Characterization of an outbreak caused by Elizabethkingia miricola using Fourier-transform infrared (FTIR) spectroscopy.

Fourier-transform infrared (FTIR) spectroscopy has the potential to be used for bacterial typing and outbreak characterization. We evaluated FTIR for the characterization of an outbreak caused by Elizabethkingia miricola. During the 2020-2021 period, 26 isolates (23 clinical and 3 environmental) were collected and analyzed by FTIR (IR Biotyper) and core-genome MLST (cgMLST), in addition to antimicrobial susceptibility testing. FTIR spectroscopy and cgMLST showed that 22 of the isolates were related to the outbreak, including the environmental samples, with only one discordance between both methods. Then, FTIR is useful for E. miricola typing and can be easily implemented in the laboratory.

Direct Detection of Carbapenemase-Producing Klebsiella pneumoniae by MALDI-TOF Analysis of Full Spectra Applying Machine Learning.

MALDI-TOF MS is considered to be an important tool for the future development of rapid microbiological techniques. We propose the application of MALDI-TOF MS as a dual technique for the identification of bacteria and the detection of resistance, with no extra hands-on procedures. We have developed a machine learning approach that uses the random forest algorithm for the direct prediction of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates, based on the spectra of complete cells. For this purpose, we used a database of 4,547 mass spectra profiles, including 715 unduplicated clinical isolates that are represented by 324 CPK with 37 different ST. The impact of the culture medium was determinant in the CPK prediction, being that the isolates were tested and cultured in the same media, compared to the isolates used to build the model (blood agar). The proposed method has an accuracy of 97.83% for the prediction of CPK and an accuracy of 95.24% for the prediction of OXA-48 or KPC carriage. For the CPK prediction, the RF algorithm yielded a value of 1.00 for both the area under the receiver operating characteristic curve and the area under the precision-recall curve. The contribution of individual mass peaks to the CPK prediction was determined using Shapley values, which revealed that the complete proteome, rather than a series of mass peaks or potential biomarkers (as previously suggested), is responsible for the algorithm-based classification. Thus, the use of the full spectrum, as proposed here, with a pattern-matching analytical algorithm produced the best outcome. The use of MALDI-TOF MS coupled with machine learning algorithm processing enabled the identification of CPK isolates within only a few minutes, thereby reducing the time to detection of resistance.

Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning.

Mycobacterium abscessus is one of the most common and pathogenic nontuberculous mycobacteria (NTM) isolated in clinical laboratories. It consists of three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Due to their different antibiotic susceptibility pattern, a rapid and accurate identification method is necessary for their differentiation. Although matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has proven useful for NTM identification, the differentiation of M. abscessus subspecies is challenging. In this study, a collection of 325 clinical isolates of M. abscessus was used for MALDI-TOF MS analysis and for the development of machine learning predictive models based on MALDI-TOF MS protein spectra. Overall, using a random forest model with several confidence criteria (samples by triplicate and similarity values >60%), a total of 96.5% of isolates were correctly identified at the subspecies level. Moreover, an improved model with Spanish isolates was able to identify 88.9% of strains collected in other countries. In addition, differences in culture media, colony morphology, and geographic origin of the strains were evaluated, showing that the latter had an impact on the protein spectra. Finally, after studying all protein peaks previously reported for this species, two novel peaks with potential for subspecies differentiation were found. Therefore, machine learning methodology has proven to be a promising approach for rapid and accurate identification of subspecies of M. abscessus using MALDI-TOF MS.

Automatic Discrimination of Species within the Enterobacter cloacae Complex Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Supervised Algorithms.

The Enterobacter cloacae complex (ECC) encompasses heterogeneous clusters of species that have been associated with nosocomial outbreaks. These species may have different acquired antimicrobial resistance and virulence mechanisms, and their identification is challenging. This study aims to develop predictive models based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles and machine learning for species-level identification. A total of 219 ECC and 118 Klebsiella aerogenes clinical isolates from three hospitals were included. The capability of the proposed method to differentiate the most common ECC species (Enterobacter asburiae, Enterobacter kobei, Enterobacter hormaechei, Enterobacter roggenkampii, Enterobacter ludwigii, and Enterobacter bugandensis) and K. aerogenes was demonstrated by applying unsupervised hierarchical clustering with principal-component analysis (PCA) preprocessing. We observed a distinctive clustering of E. hormaechei and K. aerogenes and a clear trend for the rest of the ECC species to be differentiated over the development data set. Thus, we developed supervised, nonlinear predictive models (support vector machine with radial basis function and random forest). The external validation of these models with protein spectra from two participating hospitals yielded 100% correct species-level assignment for E. asburiae, E. kobei, and E. roggenkampii and between 91.2% and 98.0% for the remaining ECC species; with data analyzed in the three participating centers, the accuracy was close to 100%. Similar results were obtained with the Mass Spectrometric Identification (MSI) database developed recently (https://msi.happy-dev.fr) except in the case of E. hormaechei, which was more accurately identified with the random forest algorithm. In short, MALDI-TOF MS combined with machine learning was demonstrated to be a rapid and accurate method for the differentiation of ECC species.

Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.

The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of moulds using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in a multicenter context. For that purpose, three Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5% and 94.8% and the score values were 1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (F. proliferatum, n = 1; T.interdigitale, n = 2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.

MALDI-TOF mass spectrometry has been improved as a diagnostic method for the rapid and reliable identification of filamentous fungi by means of the creation of an expanded database containing reference protein spectra of the most clinically impacting fungal species.

Evaluation of the Vitek Ms system for the identification of filamentous fungi.

The implementation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of fungal isolates remains challenging and has been limited to experienced laboratories in sample preparation and in-house libraries construction. However, the development of commercial kits for standardized fungal sample preparation and updated reference libraries can fill this gap. This study aimed to evaluate the performance of the commercial VITEK MS Mould Kit (bioMérieux, Marcy l'Etoile, France) and the VITEK MS system (bioMérieux) for identification using a panel of fungal species of clinical interest. Overall, 200 isolates belonging to 13 genera and 43 fungal species were analyzed with the VITEK MS system equipped with the v3.2 IVD database. Overall, 89.0% of the isolates were correctly identified, 41.5 and 43.5% at species and complex level, respectively. For an additional 4.0% of the identifications, correlation at the genus level was reported. The remaining 21 isolates (10.5%) could not be identified among which 85.0% (18/21) were species not claimed in the database. One Syncephalastrum isolate was misidentified as Rhizopus microsporus complex. Specifically, 100% of the Scedosporium/Lomentospora, 97.1% of the Fusarium, 65.7% of the Mucorales and 86.4% of the Aspergillus isolates were correctly identified at the species and complex level. The methodology described allows for an easy implementation of MALDI-TOF MS for routine identification of fungal species in a fast and reliable manner. Although further improvement in the databases is still required, an important number of fungal species can be correctly identified at the species level using this method. LAY SUMMARY: The use of MALDI-TOF for fungal identification remains a challenge. In this study, using a commercial protein extraction kit and updated database, VITEK MS system was able to identify up to 89.0% of a diverse collection of 200 filamentous fungi representing 43 fungal species.

Azole-Resistant Aspergillus fumigatus Clinical Isolate Screening in Azole-Containing Agar Plates (EUCAST E.Def 10.1): Low Impact of Plastic Trays Used and Poor Performance in Cryptic Species.

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedure's performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n = 91) and cryptic species (n = 52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multidish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harboring TR34-L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/liter. Isolates harboring G54R and TR46-Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used by clinical laboratories for identification of antibiotic-resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae isolates (112 CPK and 50 non-CPK) were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages; the first is denominated the "reproducibility stage" and the second "CPK identification." The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage to assess the final accuracy of MALDI-TOF MS for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS Data Analysis Software (Clover Biosoft, Spain) that is designed to reduce variability, guarantee interlaboratory reproducibility, and maximize the information selected from the bacterial proteome. Using the random forest (RF) algorithm, 100% of CPK isolates were correctly identified when all the peaks in the spectra were selected as input features and total ion current (TIC) normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

Evaluation of MALDI Biotyper Interpretation Criteria for Accurate Identification of Nontuberculous Mycobacteria.

Identification of mycobacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) requires not only a good protein extraction protocol but also an adequate cutoff score in order to provide reliable results. The aim of this study was to assess the cutoff scores proposed by the MALDI-TOF MS system for mycobacterial identification. A total of 693 clinical isolates from a liquid medium and 760 from a solid medium were analyzed, encompassing 67 different species of nontuberculous mycobacteria (NTM). MALDI-TOF MS identified 558 (80.5%) isolates from the liquid medium and 712 (93.7%) isolates from the solid medium with scores of ≥1.60. Among these, four (0.7%) misidentifications were obtained from the liquid medium and four (0.5%) from the solid medium. With regard to species diversity, MALDI-TOF MS successfully identified 64 (95.5%) different species, while PCR-reverse hybridization (GenoType Mycobacterium CM and AS assays) identified 24 (35.8%) different species. With MALDI-TOF MS scores of ≥2, all isolates were correctly identified, and with scores in the range from 1.60 to 1.99, most isolates were correctly identified, except for Mycobacterium angelicum, M. parascrofulaceum, M. peregrinum, M. porcinum, and M. gastri In conclusion, MALDI-TOF MS is a useful method for identifying a large diversity of NTM species. A score threshold of 1.60 proved useful for identifying almost all the isolates tested; only a few species required a higher score (≥2.00) to obtain a valid definitive identification.

Review of the impact of MALDI-TOF MS in public health and hospital hygiene, 2018.

IntroductionMALDI-TOF MS represents a new technological era for microbiology laboratories. Improved sample processing and expanded databases have facilitated rapid and direct identification of microorganisms from some clinical samples. Automated analysis of protein spectra from different microbial populations is emerging as a potential tool for epidemiological studies and is expected to impact public health.AimTo demonstrate how implementation of MALDI-TOF MS has changed the way microorganisms are identified, how its applications keep increasing and its impact on public health and hospital hygiene.MethodsA review of the available literature in PubMED, published between 2009 and 2018, was carried out.ResultsOf 9,709 articles retrieved, 108 were included in the review. They show that rapid identification of a growing number of microorganisms using MALDI-TOF MS has allowed for optimisation of patient management through prompt initiation of directed antimicrobial treatment. The diagnosis of Gram-negative bacteraemia directly from blood culture pellets has positively impacted antibiotic streamlining, length of hospital stay and costs per patient. The flexibility of MALDI-TOF MS has encouraged new forms of use, such as detecting antibiotic resistance mechanisms (e.g. carbapenemases), which provides valuable information in a reduced turnaround time. MALDI-TOF MS has also been successfully applied to bacterial typing.ConclusionsMALDI-TOF MS is a powerful method for protein analysis. The increase in speed of pathogen detection enables improvement of antimicrobial therapy, infection prevention and control measures leading to positive impact on public health. For antibiotic susceptibility testing and bacterial typing, it represents a rapid alternative to time-consuming conventional techniques.

Enoxaparin does not ameliorate liver fibrosis or portal hypertension in rats with advanced cirrhosis.

BACKGROUND & AIMS: Recent studies suggest that heparins reduce liver fibrosis and the risk of decompensation of liver disease. Here, we evaluated the effects of enoxaparin in several experimental models of advanced cirrhosis. METHODS: Cirrhosis was induced in male Sprague-Dawley (SD) rats by: (i) Oral gavage with carbon tetrachloride (CCl4ORAL ), (ii) Bile duct ligation (BDL) and (iii) CCl4 inhalation (CCl4INH ). Rats received saline or enoxaparin s.c. (40 IU/Kg/d or 180 IU/Kg/d) following various protocols. Blood biochemical parameters, liver fibrosis, endothelium- and fibrosis-related genes, portal pressure, splenomegaly, bacterial translocation, systemic inflammation and survival were evaluated. Endothelial dysfunction was assessed by in situ bivascular liver perfusions. RESULTS: Enoxaparin did not ameliorate liver function, liver fibrosis, profibrogenic gene expression, portal hypertension, splenomegaly, ascites development and infection, serum IL-6 levels or survival in rats with CCl4ORAL or BDL-induced cirrhosis. Contrarily, enoxaparin worsened portal pressure in BDL rats and decreased survival in CCl4ORAL rats. In CCl4INH rats, enoxaparin had no effects on hepatic endothelial dysfunction, except for correcting the hepatic arterial dysfunction when enoxaparin was started with the CCl4 exposure. In these rats, however, enoxaparin increased liver fibrosis and the absolute values of portal venous and sinusoidal resistance. CONCLUSIONS: Our results do not support a role of enoxaparin for improving liver fibrosis, portal hypertension or endothelial dysfunction in active disease at advanced stages of cirrhosis. These disease-related factors and the possibility of a limited therapeutic window should be considered in future studies evaluating the use of anticoagulants in cirrhosis.

Identification of Porphyromonas isolates from clinical origin using MALDI-TOF Mass Spectrometry.

Despite the wide implementation of MALDI-TOF MS for the rapid and reliable identification of most microorganisms, some taxonomic groups such as the Porphyromonas genus remain largely untested. In this study we evaluated the performance of MALDI-TOF MS on this genus using a collection of 39 isolates sent for routine identification to our institution over a 16-year period. All of them were identified by DNA-sequencing analysis of the 16S rRNA gene plus the hsp60 gene when the previous one did not yield species-level assignment. MALDI-TOF MS provided correct identification at least at the genus level of 21/39 isolates (53.9%). Twelve isolates were correctly identified at the species level with a score value ≥ 2.0 and 9 more with score values < 2.0 and ≥ 1.7. The species most represented in the database (P. gingivalis and P. somerae) lay within this category. However, the species poorly represented in this database (P. asaccharolytica and P. uenonis) were mostly identified with lower scores (1.35-1.67) or remained unidentified by MALDI-TOF MS. The addition of two P. asaccharolytica reference spectra to our in-house library allowed 72.9% of genus-level identifications with 17/37 isolates (45.9%) identified with score values ≥ 2.0. Our results showed a high level of correlation between MALDI-TOF MS and DNA-based identification for Porphyromonas spp. strains at the species level, even with score values < 2.0. The reliability provided by MALDI-TOF MS increased when the database was fed with spectra from the species poorly represented in the commercial database.

FATAL ACINETOBACTER BAUMANNII INFECTION IN THE CRITICALLY ENDANGERED EUROPEAN MINK (MUSTELA LUTREOLA).

The present study reports the first case of fatal Acinetobacter baumannii infection in the critically endangered European mink ( Mustela lutreola ). Gross examination revealed a severe, diffuse hemorrhagic pneumonia and generalized congestion as main features. Microscopically, the main lesions were an acute, severe fibrinous-hemorrhagic pneumonia associated with proliferation of coccobacilli and generalized acute-subacute congestion. Cultures yielded A. baumannii ; the species was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the strain presented a multidrug-resistant pattern. The results are not only of conservation concern but also of public health concern given A. baumannii is one of the most important pathogens implicated in nosocomial infections in humans.

Evaluation of MALDI Biotyper Mycobacteria Library v3.0 for Identification of Nontuberculous Mycobacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has demonstrated its ability to promptly identify nontuberculous mycobacteria using the Mycobacteria Library v2.0. However, some species are particularly difficult to identify reliably using this database, providing a low log(score). In this study, the identification power of an updated Mycobacteria Library (v3.0) has been evaluated. Overall, 109 NTM isolates were analyzed with both databases. The v3.0 database allowed a high-level confidence in the identification [log(score) value, ≥1.8] of 91.7% of the isolates versus 83.5% with the v2.0 version (P< 0.01).

Evaluation of MALDI-TOF MS (Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry) for routine identification of anaerobic bacteria.

Information regarding the use of MALDI-TOF MS as an alternative to conventional laboratory methods for the rapid and reliable identification of bacterial isolates is still limited. In this study, MALDI-TOF MS was evaluated on 295 anaerobic isolates previously identified by 16S rRNA gene sequencing and with biochemical tests (Rapid ID 32A system, BioMérieux). In total, 85.8% of the isolates were identified by MALDI-TOF MS at the species level vs 49.8% using the Rapid ID 32A system (p < 0.0001). None of the isolates was discordantly identified at the genus level using MALDI-TOF MS and only 9 of them could not be identified using the method. Thus, our results show that MALDI-TOF MS is a robust and reliable tool for the identification of anaerobic isolates in the microbiology laboratory. Its implementation will reduce the turnaround time for a final identification and the number of isolates that require 16S rRNA sequencing.

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nontuberculous Mycobacteria from Clinical Isolates.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of nontuberculous mycobacterial (NTM) isolates was evaluated in this study. Overall, 125 NTM isolates were analyzed by MALDI-TOF and GenoType CM/AS. Identification by 16S rRNA/hsp65 sequencing was considered the gold standard. Agreements between MALDI-TOF and GenoType CM/AS with the reference method were, respectively, 94.4% and 84.0%. In 17 cases (13.6%), results provided by GenoType and MALDI-TOF were discordant; however, the reference method agreed with MALDI-TOF in 16/17 cases (94.1%; P = 0.002).

Can MALDI-TOF mass spectrometry be used with intravascular catheters?

INTRODUCTION: The use of the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry has shown to be effective and fast in some clinical specimens for the identification of colonizing microorganisms. The objective of the study was to analyze the validity values for the prediction of colonization and catheter-related bloodstream infection (C-RBSI) of the MALDI-TOF mass spectrometry performed at all intravascular catheters that arrived in the microbiology laboratory. METHODS: Catheter tips (after performing the roll-plate technique) were tested by MALDI-TOF mass spectrometry during a period of 3-months. The gold standard for colonization and C-RBSI were, respectively: the presence of ≥15cfu/plate in the catheter tip culture; and the isolation of the same microorganism(s) in blood cultures as well as in the colonized catheter (during the 7days before or after catheter withdrawal). RESULTS: A total of 182 intravascular catheters were collected. The overall colonization rate detected by roll-plate technique and MAL-TOF mass spectrometry was 31.9% and 32.4%, respectively. Overall, there were 33 (18.1%) episodes of C-RBSI. The validity values of the MALDI-TOF mass spectrometry for the identification of colonization and C-RBSI were, respectively: sensitivity (69.0%/66.7%), specificity (84.7%/75.2%), positive predictive value (65.6%/36.1%), and negative predictive value (86.8%/92.6%). Conclusion MALDI-TOF mass spectrometry could be an alternative diagnostic tool for ruling out C-RBSI. However, despite it showing to be faster than conventional culture, future studies are required in order to improve the pre-analytical process.

Prospective study of BK virus infection in patients with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) have an immune-deficient baseline status further modulated by immunosuppressive therapy that may promote the reactivation of latent viruses such as BK virus (BKV). The aim of this prospective study was to determine the prevalence of BKV infection in IBD patients and its potential relationship with the immunosuppressive treatment. Paired urine and plasma samples from 53 consecutive patients with IBD and 53 controls were analyzed. BKV detection was performed by conventional PCR and positive samples were further quantified by real-time PCR. No viremia was detected. BKV viruria was significantly more common in IBD patients than among the controls (54.7% versus 11.3%; P < 0.0001). The only risk factor for BKV viruria in IBD was age (47.2 ± 16.3 versus 37.8 ± 15.2; P = 0.036), and there was a trend towards higher rate of viruria in outpatients (61.5% versus 38.5%; P = 0.096) and in those not receiving ciprofloxacin (59.5% versus 40.5%; P = 0.17). A clear impact of the immunosuppressive regimen on BKV infection could not be demonstrated.

Identification of Nocardia and non-tuberculous Mycobacterium species by MALDI-TOF MS using the VITEK MS coupled to IVD and RUO databases.

Identification of Nocardia and Mycobacterium species by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is still a challenging task that requires both suitable protein extraction procedures and extensive databases. This study aimed to evaluate the VITEK MS Plus system coupled with updated RUO (v4.17) and IVD (v3.2) databases for the identification of Nocardia spp. and Mycobacterium spp. clinical isolates. Sample preparation was carried out using the VITEK MS Mycobacterium/Nocardia kit for protein extraction. From 90 Nocardia spp. isolates analysed, 86 (95.6%) were correctly identified at species or complex level using IVD and 78 (86.7%) using RUO. Only two strains were misidentified as other species pertaining to the same complex. Among the 106 non-tuberculous Mycobacterium clinical isolates tested from a liquid culture medium, VITEK MS identified correctly at species or complex level 96 (90.6%) isolates in the IVD mode and 89 (84.0%) isolates in the RUO mode. No misidentifications were detected. Although the IVD mode was unable to differentiate members of the M. fortuitum complex, the RUO mode correctly discriminated M. peregrinum and M. septicum. The robustness and accuracy showed by this system allow its implementation for routine identification of these microorganisms in clinical laboratories.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.