Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

ABSTRACT Increasing resistance, recrudescences, and treatment failure have led to the replacement of chloroquine with the combination of pyrimethamine (PYR) and sulfadoxine (SDX) as the first-line antimalarial drugs for treatment of uncomplicated Plasmodium falciparum malaria in several areas where this disease is endemic. The development of resistance to PYR-SDX is favored by incomplete treatment courses or by subtherapeutic levels in plasma. PYR-SDX resistance has been associated with several single-nucleotide polymorphisms (SNPs) in the P. falciparum dihydrofolate reductase (pfdhfr) and the P. falciparum dihydropteroate synthetase (pfdhps) genes. We have established assays based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that conveniently allow the identification of SNPs associated with PYR resistance. Variants occurring at codon positions 16, 51, 59, and 108 of the pfdhfr gene were analyzed by MALDI-TOF MS in synthetic oligonucleotides to determine the detection threshold. In addition, 63 blood samples from subjects with P. falciparum parasitemia of various degrees were analyzed. The results were compared to those obtained by DNA sequencing of the respective gene fragment. The results of MALDI-TOF MS and DNA sequencing were consistent in 40 samples. In 23 samples two or three pfdhfr variants were detected by MALDI-TOF assays, whereas DNA-sequencing revealed one variant only. Simultaneous detection of two different mutations by biplex assays was, in principle, feasible. As demonstrated by the example of PYR resistance, MALDI-TOF MS allows for rapid and automated high-throughput assessment of drug sensitivity in P. falciparum malaria.

Download Full-text

Comparative evaluation of matrix-assisted laser desorption ionisation-time of flight mass spectrometry and conventional phenotypic-based methods for identification of clinically important yeasts in a UK-based medical microbiology laboratory

Journal of Clinical Pathology ◽

10.1136/jclinpath-2015-203029 ◽

2015 ◽

Vol 68 (12) ◽

pp. 1040-1042 ◽

Cited By ~ 5

Author(s):

Nita Fatania ◽

Mark Fraser ◽

Mike Savage ◽

Jason Hart ◽

Alireza Abdolrasouli

Keyword(s):

Mass Spectrometry ◽

Laser Desorption ◽

Microbiology Laboratory ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Phenotypic Methods ◽

Tof Ms ◽

Matrix Assisted Laser ◽

Matrix Assisted Laser Desorption

AimsPerformance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting.MethodsA diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene.ResultsMALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods.ConclusionsMALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods.

Download Full-text

Misidentification ofCandida guilliermondiiasC. famataamong Strains Isolated from Blood Cultures by the VITEK 2 System

BioMed Research International ◽

10.1155/2014/250408 ◽

2014 ◽

Vol 2014 ◽

pp. 1-6 ◽

Cited By ~ 10

Author(s):

Si Hyun Kim ◽

Jeong Hwan Shin ◽

Jeong Ha Mok ◽

Shine Young Kim ◽

Sae Am Song ◽

...

Keyword(s):

Gene Sequencing ◽

Candida Guilliermondii ◽

Blood Cultures ◽

Rrna Gene ◽

28S Rrna ◽

Sequencing Analysis ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Vitek 2 ◽

Tof Ms

Introduction. The aim of this study was to differentiate betweenCandida famataandCandida guilliermondiicorrectly by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and gene sequencing.Methods. Twenty-eightCandidastrains from blood cultures that had been identified asC. famata(N=25),C. famata/C. guilliermondii(N=2), andC. guilliermondii(N=1) by the VITEK 2 system using the YST ID card were included. We identified these strains by MALDI-TOF MS and gene sequencing using the 28S rRNA andITSgenes and compared the results with those obtained by the VITEK 2 system.Results. All 28 isolates were finally identified asC. guilliermondii.Sequencing analysis of the 28S rRNA gene showed 99.80%–100% similarity withC. guilliermondiifor all 28 strains. TheITSgene sequencing of the strains showed 98.34%–100% homology withC. guilliermondii.By MALDI-TOF, we could correctly identify 21 (75%) of 28C. guilliermondiiisolates.Conclusion. We should suspect misidentification whenC. famatais reported by the VITEK 2 system, and we always should keep in mind the possibility of misidentification of any organism when an uncommon species is reported.

Download Full-text

Identification of the bacterial population in manganese removal filters

Water Science & Technology Water Supply ◽

10.2166/ws.2016.184 ◽

2016 ◽

Vol 17 (3) ◽

pp. 842-850

Author(s):

Jantinus H. Bruins ◽

Branislav Petrusevski ◽

Yness M. Slokar ◽

Gerhard H. Wübbels ◽

Koen Huysman ◽

...

Keyword(s):

Dna Sequencing ◽

Bacterial Population ◽

Next Generation ◽

Filter Media ◽

Next Generation Dna Sequencing ◽

Maldi Tof Ms ◽

Manganese Removal ◽

Maldi Tof ◽

Ms Analysis ◽

Tof Ms

The aim of this study was to identify bacteria present in ripened manganese removal filters for drinking water production. The bacterial population was identified with ‘next generation’ DNA sequencing, and specific bacteria were quantified with quantitative polymerase chain reaction (qPCR) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The ‘next generation’ DNA sequencing analysis showed a bacteria population shift from the iron oxidizing species Gallionella spp. in the Fe-filter to manganese and nitrite oxidizing species Pseudomonas spp. and Nitrospira spp., respectively, present in the manganese removal filter. qPCR analysis confirmed the presence of a low concentration of the well-known Mn2+-oxidizing species Ps. putida in the manganese removal filter backwash water. Bacteria of the genus Pseudomonas, isolated from backwash water from a manganese removal filter were cultured and identified with MALDI-TOF MS analysis. Amongst others, P. gessardii, P. grimontii, and P. koreensis were identified. The presence of several manganese oxidizing bacteria species in ripened filter media supports the assumption that a microbial consortium is involved in the oxidation of manganese. Understanding the mechanisms by which manganese coating of filter media commences could endorse the creation of conditions favouring Birnessite formation, and possibly help in reducing typically long ripening periods of manganese removal filters with virgin filter media.

Download Full-text

Identification of polymorphisms within the bovine prion protein gene (Prnp) by DNA sequencing and genotyping by MALDI-TOF-MS

Neurogenetics ◽

10.1007/s10048-001-0126-0 ◽

2002 ◽

Vol 4 (1) ◽

pp. 59-60 ◽

Cited By ~ 20

Author(s):

Andreas Humeny ◽

Katrin Schiebel ◽

Silke Seeber ◽

Cord-Michael Becker

Keyword(s):

Dna Sequencing ◽

Prion Protein ◽

Protein Gene ◽

Prion Protein Gene ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Download Full-text

Implementation of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry in Routine Clinical Laboratories Improves Identification of Coagulase-Negative Staphylococci and Reveals the Pathogenic Role of Staphylococcus lugdunensis

Journal of Clinical Microbiology ◽

10.1128/jcm.00177-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2030-2036 ◽

Cited By ~ 35

Author(s):

Xavier Argemi ◽

Philippe Riegel ◽

Thierry Lavigne ◽

Nicolas Lefebvre ◽

Nicolas Grandpré ◽

...

Keyword(s):

Laser Desorption Ionization ◽

Coagulase Negative Staphylococci ◽

Ionization Time ◽

Content Type ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Phenotypic Methods ◽

Tof Ms

The use of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for identifying the main species of coagulase-negative staphylococci (CoNS) by randomly selecting 50 isolates. From 1 January 2007 to 31 August 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified asStaphylococcus aureusand 7,885 were coagulase negative staphylococci. Using MALDI-TOF MS from 1 January 2011 to 31 August 2012, 14,913 staphylococci were identified, with 5,066 asS. aureusand 9,847 as CoNS. MALDI-TOF MS allowed the identification of approximately 85% of the CoNS strains, whereas only 14% of the CoNS strains were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterizedStaphylococcusspecies, such asS. pettenkoferi,S. condimenti, andS. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of theS. lugdunensisstrains included in the analysis were associated with infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for the identification of staphylococci with clinical consequences. The species distribution reveals the occurrence of the recently identified speciesS. pettenkoferiand putative virulent species, includingS. lugdunensis.

Download Full-text

DNA sequencing by MALDI-TOF MS using alkali cleavage of RNA/DNA chimeras

Nucleic Acids Research ◽

10.1093/nar/gkm056 ◽

2007 ◽

Vol 35 (8) ◽

pp. e62-e62 ◽

Cited By ~ 13

Author(s):

F. Mauger ◽

K. Bauer ◽

C. D. Calloway ◽

J. Semhoun ◽

T. Nishimoto ◽

...

Keyword(s):

Dna Sequencing ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Download Full-text

Identifying Anaerobic Bacteria Using MALDI-TOF Mass Spectrometry: A Four-Year Experience

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.521014 ◽

2021 ◽

Vol 11 ◽

Author(s):

Luis Alcalá ◽

Mercedes Marín ◽

Adrián Ruiz ◽

Lidia Quiroga ◽

Maribel Zamora-Cintas ◽

...

Keyword(s):

Protein Extraction ◽

Anaerobic Bacteria ◽

Reference Method ◽

Rrna Gene ◽

Sequencing Analysis ◽

Gram Positive ◽

Reliable Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Because of the special culture requirements of anaerobic bacteria, their low growth-rate and the difficulties to isolate them, MALDI-TOF MS has become a reliable identification tool for these microorganisms due to the little amount of bacteria required and the accuracy of MALDI-TOF MS identifications. In this study, the performance of MALDI-TOF MS for the identification of anaerobic isolates during a 4-year period is described. Biomass from colonies grown on Brucella agar was directly smeared onto the MALDI-TOF target plate and submitted to on-plate protein extraction with 1μl of 100% formic acid. Sequencing analysis of the 16S rRNA gene was used as a reference method for the identification of isolates unreliably or not identified by MALDI-TOF MS. Overall, 95.7% of the isolates were identified to the species level using the updated V6 database vs 93.8% with previous databases lacking some anaerobic species; 68.5% of the total were reliably identified with high-confidence score values (≥2.0) and 95.0% with low-confidence values (score value ≥1.7). Besides, no differences between Gram-positive and Gram-negative isolates were detected beyond a slight decrease of correct species assignment for gram positive cocci (94.1% vs 95.7% globally). MALDI-TOF MS has demonstrated its usefulness for the identification of anaerobes, with high correlation with phenotypic and conventional methods. Over the study period, only 2.1% of the isolates could not be reliably identified and required molecular methods for a final identification. Therefore, MALDI-TOF MS provided reliable identification of anaerobic isolates, allowing clinicians to streamline the most appropriate antibiotic therapy and manage patients accordingly.

Download Full-text

A Comprehensive Analysis of MALDI-TOF MS and Ribosomal DNA Sequencing for Identification of Clinical Yeasts

Current Fungal Infection Reports ◽

10.1007/s12281-017-0297-2 ◽

2017 ◽

Vol 11 (4) ◽

pp. 184-189 ◽

Cited By ~ 3

Author(s):

Constanza Giselle Taverna ◽

Mariana Mazza ◽

Nicolas Refojo ◽

Graciela Davel

Keyword(s):

Dna Sequencing ◽

Ribosomal Dna ◽

Comprehensive Analysis ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Download Full-text

Rapid microbial identifications by MALDI-TOF – Viva la revolution!

Microbiology Australia ◽

10.1071/ma10111 ◽

2010 ◽

Vol 31 (3) ◽

pp. 111

Author(s):

Stephen A Neville ◽

Iain B Gosbell

Keyword(s):

Mass Spectrometry ◽

Bacterial Identification ◽

Maldi Tof Ms ◽

Microbial Identification ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Identification Of Species ◽

Phenotypic Methods ◽

Diagnostic Microbiology ◽

Tof Ms

Although the application of mass spectrometry to bacterial identification was proposed as far back as 1975 by John Anhalt, it was not until 1987 when Professor Franz Hillenkamp and Dr Michael Karas first pioneered Matrix Assisted Laser Desorption/Ionisation Time-of-Flight mass spectrometry (MALDI-TOF MS) for truly rapid microbial identification in diagnostic microbiology laboratories. Conventional phenotypic methods of identifying isolates of bacteria, yeasts and filamentous fungi require hours to several days to complete, depending on the type of organism involved, and may be prone to error due to bias or inexperience. Even the more recent molecular innovations have their limitations that place them outside the scope and budget of many routine laboratories. However, MALDI-TOF MS technology has changed the way we think about microbial identifications and strain differentiation by providing results from plate to name in approximately five minutes for one isolate and around 90 minutes for 60 isolates at under $2 an identification. It is likely to be particularly helpful clinically for identification of species in positive blood culture broths and for organisms that are hard to identify quickly.

Download Full-text