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Although MALDI-TOF mass spectrometry (MS) is considered as the gold standard for rapid and cost-effective identification of microorganisms in routine laboratory practices, its capability for antimicrobial resistance (AMR) detection has received limited focus. Nevertheless, recent studies explored the predictive performance of MALDI-TOF MS for detecting AMR in clinical pathogens when machine learning techniques are applied. This chapter describes a routine MALDI-TOF MS workflow for the rapid screening of AMR in foodborne pathogens, with Campylobacter spp. as a study model.

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This research focused on distinguishing distinct matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral signatures of three Enterococcus species. We evaluated and compared the predictive performance of four supervised machine learning algorithms, K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF), to accurately classify Enterococcus species. This study involved a comprehensive dataset of 410 strains, generating 1640 individual spectra through on-plate and off-plate protein extraction methods. Although the commercial database correctly identified 76.9% of the strains, machine learning classifiers demonstrated superior performance (accuracy 0.991). In the RF model, top informative peaks played a significant role in the classification. Whole-genome sequencing showed that the most informative peaks are biomarkers connected to proteins, which are essential for understanding bacterial classification and evolution. The integration of MALDI-TOF MS and machine learning provides a rapid and accurate method for identifying Enterococcus species, improving healthcare and food safety.

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BACKGROUND: Patient-derived organoids (PDOs) are multi-cellular cultures with specific three-dimensional (3D) structures. Tumor organoids (TOs) offer a personalized perspective for assessing treatment response. However, the presence of normal organoid (NO) residuals poses a potential threat to their utility for personalized medicine. There is a crucial need for an effective platform capable of distinguishing between TO and NO in cancer organoid cultures. RESULTS: We introduced a whole-mount (WM) preparation protocol for in-situ visualization of the lipidomic distribution of organoids. To assess the efficacy of this method, nine breast cancer organoids (BCOs) and six normal breast organoids (NBOs) were analyzed. Poly-l-lysine (PLL) coated slides, equipped with 12 well chambers, were utilized as a carrier for the high-throughput analysis of PDOs. Optimizing the fixation time to 30 min, preserved the integrity of organoids and the fidelity of lipid compounds. The PDOs derived from the same organoid lines exhibited similar lipidomic profiles. BCOs and NBOs were obviously distinguished based on their lipidomic signatures detected by WM autofocusing (AF) scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spectrometry imaging (MSI). SIGNIFICANCE: A whole-mount (WM) preparation protocol was developed to visualize lipidomic distributions of the organoids' surface. Using poly-l-lysine coated slides for high-throughput analysis, the method preserved organoid integrity and distinguished breast cancer organoids (BCOs) from normal breast organoids (NBOs) based on their unique lipidomic profiles using autofocusing scanning microprobe matrix-assisted laser desorption/ionization (SMALDI) mass spectrometry imaging.

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We evaluated the performance of Zybio EXS2600 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Zybio Inc., Chongqing, China) for the identification of bacteria from positive blood culture (BC) bottles using Blood Culture Positive Sample Pretreatment Kit (Zybio Inc., Chongqing, China) in comparison to an in-house saponin method. Following a positive signal by the BACTEC™ FX system, confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 94 positive BC bottles with 97 bacterial isolates were analyzed. The overall identification rates at the genus and species levels for the saponin method were 89.7% (87/97) and 74.2% (72/97), respectively. With the Zybio Kit, 88.7% (86/97) and 80.4% (78/97) of microorganisms were correctly identified to the genus and species levels, respectively. The saponin method identified 65.3% (32/49) of Gram-positive bacteria at the species level, whereas the Zybio Kit achieved a higher species-level identification rate of 79.6% (39/49) (p = 0.1153). The saponin method with additional on-plate formic acid extraction showed a significantly higher overall identification rate in comparison to the saponin method without that step for both genus (87.6% [85/97] vs. 70.1% [68/97], p = 0.0029) and species level (70.1% [68/97] vs. 46.4% [45/97], p = 0.0008). Identification rates of Gram-negative bacteria showed a higher identification rate, however, not statistically significant with additional Zybio Kit protocol step on both genus (85.4% [41/48] vs. 81.3% [39/48], p = 0.5858) and species level (77.1% [37/48] vs. 75% [36/48], p = 0.8120). Zybio Kit could offer an advantage in species-level identification, particularly for Gram-positive bacteria. The inclusion of on-plate formic acid extraction in the saponin method notably enhanced identification at both genus and species levels for Gram-positive bacteria. The extended protocol provided by the Zybio Kit could potentially offer an advantage in the identification of Gram-negative bacteria at both genus and species levels. Enhancements to the Zybio EXS2600 MALDI-TOF instrument software database are necessary.

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Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.

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PURPOSE: This study aimed to verify that aerosolization ocular surface microorganisms (AOSMs) accumulated during non-contact tonometry (NCT) measurements. METHODS: A total of 508 participants (740 eyes) were enrolled in the study. In Experiment 1, before NCT was performed on each eye, the air was disinfected, and environment air control samples were collected via Air ideal® 3P (Bio Merieux). During NCT measurements, microbial aerosol samples were collected once from each eye. In Experiment 2, we collected initial blank control samples and then repeated Experiment 1. Finally, in Experiment 3, after the background microbial aerosol investigation, we cumulatively sampled AOSMs from each 10 participants then culture once, without any interventions to interrupt the accumulation. The collected samples were incubated and identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: Pathogenic Aerococcus viridans and other microorganisms from human eyes can spread and accumulate in the air during NCT measurements. The species and quantity of AOSMs produced by NCT measurements can demonstrate an accumulation effect. CONCLUSION: AOSMs generated during NCT measurements are highly likely to spread and accumulate in the air, thereby may increase the risk of exposure to and transmission of bio-aerosols.

Pathogenic Aerococcus viridans and other species of aerosolization ocular surface microorganisms (AOSMs) can spread and accumulate with the increase of NCT measurement person times, demonstrating an accumulation effect.

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BACKGROUND: Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. METHODS: The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. RESULTS: A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. CONCLUSION: MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.

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Proteins, saccharides, and low molecular organic compounds in the blood, urine, and saliva could potentially serve as biomarkers for diseases related to diet, lifestyle, and the use of illegal drugs. Lifestyle-related diseases (LSRDs) such as diabetes mellitus (DM), non-alcoholic steatohepatitis, cardiovascular disease, hypertension, kidney disease, and osteoporosis could develop into life-threatening conditions. Therefore, there is an urgent need to develop biomarkers for their early diagnosis. Advanced glycation end-products (AGEs) are associated with LSRDs and may induce/promote LSRDs. The presence of AGEs in body fluids could represent a biomarker of LSRDs. Urine samples could potentially be used for detecting AGEs, as urine collection is convenient and non-invasive. However, the detection and identification of AGE-modified proteins in the urine could be challenging, as their concentrations in the urine might be extremely low. To address this issue, we propose a new analytical approach. This strategy employs a method previously introduced by us, which combines slot blotting, our unique lysis buffer named Takata's lysis buffer, and a polyvinylidene difluoride membrane, in conjunction with electrospray ionization-mass spectrometry (ESI)/matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). This novel strategy could be used to detect AGE-modified proteins, AGE-modified peptides, and free-type AGEs in urine samples.

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Mycobacterium abscessus (MABS) displays differential subspecies susceptibility to macrolides. Thus, identifying MABS's subspecies (M. abscessus, M. bolletii and M. massiliense) is a clinical necessity for guiding treatment decisions. We aimed to assess the potential of Machine Learning (ML)-based classifiers coupled to Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) MS to identify MABS subspecies. Two spectral databases were created by using 40 confirmed MABS strains. Spectra were obtained by using MALDI-TOF MS from strains cultivated on solid (Columbia Blood Agar, CBA) or liquid (MGIT®) media for 1 to 13 days. Each database was divided into a dataset for ML-based pipeline development and a dataset to assess the performance. An in-house programme was developed to identify discriminant peaks specific to each subspecies. The peak-based approach successfully distinguished M. massiliense from the other subspecies for strains grown on CBA. The ML approach achieved 100% accuracy for subspecies identification on CBA, falling to 77.5% on MGIT®. This study validates the usefulness of ML, in particular the Random Forest algorithm, to discriminate MABS subspecies by MALDI-TOF MS. However, identification in MGIT®, a medium largely used in mycobacteriology laboratories, is not yet reliable and should be a development priority.

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In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

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Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Mugla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.

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In this study, a comprehensive investigation was undertaken to elucidate a simple triazole compound, 5-phenyl-1-(p-tolyl)-1 H-1,2,3-triazole (PPTT), its interactions with high-abundant proteins and identification of low-abundant proteins by serum proteomics. Employing a combination of spectroscopic techniques and computational chemistry, the interactions between PPTT and three high-abundance blood globular proteins, namely human serum albumin (HSA), human immunoglobulin G (HIgG), and hemoglobin (BHb), were explored, thereby ascertaining their binding constants and thermodynamic parameters at the molecular level. Subsequently, based on the differential proteomics, utilizing two-dimensional gel electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), the research team isolated and identified differentially expressed low-abundance proteins in human blood serum samples following exposure to PPTT. The results showed that there were twenty highly expressed proteins identified from blood serum samples intervened by PPTT. Combining bioinformatics techniques, these proteins were classified, providing preliminary insights like preproprotein or precursors inhibiting the activity of elastase, defending and regulating the immune system, carrying lipid, and other functions into their biological functionalities. One of the differential proteins, apolipoprotein A-1 (ApoA-1) protein, was selected as a possible target to explore the mechanism of action of PPTT intervention on the related signaling pathways involved in human hepatocellular carcinomas(Hep G2) cells. These research findings offer scientifically sound guidance for further in-depth exploration, development, and application of the 1,2,3-triazole compound.

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Pinelliae Rhizoma (PR), a highly esteemed traditional Chinese medicinal herb, is widely applied in clinical settings due to its diverse pharmacological effects, including antitussive, expectorant, antiemetic, sedative-hypnotic, and antitumor activities. Pinellia ternata exhibits morphological variation in its leaves, with types resembling peach, bamboo, and willow leaves. However, the chemical composition differences among the corresponding rhizomes of these leaf phenotypes remain unelucidated. This pioneering research employed Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) to conduct the in situ identification and spatial profiling of 35 PR metabolites in PR, comprising 12 alkaloids, 4 organic acids, 12 amino acids, 5 flavonoids, 1 sterol, and 1 anthraquinone. Our findings revealed distinct spatial distribution patterns of secondary metabolites within the rhizome tissues of varying leaf types. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) effectively differentiated between rhizomes associated with different leaf morphologies. Furthermore, this study identified five potential differential biomarkers-methylophiopogonanone B, inosine, cytidine, adenine, and leucine/isoleucine-that elucidate the biochemical distinctions among leaf types. The precise tissue-specific localization of these secondary metabolites offers compelling insights into the specialized accumulation of bioactive compounds in medicinal plants, thereby enhancing our comprehension of PR's therapeutic potential.

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In this investigation, we detail the synthesis of silver nanoparticles (AgNPs) via a precise chemical vacuum deposition (CVD) methodology, aimed at augmenting the analytical performance of laser desorption/ionization mass spectrometry (LDI-MS) for the detection of low-molecular-weight analytes. Employing a precursor supply rate of 0.0014 mg/s facilitated the formation of uniformly dispersed AgNPs, characterized by SEM and AFM to have an average diameter of 33.5 ± 1.5 nm and a surface roughness (Ra) of 11.8 nm, indicative of their homogeneous coverage and spherical morphology. XPS and SEM-EDX analyses confirmed the metallic silver composition of the nanoparticles with Ag peak splitting, reflecting the successful synthesis of metallic Ag. Comparative analytical evaluation with traditional MALDI matrices revealed that AgNPs significantly reduce signal suppression, thereby enhancing the sensitivity and specificity of LDI-MS for low-molecular-weight compounds such as triglycerides, saccharides, amino acids, and carboxylic acids. Notably, the application of AgNPs demonstrated a superior linear response for triglyceride signals with regression coefficients surpassing 0.99, markedly outperforming conventional matrices. The study further extends into quantitative analysis through nanoparticle-based laser desorption/ionization (NALDI), where AgNPs exhibited enhanced ionization efficiency, characterized by substantially lower limits of detection (LOD) and quantification (LOQ) for tested standards. Particular attention was paid to lipids with a detailed examination of their fragmentation pathways. These results highlight the significant potential of AgNPs synthesized via CVD to transform the analytical detection and quantification of low-molecular-weight compounds using NALDI. This approach offers a promising avenue for expanding the scope of analytical applications in mass spectrometry and introducing innovative methodologies for enhanced precision and sensitivity.

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In the past few years, mass spectrometry imaging (MSI) has brought many new inspirations to plant research. However, current MSI experiments usually include only a single batch of samples, casting doubts on the reproducibility of phytochemical distribution across different batches. Consequently, MSI has seldom been applied to conduct species discrimination. In this experiment, MSI was employed to discriminate between two taxonomically similar plants, Scutellaria baicalensis Georgi and Scutellaria rehderiana Diels. A new concept termed a "spatial marker" was proposed in this article, which referred to the phytochemical marker that presented both intraspecies similarity and interspecies dissimilarity. Multiple batches of S. baicalensis and S. rehderiana were analyzed using MSI, proving that the authentication protocol using spatial markers was reliable and reproducible. The observed spatial markers were further identified using on-tissue tandem mass spectrometry and liquid chromatography coupled with mass spectrometry. Additionally, the spectral data collected from MSI were utilized to set up algorithm models for species discrimination. External validation confirmed that the established random forest model was extrapolated well to unknown samples. Overall, this investigation successfully explored the analytical applicability of MSI, facilitating rapid authentication of medicinal plants.

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Hard ticks (Acari: Ixodidae) have been historically identified by morphological methods which require highly specialized expertise and more recently by DNA-based molecular assays that involve high costs. Although both approaches provide complementary data for tick identification, each method has limitations which restrict their use on large-scale settings such as regional or national tick surveillance programs. To overcome those obstacles, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced as a cost-efficient method for the identification of various organisms, as it balances performance, speed, and high data output. Here we describe the use of this technology to validate the distinction of two closely related Dermacentor tick species based on the development of the first nationwide MALDI-TOF MS reference database described to date. The dataset obtained from this protein-based approach confirms that tick specimens collected from United States regions west of the Rocky Mountains and identified previously as Dermacentor variabilis are the recently described species, Dermacentor similis. Therefore, we propose that this integrative taxonomic tool can facilitate vector and vector-borne pathogen surveillance programs in the United States and elsewhere.

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Mesenchymal stem/stromal cell (MSC) therapies have had limited success so far in clinical trials due in part to heterogeneity in immune-responsive phenotypes. Therefore, techniques to characterize these properties of MSCs are needed during biomanufacturing. Imaging cell shape, or morphology, has been found to be associated with MSC immune responsivity-but a direct relationship between single-cell morphology and function has not been established. We used label-free differential phase contrast imaging and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to evaluate single-cell morphology and explore relationships with lipid metabolic immune response. In interferon gamma (IFN-γ)-stimulated MSCs, we found higher lipid abundances from the ceramide-1-phosphate (C1P), phosphatidylcholine (PC), LysoPC, and triglyceride (TAG) families that are involved in cell immune function. Furthermore, we identified differences in lipid signatures in morphologically defined MSC subpopulations. The use of single-cell optical imaging coupled with single-cell spatial lipidomics could assist in optimizing the MSC production process and improve mechanistic understanding of manufacturing process effects on MSC immune activity and heterogeneity.

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Azoospermia, the absence of sperm cells in semen, affects around 15% of infertile males. Sertoli cell-only syndrome (SCOS) is the most common pathological lesion in the background of non-obstructive azoospermia and is characterised by the complete absence of germinal epithelium, with Sertoli cells exclusively present in the seminiferous tubules. Studies have shown a correlation between successful spermatogenesis and male fertility with lipid composition of spermatozoa, semen, seminal plasma or testis. The aim of this research was to discover the correlation between the Johnsen scoring system and phospholipid expressions in testicular cryosections of SCOS patients. MALDI imaging mass spectrometry is used to determine spatial distributions of molecular species, such as phospholipids. Phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and sphingomyelins (SMs) are the most abundant phospholipids in mammalian cells and testis. SMs, the structural components of plasma membranes, are crucial for spermatogenesis and sperm function. Plasmalogens, are unique PCs in testis with strong antioxidative properties. This study, using imaging mass spectrometry, demonstrates the local distribution of phospholipids, particularly SMs, PCs, plasmalogens and PEs in human testicular samples with SCOS for the first time. This study found a strong relationship between the Johnsen scoring system and phospholipid expression levels in human testicular tissues. Future findings could enable routine diagnostic techniques during microTESE procedures for successful sperm extraction.

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BACKGROUND: The increased resistance rate of Salmonella to third-generation cephalosporins represented by ceftriaxone (CRO) may result in the failure of the empirical use of third-generation cephalosporins for the treatment of Salmonella infection in children. The present study was conducted to evaluate a novel method for the rapid detection of CRO-resistant Salmonella (CRS). METHODS: We introduced the concept of the ratio of optical density (ROD) with and without CRO and combined it with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) to establish a new protocol for the rapid detection of CRS. RESULTS: The optimal incubation time and CRO concentration determined by the model strain test were 2 h and 8 µg/ml, respectively. We then conducted confirmatory tests on 120 clinical strains. According to the receiver operating characteristic curve analysis, the ROD cutoff value for distinguishing CRS and non-CRS strains was 0.818 [area under the curve: 1.000; 95% confidence interval: 0.970-1.000; sensitivity: 100.00%; specificity: 100%; P < 10- 3]. CONCLUSIONS: In conclusion, the protocol for the combined ROD and MALDI-TOF MS represents a rapid, accurate, and economical method for the detection of CRS.

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To evaluate the diagnostic accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry based on nucleotide (nucleotide MALDI-TOF MS) on bronchoalveolar lavage fluid (BALF) from suspected pulmonary tuberculosis (PTB) patients. A retrospective study was conducted on suspected PTB patients (total of 960) admitted to Chongqing Public Health Medical Center between May 2021 and January 2022. The sensitivity, specificity, positive predictive value, negative predictive value (NPV) and area under the curve values of nucleotide MALDI-TOF MS as well as smear microscopy, Mycobacterium Growth Indicator Tube 960 culture (MGIT culture), and Xpert MTB/RIF were calculated and compared. Total of 343 presumed PTB cases were enrolled. Overall, using the clinical diagnosis as reference, the sensitivity and NPV of nucleotide MALDI-TOF MS was 71.5% and 43.1%, respectively, significantly higher than smear microscopy (22.6%, 23.2%), MGIT culture (40.6%, 18.9%), Xpert MTB/RIF (40.8%, 27.9%). Furthermore, nucleotide MALDI-TOF MS also outperformed over Xpert MTB/RIF and MGIT culture on smear-negative BALFs. Approximately 50% and 30% of patients benefited from nucleotide MALDI-TOF MS compared with smear and MGIT culture or Xpert MTB/RIF, respectively. This study demonstrated that the analysis of BALF with nucleotide MALDI-TOF MS provided an accurate and promising tool for the early diagnosis of PTB.

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