Use Of Matrix-assisted Laser Desorption Ionization-time Of Flight Research Articles

Identification and Antifungal Drug Susceptibility Pattern of Candida auris in India.

Candida auris has turned up as a multidrug-resistant nosocomial agent with outbreaks reported worldwide. The present study was conducted to evaluate the antifungal drug susceptibility pattern of C. auris. Isolates of C. auris were obtained from clinically suspected cases of candidemia from January 2019 to June 2021. Identification was done with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and panfungal DNA polymerase chain reaction (PCR), followed by sequencing. Antifungal susceptibility testing was performed with broth microdilution method. Out of 50 isolates C. auris, 49 were identified by MALDI-TOF and one isolate was identified with panfungal DNA PCR followed by sequencing. For fluconazole, 84% (n = 42) isolates were found to be resistant and 16% (n = 8) isolates were susceptible (minimum inhibitory concentrations [MICs] range 0.5-16). Posaconazole exhibited potent activity, followed by itraconazole. For amphotericin B, only 6% (n = 3) isolates were resistant with MICs ≥2 μg/mL. Only 4% (n = 2) isolates exhibited resistance to caspofungin. No resistance was noted for micafungin and anidulafungin. One (2%) isolate was found to be panazole resistant. One (2%) isolate was resistant to fluconazole, amphotericin B, and caspofungin. Correct identification of C. auris can be obtained with the use of MALDI-TOF and sequencing methods. A small percentage of fluconazole-sensitive isolates are present. Although elevated MICs for amphotericin B and echinocandins are not generally observed, the possibility of resistance with the irrational use of these antifungal drugs cannot be denied. Pan azole-resistant and pan drug-resistant strains of C. auris are on rise.

New assessment of Anopheles vector species identification using MALDI-TOF MS

BackgroundAnopheles species identification is essential for an effective malaria vector control programme. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been developed to identify adult Anopheles species, using the legs or the cephalothorax. The protein repertoire from arthropods can vary according to compartment, but there is no general consensus regarding the anatomic part to be used.MethodsTo determine the body part of the Anopheles mosquitoes best suited for the identification of field specimens, a mass spectral library was generated with head, thorax with wings and legs of Anopheles gambiae, Anopheles arabiensis and Anopheles funestus obtained from reference centres. The MSL was evaluated using two independent panels of 52 and 40 An. gambiae field-collected in Mali and Guinea, respectively. Geographic variability was also tested using the panel from Mali and several databases containing added specimens from Mali and Senegal.ResultsUsing the head and a database without specimens from the same field collection, the proportion of interpretable and correct identifications was significantly higher than using the other body parts at a threshold value of 1.7 (p < 0.0001). The thorax of engorged specimens was negatively impacted by the blood meal after frozen storage. The addition of specimens from Mali into the database significantly improved the results of Mali panel (p < 0.0001), which became comparable between head and legs. With higher identification scores, the using of the head will allow to decrease the number of technical replicates of protein extract per specimen, which represents a significant improvement for routine use of MALDI-TOF MS.ConclusionsThe using of the head of Anopheles may improve the performance of MALDI-TOF MS. Region-specific mass spectrum databases will have to be produced. Further research is needed to improve the standardization in order to share online spectral databases.

In-house protocol and performance of MALDI-TOF MS in the early diagnosis of bloodstream infections in a fourth-level hospital in Colombia: Jumping to full use of this technology

BackgroundBloodstream infections (BSIs) are a major cause of mortality in hospitalized patients. Rapid diagnosis is crucial because any delay in the antimicrobial treatment is associated with an increase in adverse patient outcomes. The application of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology directly to blood cultures permits earlier identification of BSIs and facilitates treatment management. MethodsA total of 470 positive blood cultures from patient samples were analyzed using Standard Aerobic/F and Anaerobic/F blood culture media. Isolates were identified using conventional identification methods and by the direct method using the MALDI-TOF MS system. ResultsIn 470 blood cultures, the direct method showed good identification results (420/470, 89%); specifically, accurate species and genus identification in 283/470 (60%), and only correct genus identification in 137/470 (29%). The direct protocol had better performance for Gram-negative compared to Gram-positive bacteria (97% vs 76%) and was unable to identify the positive blood cultures for both yeasts and some bacteria, mostly Gram-positive (50/470). ConclusionsThe protocol used here gave good and reliable results, being available up to 24 h earlier, while also leading to better use of MALDI-TOF.

536. Challenges in Using MALDI-TOF Technology to Assess for KPC Resistance in Klebsiella pneumoniae isolates in America

BackgroundThe emergence of Klebsiella pneumoniae Carbapenemase-producing Enterobacteriaceae (KPC-E) has created a major public health concern. In clinical practice, rapid identification of KPC-KP has important implications for clinical management and infection control. In some settings matrix-assisted laser desorption ionization time of flight (MALDI-TOF) software has been used for rapid detection of KPC producing K. pneumoniae with high sensitivity and specificity. Genomic sequencing has determined that the 11.09 m/z peak is related to protein expression from the P109 gene found mostly in Tna4401a isoform among KPC-E. In our study, we evaluated the use of MALDI-TOF automated detection software to evaluate for KPC detection among a diverse group of KPC-E isolates.MethodsWe tested 52 KPC-E isolates from various hospitals in Connecticut and the Centers for Disease Control and Prevention (CDC) Antibiotic Resistance (AR) Bank. All specimens were verified as KPC-producing strains by detection of the blaKPCgene through polymerase chain reaction. Protein extraction using the standard extraction method was performed on sub-cultured isolates. Each isolate was tested three times on MALDI-TOF MS with the incorporated bio-subtype KPC module. An organism confidence or log score value of 2 or higher was considered valid.ResultsAmong 52 tested KPC-K. pneumoniae isolates, 44 (85%) were from various hospitals in Connecticut, eight (15%) came from the AR Bank. Only 15 (25.1%) of the isolates were detected as KPC-producing using the MALDI-TOF KPC module. Further investigation by peak analysis confirmed all 15 isolates detected positive demonstrated a peak at 11.09 m/z. The 11.09 m/z peak was not found in the 37 specimens that were not detected.ConclusionThe results from our study suggest low sensitivity using this software and contradicts results seen in previous European studies. The Tna4401a isoform is often seen in KPC-2 strains, which may be less prevalent in our sample of isolates, explaining the poor sensitivity of MALDI-TOF. Further study is needed to explore this finding and potential opportunities for MALDI-TOF for rapid identification of KPC-KP.Disclosures All authors: No reported disclosures.

Impact of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight for the Identification of Gram-Positive Bloodstream Pathogens without Antimicrobial Stewardship Intervention in Hospitalized Children

AbstractWe performed a 2-year retrospective cohort study of inpatient children with growth of gram-positive bacteria from blood cultures identified with and without the use of matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) in the absence of an antimicrobial stewardship program. Use of MALDI-TOF reduced the time to organism identification by 15.7 hours (p &lt; 0.0001). However, no differences in length of stay, time to optimization of antimicrobial therapy, or time to discontinuation of antibiotics for growth of contaminants were documented, suggesting that such potential patient-oriented benefits of MALDI-TOF may only be evident in the presence of a concurrent antimicrobial stewardship program.

Differentiation of Flavobacterium psychrophilum from Flavobacterium psychrophilum-like species by MALDI-TOF mass spectrometry

Rainbow trout fry syndrome (RTFS) is an important infectious disease caused by Flavobacterium psychrophilum affecting farmed salmonids worldwide. Other Flavobacterium psychrophilum-like species (F. plurextorum, F. oncorhynchi, F. tructae, F. collinsii and F. piscis) have been isolated from diseased rainbow trout fry suspected of RTFS although the epidemiological and clinical relevance of these pathogens are unknown. The objective of this study was to evaluate the potential use of MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight) Mass Spectrometry as method for specific identification of F. psychrophilum and its differentiation from other F. psychrophilum-like species isolated from diseased fish. Fifty-three isolates were analyzed after the creation of the Main Spectrum Profile (MSP) of reference strains of each of abovementioned species. F. psychrophilum exhibited a mass spectra very different from those of F. psychrophilum-like species, with five peaks (m/z 3654, 4585, 5388, 6730 and 7310) present only in F. psychrophilum isolates, and three peaks (m/z 6170, 7098 and 9241) absent in F. psychrophilum but present in all F. psychrophilum-like species. All F. psychrophilum isolates were correctly identified and differentiated from the F. psychrophilum-like species by MALDI-TOF. Although this approach showed a limited ability to differentiate among F. psychrophilum-like species, its complementation with a few simple biochemical tests may represent an alternative approach for the routine identification of the Flavobacterium psychrophilum-like species.

Impact of Matrix-Assisted Laser Desorption and Ionization Time-of-Flight and Antimicrobial Stewardship Intervention on Treatment of Bloodstream Infections in Hospitalized Children

Early definitive identification of infectious pathogens coupled with antimicrobial stewardship interventions allow for targeted and timely administration of antimicrobials. We investigated the combined impact of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) technology and an antimicrobial stewardship program (ASP) in pediatric patients with blood stream infections (BSIs). This is a single-center study comparing a control group of patients from October 2009 to July 2010 with BSIs to a cohort of patients postimplementation of MALDI-TOF and an ASP, from October 2013 to July 2014. Primary outcome was time to optimal therapy. Secondary outcomes included time to effective therapy, 30-day all-cause mortality, 30-day readmission rate, hospital length of stay, and intensive care admission. One hundred episodes of BSIs were identified in the preintervention period, and 121 episodes were identified in the postintervention period. Time from blood culture collection to organism identification was significantly reduced in the prospective cohort compared with historical controls (18.8 vs 43.7 hours, respectively). A total of 73 ASP interventions were made on the treatment of BSIs in the postintervention period. Combined use of MALDI-TOF and ASP significantly reduced time to optimal therapy (77.0 to 54.2 hours, P < .001). In the subgroup analysis of Gram-negative bacteremia, time to effective and optimal therapy were significantly reduced (2.0 vs 0.7 hours and 146.8 vs 48.0 hours, respectively). There were no significant differences in clinical outcomes. The combined use of MALDI-TOF and ASP allows early optimization of antimicrobial therapy in pediatric inpatients with BSIs.

Aplicación de la espectrometría de masas en la identificación de bacterias

Correct and rapid identification of bacteria is essential for the correct diagnosis and treatment of infected patients. Until a few years ago, biochemical, colorimetric or even antibiotic sensitivity tests were used to identify genera and species. The main limitations of these methods were the time needed for their performance and the difficulty of distinguishing between microorganisms that were little reactive, highly similar, or difficult to culture. Many of these problems have been solved by the introduction of mass spectrometry (MS) in the laboratory with the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight). Knowledge of the strengths and weaknesses of this technology is essential to be able to take maximum advantage of this technique. Not all microorganisms can be identified with the same ease and reliability by MALDI-TOF and microbiologists need to know how to interpret the results obtained with this technique and the available alternatives in order to identify the microorganisms causing the most problems. This article aims to summarise the available information on the correct identification of the main human pathogenic bacteria through the use of MALDI-TOF MS, focusing on Gram-negative, Grampositive and anaerobic microorganisms. The main factors that must be taken into account for the reliable identification of any bacterium are the conditions for culture, sample preparation with the ideal extraction method and especially the use of a correct and updated database.

Estudios de coste-efectividad con MALDI-TOF e impacto clínico

In general, new technologies usually increase laboratory costs due to the need for an initial investment. However, as occurred with MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry, this increase is subsequently offset by the discontinued use of traditional technologies and by the benefits to patients of the new information generated. In the clinical microbiology laboratory, the identification time is reduced with the use of MALDI-TOF (by at least 24 hours) and turnaround is improved, allowing faster production of the microbiological report. This beneficial effect has mainly been studied with blood cultures in patients with bacteraemia. In these patients, the length of hospital stay has been reduced by 1.6-6.6 days, depending on the type of patient and the appropriateness of treatment. This leads to better antimicrobial use and a reduction in total hospital cost of up to 43% per patient. Another factor that has been analysed is the decrease in mortality due to better management of antimicrobial therapy. Future multicentre studies should include other factors such as hospital organisation changes and clinical activity arising in response to the efforts of the clinical microbiology laboratory to rapidly obtain information of clinical value.

Aplicación de la espectrometría de masas en micobacterias

To date, more than 170 species of mycobacteria have been described, of which more than one third may be pathogenic to humans, representing a significant workload for microbiology laboratories. These species must be identified in clinical practice, which has long been a major problem due to the shortcomings of conventional (phenotypic) methods and the limitations and complexity of modern methods largely based on molecular biology techniques. The aim of this review was to briefly describe different aspects related to the use of MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight) mass spectrometry (MS) for the identification of mycobacteria. Several difficulties are encountered with the use of this methodology in these microorganisms mainly due to the high pathogenicity of some mycobacteria and the peculiar structure of their cell wall, requiring inactivation and special protein extraction protocols. We also analysed other relevant aspects such as culture media, the reference methods employed (gold standard) in the final identification of the different species, the cut-off used to accept data as valid, and the databases of the different mass spectrometry systems available. MS has revolutionized diagnosis in modern microbiology; however, specific improvements are needed to consolidate the use of this technology in mycobacteriology.

MALDI-TOF is not useful in the diagnosis of catheter colonization based on superficial cultures: results from an in vitro study

We compared in an vitro model the yields of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and conventional culture (CC) for the detection of catheter colonization with superficial catheter samples (SS). We used blood culture bottles (BCB) with an inserted cannula and incubated at 37 °C. The BCB were manipulated with different contaminations and when a BCB turned positive, SS were obtained to perform both techniques. To compare both techniques we analyzed the mean time to colonization (MTC) and the mean time to a result (MTR). The MTC (SD, days) by CC and MALDI-TOF was as follows: hub, 0.59 (0.79) versus 1.07 (1.39), P=0.06; surface: 0.62 (0.67) versus 0.82 (0.81), P<0.001. The MTR (SD, days) of CC and MALDI-TOF was as follows: hub: 1.58 (0.79) versus 2.25 (1.48), P=0.04; surface: 1.62 (0.67) versus 1.95 (0.80), P<0.001. In general, the use of MALDI-TOF performed directly with SS was no better than CC and did not anticipate colonization results.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology

Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.

Molecular and Epidemiological Analysis of Nosocomial Carbapenem-Resistant Klebsiella spp. Using Repetitive Extragenic Palindromic-Polymerase Chain Reaction and Matrix-Assisted Laser Desorption/Ionization-Time of Flight

Infections with carbapenem-resistant enterobacteria are an emerging threat. This study reports the microbiologic, clinical, and epidemiologic features and the therapeutic outcomes of the infections caused by carbapenem- and pandrug-resistant Klebsiella emerged in our hospital. Fingerprinting analyses by automated repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry are also compared. Carbapenem-resistant Klebsiella spp. affecting 13 patients were investigated using automated rep-PCR (DiversiLab System) and MALDI-TOF. Species identification was performed by Vitek 2 System and MALDI-TOF. Antimicrobial susceptibility testing was made using Vitek 2 System and Etest. Screening for extended spectrum beta-lactamase (ESBL) and carbapenemase production was made by double disk synergy and Hodge tests, respectively. Synergy studies were performed using Etest. DNA array was used for detection of KPC and ESBLs. bla(VIM-1) gene was amplified by PCR and sequencing. Use of carbapenems in the hospital was studied. A total of 13 patients were found to be colonized/infected with carbapenem-resistant Klebsiella. All patients were previously submitted to surgery and/or presented with severe underlying disease. After carbapenem-resistant Klebsiella isolation, the majority of the patients were treated with amikacin plus carbapenem, tigecycline, or fosfomycin. All Klebsiella isolates (n = 14), except two, had the bla(VIM-1) gene and all Klebsiella pneumoniae also had bla(SHV) gene associated with ESBL production. DiversiLab system showed higher discriminatory power than MALDI-TOF for strain typing. The risk of a rapid dissemination and the persistence of these multidrug-resistant strains through the time determine the need to implement routine procedures for metallo-beta-lactamase detection and measures for prevention of the spread of these microorganisms. The combined use of MALDI-TOF for species identification and DiversiLab System for clonal strain typing may be a useful tool for fast and accurate management of nosocomial outbreaks. The potential clinical utility of fosfomycin in this matter should be considered in future studies.