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

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
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.

scholarly journals The clinical utility of two high-throughput 16S rRNA gene sequencing workflows for taxonomic assignment of unidentifiable bacterial pathogens in MALDI-TOF MS

2021 ◽  
Author(s):  
Hiu-Yin Lao ◽  
Timothy Ting Leung Ng ◽  
Ryan Yik Lam Wong ◽  
Celia Sze Ting Wong ◽  
Chloe Toi Mei Chan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Reference Standard ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming and low-throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests, which are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by respective built-in analysis programs (MiSeq Reporter Software and Epi2me) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a comparatively higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of optimized Nanopore 16S was 96.36%, which was identical to Sanger 16S and was better than Illumina 16S (71.52%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78h and 8.25h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

scholarly journals Epidemiology of Nontuberculous Mycobacteria in French Polynesia

2015 ◽  
Vol 53 (12) ◽  
pp. 3798-3804 ◽  
Author(s):  
Michael Phelippeau ◽  
Djaltou Aboubaker Osman ◽  
Didier Musso ◽  
Michel Drancourt
Keyword(s):  
Nontuberculous Mycobacteria ◽  
Sequence Similarity ◽  
Gene Sequencing ◽  
French Polynesia ◽  
Rrna Gene ◽  
Mycobacterium Fortuitum ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

As few data are available in the Pacific countries and territories of the Oceania region regarding nontuberculous mycobacteria, we retrospectively identified 87 such isolates from French Polynesia from 2008 to 2013 by hybridization using DNA-strip, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and partialrpoBgene sequencing. PartialrpoBgene sequencing classified 42/87 (48.3%) isolates in theMycobacterium fortuitumcomplex, 28 (32.2%) in theMycobacterium abscessuscomplex, 8 (9.2%) in theMycobacterium mucogenicumcomplex, and 5 (5.7%) in theMycobacterium aviumcomplex. Two isolates were identified asMycobacterium acapulcensisandMycobacteriumcosmeticumby partial 16S rRNA gene sequencing. One isolate, unidentified by MALDI-TOF MS and yielding less than 92% and 96% sequence similarity withrpoBandhsp65reference sequences, respectively, was regarded as a potentially new species. Samples from three patients exhibiting ≥2Mycobacterium porcinumisolates and from one patient with emphysema and a lung abscess exhibiting 2Mycobacterium senegalenseisolates fulfilled the American Thoracic Society microbiological criteria for nontuberculous mycobacterial lung infection. Remote geographic areas, such as French Polynesia, are potential sources for the discovery of new mycobacterial species.

Matrix-Assisted Laser Desorption and Ionization–Time-of-Flight Mass Spectrometry, 16S rRNA Gene Sequencing, and API 32E for Identification of Cronobacter spp.: A Comparative Study

2011 ◽  
Vol 74 (12) ◽  
pp. 2182-2187 ◽  
Author(s):  
SHA ZHU ◽  
STEFAN RATERING ◽  
SYLVIA SCHNELL ◽  
RON WACKER
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Twenty-two isolates of the family Enterobacteriaceae, with focus on Cronobacter isolated from infant formula and the environment of milk powder plants, were comparatively identified using API 32E (bioMérieux, Marcy l'Etoile, France), 16S rRNA gene sequencing (Accugenix, Newark, USA), and matrix-assisted laser desorption and ionization–time-of-flight mass spectrometry (MALDI-TOF MS; Mabritec, Riehen, Switzerland and AnagnosTec, Potsdam, Germany). With API 32E, 22% of the isolates were assigned to species, 64% were assigned to a genus, and 14% could not be discriminated at any taxonomic level. Both 16S rRNA gene sequencing and MALDI-TOF MS assigned 100% of the isolates to species, but the identifications based on MALDI-TOF MS results were more discriminating and unequivocal. Our data indicate that MALDI-TOF MS provides the most rapid and unambiguous identification of Cronobacter and closely related Enterobacteriaceae isolates.

scholarly journals Research on the establishment and application of protein fingerprint spectrum database of Burkholderia pseudomallei in Hainan Province China

2020 ◽  
Author(s):  
Xu-Ming Wang ◽  
Ling-Li Liu ◽  
Hua Wu ◽  
Mei-Hui Huang
Keyword(s):  
16S Rrna ◽  
Burkholderia Pseudomallei ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Hainan Province ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Abstract Background: The aim of this study was to establish a SuperSpectrum of Burkholderia pseudomallei(B. pseudomallei) in Hainan and evaluate its application value in the rapid identification of clinical isolates of B. pseudomallei.Methods: Using a collection of 167 isolates of B. pseudomallei from June 2010 to May 2019 in different regions of Hainan province, multilocus sequence typing (MLST) was performed, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for spectrum acquisition. A SuperSpectrum was created based on the selection of 80 representative average spectra. In a second step, we validated the SuperSpectra with 137 strains of B. pseudomallei, 8 strains of Burkholderia thailandensis(B. thailandensis), 2 strains of Burkholderia cepacia(B. cepacia), 1 strain of Burkholderia cenocepacia(B. cenocepacia) and 1 strain of Burkholderia multivorans(B. multivorans), as well as 1 strain of Burkholderia gladioli(B. gladioli) identified by MLST typing and 16S rRNA gene sequencing.Results: The results showed that there was 100% agreement between the validation strains analyzed by MALDI-TOF MS and those evaluated by MLST typing and 16S rRNA gene-sequencing analysis methods. Protein fingerprints spectra showed that specific peaks occurred in B. pseudomallei from the Hainan region. The result of clustering typing indicated that B. pseudomallei and its closely related species could be well classified by MALDI-TOF MS at the protein level.Conclusions: MALDI-TOF MS is a promising, rapid, and economical method to monitor the outbreaks and spread of B. pseudomallei isolates.The establishment of an accurate and objective SuperSpectrum database can provide a new platform for the clinical rapid diagnosis of melioidosis.

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

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.

scholarly journals The clinical utility of two high-throughput 16S rRNA gene sequencing workflows for taxonomic assignment of unidentifiable bacterial pathogens in MALDI-TOF MS

2021 ◽  
Author(s):  
Hiu-Yin Lao ◽  
Timothy Ting-Leung Ng ◽  
Ryan Yik-Lam Wong ◽  
Celia Sze-Ting Wong ◽  
Lam-Kwong Lee ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Reference Standard ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Rrna Gene Sequencing ◽  
Tof Ms

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming and low-throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests, which are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by respective built-in programs (MiSeq Reporter Software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to Sanger 16S and was better than Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78h and 8.25h respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

Sign in / Sign up

Export Citation Format

Share Document