Practical Guidance for Clinical Microbiology Laboratories: Updated guidance for processing respiratory tract samples from people with cystic fibrosis.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus, although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices.

Advances in mass spectrometry imaging for plant metabolomics-Expanding the analytical toolbox.

Mass spectrometry imaging (MSI) has become increasingly popular in plant science due to its ability to characterize complex chemical, spatial, and temporal aspects of plant metabolism. Over the past decade, as the emerging and unique features of various MSI techniques have continued to support new discoveries in studies of plant metabolism closely associated with various aspects of plant function and physiology, spatial metabolomics based on MSI techniques has positioned it at the forefront of plant metabolic studies, providing the opportunity for far higher resolution than was previously available. Despite these efforts, profound challenges at the levels of spatial resolution, sensitivity, quantitative ability, chemical confidence, isomer discrimination, and spatial multi-omics integration, undoubtedly remain. In this Perspective, we provide a contemporary overview of the emergent MSI techniques widely used in the plant sciences, with particular emphasis on recent advances in methodological breakthroughs. Having established the detailed context of MSI, we outline both the golden opportunities and key challenges currently facing plant metabolomics, presenting our vision as to how the enormous potential of MSI technologies will contribute to progress in plant science in the coming years.

A risk assessment framework for multidrug-resistant Staphylococcus aureus using machine learning and mass spectrometry technology.

The emergence of multidrug-resistant bacteria is a critical global crisis that poses a serious threat to public health, particularly with the rise of multidrug-resistant Staphylococcus aureus. Accurate assessment of drug resistance is essential for appropriate treatment and prevention of transmission of these deadly pathogens. Early detection of drug resistance in patients is critical for providing timely treatment and reducing the spread of multidrug-resistant bacteria. This study aims to develop a novel risk assessment framework for S. aureus that can accurately determine the resistance to multiple antibiotics. The comprehensive 7-year study involved ˃20 000 isolates with susceptibility testing profiles of six antibiotics. By incorporating mass spectrometry and machine learning, the study was able to predict the susceptibility to four different antibiotics with high accuracy. To validate the accuracy of our models, we externally tested on an independent cohort and achieved impressive results with an area under the receiver operating characteristic curve of 0. 94, 0.90, 0.86 and 0.91, and an area under the precision-recall curve of 0.93, 0.87, 0.87 and 0.81, respectively, for oxacillin, clindamycin, erythromycin and trimethoprim-sulfamethoxazole. In addition, the framework evaluated the level of multidrug resistance of the isolates by using the predicted drug resistance probabilities, interpreting them in the context of a multidrug resistance risk score and analyzing the performance contribution of different sample groups. The results of this study provide an efficient method for early antibiotic decision-making and a better understanding of the multidrug resistance risk of S. aureus.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2021-2022.

The use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of carbohydrates and glycoconjugates is a well-established technique and this review is the 12th update of the original article published in 1999 and brings coverage of the literature to the end of 2022. As with previous review, this review also includes a few papers that describe methods appropriate to analysis by MALDI, such as sample preparation, even though the ionization method is not MALDI. The review follows the same format as previous reviews. It is divided into three sections: (1) general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, quantification and the use of computer software for structural identification. (2) Applications to various structural types such as oligo- and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals, and (3) other general areas such as medicine, industrial processes, natural products and glycan synthesis where MALDI is extensively used. Much of the material relating to applications is presented in tabular form. MALDI is still an ideal technique for carbohydrate analysis, particularly in its ability to produce single ions from each analyte and advancements in the technique and range of applications show little sign of diminishing.

Metabolic insights from mass spectrometry imaging of biofilms: A perspective from model microorganisms.

Biofilms are dense aggregates of bacterial colonies embedded inside a self-produced polymeric matrix. Biofilms have received increasing attention in medical, industrial, and environmental settings due to their enhanced survival. Their characterization using microscopy techniques has revealed the presence of structural and cellular heterogeneity in many bacterial systems. However, these techniques provide limited chemical detail and lack information about the molecules important for bacterial communication and virulence. Mass spectrometry imaging (MSI) bridges the gap by generating spatial chemical information with unmatched chemical detail, making it an irreplaceable analytical platform in the multi-modal imaging of biofilms. In the last two decades, over 30 species of biofilm-forming bacteria have been studied using MSI in different environments. The literature conveys both analytical advancements and an improved understanding of the effects of environmental variables such as host surface characteristics, antibiotics, and other species of microorganisms on biofilms. This review summarizes the insights from frequently studied model microorganisms. We share a detailed list of organism-wide metabolites, commonly observed mass spectral adducts, culture conditions, strains of bacteria, substrate, broad problem definition, and details of the MS instrumentation, such as ionization sources and matrix, to facilitate future studies. We also compared the spatial characteristics of the secretome under different study designs to highlight changes because of various environmental influences. In addition, we highlight the current limitations of MSI in relation to biofilm characterization to enable cross-comparison between experiments. Overall, MSI has emerged to become an important approach for the spatial/chemical characterization of bacterial biofilms and its use will continue to grow as MSI becomes more accessible.

Prospective on Imaging Mass Spectrometry in Clinical Diagnostics.

Imaging mass spectrometry (IMS) is a molecular technology utilized for spatially driven research, providing molecular maps from tissue sections. This article reviews matrix-assisted laser desorption ionization (MALDI) IMS and its progress as a primary tool in the clinical laboratory. MALDI mass spectrometry has been used to classify bacteria and perform other bulk analyses for plate-based assays for many years. However, the clinical application of spatial data within a tissue biopsy for diagnoses and prognoses is still an emerging opportunity in molecular diagnostics. This work considers spatially driven mass spectrometry approaches for clinical diagnostics and addresses aspects of new imaging-based assays that include analyte selection, quality control/assurance metrics, data reproducibility, data classification, and data scoring. It is necessary to implement these tasks for the rigorous translation of IMS to the clinical laboratory; however, this requires detailed standardized protocols for introducing IMS into the clinical laboratory to deliver reliable and reproducible results that inform and guide patient care.

A primer for ZooMS applications in archaeology.

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

Mass spectrometry imaging to explore molecular heterogeneity in cell culture.

Molecular analysis on the single-cell level represents a rapidly growing field in the life sciences. While bulk analysis from a pool of cells provides a general molecular profile, it is blind to heterogeneities between individual cells. This heterogeneity, however, is an inherent property of every cell population. Its analysis is fundamental to understanding the development, function, and role of specific cells of the same genotype that display different phenotypical properties. Single-cell mass spectrometry (MS) aims to provide broad molecular information for a significantly large number of cells to help decipher cellular heterogeneity using statistical analysis. Here, we present a sensitive approach to single-cell MS based on high-resolution MALDI-2-MS imaging in combination with MALDI-compatible staining and use of optical microscopy. Our approach allowed analyzing large amounts of unperturbed cells directly from the growth chamber. Confident coregistration of both modalities enabled a reliable compilation of single-cell mass spectra and a straightforward inclusion of optical as well as mass spectrometric features in the interpretation of data. The resulting multimodal datasets permit the use of various statistical methods like machine learning-driven classification and multivariate analysis based on molecular profile and establish a direct connection of MS data with microscopy information of individual cells. Displaying data in the form of histograms for individual signal intensities helps to investigate heterogeneous expression of specific lipids within the cell culture and to identify subpopulations intuitively. Ultimately, t-MALDI-2-MSI measurements at 2-µm pixel sizes deliver a glimpse of intracellular lipid distributions and reveal molecular profiles for subcellular domains.

Identification of the native Torpedo californica nicotinic acetylcholine receptor's glycan composition after a multi-step sequential purification method using MALDI-ToF MS.

The Cys-loop pentameric ligand-gated ion channels comprise a dynamic group of proteins that have been extensively studied for decades, yielding a wealth of findings at both the structural and functional levels. The nicotinic acetylcholine receptor (nAChR) is no exception, as it is part of this large protein family involved in proper organismal function. Our efforts have successfully produced a highly pure nAChR in detergent complex (nAChR-DC), enabling more robust studies to be conducted on it, including beginning to experiment with high-throughput crystallization. Our homogeneous product has been identified and extensively characterized with 100% identity using Nano Lc MS/MS and MALDI ToF/ToF for each nAChR subunit. Additionally, the N-linked glycans in the Torpedo californica-nAChR (Tc-nAChR) subunits have been identified. To study this, the Tc-nAChR subunits were digested with PNGase F and the released glycans were analyzed by MALDI-ToF. The MS results showed the presence of high-mannose N-glycan in all native Tc-nAChR subunits. Specifically, the oligommanose population Man8-9GlcNac2 with peaks at m/z 1742 and 1904 ([M + Na]+ ions) were observed.

Advancing rare cancer research by MALDI mass spectrometry imaging: Applications, challenges, and future perspectives in sarcoma.

MALDI mass spectrometry imaging (MALDI imaging) uniquely advances cancer research, by measuring spatial distribution of endogenous and exogenous molecules directly from tissue sections. These molecular maps provide valuable insights into basic and translational cancer research, including tumor biology, tumor microenvironment, biomarker identification, drug treatment, and patient stratification. Despite its advantages, MALDI imaging is underutilized in studying rare cancers. Sarcomas, a group of malignant mesenchymal tumors, pose unique challenges in medical research due to their complex heterogeneity and low incidence, resulting in understudied subtypes with suboptimal management and outcomes. In this review, we explore the applicability of MALDI imaging in sarcoma research, showcasing its value in understanding this highly heterogeneous and challenging rare cancer. We summarize all MALDI imaging studies in sarcoma to date, highlight their impact on key research fields, including molecular signatures, cancer heterogeneity, and drug studies. We address specific challenges encountered when employing MALDI imaging for sarcomas, and propose solutions, such as using formalin-fixed paraffin-embedded tissues, and multiplexed experiments, and considerations for multi-site studies and digital data sharing practices. Through this review, we aim to spark collaboration between MALDI imaging researchers and clinical colleagues, to deploy the unique capabilities of MALDI imaging in the context of sarcoma.

Spatial Omics Reveals that Cancer-Associated Glycan Changes Occur Early in Liver Disease Development in a Western Diet Mouse Model of MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disease and comprises different stages of liver damage; it is significantly associated with obese and overweight patients. Untreated MASLD can progress to life-threatening end-stage conditions, such as cirrhosis and liver cancer. N-Linked glycosylation is one of the most common post-translational modifications in the cell surface and secreted proteins. N-Linked glycan alterations have been established to be signatures of liver diseases. However, the N-linked glycan changes during the progression of MASLD to liver cancer are still unknown. Here, we induced different stages of MASLD in mice and liver-cancer-related phenotypes and elucidated the N-glycome profile during the progression of MASLD by quantitative and qualitative profiling in situ using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Importantly, we identified specific N-glycan structures including fucosylated and highly branched N-linked glycans at very early stages of liver injury (steatosis), which in humans are associated with cancer development, establishing the importance of these modifications with disease progression. Finally, we report that N-linked glycan alterations can be observed in our models by MALDI-IMS before liver injury is identified by histological analysis. Overall, we propose these findings as promising biomarkers for the early diagnosis of liver injury in MASLD.

Feasibility of MALDI-MSI-Based Proteomics Using Bouin-Fixed Pathology Samples: Untapping the Goldmine of Nephropathology Archives.

The application of innovative spatial proteomics techniques, such as those based upon matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technology, has the potential to impact research in the field of nephropathology. Notwithstanding, the possibility to apply this technology in more routine diagnostic contexts remains limited by the alternative fixatives employed by this ultraspecialized diagnostic field, where most nephropathology laboratories worldwide use bouin-fixed paraffin-embedded (BFPE) samples. Here, the feasibility of performing MALDI-MSI on BFPE renal tissue is explored, evaluating variability within the trypsin-digested proteome as a result of different preanalytical conditions and comparing them with the more standardized formalin-fixed paraffin-embedded (FFPE) counterparts. A large proportion of the features (270, 68.9%) was detected in both BFPE and FFPE renal samples, demonstrating only limited variability in signal intensity (10.22-10.06%). Samples processed with either fixative were able to discriminate the principal parenchyma regions along with diverse renal substructures, such as glomeruli, tubules, and vessels. This was observed when performing an additional "stress test", showing comparable results in both BFPE and FFPE samples when the distribution of several amyloid fingerprint proteins was mapped. These results suggest the utility of BFPE tissue specimens in MSI-based nephropathology research, further widening their application in this field.

Low-Invasive Sampling Method with Tape-Disc Sampling for the Taxonomic Identification of Archeological and Paleontological Bones by Proteomics.

Collagen from paleontological bones is an important organic material for isotopic measurement, radiocarbon analysis, and paleoproteomic analysis to provide information on diet, dating, taxonomy, and phylogeny. Current paleoproteomic methods are destructive and require from a few milligrams to several tens of milligrams of bone for analysis. In many cultures, bones are raw materials for artifacts that are conserved in museums, which hampers damage to these precious objects during sampling. Here, we describe a low-invasive sampling method that identifies collagen, taxonomy, and post-translational modifications from Holocene and Upper Pleistocene bones dated to 130,000 and 150 BC using dermatological skin tape discs for sampling. The sampled bone micropowders were digested following our highly optimized enhanced filter-aided sample preparation protocol and then analyzed by MALDI FTICR MS and LC-MS/MS for identifying the genus taxa of the bones. We show that this low-invasive sampling does not deteriorate the bones and achieves results similar to those obtained by more destructive sampling. Moreover, this sampling method can be carried out at archeological sites or in museums.

N-glycan antennal modifications are altered in Caenorhabditis elegans lacking the HEX-4 N-acetylgalactosamine-specific hexosaminidase.

Simple organisms are often considered to have simple glycomes, but plentiful paucimannosidic and oligomannosidic glycans overshadow the less abundant N-glycans with highly variable core and antennal modifications; Caenorhabditis elegans is no exception. By use of optimized fractionation and assessing wildtype in comparison to mutant strains lacking either the HEX-4 or HEX-5 ß-N-acetylgalactosaminidases, we conclude that the model nematode has a total N-glycomic potential of 300 verified isomers. Three pools of glycans were analyzed for each strain: either PNGase F released and eluted from a reversed-phase C18 resin with either water or 15% methanol or PNGase Ar released. While the water-eluted fractions were dominated by typical paucimannosidic and oligomannosidic glycans and the PNGase Ar-released pools by glycans with various core modifications, the methanol-eluted fractions contained a huge range of phosphorylcholine-modified structures with up to three antennae, sometimes with four N-acetylhexosamine residues in series. There were no major differences between the C. elegans wildtype and hex-5 mutant strains, but the hex-4 mutant strains displayed altered sets of methanol-eluted and PNGase Ar-released pools. In keeping with the specificity of HEX-4, there were more glycans capped with N-acetylgalactosamine in the hex-4 mutants, as compared with isomeric chito-oligomer motifs in the wildtype. Considering that fluorescence microscopy showed that a HEX-4::enhanced GFP fusion protein colocalizes with a Golgi tracker, we conclude that HEX-4 plays a significant role in late-stage Golgi processing of N-glycans in C. elegans. Furthermore, finding more "parasite-like" structures in the model worm may facilitate discovery of glycan-processing enzymes occurring in other nematodes.

Hakai, a novel Runx2 interacting protein, augments osteoblast differentiation by rescuing Runx2 from Smurf2-mediated proteasome degradation.

Runt-related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2-deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown-based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme-like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)-mediated proteasome degradation. Wild-type Hakai but not Hakai-C109A inhibited Smurf2 protein levels through proteasome-mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2-mediated ubiquitin-proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis.

Proteome and Peptidome Changes and Zn Concentration in Chicken after In Ovo Stimulation with a Multi-Strain Probiotic and Zn-Gly Chelate: Preliminary Research.

The aim of the study was to determine differences in the proteome and peptidome and zinc concentrations in the serum and tissues of chickens supplemented with a multi-strain probiotic and/or zinc glycine chelate in ovo. A total of 1400 fertilized broiler eggs (Ross × Ross 708) were divided into four groups: a control and experimental groups injected with a multi-strain probiotic, with zinc glycine chelate, and with the multi-strain probiotic and zinc glycine chelate. The proteome and peptidome were analyzed using SDS-PAGE and MALDI-TOF MS, and the zinc concentration was determined by flame atomic absorption spectrometry. We showed that in ovo supplementation with zinc glycine chelate increased the Zn concentration in the serum and yolk sac at 12 h post-hatch. The results of SDS-PAGE and western blot confirmed the presence of Cu/Zn SOD in the liver and in the small and large intestines at 12 h and at 7 days after hatching in all groups. Analysis of the MALDI-TOF MS spectra of chicken tissues showed in all experimental groups the expression of proteins and peptides that regulate immune response, metabolic processes, growth, development, and reproduction.

Characterisation of glucose-induced protein fragments among the order Enterobacterales using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

To characterise the glucose-induced protein fragments by MALDI-TOF MS analysis, we compared data for samples from Escherichia coli cultured in media with or without glucose. Characteristic peaks were observed in the presence of glucose, and MS/MS revealed Asr-specific fragments. The amino acid sequences of the fragments suggested sequence-specific proteolysis. Blast-analysis revealed that numerous Enterobacterales harbored genes encoding Asr as well as E. coli. Here, we analysed 32 strains from 20 genera and 25 species of seven Enterobacterales families. We did not detect changes in the mass spectra of four strains of Morganellaceae lacking asr, whereas peaks of Asr-specific fragments were detected in the other 28 strains. We therefore concluded that the induction of Asr production in the presence of glucose is common among the Enterobacterales, except for certain Morganellaceae species. In members of family Budviciaceae, unfragmented Asr was detected. Molecular genetic information suggested that the amino acid sequences of Asr homologs are diverse, with fragments varying in number and size, indicating that Asr may serve as a discriminative biomarker for identifying Enterobacterales species.

Comparative genomics of four lactic acid bacteria identified with Vitek MS (MALDI-TOF) and whole-genome sequencing.

Lactic acid bacteria (LAB) can be used as a probiotic or starter culture in dairy, meat, and vegetable fermentation. Therefore, their isolation and identification are essential. Recent advances in omics technologies and high-throughput sequencing have made the identification and characterization of bacteria. This study firstly aimed to demonstrate the sensitivity of the Vitek MS (MALDI-TOF) system in the identification of lactic acid bacteria and, secondly, to characterize bacteria using various bioinformatics approaches. Probiotic potency-related genes and secondary metabolite biosynthesis gene clusters were examined. The Vitek MS (MALDI-TOF) system was able to identify all of the bacteria at the genus level. According to whole genome sequencing, the bacteria were confirmed to be Lentilactobacillus buchneri, Levilactobacillus brevis, Lactiplantibacillus plantarum, Levilactobacillus namurensis. Bacteria had most of the probiotic potency-related genes, and different toxin-antitoxin systems such as PemIK/MazEF, Hig A/B, YdcE/YdcD, YefM/YoeB. Also, some of the secondary metabolite biosynthesis gene clusters, some toxic metabolite-related genes, and antibiotic resistance-related genes were detected. In addition, Lentilactobacillus buchneri Egmn17 had a type II-A CRISPR/Cas system. Lactiplantibacillus plantarum Gmze16 had a bacteriocin, plantaricin E/F.

Performance of MALDI-TOF MS, real-time PCR, antigen detection, and automated biochemical testing for the identification of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease highly endemic to Southeast Asia and northern Australia, though the area of endemicity is expanding. Cases may occur in returning travelers or, rarely, from imported contaminated products. Identification of B. pseudomallei is challenging for laboratories that do not see this organism frequently, and misidentifications by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and automated biochemical testing have been reported. The in vitro diagnostic database for use with the Vitek MS has recently been updated to include B. pseudomallei and we aimed to validate the performance for identification in comparison to automated biochemical testing with the Vitek 2 GN card, quantitative real-time polymerase chain reaction (qPCR) targeting the type III secretion system, and capsular polysaccharide antigen detection using a lateral flow immunoassay (LFA). We tested a "derivation" cohort including geographically diverse B. pseudomallei and a range of closely related Burkholderia species, and a prospective "validation" cohort of B. pseudomallei and B. cepacia complex clinical isolates. MALDI-TOF MS had a sensitivity of 1.0 and specificity of 1.0 for the identification and differentiation of B. pseudomallei from related Burkholderia species when a certainty cutoff of 99.9% was used. In contrast, automated biochemical testing for B. pseudomallei identification had a sensitivity of 0.83 and specificity of 0.88. Both qPCR and LFA correctly identified all B. pseudomallei isolates with no false positives. Due to the high level of accuracy, we have now incorporated MALDI-TOF MS into our laboratory's B. pseudomallei identification workflow.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a disease associated with high morbidity and mortality that disproportionately affects rural areas in Southeast Asia and northern Australia. The known area of endemicity is expanding and now includes the continental United States. Laboratory identification can be challenging which may result in missed or delayed diagnoses and poor patient outcomes. In this study, we compared mass spectrometry using an updated spectral database with multiple other methods for B. pseudomallei identification and found mass spectrometry highly accurate. We have therefore incorporated this fast and cost-effective method into our laboratory's workflow for B. pseudomallei identification.

Diagnostic performance of an automated robot for MALDI target preparation in microbial identification.

The MBT Pathfinder is an automated colony-picking robot designed for efficient sample preparation in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This article presents results from three key experiments evaluating the instrument's performance in conjunction with MALDI Biotyper instrument. The method comparison experiment assessed its clinical performance, demonstrating comparable results with gram-positive, gram-negative, and anaerobic bacteria (scores larger than 2.00) and superior performance over simple direct yeast transfer (score: 1.80) when compared to samples prepared manually. The repeatability experiment confirmed consistent performance over multiple days and labs (average log score: 2.12, std. deviation: 0.59). The challenge panel experiment showcased its consistent and accurate performance across various samples and settings, yielding average scores between 1.76 and 2.19. These findings underline the MBT Pathfinder as a reliable and efficient tool for MALDI-TOF mass spectrometry sample preparation in clinical and research applications.