Effect of humidity during sample preparation on bacterial identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a highly reliable and efficient technology for the identification of microbial pathogens. We previously found that 40% humidity was the optimal condition for the preparation of samples (co-crystallization of the sample and matrix) for serum peptidomic analysis via MALDI-TOF MS profiling. This optimum temperature was applied to obtain the highest reproducibility and throughput and greatest number of peaks. We therefore hypothesized that humidity control was also essential for MALDI-TOF MS bacterial identification. In this study, we constructed a simple sample preparation device that enables humidity control and used it for co-crystallization of the sample and matrix. Identification scores for five Gram-negative bacteria and six Gram-positive bacteria were determined using the MALDI BioTyper® system at three humidity ranges (10-20%, 30-40%, and 50-60%). As a result, higher identification scores were obtained at 30-40% humidity than at 10-20% or 50-60% humidity. At 30-40% humidity, 517/550 (94.0%) isolates scored greater than 2.0, indicating the success of species-level identification. Similarly, 537/550 (97.6%) isolates scored greater than 1.7, indicating the success of genus-level identification. Thus, 30-40% humidity generated optimal MALDI-TOF MS identification scores and the highest percentage of correct identifications. These results could lead to further improvements in the accuracy of MALDI-TOF MS bacterial identification.

Comparison of novel approaches for expedited pathogen identification and antimicrobial susceptibility testing against routine blood culture diagnostics.

Blood stream infections pose a major challenge for clinicians as the immediate application of an appropriate antibiotic treatment is the vital factor to safe the patients' lives. This preliminary study compares three different systems promising fast pathogen identification and susceptibility testing in comparison to conventional blood culture (BC): (i) the rapid antimicrobial susceptibility testing protocol according to EUCAST in combination with the Sepsityper® kit (sRAST), (ii) the direct inoculation method on the VITEK® 2 system (dVIT) and (iii) testing with the Accelerate Pheno® system (AccPh). All methods were assessed in terms of accuracy, time to result and usability. Twenty-three BC samples obtained from patients suffering from proven sepsis were analysed in detail. Pathogen identification was successful in 95·6, 91·3 and 91·3% in sRAST, dVIT and AccPh, respectively. Categorical agreement in antimicrobial susceptibility testing was 89·5, 96 and 96·6%, respectively. Time to result from sample entry to reporting ranged from an average of 4·6 h for sRAST and 6·9 h for AccPh to 10·6 h for dVIT. These results imply a significant shortening of reporting times at considerably high agreement rates for these new diagnostic approaches.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory.

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

The MPB64 immunochromatography assay: an analysis of doubtful results.

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth indicator tube (MGIT), endorsed by the World Health Organization (WHO), is widely used. Further identification of a positive culture is done with the help of an immunochromatography assay, which often shows faint bands that are difficult to interpret. We analysed 125 BACTEC MGIT culture positive results, of which 11/16 (68.7%) of the doubtful assays, analysed by MGIT™ TBc Identification test (TBcId), were positive for Mycobacterium tuberculosis complex (MTBC), the remaining being non-tuberculous mycobacteria as determined by an in-house duplex polymerase chain reaction and line probe assay. Guidelines on faint or doubtful bands in immunochromatography assays are important so as not to overlook true-positive cases of TB.

Comparison of pulsed-field gel electrophoresis and whole-genome-sequencing-based typing confirms the accuracy of pulsed-field gel electrophoresis for the investigation of local Pseudomonas aeruginosa outbreaks.

AIM: To determine whether pulsed-field gel electrophoresis (PFGE) accurately recognizes isolates belonging to clusters defined by techniques based on whole-genome sequencing (WGS) using Pseudomonas aeruginosa as a model. METHODS: We selected 65 isolates of ST395 P. aeruginosa isolated in seven European hospitals between 1998 and 2012. Isolates were typed by PFGE and sequenced by WGS. A core genome multi-locus sequence typing (cgMLST) analysis based on 3831 genes was performed with a homemade pipeline. FINDINGS: PFGE identified eight pulsotypes and cgMLST differentiated nine clusters and nine singletons. Five cgMLST clusters and pulsotypes (31/65 isolates) coincided perfectly. Isolates without evident epidemiological links grouped by PFGE were separated by cgMLST (16/65 isolates) differentiating cities, suggesting that PFGE should be kept for the investigation of local outbreaks. Importantly, hypermutator isolates still shared the pulsotype with their parents (16/65 isolates), whereas they were not recognized by cgMLST. This shows that PFGE was less affected than WGS-based typing by the accelerated genetic drift that occurs in epidemic P. aeruginosa. CONCLUSIONS: although WGS-based typing has logically become the new reference standard, we show here that the PFGE can be used with confidence for the investigation of local outbreaks caused by P. aeruginosa.

Species identification of Enterococcus spp: Whole genome sequencing compared to three biochemical test-based systems and two Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) systems.

AIM: Here, we evaluated the performance of two commercial MALDI-TOF MS systems and three biochemical-based systems and compared them to WGS as the gold standard for identifying isolates of vancomycin-resistant enterococci (VRE). METHODS: A total of 87 VRE clinical isolates were included. The mass spectrometers were the Microflex system with Biotyper software 3.1 and the Vitek MS system. The biochemical-based systems included the Vitek 2, Phoenix, and MicroScan WalkAway systems. WGS was performed on an Illumina MiSeq instrument using the MiSeq v3 reagent kit. Vancomycin resistance was determined according to CLSI criteria. RESULTS: Among the 87 VRE, 71 and 16 were identified as Enterococcus faecium and Enterococcus faecalis by WGS. All 71 E faecium were correctly identified by both mass spectrometers, as well as the Vitek 2 and Phoenix instruments. However, only 51 E faecium isolates were correctly identified by the MicroScan system. The most frequent misidentification was Enterococcus casseliflavus (n = 20). For vancomycin-resistant E faecium, the Microflex Biotyper system had the highest sensitivity (85.54%), and all instruments (except for the Microscan) had a 100% specificity and PPV. Up to 87% of E faecalis isolates were misidentified by VITEK MS and VITEK2, 81% by Microscan and Phoenix, and 75% by Bruker biotyper. CONCLUSION: As the coverage of type strain-genome sequence database continues to grow and the cost of DNA sequencing continues to decrease, genome-based identification can be a useful tool for diagnostic laboratories, with its superior accuracy even over MALDI-TOF and database-driven operations.

Comparison of Rapid and Routine Methods of Identification and Antibiotic Susceptibility Testing of Microorganisms from Blood Culture Bottles.

Reporting of the results of routine laboratory blood culture tests to clinicians is vital to the patients' early treatment. This study aimed to perform identification and antibiotic susceptibility tests of the blood cultures showing positive signals of microbial growth in the first 12 hours of incubation by using centrifugation and Gram staining of 5 ml of liquid from the vial, thus achieving faster results. This study included 152 consecutively incubated blood culture samples showing positive microbial growth signals in the first 12 hours. The samples were centrifuged and then categorized into two groups (Gram-positive and Gram-negative) using Gram staining. Identification and antibiotic susceptibility tests were performed using an automated culture antibiogram device. For routine processing, media inoculated with positive blood culture were kept in the incubator for at least 24 hours. To compare the two methods in terms of the bacteria identification, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the growing colony was studied. By Gram staining, the same bacterial strains were obtained for 138 (92%) of the 152 samples, similar to the results of the procedures mentioned earlier. With the samples tested with both methods, the antibiotic susceptibility profiles were compared using the antibiogram results for 1,984 samples that underwent the antibiotic testing. A 97.4% (for 1,934 antibiotic susceptibility assays) agreement was observed between the two methods. Comparing the results of the post-centrifugation Gram staining to those obtained for the specimens using routine procedures, the clinicians reported a high success rate (approximately 97%).Reporting of the results of routine laboratory blood culture tests to clinicians is vital to the patients' early treatment. This study aimed to perform identification and antibiotic susceptibility tests of the blood cultures showing positive signals of microbial growth in the first 12 hours of incubation by using centrifugation and Gram staining of 5 ml of liquid from the vial, thus achieving faster results. This study included 152 consecutively incubated blood culture samples showing positive microbial growth signals in the first 12 hours. The samples were centrifuged and then categorized into two groups (Gram-positive and Gram-negative) using Gram staining. Identification and antibiotic susceptibility tests were performed using an automated culture antibiogram device. For routine processing, media inoculated with positive blood culture were kept in the incubator for at least 24 hours. To compare the two methods in terms of the bacteria identification, matrix­assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI­TOF MS) of the growing colony was studied. By Gram staining, the same bacterial strains were obtained for 138 (92%) of the 152 samples, similar to the results of the procedures mentioned earlier. With the samples tested with both methods, the antibiotic susceptibility profiles were compared using the antibiogram results for 1,984 samples that underwent the antibiotic testing. A 97.4% (for 1,934 antibiotic susceptibility assays) agreement was observed between the two methods. Comparing the results of the post-centrifugation Gram staining to those obtained for the specimens using routine procedures, the clinicians reported a high success rate (approximately 97%).

Proposed nomenclature or classification changes for bacteria of medical importance: taxonomic update 5.

A key aspect of medical, public health, and diagnostic microbiology laboratories is the accurate identification and rapid reporting and communication to medical staff regarding patients with infectious agents of clinical importance. Microbial taxonomy continues to change at a very rapid rate in the era of molecular diagnostics including whole genome sequencing. This update focuses on taxonomic changes and proposals that may be of medical importance from 2018 to 2020.

Workflow optimization for syndromic diarrhea diagnosis using the molecular Seegene Allplex™ GI-Bacteria(I) assay.

Syndromic panel-based molecular testing has been suggested to improve and accelerate microbiological diagnosis. We aimed to analyze workflow improvements when using the multiplex Seegene Allplex™ GI-Bacteria(I) assay as a first-line assay for bacterial diarrhea. Technical assay evaluation was done using spiked stool samples and stored patient samples. After implementation of the assay in the routine clinical workflow, an analysis of 5032 clinical samples analyzed by the Seegene assay and 4173 control samples examined by culture in a similar time period 1 year earlier was performed. Sensitivity of the assay was shown to be between 0.4 and 95.9 genome equivalents/PCR. For 159 positive patient samples with a composite reference of culture and/or a molecular assay, the sensitivity of the assay was 100% for Campylobacter, 92% for Salmonella, 89% for Aeromonas, and 83% for Shigella. Sensitivity for C. difficile toxin B detection was 93.9%. The comparison of clinical samples obtained in two 8-month periods showed increased detection rates for Aeromonas (2.90%vs. 0.34%), Campylobacter spp. (2.25% vs. 1.34%), Shigella spp. (0.42% vs. 0.05%) whereas detection of Salmonella was slightly decreased (0.46% vs. 0.67%) when using the Seegene assay. An analysis of the time-to-result showed that the median dropped from 52.7 to 26.4 h when using the molecular panel testing. The Seegene Allplex™ GI-Bacteria(I) assay allows accelerated, reliable detection of major gastrointestinal bacteria roughly within 1 day. Workload is reduced, specifically in a low-prevalence setting.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

Comparison of standard and on-plate extraction protocols for identification of mastitis-causing bacteria by MALDI-TOF MS.

The objective was to compare standard versus on-plate sample preparation protocols for identification of mastitis bacteria by MALDI-TOF MS. A total of 186 bacterial isolates from cows with subclinical mastitis were identified by MALDI-TOF MS after preparation using two extraction protocols. On-plate protocol was performed by applying the bacterial colony directly from the culture plate onto the plate spot. For the standard protocol, lysis of bacterial colonies using reagents was performed in a cryotube, and the resulting extract was applied onto the plate spot for analysis. The on-plate protocol showed a similar bacteria identification rate (91.4%, n = 170/186) in comparison to the standard (94.6%, n = 176/186). Identification was higher for both protocols when scores used for species-level identification (≥ 2.0) was reduced to genus-level (≥ 1.7); genus-level identification score rate increased from 94.6 to 100% when using the standard protocol, and from 91.4 to 94.6% when using the on-plate protocol. However, when compared standard (as gold standard) versus on-plate protocol, genus-level identification score rate ranged from 87.1 to 89.8%. Therefore, when the on-plate protocol fails to identify any specie, the standard extraction may be more suitable as a reference protocol for use. Strategy for increasing identification with the on-plate protocol may include upgrading the reference database library. Choice of protocol for preparation may be influenced by the bacterial type to be identified. Standard and on-plate extraction protocols of bacterial ribosomal proteins associated with MALDI-TOF MS might be alternatives to conventional microbiology methods for identification of subclinical mastitis pathogens.

Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity.

We present here a new passive-filtration-based culture device combined with rapid identification with a new electron microscope (Hitachi TM4000) for the detection and culture of Treponema species from the human oral cavity. Of the 44 oral samples cultivated, 15 (34%) were found to be positive for Treponema using electron microscopy and were also culture positive. All were subcultured on agar plates; based on genome sequencing and analyses, 10 were strains of Treponema pectinovorum and 5 were strains of Treponema denticola The 29 samples that were negative for Treponema remained culture negative. In addition, 14 Treponema species ordered from the DSMZ collection were cultured in the T-Raoult culture medium optimized here. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used and 30 novel spectra were added to the MALDI-TOF MS database. We have successfully developed a new and effective method for treponemal detection, culture, and identification.

Cost and operational impact of promoting upfront GeneXpert MTB/RIF test referrals for presumptive pediatric tuberculosis patients in India.

BACKGROUND: Outreach and promotion programs are essential to ensuring uptake of new public health interventions and guidelines. We assessed the costs and operation dynamics of outreach and promotion efforts for up front Xpert MTB/RIF (Xpert) testing for pediatric presumptive tuberculosis (TB) patients in four major Indian cities. METHODS: Xpert test costs were assessed as weighted average per-test costs based on the daily workload dynamics matched by test volume specific Xpert unit cost at each study site. Costs of outreach programs to recruit health providers to refer pediatric patients for Xpert testing were assessed as cost per referral for each quarter based on total program costs and referral data. All costs were assessed in the health service provider's perspective and expressed in 2015 USD. RESULTS: Weighted average per-test costs ranged from $14.71 to $17.81 at the four laboratories assessed. Differences between laboratories were associated with unused testing capacity and/or frequencies of overtime work to cope with increasing demand and same-day testing requirements. Outreach activities generated between 825 and 2,065 Xpert testing referrals on average each quarter across the four study sites, translating into $0.63 to $2.55 per patient referred. Overall outreach costs per referral decreased with time, stabilizing at an average cost of $1.10, and demonstrated a clear association with increased referrals. CONCLUSIONS: Xpert test and outreach program costs within and across study sites were mainly driven by the dynamics of Xpert testing demand resulting from the combined outreach activities. However, these increases in demand required considerable overtime work resulting in additional costs and operational challenges at the study laboratories. Therefore, careful laboratory operational adjustment should be evaluated at target areas in parallel to the anticipated demand from the Xpert referral outreach program scale-up in other Indian regions.

Comparison of Three Nucleic Acid Amplification Tests and Culture for Detection of Group B Streptococcus from Enrichment Broth.

Colonization of the gastrointestinal and genitourinary tracts of pregnant women with group B Streptococcus (GBS) can result in vertical transmission to neonates during labor/delivery. GBS infections in neonates can cause severe complications, such as sepsis, meningitis, and pneumonia. Accurate detection is critical because administration of intrapartum antibiotics can significantly reduce transmission. We compared the clinical sensitivities of three nucleic acid amplification tests (NAATs), the Hologic Panther Fusion GBS, Luminex Aries GBS, and Cepheid Xpert GBS LB assays, to that of the standard of care culture method recommended for GBS screening using 500 vaginal-rectal swab specimens after 18 to 24 h of broth enrichment. We identified 108 positive specimens (21.6%) by culture, while at least 1 of the 3 NAATs was positive for GBS in 155 specimens (31.0%). All 108 specimens positive by culture were also detected by the Panther Fusion assay, while 107/108 (99.1%) were detected by the Cepheid Xpert and Luminex Aries assays. Of the 61 specimens positive by at least 1 NAAT but negative by culture, 24 (39.3%) were positive by all 3 NAATs, suggesting that they represent true positives (TPs). NAATs offer less hands-on time, greater throughput, faster time to result, and potentially greater sensitivity than culture methods, and they should be considered the new gold standard for intrapartum GBS screening.

Factors That Influence Confirmation of Neisseria gonorrhoeae Positivity by Molecular Methods.

Several Neisseria gonorrhoeae nucleic acid amplification tests (NAATs) with high sensitivity exist. However, the specificity of N. gonorrhoeae NAATs may be suboptimal, particularly for extragenital biospecimens. Consequently, confirmation with a second NAAT is common, although this represents a burden on resources. Furthermore, the rationale for confirmation is contentious. The objective of this work was to assess N. gonorrhoeae confirmation in over 13,000 N. gonorrhoeae screen-positive samples representing various biospecimens and three separate screening assays, the Abbott RealTime CT/NG (Abbott Molecular, Inc., Des Plaines, IL), the Cobas CT/NG test (Roche Molecular Systems Inc., Alameda, CA), and the BD ProbeTec ET CT/GC amplified DNA assay (BD Diagnostics, Sparks, MD). Factors predictive of confirmation were determined via logistic regression involving sex, year, whether the sample was formally validated, and sample site. Level of confirmation varied according to screening assay (96.2%, 86.0%, and 73.9% for the Abbott, Roche, and BD tests, respectively) in sample types formally included according to the manufacturers' instructions (i.e., validated). Sex did not affect confirmation for 2/3 assays, and the likelihood of confirmation of samples not formally included in manufacturer instructions (i.e., nonvalidated) was 89.1%, 82.1%, and 59.2% for the Abbott, Roche, and BD tests, respectively. Rectal swabs, which are nonvalidated samples, confirmed in 91.5%, 90.1%, and 87.4% of samples initially tested with the respective assays. The requirement to confirm N. gonorrhoeae in validated samples is not required for all NAATs, although initial assay-specific evaluation is justified given observed variability. Rectal samples represent robust biospecimens for N. gonorrhoeae NAAT testing and may not require confirmation when screened with the assays described.

Laboratory validation of a KPC-producing strain identification method based on the detection of a specific 11,109 Da peak via Maldi-Tof-Vitek MS in an endemic area.

Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Vitek MS system is a useful technique to identify bacteria strains isolated in clinical samples. In this paper, we applied this method to KPC-producing Enterobacteriaceae detection through the determination of a specific 11,109 (±8) Da peak. We assayed the presence, specificity and reliability of this peak on routine workflow through the analysis of 183 Enterobacteriaceae strains isolated from clinical samples and characterized by classical approaches. The peak was detected in 95.5% (129/135) of carbapenemase-producing strains spectra compared with the 48 extended spectrum beta-lactamase producing controls strains, which all lacked this peak. Hence, this 11,109 Da peak determination showed a Positive Predictive Value (PPV) of 100% and a Negative Predictive Value (NPV) of 94.4%. The characterization of this specific peak in a MALDI-TOF Vitek MS system might be considered a valuable tool to reveal KPC-producing Enterobacteriaceae especially in KPC endemic region.

Comparative Evaluation of Enteric Bacterial Culture and a Molecular Multiplex Syndromic Panel in Children with Acute Gastroenteritis.

Although enteric multianalyte syndromic panels are increasingly employed, direct comparisons with traditional methods and the inclusion of host phenotype correlations are limited. Luminex xTAG gastrointestinal pathogen panel (GPP) and culture results are highly concordant. However, phenotypic and microbiological confirmatory testing raises concerns regarding the accuracy of the GPP, especially for Salmonella spp. A total of 3,089 children with gastroenteritis submitted stool specimens, rectal swab specimens, and clinical data. The primary outcome was bacterial pathogen detection agreement for shared targets between culture and the Luminex xTAG GPP. Secondary analyses included phenotype assessment, additional testing of GPP-negative/culture-positive isolate suspensions with the GPP, and in-house and commercial confirmatory nucleic acid testing of GPP-positive/culture-negative extracts. The overall percent agreement between technologies was >99% for each pathogen. Salmonella spp. were detected in specimens from 64 participants: 12 (19%) by culture only, 9 (14%) by GPP only, and 43 (67%) by both techniques. Positive percent agreement for Salmonella spp. was 78.2% (95% confidence interval [CI], 64.6%, 87.8%). Isolate suspensions from the 12 participants with specimens GPP negative/culture positive for Salmonella tested positive by GPP. Specimens GPP positive/culture negative for Salmonella originated in younger children with less diarrhea and more vomiting. GPP-positive/culture-negative specimen extracts tested positive using additional assays for 0/2 Campylobacter-positive specimens, 0/4 Escherichia coli O157-positive specimens, 0/9 Salmonella-positive specimens, and 2/3 Shigella-positive specimens. For both rectal swab and stool samples, the median cycle threshold (CT ) values, determined using quantitative PCR, were higher for GPP-negative/culture-positive samples than for GPP-positive/culture-positive samples (for rectal swabs, 36.9 [interquartile range {IQR}, 33.7, 37.1] versus 30.0 [IQR, 26.2, 33.2], respectively [P = 0.002]; for stool samples, 36.9 [IQR, 33.7, 37.1] versus 29.0 [IQR, 24.8, 30.8], respectively [P = 0.001]). GPP and culture have excellent overall agreement; however, for specific pathogens, GPP is less sensitive than culture and, notably, identifies samples false positive for Salmonella spp.

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation.

Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

Use of MALDI-TOF mass spectrometry to detect nosocomial outbreaks of Serratia marcescens and Citrobacter freundii.

MALDI-TOF mass spectrometry (MS) may be used as a rapid typing method for nosocomial pathogens. Here, we evaluated MALDI-TOF MS for discrimination of hospital outbreak-related clusters of Serratia marcescens and carbapenemase-producing Citrobacter freundii. Thirty-three S. marcescens isolates collected from neonatal intensive care unit (NICU) patients, and 23 C. freundii isolates including VIM-positive isolates from a hospital colonization outbreak were measured by Vitek MS. Consensus spectra of each isolate were clustered using SARAMIS software. Genotyping was performed by whole-genome sequencing (WGS). First, a set of 21 S. marcescens isolates from 2014 with seven genotypes including three monoclonal clusters was used for the evaluation of MALDI-TOF typing. MS clustering was largely in agreement with genotyping results when the similarity cut-off for clonal identity was set on 90%. MALDI-TOF cluster analysis was then investigated for the surveillance of S. marcescens in the NICU in 2017 and demonstrated the introduction of new strains into the hospital and nosocomial transmissions. MS analysis of the C. freundii outbreak in 2016 revealed a monoclonal cluster of VIM-positive isolates and the separation of epidemiologically non-related VIM-positive and negative isolates. Two additional VIM-positive Citrobacter isolates from food samples were closely related to the large monoclonal cluster. WGS confirmed the MS results. MALDI-TOF MS may be used as a first-line typing tool for S. marcescens and C. freundii to detect transmission events in the hospital because isolates of an identical WGS type were grouped into the same MS cluster.

Application of MALDI Biotyper System for Rapid Identification of Bacteria Isolated from a Fresh Produce Market.

MALDI-TOF MS has revolutionized the identification of microorganisms and has become an indispensable part of routine diagnostics in the clinical microbiological laboratory. However, application of this technique in microbial surveillance outside of clinical settings is limited. In this study, we have evaluated the performance of a Bruker MALDI Biotyper System for the identification of bacteria isolated from the hand palms of fresh produce handlers and their surrounding environments in a wholesale fresh produce market in Doha, Qatar. The accuracy was verified against the results obtained by bacterial 16S rRNA gene sequencing. A total of 105 isolates were tested, of which 67 (64%) isolates were identified by MALDI-TOF MS and 101 isolates (96%) were identified by 16S rRNA gene sequencing, either at the genus level or species level. However, MALDI-TOF MS identified more isolates (41%) at the species level than 16S rRNA gene sequencing (28%). MALDI-TOF MS was particularly useful in the species level identification of Enterobacteriaceae. MALDI-TOF MS successfully identified most known human pathogens in a rapid and cost-effective manner but failed to identify a significant number of isolates that were of environmental origin, suggesting room for further expansion of the reference database.