submitted to standard culture (SC) method on ChromID™CPS® plate by semi-quantitative technique using 1 µL disposable loop, incubated at 35–37°C and the colony counting were read after 24 and 48h of incubation. Bacterial isolates from positive cultures were identified by Vitek®2 System, according to the manufacture’s recommendations. Cultures with the presence of three or more microorganisms were considered negative. Results Out of 334 clinical samples, 261 (78.1%) were negative for the growth by UQ system and for the presence of microorganisms by SC, 78 (21.9%) samples were positive with growth as ≥104 CFU/mL by UQ system, of which 32 samples were also positive with microorganism isolated by SC. Among the 78 microorganisms detected on the samples with growth curve by UQ system, 74 (94.8%) were identified to the species level by the mass spectrometry and 4 (5.2%) were not identified. The microorganisms were Escherichia coli (n = 40), Enterococcus faecalis (n = 12), Klebsiella pneumoniae (n = 6), Proteus mirabilis (n = 4), Citrobacter koseri (n = 4), Streptococcus agalactiae (n = 3), Morganella morganii (n = 2), Enterobacter cloacae (n = 1), Citrobacter freundii (n = 1) and Gardnerella vaginalis (n = 1). Among the 32 microorganisms detected by SC, 28 showed the same identification at species level when was compared with direct identification by Vitek-MS. Conclusion This study showed that both UQ and CW methods by Vitek-MS technology used together were able to provides a rapid result (<4 h) for bacterial identification directly from urine samples, that can be useful in clinical practices, especially in severe infectious cases like pyelonephritis. So, future studies should address with a larger number of samples to improve the sensibility of this test for different groups of microorganisms.

A Method to Directly Identify Cronobacter sakazakii in Liquid Medium by MALDI-TOF MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been widely used as an emerging technology for the rapid identification of microorganisms. Cronobacter sakazakii (C. sakazakii) is a food-borne pathogen of particular importance to the powdered infant formula (PIF) processing environment due to its high lethality in infants. However, the traditional solid spotting detection method of pretreating samples for MALDI-TOF MS leads only to qualitative detection of C. sakazakii. We developed a new, low-cost, robust liquid spotting pretreatment method and used a response surface methodology to optimize its parameters. The applicability, accuracy, and quantitative potential were measured for different types of samples. The optimal parameters of this method were as follows: a volume of 70% formic acid of 25 μL, treatment with ultrasound at 350 W for 3 min, and a volume of acetonitrile added of 75 μL. These conditions led to the highest identification score for C. sakazakii (1926.42 ± 48.497). This method was found to detect bacteria accurately and reproducibly. When 70 strains of C. sakazakii isolates were analyzed with this method, the identification accuracy was 100%. The detection limit of C. sakazakii in environmental and PIF samples was 4.1 × 101 cfu/mL and 2.72 × 103 cfu/mL, respectively.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in veterinary medicine: Recent advances (2019-present).

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a valuable laboratory tool for rapid diagnostics, research, and exploration in veterinary medicine. While instrument acquisition costs are high for the technology, cost per sample is very low, the method requires minimal sample preparation, and analysis is easily conducted by end-users requiring minimal training. Matrix-assisted laser desorption ionization-time-of-flight MS has found widespread application for the rapid identification of microorganisms, diagnosis of dermatophytes and parasites, protein/lipid profiling, molecular diagnostics, and the technique demonstrates significant promise for 2D chemical mapping of tissue sections collected postmortem. In this review, an overview of the MALDI-TOF technique will be reported and manuscripts outlining current uses of the technology for veterinary science since 2019 will be summarized. The article concludes by discussing gaps in knowledge and areas of future growth.

Clinical and Economic Impact of Rapid Blood Pathogen Identification Via Verigene.

Bloodstream infections (BSIs) are associated with increased morbidity and mortality if not treated appropriately. Rapid identification of microorganisms will allow clinicians the opportunity to modify initial broad-spectrum antibiotic therapy and improve patient outcomes in bacteremia. We aim to evaluate the impact of the Verigene Gram-positive blood culture (BC-GP) technology on time to modification of antibiotic therapy by clinicians. This was a retrospective research study conducted at Corpus Christi Medical Center. Verigene BC-GP technology was employed to rapidly identify microorganisms in patients with suspected Gram-positive bacteremia. Empiric antibiotic therapy was modified via de-escalation or escalation when culture results became available. The primary outcome for this study was the mean time to modification of antibiotic therapy after Verigene BC-GP results became available. Data analysis was conducted from data collected between January 2015 and August 2017 to assess the clinical and pharmacoeconomic impact of BC-GP. Data were collected on 159 patients, with 123 of 159 (77%) meeting the inclusion criteria. The mean age was 66 ± 14.9 years, with 53/123 (43%) females and 70/123 (57%) males. Positive cultures identified were as follows:Streptococcusspecies (34), Staphylococcus species (72), 31/72 (43%) were MRSA, andEnterococcusspecies (19), 4/19 (21%) were Vancomycin-resistant Enterococcus (VRE). Antibiotic therapies in 31 of 123 patients (25%) were escalated, and 29 of 123 (24%) were de-escalated. Therapy was determined to be appropriate based on culture results in 63 of 123 (51%) patients, and thus therapy was not modified in this group. The mean time to escalate therapy was 6.2 ± 6 h and 9.2 ± 12.1 h to de-escalate. The average time for modification of antibiotic therapy was 7.6 ± 9.5 h. The conventional approach would take approximately 24-72 h for pathogen identification. Data on cost savings per intervention is estimated to be approximately $4000 per intervention. Based on this model, we estimate approximately $240,000 in cost savings from the 60 cases where interventions occurred. There is a significant time advantage to pathogen identification, therapy modification as well as a pharmacoeconomic benefit associated with the Verigene GC-GP system as compared to the conventional approach, which translates to positive patient outcomes.

Impact of an Antimicrobial Stewardship Program Intervention Associated with the Rapid Identification of Microorganisms by MALDI-TOF and Detection of Resistance Genes in ICU Patients with Gram-Negative Bacteremia.

Combination of strategies for rapid diagnostics tests (RDT) with real-time intervention could improve patient outcomes. We aimed to assess the impact on clinical outcomes, antimicrobial consumption, and costs in patients with gram-negative bacteremia. We designed a quasi-experimental study among 216 episodes of gram-negative bacteremia using RDT (MALDI-TOF and detection of resistance genes) directly from blood culture bottles combined with real-time communication of results. Our study did not demonstrate impact on 30-day mortality (25% vs. 35%; p = 0.115). Hospital and ICU length of stay were significantly lower in the intervention period ((44 days vs. 39 days; p = 0.005) and (17 days vs. 13 days; p = 0.033)), respectively. The antimicrobial consumption was 1381 DOT/1000 days in the pre-intervention period compared to 1262 DOT/1000 days in the intervention period (p = 0.032). Antimicrobials against gram-positive and carbapenems had a significantly reduced consumption in the intervention period. Our intervention showed no impact on 30 days-mortality, but demonstrated an impact on hospital and ICU length of stay, as well as antimicrobials consumption and costs. Knowledge of resistance genes adds value and information for safe decision making that can result in direct and indirect benefits related to the economic burden of antibiotic overuse and bacterial resistance.

Rapid identification of new isolates of Acidipropionibacterium acidipropionici by fluorescence in situ hybridization (FISH)

Acidipropionibacterium acidipropionici is widely used for many applications, such as propionic acid production, cereal silage, and also as probiotic. Due to this plethora of applications, new isolates of A. acidipropionici with improved features are being searched for. These new isolates must be accurately identified, however, most approaches become expensive and time-consuming when the number of isolates is high. On the contrary, fluorescence in situ hybridization allows the affordable, reliable, and rapid identification of microorganisms in pure cultures and environmental and medical samples. Therefore, the aim of this work was to apply a fluorescent in situ hybridization probe for the reliable identification of new A. acidipropionici isolates. To this end, probe Pap446, specific for A. acidipropionici, was validated by hybridization assays with strains of this species from different origins, other species of the same genus or family, and unrelated genera. Eight isolates with propionibacterium characteristics were obtained from milk and feces of cows. Probe Pap446, hybridized only with isolates III and VI. The identity of these isolates was further confirmed by PCR using group and species-specific primers for propionibacteria and 16S rDNA sequencing.

Fiber Probe-Based Raman Spectroscopic Identification of Pathogenic Infection Microorganisms on Agar Plates.

Rapid identification of microorganisms is clinically meaningful, and it helps to decelerate the spread of drug resistance and improve patient treatment. In this study, we present a rapid fiber probe-based Raman technique with an excitation wavelength of 785 nm, which is applied to classify and identify nine different species of microorganisms. The cost-effective fiber probe compresses the dimension of the system and provides a more reliable and stable database. All microorganisms were simply cultivated on Luria-Bertani (LB) agar, and Raman spectra were obtained directly from the microbial colonies with the fiber probe within 30 s. The classification model consists of principal component analysis (PCA) in combination with linear discriminant analysis (LDA) and was examined by applying leave-one-batch-out cross-validation (LOBOCV). This model achieved an accuracy of 98.9%. In addition, the validation and identification processes based on independent replicates achieved accuracies of 99.8% and 100%, respectively. The results demonstrated that fiber probe Raman spectroscopy in combination with chemometric analysis allowed a rapid classification and identification of microorganisms only with a normal culture. Therefore, it is promising especially for medical applications and could moreover be helpful to investigate and identify microorganisms rapidly in further studies.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for Identification of Microorganisms in Clinical Urine Specimens after Two Pretreatments.

Rapid identification of microorganisms in urine is essential for patients with urinary tract infections (UTIs). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a method for the direct identification of urinary pathogens. Our purpose was to compare centrifugation-based MALDI-TOF MS and short-term culture combined with MALDI-TOF MS for the direct identification of pathogens in urine specimens. We collected 965 urine specimens from patients with suspected UTIs, 211/965 isolates were identified as positive by conventional urine culture. Compared with the conventional method, the results of centrifugation-based MALDI-TOF MS were consistent in 159/211 cases (75.4%), of which 135/159 (84.9%) had scores ≥ 2.00; 182/211 cases (86.3%) were detected using short-term culture combined with MALDI-TOF MS, of which 153/182 (84.1%) had scores ≥ 2.00. There were no apparent differences among the three methods (p = 0.135). MALDI-TOF MS appears to accelerate the microbial identification speed in urine and saves at least 24 to 48 hours compared with the routine urine culture. Centrifugation-based MALDI-TOF MS is characterized by faster identification speed; however, it is substantially affected by the number of bacterial colonies. In contrast, short-term culture combined with MALDI-TOF MS has a higher detection rate but a relatively slow identification speed. Combining these characteristics, the two methods may be effective and reliable alternatives to traditional urine culture.

Evaluation of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical and subclinical mastitis

This study aimed to evaluate the diagnostic performance (specificity, Sp; sensitivity, Se; accuracy; positive predictive value; negative predictive value; and Cohen's kappa coefficient, κ, of agreement) of chromogenic culture media for rapid identification of microorganisms isolated from cows with clinical (CM) and subclinical mastitis (SCM). For this, 2 experiments were carried out: evaluation of (1) biplate, and (2) triplate of chromogenic culture media for rapid identification of mastitis-causing microorganisms. For the evaluation of diagnostic performance, identification of microorganisms by MALDI-TOF mass spectrometry was considered the standard methodology. In experiment 1, 476 milk samples collected from cows with CM and 660 from cows with SCM were evaluated by inoculation in 2 selective chromogenic culture media (CHROMagar) for gram-positive bacteria and another for gram-negative bacteria. In experiment 2, 476 milk samples from cows with CM and 500 from cows with SCM were evaluated by inoculation in triplate chromogenic culture media (Smartcolor2, Onfarm), selective for Streptococcus and Strep-like organisms, Staphylococcus, and gram-negative bacteria. In experiment 1 for the CM samples, the use of biplates with gram-positive and gram-negative culture media showed Se that ranged from 0.56 (0.32-0.81; Staphylococcus aureus) to 0.90 (0.80-0.99 Streptococcus uberis), Sp varied from 0.94 (0.92-0.96; Strep. uberis) to 1.00 (Prototheca spp. or yeast), and κ ranged from 0.47 (0.26-0.67; Staph. aureus) to 0.84 (0.78-0.9; Escherichia coli). The Se of biplates for SCM samples ranged from 0.50 (0.15-0.85; E. coli) to 0.94 (0.87-1.00; Staph. aureus), Sp varied from 0.95 (0.93-0.97; Strep. uberis) to 0.99 (0.98-1.00; Staph. aureus and Strep. Agalactiae or dysgalactiae), and κ ranged from 0.18 (0.00-0.40; Escherichia coli) to 0.88 (0.80-0.95; Staph. aureus). In experiment 2, the Se of the triplate chromogenic media in CM samples ranged from 0.09 (0.00-0.26; Serratia spp.) to 0.94 (0.85-1.00; Klebsiella spp. and Enterobacter spp.), Sp varied from 0.94 (0.92-0.96; Strep. agalactiae and Strep. dysgalactiae) to 1.00 (Serratia spp.) and κ ranged from 0.07 (0.00-0.24; Serratia spp.) to 0.85 (0.75-0.94; Klebsiella spp. and Enterobacter spp.). For SCM samples, the use of the triplate with the chromogenic culture media showed Se that varied from 0.25 (0.10-0.40; Lactococcus spp.) to 1.00 (Strep. Agalactiae or dysgalactiae), Sp ranged from 0.92 (0.90-0.94; Strep. Agalactiae and Strep. dysgalactiae) to 0.99 (0.98-1.00; Klebsiella spp. and Enterobacter spp.), and κ varied from 0.28 (0.00-0.72; E. coli) to 0.72 (0.60-0.82; Staph. aureus). Our results suggest that the diagnostic accuracy of the biplate and triplate of chromogenic culture media varies according to pathogen, and the results of chromogenic culture media may be useful for rapid decision-making on mastitis treatment protocols of the main mastitis-causing microorganisms, but their use for implementation of mastitis control measures will depend on each farm specific needs.

Efficacy of 16S rRNA variable regions high-resolution melt analysis for bacterial pathogens identification in periprosthetic joint infections

BackgroundAccurate and rapid identification of microorganisms causing periprosthetic joint infections (PJIs) are necessary for choosing an appropriate antibiotic therapy. Therefore, molecular techniques are suggested for diagnosis in suspected PJIs. The Broad-range PCR and High-Resolution Melt Analysis (HRMA) were evaluated for the identification of causative organisms of PJIs in this study.ResultsFor 47 of 63 specimens, both the culture and broad-range PCR were positive. The culture was found to be able of organism’s detection in 74.6% (47/63) of patients. Of 47 positive cultures, 11 (23.4%) were polymicrobial and 36 (76.59%) were monomicrobial cultures, in which 34 (91.89%) cases were detected by HRM assay. The sensitivity, specificity of HRMA vs monomicrobial culture were 91.89, 93.75%, respectively. The sensitivity, specificity of total HRMA (mono + poly) vs culture were 82.92, 93.75%.ConclusionsHRM assay coupled with broad-range PCR are effective screening, rapid, and relatively cost-effective methods for discrimination of PJIs especially in aiding culture method. Using computer programs such as the Matlab-2018b program for HRM data analysis is also valuable and helpful in diagnosis.

Evaluation of a Rapid and Simplified Protocol for Direct Identification of Microorganisms From Positive Blood Cultures by Using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

Early and rapid identification of microorganisms is critical for reducing the mortality rate caused by bloodstream infections (BSIs). The accuracy and feasibility of directly identifying pathogens in positive blood cultures by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been intensely confirmed. In this study, we combined density centrifugation and extra chemical lysis-extraction to develop an optimized method in the blood culture process, which significantly improved the effectiveness of direct identification by MALDI-TOF MS. The accuracy was evaluated by 2,032 positive blood culture samples (115 species of microorganism). The overall MALDI-TOF MS based identification rate with scores ≥ 1.700 was 87.60%. 94.06% of gram-negative bacteria were identified consistently to the genus level, followed by anaerobes (93.33%), gram-positive bacteria (84.46%), and fungi (60.87%). This protocol could obtain results within 10–20 min at a cost of less than $0.1 per sample, which saved up to 24 h in identifying 87.60% of the microorganism from positive blood cultures. This rapid and simplified protocol facilitates the direct identification of microorganism in positive blood cultures, and exhibits the advantages of cost-effective, time-saving, and easy-to-use. It could provide the causative organism of the patient to clinicians in time for targeted treatment and reduce mortality.

76215 Implementation of Proteomics as a Diagnostic tool for Nontuberculous mycobacteria (NTM) Infection

ABSTRACT IMPACT: Implementation of proteomics as a diagnostic tool for Nontuberculous mycobacteria (NTM) infection can provide a more accurate, efficient and cost-effective means for effectively diagnosing disease and enacting timely management decisions which can revolutionize patient care. OBJECTIVES/GOALS: Proteomic analysis is a proven diagnostic modality enabling rapid identification of microorganisms. We sought to apply proteomics to detect proteins unique to the most clinically relevant NTM. We then determined whether these unique proteomes could be used to successfully identify NTM species from in vitro cocktail preparations. METHODS/STUDY POPULATION: NTM reference strains for M. avium, m. intracellulare, m.chimaera, m. abscessus abscessus, m. abscessus massiliense and m. abscessus boletti were cultured in vitro and subjected to proteomic analysis using Liquid Chromatography tandem-Mass Spectrometry (LCMS). Tandem Mass Tag (TMT) data acquisition utilized an MS3 approach for data collection using Proteome Discoverer 2.4.A comparative analysis of the proteome of each of these six species was performed quantitatively using LCMS. The process was repeated for three technical replicates and analyzed using the SEQUEST algorithm. Only high scoring peptides were considered utilizing a false discovery rate (FDR) of 1%. Once species-specific proteins were identified, we validated detection in individual and mixed samples of the six reference strains. RESULTS/ANTICIPATED RESULTS: The proteomic profiling of the six NTM reference strains successfully demonstrated proteins unique to each of the MAC species and MABC subspecies. Proteomic MAC species analysis produced between 327 to 2,540 unique peptides for each of the 3 species. MABC proteomic analysis identified between 17-74 unique peptides for each of the 3 subspecies. Fifteen different mixed preparations of MAC and MABC were then subjected to LCMS analysis and compared against the proteome profiles already curated for the six strains. We accurately identified at least one NTM in the majority of the samples (10/15). In three samples (3/15), the NTM was not correctly identified; in two of the samples (2/15) we were unable to determine the identity of NTM within the preparation. Further database curation will be performed to hone these results. DISCUSSION/SIGNIFICANCE OF FINDINGS: Proteomic analysis of in vitro reference strains successfully demonstrated protein fingerprints specific to six common disease-causing strains of NTM. Such findings can be used to evaluate clinical samples enabling more efficient diagnostic specificity. Further research will focus on identification of NTM in sputum samples of infected patients.

Usefulness of the MALDI-TOF MS technology with membrane filter protocol for the rapid identification of microorganisms in perioperative drainage fluids of hepatobiliary pancreatic surgery.

Surgical site infections (SSIs) are significant and frequent perioperative complications, occurring due to the contamination of the surgical site. The late detection of SSIs, especially organ/space SSIs which are the more difficult to treat, often leads to severe complications. An effective method that can identify bacteria with a high accuracy, leading to the early detection of organ/space SSIs, is needed. Ninety-eight drainage fluid samples obtained from 22 patients with hepatobiliary pancreatic disease were analyzed to identify microorganisms using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) with a new membrane filtration protocol and rapid BACpro® pretreatment compared to sole rapid BACpro® pretreatment. The levels of detail of rapid BACpro® pretreatment with or without filtration were also evaluated for the accuracy of bacterial identification. We found that reliable scores for E. coli and E. faecalis were obtained by inoculation with 1.0 × 104 CFU/ml after preparation of the membrane filter with rapid BACpro®, indicating approximately 10-folds more sensitive compared to sole rapid BACpro® pretreatment in drainage fluid specimens. Among 60 bacterial positive colonies in drainage fluid specimens, the MALDI-TOF MS and the membrane filtration with rapid BACpro® identified 53 isolates (88.3%) with a significantly higher accuracy, compared to 25 isolates in the rapid BACpro® pretreatment group (41.7%) (p < 0.001). Among the 78 strains, 14 enteric Gram-negative bacteria (93.0%) and 55 Gram-positive cocci (87.3%) were correctly identified by the membrane filtration with rapid BACpro® with a high reliability. This novel protocol could identify bacterial species within 30 min, at $2-$3 per sample, thus leading to cost and time savings. MALDI-TOF MS with membrane filter and rapid BACpro® is a quick and reliable method for bacterial identification in drainage fluids. The shortened analysis time will enable earlier selection of suitable antibiotics for treatment of organ/space SSIs to improve patients' outcomes.

Application of MALDI-TOF MS for Rapid Identification of Microorganisms in Food Microbiology

Application of MALDI-TOF MS for Rapid Identification of Microorganisms in Food Microbiology

Implementation of MALDI-TOF Mass Spectrometry and Peak Analysis: Application to the Discrimination of Cryptococcus neoformans Species Complex and Their Interspecies Hybrids.

Matrix-assisted laser desorption–ionization/time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for the rapid identification of microorganisms. Although most bacteria, yeasts and filamentous fungi can be accurately identified with this method, some closely related species still represent a challenge for MALDI-TOF MS. In this study, two MALDI-TOF-based approaches were applied for discrimination at the species-level of isolates belonging to the Cryptococcus neoformans complex, previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region: (i) an expanded database was built with 26 isolates from the main Cryptococcus species found in our setting (C. neoformans, C. deneoformans and AFLP3 interspecies hybrids) and (ii) peak analysis and data modeling were applied to the protein spectra of the analyzed Cryptococcus isolates. The implementation of the in-house database did not allow for the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of supervised classifiers (partial least squares-discriminant analysis and support vector machine) in a two-step analysis allowed for the 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. In addition, PCA analysis prior to support vector machine (SVM) provided 98.45% correct discrimination of the three analyzed species in a one-step analysis. This novel method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS) is not suitable for rapid bacterial identification in peritoneal dialysis effluent.

Peritoneal dialysis (PD)-related peritonitis is a serious complication of PD, but routine microbiological culture is slow and could not identify the organism in 15% cases. We examine the accuracy of polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS), a PCR-based method developed for the direct detection of bacteria in blood, for rapid identification of microorganisms from PD effluent. We recruited 73 consecutive patients with PD-related peritonitis. Dialysis effluent was collected for routine bacterial culture, PCR/ESI-MS, and bacterial DNA quantification before initiation of antibiotic therapy. By digital PCR with universal bacterial primers, bacterial DNA was detectable in all PD effluent specimens. For the entire cohort, taking standard bacterial culture as the gold standard, the PCR/ESI-MS assay correctly identified 34.3% of the causative organisms, failed to identify any organism in 52.1% cases, and identified a different organism in 8.2% cases. For the 14 episodes of peritonitis that were culture negative by conventional bacterial culture, the PCR/ESI-MS assay identified an organism in only four cases. The detection rate of the IRIDICA BAC BSI assay was not affected by the use of biocompatible PD solution or concomitant exit-site infection. The PCR/ESI-MS assay could not identify the causative organism in over 50% of the PD effluent samples in patients with PD-related peritonitis and should be not used for such purpose. The reason for the poor performance needs further investigation.

Rapid identification of microorganisms from positive blood cultures in pediatric patients by MALDI-TOF MS: Sepsityper kit versus short-term subculture

Background and aimThe rapid diagnosis of bloodstream infection (BSI) often leads to better clinical outcomes. The present study was conducted to compare two rapid protocols (Sepsityper kit and short-term subculture) for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based identification of microorganisms from positive blood cultures in pediatric patients. MethodsThis study was conducted between May 1, 2018, and April 30, 2019, at a tertiary children's hospital in eastern China. Only monomicrobial blood cultures included in this study were used to conduct the Sepsityper kit protocol and short-term subculture protocol at the same time. ResultsIn total, 115 monomicrobial blood cultures were included in this study. For the Sepsityper kit protocol, 85.2% and 64.3% of microorganisms were correctly identified to the genus (score ≥ 1.700) and species levels (score ≥ 2.000), respectively. For the short-term subculture protocol, 89.6% and 70.4% of microorganisms were correctly identified to the genus and species levels, respectively. At the genus level (P = .321) or the species level (P = .325), there was no significant difference between the Sepsityper kit protocol and the short-term subculture protocol. Moreover, the short-term subculture protocol exhibited similar performance between Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB) (the genus level: 93.7% (GPB) versus 87.9% (GNB), P = .518; the species level: 68.4% (GPB) versus 81.8% (GNB), P = .147). In addition, the Sepsityper kit protocol exhibited similar performance between GPB and GNB at the genus level (86.1% (GPB) versus 84.8% (GNB), P = 1.000). However, the Sepsityper kit protocol exhibited better performance in GNB at the species level (58.2% (GPB) versus 81.8% (GNB), P = .017). The rates of yeast-like fungi were correctly identified to the genus level (0.0%) or the species level (0.0%) for short-term subculture protocol were significantly lower than those of other microorganisms (the genus level: 92.0%, P = .001; the species level: 72.3%, P = .024). However, a similar result of identification was not found using the Sepsityper kit protocol (the genus level: P = .384; the species level: P = .599). In addition, the two rapid protocols both exhibited better performance at the genus level when the time to positivity (TTP) of blood cultures <19 h (the Sepsityper kit protocol: 91.8% (TTP < 19 h) versus 77.8% (TTP ≥ 19 h), P = .034; the short-term subculture protocol: 95.1% (TTP < 19 h) versus 83.3% (TTP ≥ 19 h), P = .040). In addition, the two rapid protocols both exhibited better performance at the species level when the TTP of blood cultures was <19 h (the Sepsityper kit protocol: 78.7% (TTP < 19 h) versus 48.1% (TTP ≥ 19 h), P = .000; the short-term subculture protocol: 83.6% (TTP < 19 h) versus 55.6% (TTP ≥ 19 h), P = .001). ConclusionThe Sepsityper kit protocol and short-term subculture protocol are both reliable and rapid methods for the identification of most microorganisms from positive blood cultures in pediatric patients. The performance of these two rapid protocols is associated with the TTP of blood cultures.

Development of MALDI-TOF mass spectrometry for the identification of lice isolated from farm animals.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now routinely used for the rapid identification of microorganisms isolated from clinical samples and has been recently successfully applied to the identification of arthropods. In the present study, this proteomics tool was used to identify lice collected from livestock and poultry in Algeria. The MALDI-TOF MS spectra of 408 adult specimens were measured for 14 species, including Bovicola bovis, B. ovis, B. caprae, Haematopinus eurysternus, Linognathus africanus, L. vituli, Solenopotes capillatus, Menacanthus stramineus, Menopon gallinae, Chelopistes meleagridis, Goniocotes gallinae, Goniodes gigas, Lipeurus caponis and laboratory reared Pediculus humanus corporis. Good quality spectra were obtained for 305 samples. Spectral analysis revealed intra-species reproducibility and inter-species specificity that were consistent with the morphological classification. A blind test of 248 specimens was performed against the in-lab database upgraded with new spectra and validated using molecular tools. With identification percentages ranging from 76% to 100% alongside high identification scores (mean = 2.115), this study proposes MALDI-TOF MS as an effective tool for discriminating lice species.

A new culture-based method for rapid identification of microorganisms in polymicrobial blood cultures by MALDI-TOF MS

BackgroundThe application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to microbial identification has allowed the development of rapid methods for identification of microorganisms directly in positive, blood cultures (BCs). These methods can yield accurate results for monomicrobial BCs, but often fail to identify multiple microorganisms in polymicrobial BCs. The present study was aimed at establishing a rapid and simple method for identification of bacteria and yeast in polymicrobial BCs from patients with bloodstream infection.ResultsThe rapid method herein proposed is based on short-term culture in liquid media allowing selective growth of microorganisms recovered from polymicrobial BCs, followed by rapid identification by MALDI-TOF MS. To evaluate the accuracy of this method, 56 polymicrobial BCs were comparatively analyzed with the rapid and routine methods. The results showed concordant identification for both microbial species in 43/50 (86%) BCs containing two different microorganisms, and for two microbial species in six BCs containing more than two different species. Overall, 102/119 (85.7%) microorganisms were concordantly identified by the rapid and routine methods using a cut-off value of 1.700 for valid identification. The mean time to identification after BC positivity was about 4.2 h for streptococci/enterococci, 8.7 h for staphylococci, 11.1 h for Gram-negative bacteria, and 14.4 h for yeast, allowing a significant time saving compared to the routine method.ConclusionsThe proposed method allowed rapid and reliable microbial identification in polymicrobial BCs, and could provide clinicians with timely, useful information to streamline empirical antimicrobial therapy in critically ill patients.