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Showing papers on "Typing published in 2019"


Journal ArticleDOI
TL;DR: As WGS provides more information, such as antimicrobial resistance and virulence of the tested bacteria in comparison to PFGE, more and more laboratories are currently transitioning from PFGE to WGS for bacteria typing, ensuring PFGE will remain an affordable and relevant technique for small laboratories and hospitals in years to come.

88 citations


Journal ArticleDOI
TL;DR: FTIR spectroscopy is a promising tool for strain typing of clinical E. cloacae complex strains due to its low costs and fast turnaround times, and presents a valuable tool for real-time surveillance as well as outbreak analysis.
Abstract: Members of the Enterobacter (E.) cloacae complex have emerged as important pathogens frequently encountered in nosocomial infections. Several outbreaks with E. cloacae complex have been reported in recent years, especially in neonatal units. Fast and reliable strain typing methods are crucial for real-time surveillance and outbreak analysis to detect pathogen reservoirs and transmission routes. The aim of this study was to evaluate the performance of Fourier-transform infrared (FTIR) spectroscopy as a fast method for typing of clinical E. cloacae complex isolates, when whole genome sequencing (WGS) analysis was used as reference. First, the technique was used retrospectively on 24 first isolates of E. cloacae complex strains from neonatal patients and showed good concordance with SNP-based clustering (Adjusted Rand Index [ARI] = 0.818) and with the sequence type (ST) (ARI = 0.801). 29 consecutive isolates from the same patients were shown by WGS analysis to almost always belong to the same SNP cluster as the first isolates, which was only inconsistently recognized by FTIR spectroscopy. Training of an artificial neural network (ANN) with all FTIR spectra from sequenced strains markedly improved the recognition of related and unrelated isolate spectra. In a second step, FTIR spectroscopy was applied on 14 strains during an outbreak with E. cloacae complex and provided fast typing results that were confirmed by WGS analysis. In conclusion, FTIR spectroscopy is a promising tool for strain typing of clinical E. cloacae complex strains. Discriminatory power can be improved by implementing an ANN for spectrum analysis. Due to its low costs and fast turnaround times, the method presents a valuable tool for real-time surveillance as well as outbreak analysis.

46 citations


01 Jan 2019
TL;DR: The Meningococcal Antigen Typing System (MATS) was developed to identify meningococcus group B strains with a high likelihood of being covered by the 4CMenB vaccine, but is limited by the requirement for viable isolates from culture confirmed cases as discussed by the authors.
Abstract: BACKGROUND The Meningococcal Antigen Typing System (MATS) was developed to identify meningococcus group B strains with a high likelihood of being covered by the 4CMenB vaccine, but is limited by the requirement for viable isolates from culture-confirmed cases. We examined if antigen genotyping could complement MATS in predicting strain coverage by the 4CMenB vaccine. METHODS From a panel of 3912 MATS-typed invasive meningococcal disease isolates collected in England and Wales in 2007-2008, 2014-2015 and 2015-2016, and in 16 other countries in 2000-2015, 3481 isolates were also characterized by antigen genotyping. Individual associations between antigen genotypes and MATS coverage for each 4CMenB component were used to define a genetic MATS (gMATS). gMATS estimates were compared with England and Wales human complement serum bactericidal assay (hSBA) data and vaccine effectiveness (VE) data from England. RESULTS Overall, 81% of the strain panel had genetically predictable MATS coverage, with 92% accuracy and highly concordant results across national panels (Lin's accuracy coefficient, 0.98; root-mean-square deviation, 6%). England and Wales strain coverage estimates were 72-73% by genotyping (66-73% by MATS), underestimating hSBA values after four vaccine doses (88%) and VE after two doses (83%). The gMATS predicted strain coverage in other countries was 58-88%. CONCLUSIONS gMATS can replace MATS in predicting 4CMenB strain coverage in four out of five cases, without requiring a cultivable isolate, and is open to further improvement. Both methods underestimated VE in England. Strain coverage predictions in other countries matched or exceeded England and Wales estimates.

46 citations


Journal ArticleDOI
TL;DR: Genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution, providing an international standardized nomenclature suitable for surveillance approaches in various sectors, linking public health, veterinarypublic health, and food safety in a true One Health fashion.
Abstract: Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

42 citations


Journal ArticleDOI
TL;DR: The present study was conducted from July to August 2018 on milk samples taken at dairy farms in the Northern Province and Kigali District of Rwanda in order to identify Staphylococcus spp.
Abstract: The present study was conducted from July to August 2018 on milk samples taken at dairy farms in the Northern Province and Kigali District of Rwanda in order to identify Staphylococcus spp. associated with bovine intramammary infection. A total of 161 staphylococcal isolates originating from quarter milk samples of 112 crossbred dairy cattle were included in the study. Antimicrobial susceptibility testing was performed and isolates were examined for the presence of various resistance genes. Staphylococcus aureus isolates were also analyzed for the presence of virulence factors, genotyped by spa typing and further phenotypically subtyped for capsule expression using Fourier Transform Infrared (FTIR) spectroscopy. Selected S. aureus were characterized using DNA microarray technology, multi-locus sequence typing (MLST) and whole-genome sequencing. All mecA-positive staphylococci were further genotyped using dru typing. In total, 14 different staphylococcal species were detected, with S. aureus being most prevalent (26.7%), followed by S. xylosus (22.4%) and S. haemolyticus (14.9%). A high number of isolates was resistant to penicillin and tetracycline. Various antimicrobial and biocide resistance genes were detected. Among S. aureus, the Panton-Valentine leukocidin (PVL) genes, as well as bovine leukocidin (LukM/LukF-P83) genes, were detected in two and three isolates, respectively, of which two also carried the toxic shock syndrome toxin gene tsst-1 bovine variant. t1236 was the predominant spa type. FTIR-based capsule serotyping revealed a high prevalence of non-encapsulated S. aureus isolates (89.5%). The majority of the selected S. aureus isolates belonged to clonal complex (CC) 97 which was determined using DNA microarray based assignment. Three new MLST sequence types were detected.

32 citations


Journal ArticleDOI
TL;DR: The role of high‐resolution 2‐field HLA typing (HR‐2F) in SOT is assessed by retrospectively evaluating NGS‐typed pre‐ and post‐SOT cases, whereby 21% of the cases required HR‐ 2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti‐HLA antibody issues.

32 citations


Journal ArticleDOI
TL;DR: The limitations of multilocus sequence typing are demonstrated using a robust genome phylogeny for typing Acinetobacter baumannii, showing it cannot reflect the true relationships of isolates for species with very dynamic genomes.
Abstract: Multilocus sequence typing has been useful for genotyping pathogens in surveillance and epidemiologic studies. However, it cannot reflect the true relationships of isolates for species with very dynamic genomes. Using a robust genome phylogeny, we demonstrated the limitations of this method for typing Acinetobacter baumannii.

30 citations


Journal ArticleDOI
TL;DR: The recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing is extended to whole exome sequencing (WES).

27 citations


Journal ArticleDOI
TL;DR: Most parasite haplotypes from patients were identical or similar to those for Triatoma dimidiata from the same region, confirming their local circulation and should be taken into account to evaluate associations between parasite diversity and clinical aspects of T. cruzi infections.
Abstract: The diversity of Trypanosoma cruzi parasites infecting humans is still poorly understood. We used deep sequencing to analyze this diversity in chagasic patients from Mexico. Such information is crucial to understand transmission cycles and to identify determinants of epidemiological and clinical characteristics of the infection. We analyzed parasite mini-exon spliced-leader sequences following amplification of blood DNA by polymerase chain reaction and deep sequencing. Chagasic patients presented a diverse assemblage of parasite haplotypes covering TcI, TcII, TcV, and TcVI discrete typing units, with a mean (±SEM) of 3.9 ± 0.7 haplotypes/patient, and 47% harbored infections with multiple discrete typing units. Most parasite haplotypes from patients were identical or similar to those for Triatoma dimidiata from the same region, confirming their local circulation. Infection with multiple T. cruzi strains may influence serological diagnostic test results and disease progression in patients and should be taken into account to evaluate associations between parasite diversity and clinical aspects of T. cruzi infections.

27 citations


Journal ArticleDOI
01 Sep 2019
TL;DR: This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single‐pass NGS HLA typing method has direct application for solid organ transplantation.
Abstract: Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.

26 citations


Journal ArticleDOI
TL;DR: The human leukocyte antigen (HLA) system is a highly polymorphic family of genes involved in immunity and responsible for identifying self versus non-self and is essential for solid organ and bone marrow transplantation as well as in non-transplant settings such as disease association and pharmacogenomics.
Abstract: The human leukocyte antigen (HLA) system is a highly polymorphic family of genes involved in immunity and responsible for identifying self versus non-self. HLA typing is essential for solid organ and bone marrow transplantation as well as in non-transplant settings such as disease association and pharmacogenomics. Typing of HLA genes differs from most molecular testing as, rather than evaluating differences from an accepted "wild-type" gene, it must distinguish between thousands of similar, but distinct alleles. This article will describe the HLA system and nomenclature. We will then discuss clinical uses of HLA typing including solid organ transplantation, hematopoietic stem cell transplantation, evaluation of platelet refractory patients, disease association, and pharmacogenetics. Finally, we describe common molecular methods of HLA typing.

Journal ArticleDOI
TL;DR: Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years, and type 1 became more common, and MLVA type 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011.
Abstract: Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains’ antigenic properties have been suggested as contributing factors. M. pneumoniae PCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden’s population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden.

Journal ArticleDOI
TL;DR: In light of the observed MRSA diversity the spa typing could constitute a preferable approach for MRSA typing.

Journal ArticleDOI
09 Jan 2019-PeerJ
TL;DR: This study proposes the establishment of a single MLST of T. p.
Abstract: Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with worldwide prevalence. Several different molecular typing schemes are currently available for this pathogen. To enable population biology studies of the syphilis agent and for epidemiological surveillance at the global scale, a harmonized typing tool needs to be introduced. Recently, we published a new multi-locus sequence typing (MLST) with the potential to significantly enhance the epidemiological data in several aspects (e.g., distinguishing genetically different clades of syphilis, subtyping inside these clades, and finally, distinguishing different subspecies of non-cultivable pathogenic treponemes). In this short report, we introduce the PubMLST database for treponemal DNA data storage and for assignments of allelic profiles and sequencing types. Moreover, we have summarized epidemiological data of all treponemal strains (n = 358) with available DNA sequences in typing loci and found several association between genetic groups and characteristics of patients. This study proposes the establishment of a single MLST of T. p. pallidum and encourages researchers and public health communities to use this PubMLST database as a universal tool for molecular typing studies of the syphilis pathogen.

Journal ArticleDOI
TL;DR: A high prevalence of MRSP amongst clinical samples from pets in Italy is confirmed and these isolates show multidrug resistance features that are of concern both in veterinary and human medicine for clinical and epidemiological reasons.
Abstract: The aim of this study was to assess the prevalence, the genotypic diversity, the antimicrobial resistance traits of canine and feline clinical methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates in a diagnostic laboratory in Italy during 2015-2016. All isolates were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome (SCC)- mec typing and staphylococcal protein A ( spa )-typing. The resistance profiles were assessed by antimicrobial susceptibility testing and confirmed genotypically by the detection of mecA gene and by microarray analyses. The prevalence of MRSP isolates was high (31.6%). All the strains were multidrug resistant and the most frequent clone was ST71-SCC mec type II-III. These results confirm a high prevalence of MRSP amongst clinical samples from pets in Italy. These isolates show multidrug resistance features that are of concern both in veterinary and human medicine for clinical and epidemiological reasons.

Journal ArticleDOI
TL;DR: A multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE.
Abstract: We developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the E. coli reference (ECOR) collection ( n = 72), O1:K1 isolates causing neonatal meningitis ( n = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone ( n = 38), Shiga toxin-producing E. coli (STEC) isolates of serogroups O157:H7 ( n = 21) and O26 ( n = 16, 8 of which belonged to an outbreak), 27 Shigella isolates (22 Shigella sonnei isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 Shigella isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of E. coli/ Shigella isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE. IMPORTANCE Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all E. coli isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).

Journal ArticleDOI
TL;DR: Some erythromycin-resistant isolates harboured a diverse array of resistance mechanisms, including the presence of ermB, 23S rRNA A2074G mutation, and point mutations the rplD and rplV ribosomal genes, and the cmeABC multidrug efflux pump genes.
Abstract: The aim of this study was to better understand the molecular epidemiology of Campylobacter coli isolated from multiple sources in Belgium, by studying the genotypic diversity and antimicrobial resistance phenotypes and resistance mechanisms of 59 C. coli isolates. Isolates from broiler carcasses and human cases were genotyped using multilocus sequence typing (MLST), porA typing, flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, and by PCR binary typing (P-BIT). Thirty-two MLST sequence types, 24 flaA types, 31 porA alleles, and 29 P-BIT types were identified among the screened isolates. Some types and alleles were shared among strains recovered from both broiler carcasses and diarrhoeal patients. Both porA typing and MLST revealed a similar discriminatory power (0.969), which was the highest discriminatory power when compared to other methods. Minimum inhibitory concentrations against seven different antibiotics (ciprofloxacin, chloramphenicol, nalidixic acid, streptomycin, tetracycline, gentamicin, and erythromycin) were analysed. Strains were most frequently resistant to tetracycline (81.4%), followed by: ciprofloxacin and nalidixic acid (76.3%); streptomycin (33.9%); erythromycin (27.1%); and chloramphenicol (3.4%). All isolates were sensitive to gentamicin. Multidrug resistance was observed in 24 of 59 C. coli isolates (40.7%). Molecular screening of antimicrobial resistance mechanisms revealed the predominance of the gyrA T86I substitution among ciprofloxacin- and nalidixic acid-resistant isolates, the tet(O) gene among tetracycline-resistant isolates and the 23S rRNA A2075G mutation among erythromycin- resistant isolates. Furthermore, some erythromycin-resistant isolates harboured a diverse array of resistance mechanisms, including the presence of ermB, 23S rRNA A2074G mutation, and point mutations the rplD and rplV ribosomal genes, and the cmeABC multidrug efflux pump genes.

Journal ArticleDOI
TL;DR: Subtyping and epidemiological evidence suggests that the outbreak of yersiniosis in the Bay of Plenty region between October and December 2016 could be attributed to a point source, however, subtyping results further suggest that the same clone was isolated from several regions between August 2016 and March 2017.
Abstract: Incidence of human yersiniosis in New Zealand has increased between 2013 and 2017. For surveillance and outbreak investigations it is essential that an appropriate level of discrimination between pathogenic Yersinia enterocolitica isolates is provided, in order to support epidemiological linking of connected cases. Subtyping of 227 Y. enterocolitica isolates was performed using a range of different typing methods, including biotyping, serotyping and seven loci multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, core genome single-nucleotide polymorphism (core SNP) analysis and multi-locus sequence typing were performed on a subset of 69 isolates. Sixty-seven different MLVA types were identified. One MLVA profile was associated with an outbreak in the Bay of Plenty region, supported by epidemiological data. Core SNP analysis showed that all the outbreak-related isolates clustered together. The subtyping and epidemiological evidence suggests that the outbreak of yersiniosis in the Bay of Plenty region between October and December 2016 could be attributed to a point source. However, subtyping results further suggest that the same clone was isolated from several regions between August 2016 and March 2017. Core SNP analysis and MLVA typing failed to differentiate between Y. enterocolitica biotype 2 and biotype 3. For this reason, we propose that these biotypes should be reported as a single type namely: Y. enterocolitica biotype 2/3 and that the serotype should be prioritised as an indicator of prevalence.

Journal ArticleDOI
TL;DR: WGS by cgMLST is a valuable tool, better suited for prompt epidemiological investigations than traditional typing methods because of its higher discriminatory ability in determining clonal relatedness.
Abstract: Objectives Carbapenem-resistant Acinetobacter baumannii (CRAB) is a serious nosocomial pathogen that causes a variety of serious, often life-threatening, infections and outbreaks. This study aimed to investigate the molecular epidemiology of clinical CRAB isolates from an outbreak that occurred in the intensive care unit (ICU) of an Italian hospital. Methods From December 2016 to April 2017, 13 CRAB isolates were collected from seven patients treated in the ICU at ‘L. Spallanzani’ Hospital (Rome, Italy). Typing was performed by repetitive extragenic palindromic PCR (rep-PCR) using a DiversiLab® system. Whole-genome sequencing (WGS) data were used for in silico analysis of traditional multilocus sequence typing (MLST) results, to identify resistance genes and for core genome MLST (cgMLST) analysis. Epidemiological data were obtained from hospital records. Results All isolates showed a carbapenem-resistant profile and carried the blaOXA-23 carbapenemase gene. Typing performed by rep-PCR and MLST showed that the isolates clustered into one group, whilst the cgMLST approach, which uses 2390 gene targets to characterise the gene-by-gene allelic profile, highlighted the presence of two cluster types. These results allowed us to identify two patients who were likely to be the source of two separate transmission chains. Conclusion These results show that WGS by cgMLST is a valuable tool, better suited for prompt epidemiological investigations than traditional typing methods because of its higher discriminatory ability in determining clonal relatedness.

Journal ArticleDOI
TL;DR: Despite lower resolution compared to PFGE, MALDI-TOF may provide an efficient mean for real-time monitoring spread of infection, as it is shown to provide a strong correlation with PFGE.
Abstract: Outbreaks of multidrug resistant bacteria including vancomycin-resistant enterococci (VRE) in healthcare institutions are increasing in Norway, despite a low level of resistance compared to other European countries. In this study, we describe epidemiological relatedness of vancomycin-resistant Enterococcus faecium isolated during an outbreak at a Norwegian hospital in 2012–2013. During the outbreak, 9454 fecal samples were screened for VRE by culture and/or PCR. Isolates from 86 patients carrying the vanA resistance gene were characterized using pulsed-field gel electrophoresis (PFGE), MALDI-TOF mass spectrometry and single nucleotide polymorphism typing. PFGE revealed two main clusters, the first comprised 56 isolates related to an initial outbreak strain, and the second comprised 21 isolates originating from a later introduced strain, together causing two partly overlapping outbreaks. Nine isolates, including the index case were not related to the two outbreak clusters. In conclusion, the epidemiological analyses show that the outbreak was discovered by coincidence, and that infection control measures were successful. All typing methods identified the two outbreak clusters, and the experiment congruence between the MALDI-TOF and the PFGE clustering was 63.2%, with a strong correlation (r = 72.4%). Despite lower resolution compared to PFGE, MALDI-TOF may provide an efficient mean for real-time monitoring spread of infection.

Journal ArticleDOI
TL;DR: Real-life data is presented from the DKMS Germany donor file regarding discrepancies between HLA typing results at donor recruitment and CT results and this letter focuses on donors who were typed by NGS-based methods at theDKMS Life Science Lab (Dresden, Germany).
Abstract: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for severe blood diseases. According to the EBMT activity survey, unrelated stem cell donors provided 52% of all allogeneic stem cell products in 2015 [1]. Unrelated donor registries inform, recruit and administer potential donors and have built up a global pool of currently more than 32 million donors since the 1970s [2]. As an essential part of the donor recruitment process, the potential donor’s HLA genotype is determined. The specification of typing scope (number of loci, resolution) and typing method (e.g., Sanger sequencing, next-generation sequencing (NGS)) is made by donor registries under consideration of limited resources for donor registry operations. NGS technologies offer high-throughput high-resolution HLA typing of new donors at unprecedented low costs [3–6]. The process leading to stem cell donation currently includes a mandatory confirmatory typing (CT) (sometimes also named verification typing (VT)) step. Main purpose of the CT step is the confirmation of the potential donor’s HLA typing result by typing a new donor sample, generally at the same lab that executed the prospective transplant recipient’s HLA typing. In many, albeit not all, cases of discrepancies between the original and confirmatory typing results, the donor registry administering the respective donor and providing the CT sample is informed accordingly. At DKMS, we resolve cases with at least one discrepant HLA locus, e.g., by collecting a third donor sample and mandating a “neutral” HLA lab (different from the labs carrying out the original typing and the CT) to type the sample. Based on the result, either the original typing result or the CT result is classified as erroneous. Discrepancies without relevance for donor-patient HLA matching (e.g., silent mutations outside exons coding for the antigen recognition domain) sometimes remain unresolved. For the analyses in this letter, only samples and loci were considered for which recruitment typing and CT typing results were available. Apart from confirmation or falsification of the donor’s original HLA typing result, the CT step also comprises a phone contact with the potential donor including the provision of detailed information on the two stem cell donation methods, the clarification of the donor’s continued interest to donate, the analysis of infectious disease markers (IDM), and a questionnaire-based health screening. In urgent cases, the CT step can be merged with the following step in the process leading to stem cell donation, the so-called donor work-up (WU). Donor WU includes, for example, the donor’s final medical clearing at the collection center. In 2016, DKMS Germany received 27,232 CT requests, 6825 work-up requests and 412 combined requests (named “CT/work-up parallel requests” (CWP requests) in the following). Of the CT requests, 4924 (18.1%) finally led to a donation while further 116 requests (0.4%) were still open at the time of data retrieval. Furthermore, 227 CWP requests (55.1%) resulted in a donation with no requests open anymore. In this letter, we present real-life data from the DKMS Germany donor file regarding discrepancies between HLA typing results at donor recruitment and CT results. Primarily, we focus on donors who were typed by NGS-based methods at the DKMS Life Science Lab (Dresden, Germany) where NGS had been established as high-throughput method for donor typing at recruitment in 2013 [3, 6]. Until 31 March 2017, 2,055,028 newly registered DKMS German donors * Jürgen Sauter sauter@dkms.de

Journal ArticleDOI
13 Aug 2019-Anaerobe
TL;DR: The results suggest that a specific RT 018 clone has spread in the geriatric unit and has evolved slowly over time, and is likely to be responsible for the C. difficile infection in France.

Journal ArticleDOI
TL;DR: A new two-locus typing method (blaOXA-51-like and ampC) method for the rapid identification of clonal complexes (CCs) that has more discrimination than the application of each method separately and it could be applied forThe rapid determination of the CC without performing MLST.
Abstract: Purpose Multi-drug resistant (MDR) Acinetobacter baumannii has introduced a worldwide health crisis. The purposes of this study were to characterize the clonal relatedness among MDR clinical strains and to introduce a new two-locus typing method confirmed by multi-locus sequence typing (MLST). Methodology In this study, we determined antimicrobial resistance, detected genes associated with carbapenem resistance and characterized clonal relatedness among 99 clinical isolates extracted from 82 hospitalized inpatients in a university hospital. Results Of the 99 A. baumannii isolates, 92.9% (92/99) were resistant to imipenem and 97.9% (97/99) had an MDR profile. We found that the high prevalence of blaVIM [94.9% (94/99)] and blaOXA-23-like [93.93% (93/99)] is the main mechanism of carbapenem resistance. This study proposes a new two-locus typing (blaOXA-51-like and ampC) method for the rapid identification of clonal complexes (CCs). The results of this method and confirmation by MLST show that clinical isolates carry blaOXA-68 as well as ampC-10 or ampC-20 genes belonging to CC10 (ST10); blaOXA-66 and ampC-2 belonging to CC2 (ST2); and blaOXA-71 and ampC-3 belonging to CC3 (ST3). One isolate had blaOXA-90 with an undetermined allele number of ampC belonging to ST513. Conclusion The high prevalence of MDR strains and the circulation of four limited clones, including ST10 (45/99), ST2 (41/99), ST3 (12/99) and ST513 (1/99), in the clinical setting highlights the importance of a rigorous infection control programme. The two-locus typing method has more discrimination than the application of each method separately and it could be applied for the rapid determination of the CC without performing MLST.

Journal ArticleDOI
TL;DR: While the current agglutination‐based RBC‐typing methods are reliable for testing a selected number of antigens, they are not easily adaptable for high‐throughput multiplex blood typing beyond the current standard.

Journal ArticleDOI
TL;DR: An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, and four markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.

Journal ArticleDOI
TL;DR: In this article, the authors extended the existing scheme from PubMLST using whole-genome data for M. abscessus by extracting data for 15 genetic regions within the M.Abscessus genome.
Abstract: Background:Mycobacterium abscessus is a rapid growing nontuberculous mycobacteria (NTM) and a clinically significant pathogen capable of causing variable infections in humans that are difficult to treat and may require months of therapy/surgical interventions. Like other NTMs, M. abscessus can be associated with outbreaks leading to complex investigations and treatment of affected cases. Typing schemes for bacterial pathogens provide numerous applications; including identifying chain of transmission and tracking genomic evolution, are lacking or limited for many NTMs including M. abscessus. Methods: We extended the existing scheme from PubMLST using whole-genome data for M. abscessus by extracting data for 15 genetic regions within the M. abscessus genome. A total of 168 whole genomes and 11 gene sequences were used to build this scheme (MAB-multilocus sequence typing [MLST]). Results: All seven genes from the PubMLST scheme, namely argH, cya, gnd, murC, pta, purH, and rpoB, were expanded by 10, 14, 13, 10, 13, 10, and 9 alleles, respectively. Another eight novel genes were added including hsp 65, erm(41), arr, rrs, rrl, gyrA, gyrB, and recA with 16, 16, 25, 7, 32, 35, 29, and 15 alleles, respectively, with 85 unique sequence types identified among all isolates. Conclusion: MAB-MLST can provide identification of M. abscessus complex to the subspecies level based on three genes and can provide antimicrobial resistance susceptibility prediction based on results from seven genes. MAB-MLST generated a total of 85 STs, resulting in subtyping of 90 additional isolates that could not be genotyped using PubMLST and yielding results comparable to whole-genome sequencing (WGS). Implementation of a Galaxy-based data analysis tool, MAB-MLST, that simplifies the WGS data and yet maintains a high discriminatory index that can aid in deciphering an outbreak has vast applicability for routine diagnostics.

Journal ArticleDOI
TL;DR: BOX-PCR provides a suitable molecular typing method for discriminating genetic relatedness among Salmonella spp.
Abstract: Salmonella enterica Serotype 4,[5],12:i:-, a monophasic variant of S. Typhimurium, with high virulence and multidrug resistance is distributed globally causing pathogenicity to both humans and domesticated animals. BOX-A1R-based repetitive extragenic palindromic-PCR (BOX)-PCR proved to be superior to three other repetitive element-based PCR typing methods, namely, enterobacterial repetitive intergenic consensus (ERIC)-, poly-trinucleotide (GTG)5-, and repetitive extragenic palindromic (REP)-PCR (carried out under a single optimized amplification condition), in differentiating genetic relatedness among S. 4,[5],12:i:- isolates from feces of hospitalized patients (n=12) and isolates from minced pork samples of S. 4,[5],12:i:- (n=6), S. Typhimurium (n=6), and Salmonella Serogroup B (n=4) collected from different regions of northern Thailand. Construction of phylogenetic trees from amplicon size patterns allowed allocation of Salmonella isolates into clusters of similar genetic relatedness, with BOX-PCR generating more unique clusters for each serotype than the other three typing methods. BOX-, (GTG)5-, and REP-PCR indicated significant genetic relatedness between S. 4,[5],12:i:- isolates 1 and 9 from hospitalized patients and S. 4,[5],12:i:- isolate en 29 from minced pork, suggesting a possible route of transmission. Thus, BOX-PCR provides a suitable molecular typing method for discriminating genetic relatedness among Salmonella spp. of the same and different serotypes and should be suitable for application in typing and tracking route of transmission in Salmonella outbreaks.

Journal ArticleDOI
23 Nov 2019
TL;DR: To detect these secondary resistant strains, prevent reinfections, and improve the control of M. genitalium infections, tests of cure and contact tracing of sexual partners should be mandatory.
Abstract: Mycoplasma genitalium causes a sexually transmitted infection that sometimes persists or recurs despite adequate antibiotic treatment. Between 2014 and 2018, molecular typing was applied to 75 M. genitalium-positive samples from 48 patients with repeated infection and/or couples/groups of other infected sexual contacts. MG191 adhesin, MG309 lipoprotein, and the rRNA operon were amplified, sequenced, and typed using phylogenetic, variable number tandem repeat, and single-nucleotide polymorphism analysis, respectively. Amplicons were obtained in 74/75 samples, and the combination of locus patterns gave 44 different genetic profiles (discriminatory index of 0.987), with 43 considering only MG191 and MG309. Interestingly, 15/17 patients who presented a first sample sensitive and a second resistant to macrolides had the same genetic variant in the samples (persistence of the same strain). In 2/17 patients, discordant variants (one mixed infection and one recurrence due to incomplete contact tracing) were detected. In 31 additional not related and randomly distributed samples, MG191 typing obtained 23 different genotypes, with no appreciable clustering over time. The typing method allowed persistent and recurrent infections to be distinguished, indicating that macrolide resistance-associated mutations mostly developed during treatment. To detect these secondary resistant strains, prevent reinfections, and improve the control of M. genitalium infections, tests of cure and contact tracing of sexual partners should be mandatory.

Journal ArticleDOI
TL;DR: An updated review on A. baumannii typing methods recapping the added value of well-established techniques previously applied and detailing new ones (as clustered regularly interspaced short palindromic repeats-based typing) with a special focus on whole genome sequencing.
Abstract: The outstanding ability of Acinetobacter baumannii to cause outbreaks and acquire multidrug resistance motivated the development of a plethora of typing techniques, which can help infection preventionists and hospital epidemiologists to more efficiently implement intervention controls. Nowadays, the world is witnessing a gradual transition from traditional typing methodology to whole genome sequencing-based approaches. Such approaches are opening new prospects and applications never achieved by existing typing methods. Herein, we provide the reader with an updated review on A. baumannii typing methods recapping the added value of well-established techniques previously applied for A. baumannii and detailing new ones (as clustered regularly interspaced short palindromic repeats-based typing) with a special focus on whole genome sequencing.

Journal ArticleDOI
TL;DR: Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.
Abstract: Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter, however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii, C. malonaticus, and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.