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In some industrial applications, it is required to do off-line multiobjective optimization in embedded systems. Due to their limited computing and memory capability, embedded processor may not be able to run conventional multiobjective optimization evolutionary algorithms (MOEAs). This article proposes a micro MOEA with piecewise strategy $(\mu $ MOEA) for industrial optimization in embedded processor. $\mu $ MOEA introduces an improved piecewise strategy based on the MOEA/D framework, which serially optimizes subclusters to be compatible with embedded processor under limited computing power. For the purpose of further enhancing $\mu $ MOEA, a dynamic and flexible weight vector update trigger mechanism is proposed, so that the algorithm can save and utilize the computing resources of the embedded processor as much as possible. Abundant artificial test problems are carrying out to test the performance of $\mu $ MOEA. Through various experiments, it can be found that $\mu $ MOEA has outstanding performance in ZDT, DTLZ, SMOP, and MaF problems. Last and most importantly, $\mu $ MOEA is successfully applied to two specific application scenarios of industrial optimization on embedded processor for simulation, such as two different types of semi-autogenous grinding optimization problems and micro-grid energy optimization problem, which prove the feasibility of applying MOEA to embedded processor.

This study aimed to build an in-situ monitoring system based on a multi-sensing diagnosis strategy combined with a machine learning model for laser hot wire cladding applications. A high speed camera and spectrometer were mounted to obtain in situ synchronization information. Eight process signal features were extracted based on the molten pool and plasma. The silica brittle inclusions result in a higher, narrower, and rougher clad layer with higher and more uneven hardness than the normal clad layer and eventually caused undercut defects. Three quality features based on a seam tracker based surface profiling technique were extracted to label the process signal features, and an empirical database was produced based on the quantified experimental results. The minimum redundancy maximum relevance algorithm was then used to rank the importance of the process signal features, through which, a new feature vector was created. Finally, the eXtreme gradient boosting (XGBoost) model was used to develop an in-situ monitoring system for the laser hot wire cladding process. This MRMR-XGBoost based system reduces the redundancy of process information and achieved the cladding quality classification with an accuracy of 0.9727 and identify time of 7.979 ms.

Due to the inherent hysteresis and nonlinearity of giant magnetostrictive material (GMM), the measurement waveform of optical fiber current transformer based on GMM material was distorted, which seriously affects the instantaneous accuracy of measurement results. In this paper, a correction method to data signal was proposed based on the law of graphics. The nonlinearity characteristic of the experimental system was analyzed, the transfer model corresponding to the unknown measured current was derived, and the measurement results were corrected by the inverse model. The comparison with experimental results showed that, this correction method could effectively reduce the influence of the nonlinear and hysteresis characteristics of GMM on the measurement results, at same time it greatly improved the measurement accuracy of the GMM current transformer.

•A modified DIC method was used to study tensile behaviors of laser welded Al alloy.•Porosities within welded joints were detected by an X-ray computed microtomography.•Various DIC-based material models were extracted for nonlinear FE modeling.•Welding parameters dominate the mechanical properties of laser welds.•Joint size and porosities substantially affected tensile behaviors of Al welds.

Aim:We investigated the interactions between macrophage and different strains of Prototheca. Materials & method:J774A.1 macrophages were infected with clinical isolates of Prototheca ciferrii18125 and P. ciferrii50779 and environmental isolate of P. ciferriiN71. Phagocytosis activities were compared by colony-forming unit assays at 3, 6 and 9 h after infection. Cytokine levels were detected by RT-PCR and ELISA. iNOS protein expression was examined by western blotting. Results:All P. ciferriistrains were phagocytized by macrophages but induced different levels of cytokines in macrophages. Moreover, infected by P. ciferriiN71 upregulated much higher iNOS protein expression in J774A.1 than that infected by the clinical strains. Conclusion:Clinical and environmental P. ciferriistrains show differences in their interactions with macrophages, which may be attributed to their virulence.

The process monitoring of the top side and the evaluation of surface quality were applied in adjustable ring mode (ARM) laser welding of 316 stainless steel by using visual monitoring and confocal sensor technology. Experiments were conducted in different power arrangements of an ARM laser. In this study, the process status was evaluated based on the area of keyhole entrance and the width of the pool. The geometric characteristics of the topside weld were measured by a confocal sensor. The shape of the fusion zone was analyzed based on its cross section. The process mechanism of a different mode of ARM laser welding was investigated by analyzing the process status and geometric characteristics of the surface bead. Experimental results showed that dual-mode laser welding could stabilize the keyhole entrance with a uniform weld surface formation in comparison to the pure ring-mode laser weld. The dual-mode laser also generated a wider molten pool with a wider fusion zone than the pure center laser weld.

Clostridium difficileinfection (CDI) is a significant cause of morbidity and mortality in both the acute care setting and the wider healthcare system. The purpose of this study was to evaluate the in vitroactivity of fidaxomicin against C.difficileisolates from a university teaching hospital in China.

Aim:Distinction of species within the Candida glabratacomplex (i.e., C. glabrata sensu stricto, Candida nivariensisand Candida bracarensis) is relevant for epidemiological purposes and antifungal management. Materials & methods:Two commercial matrix-assisted laser desorption ionization–time of flight mass spectrometry systems were comprehensively evaluated for the identification of fungi within this complex. Results:None of the species (C. nivariensisand C. bracarensis) were identified correctly by Vitek mass spectrometry (MS®) v2.0 In VitroDiagnosis system and Bruker Biotyper MS®v3.1, but all were correct by the Vitek MS®Research Use Only system. The Bruker ClinProTools software showed 100 recognition capability and cross validation for the discrimination of C. nivariensisand C. bracarensis. Conclusion:Using Vitek MS Research Use Only and Bruker ClinProTools can overcome limitations of the Vitek MS In VitroDiagnosis and Bruker Biotyper databases in the identification of C. glabratacomplex.

ABSTRACTCandida haemuloniicomplex (Candida haemulonii, Candida haemuloniivar. vulnera, and Candida duobushaemulonii) consists of emerging pathogens. Thirty-one isolates from 14 hospitals in China were studied for their species classification and antifungal susceptibilities. Performances of molecular (i.e., ribosomal DNA [rDNA] internal transcribed spacer [ITS] sequencing, D1/D2 sequencing, and ITS sequencer-based capillary gel electrophoresis [SCGE]) and phenotypic identification methods in species identification were compared. Twenty-six (83.9%) of 31 isolates were identified as C. haemuloniiand 5 isolates were identified as C. duobushaemuloniiby ITS sequencing as the reference method; results obtained by D1/D2 sequencing and ITS SCGE were concordant with those obtained by ITS sequencing for all (100%) of the isolates. All 31 isolates were identified as C. haemuloniiby the Vitek matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system (bioMérieux, France), whereas the Bruker MS system (Bruker Daltoniks, Germany) correctly provided species identification for 77.4% and 100% of isolates using cutoff scores for species of =2.0 and =1.70, respectively. The Vitek 2 compact (bioMérieux) only identified 9 (29%) of 31 isolates. All isolates showed high MICs for amphotericin B (range, 2 to >8 µg/ml) and fluconazole (=128 µg/ml) but low MICs (=0.5 µg/ml) for the echinocandins. Our results reinforce the need for MALDI-TOF MS and/or molecular differentiation of species within the C. haemuloniicomplex. The multiresistant antifungal susceptibility profile of these isolates represents a challenge to therapy.

Chaetomium atrobrunneumhas never been reported to be associated with pneumonia. We report the isolation of C. atrobrunneumand Aspergillus fumigatusfrom a Chinese elderly patient with fatal pneumonia. Branched, long, and septate hyphae were observed in potassium hydroxide preparations and Gram-stained smears, and confluent C. atrobrunneumgrowth and a few A. fumigatuscolonies were found in tracheal aspirates (nine separate occasions). These isolates were identified by conventional morphological methods and by sequencing the internal transcribed spacer and the D1/D2 domain of the 26S rRNA gene. The patient responded poorly to the combination therapy of amphotericin B and caspofungin. This report adds C. atrobrunneumto the list of fungal pneumonia in immunocompromised hosts. This case report also illustrated the presence of a growth symbiosis between Chaetomiumspecies and A. fumigatus.

ABSTRACTAmong the 2,683 yeast isolates representing 41 different species (25 Candidaand Candida-related species and 16 non-Candidayeast species) collected in the National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program (2012 to 2013), the Bruker Biotyper MS matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system exhibited significantly higher accuracy rates than the Vitek MS system for identification of all yeast isolates (98.8% versus 95.4%, P<0.001 by Pearson's chi-square test) and for all Candidaand Candida-related species isolates (99.4% versus 95.5%, P< 0.001).

Abstract: Recently, heterogeneous single-atom catalysts (SACs) have attracted enormous attention in electrochemical applications owing to their advantages of high metal utilization, well-defined active sites, tunable selectivity, and excellent activity. To avoid the aggregation of atomically dispersed metal sites, an appropriate support has to be adopted to reduce the surface free energy of catalysts. Graphene with a high surface area, outstanding conductivity, and unique electronic properties has generally been utilized as the substrate for SACs. Moreover, the correlations between metal–support interactions and the electrocatalytic performance at the atomic scale can be studied on graphene-supported single-atom catalyst (G-SAC) nanoplatforms. In this review, we start from an overview of the synthetic methods for G-SACs. Subsequently, several advanced and effective characterization techniques are discussed. Then, we present a comprehensive summary of recent progress in G-SACs for a variety of electrochemical applications. Finally, we present challenges for and an outlook on the development of G-SACs with outstanding catalytic activity, stability, and selectivity. Graphic Abstract:

ABSTRACTForty-two putative Cryptococcus laurentiiisolates identified by the Vitek 2 system were collected in China. The gold standard, internal transcribed spacer (ITS) sequencing, confirmed that only two isolates were genuine C. laurentii. Bruker Biotyper matrix-assisted laser desorption ionization–time of flight mass spectrometry was able to identify the C. laurentiiisolates with an expanded custom database.

ABSTRACTWe investigated three nosocomial Candida quercitrusacandidemia cases occurring within 2 months in a Chinese hospital. Isolates were identifiable only by DNA sequencing of the rRNA genes. Genetic (via random amplified polymorphic DNA [RAPD]) and protein mass spectral (via matrix-assisted laser desorption ionization–time of flight mass spectrometry [MALDI-TOF MS]) analyses yielded identical profiles suggesting an outbreak. The fluconazole MICs of all the strains were 16 to 32 µg/ml.

ABSTRACTRapid and accurate detection of multidrug resistance (MDR) in Mycobacterium tuberculosisis essential to improve treatment outcomes and reduce global transmission but remains a challenge. Rifampin (RIF) resistance is a reliable marker of MDR tuberculosis (TB) since by far the majority of RIF-resistant strains are also isoniazid (INH) resistant. We have developed a rapid, sensitive, and specific method for detecting the most common mutations associated with RIF resistance, in the RIF resistance determining region (RRDR) of rpoB, using a cocktail of six padlock probes and rolling circle amplification (RCA). We used this method to test 46 stored M. tuberculosisclinical isolates with known RIF susceptibility profiles (18 RIF resistant, 28 susceptible), a standard susceptible strain (H37Rv, ATCC 27294) and 78 M. tuberculosisculture-positive clinical (sputum) samples, 59 of which grew RIF-resistant strains. All stored clinical isolates were correctly categorized, by the padlock probe/RCA method, as RIF susceptible or resistant; the sensitivity and specificity of the method, for direct detection of phenotypically RIF-resistant M. tuberculosisin clinical specimens, were 96.6 and 89.5%, respectively. This method is rapid, simple, and inexpensive and has the potential for high-throughput routine screening of clinical specimens for MDR M. tuberculosis, particularly in high prevalence settings with limited resources.

ABSTRACTSequence analysis of the internal transcribed spacer (ITS) region was employed as the gold standard method for yeast identification in the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). It has subsequently been found that matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is potentially a more practical approach for this purpose. In the present study, the performance of the Vitek MS v2.0 system for the identification of yeast isolates collected from patients with invasive fungal infections in the 2011 CHIF-NET was evaluated. A total of 1,243 isolates representing 31 yeast species were analyzed, and the identification results by the Vitek MS v2.0 system were compared to those obtained by ITS sequence analysis. By the Vitek MS v2.0 system, 96.7% (n= 1,202) of the isolates were correctly assigned to the species level and 0.2% (n= 2) of the isolates were identified to the genus level, while 2.4% (n= 30) and 0.7% (n= 9) of the isolates were unidentified and misidentified, respectively. After retesting of the unidentified and misidentified strains, 97.3% (n= 1,209) of the isolates were correctly identified to the species level. Based on these results, a testing algorithm that combines the use of the Vitek MS system with selected supplementary ribosomal DNA (rDNA) sequencing was developed and validated for yeast identification purposes. By employing this algorithm, 99.7% (1,240/1,243) of the study isolates were accurately identified with the exception of two isolates of Candida fermentatiand one isolate of Cryptococcus gattii. In conclusion, the proposed identification algorithm could be practically implemented in strategic programs of fungal infection surveillance.

ABSTRACTThe global spread of methicillin-resistant Staphylococcus aureus(MRSA) is a serious problem, particularly in mainland China. In order to better understand the national molecular epidemiology and resistance profiles of hospital-associated MRSA (HA-MRSA) in China, a laboratory-based multicenter surveillance study was conducted. Sixty-nine hospitals in 45 large cities in 27 provinces were involved, and a total of 1,141 HA-MRSA isolates were collected during the 6-month study period in 2011. All MRSA isolates were characterized by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec(SCCmec) typing, spatyping, detection of the Panton-Valentine leukocidin (PVL) locus (lukS-PVand lukF-PV), and antibiogram analysis. ST239-III-t030, ST239-III-t037, and ST5-II-t002 were the predominant HA-MRSA clones (overall prevalence rates, 57.1%, 12.9%, and 8.1%, respectively), although the prevalence rates of these major clones varied markedly in different administrative regions. Of note, 6.6% of the HA-MRSA isolates were found to belong to ST59, which had typical community-associated MRSA (CA-MRSA) features, including carriage of SCCmectype IV or V and PVL and less antimicrobial resistance than other major HA-MRSA clones. Moreover, among 36 MLST sequence types (STs) identified, 15 STs, accounting for 3.5% of total isolates, were novel. A novel ST designated ST2590, which is a single-locus variant of ST5-II-t002, was identified in three hospitals in two large cities, with a total of 17 isolates. To further monitor trends in HA-MRSA prevalence, epidemic clonal shifts, clone emergence, and transmission between community and health care settings, longitudinal national MRSA surveillance is required.

ABSTRACTThe recent emergence of multidrug-resistant Escherichia colisequence type 131 (ST131) has coincided with an increase in general antibiotic resistance of E. coli, suggesting that ST131 has a contributing role in resistance. However, there is little information about the contribution of ST131 to different clinical syndromes or the basis for its impressive emergence and epidemic spread. To investigate this, we studied 953 E. coliisolates from women of reproductive age in the central west region of New South Wales, Australia, including 623 urinary isolates from patients with cystitis (cystitis isolates) (n= 322) or pyelonephritis (pyelonephritis isolates) (n= 301) and 330 fecal isolates from healthy controls. The characteristics studied included ST131 clonal group status, resistance to different antibiotics, presence of virulence factor (VF) genes, and biofilm production. As expected, fecal isolates differed significantly from urinary (cystitis and pyelonephritis) isolates in most of the studied characteristics. Antibiotic resistance was significantly more common in ST131 than in non-ST131 isolates. Both antibiotic resistance and ST131 were more common in pyelonephritis than cystitis isolates and least so among fecal isolates. Within each source group, individual VF genes were more prevalent and VF scores were higher for ST131 than for non-ST131 isolates. For ST131 only, the prevalences of most individual VF genes and VF scores were the lowest in the fecal isolates, higher in the cystitis isolates, and highest in the pyelonephritis isolates. Biofilm production was strongly associated with ST131 status and antibiotic resistance. These results clarify the distribution of the ST131 clonal group and its epidemiological associations in our region and suggest that it exhibits both enhanced virulence and increased antibiotic resistance compared with those of other urinary tract infection (UTI) and fecal E. coliisolates from women of reproductive age.

Hybrid laser/gas metal arc welding was applied to join the thick high-strength steel in square butt joints with narrow gaps. An X-ray diffraction technique was used to measure the surface residual stress distribution of high-strength steel by a hybrid laser/gas metal arc welding in a butt joint configuration. The influence of welding parameters, including welding speed and wire feeding rate, on the residual stress distribution and mechanical properties of the hybrid weld was experimentally studied. The obtained results show that there exist high compressive transverse and longitudinal residual stresses along the centerline of the weld. However, high tensile transverse and longitudinal stresses are located in the nearby heat-affected zone. An increase in the welding speed definitely decreases the residual stress concentration. A good weld quality of high-strength steel butt welds without cracking or defects can be achieved by a hybrid laser/gas metal arc welding technique.

Based on the relationship between Clostridium difficilesurface layer protein A (slpA) sequence types (STs) and PCR-ribotypes (RTs), a multiplex polymerase chain reaction (mPCR) assay was developed to rapidly confirm C. difficiletoxigenicity and, simultaneously, to identify any of five slpASTs, gr, hr, fr, gc8 and 078, that usually correspond with globally distributed RTs, 001,014, 017, 027 and 078, respectively.

Laser welding assisted by a cold filler wire (LWCFW) and hybrid laser–gas metal arc (HLGMA) welding were applied to execute the welding of 6.63-mm-thick high-strength steel (HSS) plates. The corresponding hardness distribution, tensile strength, and micrographs of the welded HSS were experimentally obtained. Results show that LWCFW can achieve a good weld quality without the groove preparation that is usually required in HLGMA welding. A narrower fusion zone and heat-affected zone can be achieved by LWCFW as compared to HLGMA welding. However, the size of the gap in a butt joint in LWCFW is limited. It was found that when a gap of about 1 mm is used in joining 6.63-mm-thick plates in a square butt joint with 4 kW of fiber laser power and a wire diameter of 0.9 mm, an acceptable weld quality could be achieved.

Urinary tract infections (UTI) are common in children and contribute significantly to morbidity and mortality. The major cause is Escherichia coli, carrying multiple virulence-associated factors (VFs). However, the specific traits that distinguish childhood uropathogenic E. colifrom fecal commensals of healthy children are incompletely defined.

ABSTRACTWe conducted active, laboratory-based surveillance for isolates from patients with invasive infections across China from August 2009 to July 2010. DNA sequencing methods were used to define species, and susceptibility to fluconazole and voriconazole was determined by the Clinical and Laboratory Standards Institute M44-A2 disk diffusion method but using up-to-date clinical breakpoints or epidemiological cutoff values. Candidaspp. made up 90.5% of the 814 yeast strains isolated, followed by Cryptococcus neoformans(7.7%) and other non-Candidayeast strains (1.7%). Bloodstream isolates made up 42.9% of the strains, isolates from ascitic fluid made up 22.1%, but pus/tissue specimens yielded yeast strains in <5% of the cases. Among the Candidaisolates, Candida albicanswas the most common species from specimens other than blood (50.1%) but made up only 23% of the bloodstream isolates (P< 0.001). C. parapsilosiscomplex species were the most common Candidaisolates from blood (33.2%). Uncommon bloodstream yeast strains included Trichosporonspp., C. pelliculosa, and the novel species C. quercitrusa, reported for the first time as a cause of candidemia. Most (>94%) of the isolates of C. albicans, C. tropicalis, and the C. parapsilosiscomplex were susceptible to fluconazole and voriconazole, as were all of the Trichosporonstrains; however, 12.2% of the C. glabrata sensu strictoisolates were fluconazole resistant and 17.8% had non-wild-type susceptibility to voriconazole. Seven C. tropicalisstrains were cross-resistant to fluconazole and voriconazole; six were from patients in the same institution. Resistance to fluconazole and voriconazole was seen in 31.9% and 13.3% of the uncommon Candidaand non-Candidayeast strains, respectively. Causative species and azole susceptibility varied with the geographic region. This study provided clinically useful data on yeast strains and their antifungal susceptibilities in China.

ABSTRACTPCR ribotyping is the most commonly used Clostridium difficilegenotyping method, but its utility is limited by lack of standardization. In this study, we analyzed four published whole genomes and tested an international collection of 21 well-characterized C. difficileribotype 027 isolates as the basis for comparison of two capillary gel electrophoresis (CGE)-based ribotyping methods. There were unexpected differences between the 16S-23S rRNA intergenic spacer region (ISR) allelic profiles of the four ribotype 027 genomes, but six bands were identified in all four and a seventh in three genomes. All seven bands and another, not identified in any of the whole genomes, were found in all 21 isolates. We compared sequencer-based CGE (SCGE) with three different primer pairs to the Qiagen QIAxcel CGE (QCGE) platform. Deviations from individual reference/consensus band sizes were smaller for SCGE (0 to 0.2 bp) than for QCGE (4.2 to 9.5 bp). Compared with QCGE, SCGE more readily distinguished bands of similar length (more discriminatory), detected bands of larger size and lower intensity (more sensitive), and assigned band sizes more accurately and reproducibly, making it more suitable for standardization. Specifically, QCGE failed to identify the largest ISR amplicon. Based on several criteria, we recommend the primer set 16S-USA/23S-USA for use in a proposed standard SCGE method. Similar differences between SCGE and QCGE were found on testing of 14 isolates of four other C. difficileribotypes. Based on our results, ISR profiles based on accurate sequencer-based band lengths would be preferable to agarose gel-based banding patterns for the assignment of ribotypes.

Three reference and 45 clinical isolates of Trichosporon were analyzed by conventional phenotypic and molecular methods to determine the species and genotypes of Trichosporon isolates from China. Target loci for molecular methods included the internal transcribed spacer (ITS) region, the D1/D2 domain of the 26S rRNA gene, and the intergenic spacer 1 (IGS1) region. Identification of eight Trichosporon species was achieved, of which Trichosporon asahii was the most common. Of the sequence-based molecular methods, the one targeting the D1/D2 domain assigned 97.9% (47/48) of isolates (seven species) correctly, while tests targeting both the ITS and IGS1 regions correctly identified all 48 isolates. The commercial API 20C AUX and Vitek 2 Compact YST systems correctly identified 91.9% and 73% of isolates when their biochemical profiles were queried against those of species contained in the databases, respectively, and misidentified 63.6% and 36.4% of isolates of species that were unclaimed by the databases, respectively. The predominant genotype among T. asahii clinical isolates, genotype 4 (51.4%), is rarely found in other countries. Voriconazole and itraconazole were the most active drugs in vitro against all the Trichosporon species tested, while caspofungin and amphotericin B demonstrated poor activity.

ABSTRACTA high-resolution melting analysis (HRMA) assay was developed to detect isoniazid, rifampin, and ofloxacin resistance in Mycobacterium tuberculosisby targeting resistance-associated mutations in the katG, mabA-inhApromoter, rpoB, and gyrAgenes. A set of 28 (17 drug-resistant and 11 fully susceptible) clinical M. tuberculosisisolates was selected for development and evaluation of HRMA. PCR amplicons from the katG, mabA-inhApromoter, rpoB, and gyrAgenes of all 28 isolates were sequenced. HRMA results matched well with 18 mutations, identified by sequencing, in 17 drug-resistant isolates and the absence of mutations in 11 susceptible isolates. Among 87 additional isolates with known resistance phenotypes, HRMA identified katGand/or mabA-inhApromoter mutations in 66 of 69 (95.7%) isoniazid-resistant isolates, rpoBmutations in 51 of 54 (94.4%) rifampin-resistant isolates, and gyrAmutations in all of 41 (100%) ofloxacin-resistant isolates. All mutations within the HRMA primer target regions were detected as variant HRMA profiles. The corresponding specificities were 97.8%, 100%, and 98.6%, respectively. Most false-positive results were due to synonymous mutations, which did not affect susceptibility. HRMA is a rapid, sensitive method for detection of drug resistance in M. tuberculosiswhich could be used routinely for screening isolates in countries with a high prevalence of tuberculosis and drug resistance or in individual isolates when drug resistance is suspected.

Human adenoviruses are common pathogens associated with a broad spectrum of disease. There is a growing clinical interest in typing clinical isolates since it is becoming increasingly clear that individual serotypes are associated with different disease spectra, virulence, severity of consequences, and outbreaks. Current methods cannot detect all known adenoviruses simultaneously and rapidly. We designed a practical adenovirus typing method with polymerase chain reaction (PCR)-based reverse line blot hybridisation assay (RLB) using hypervariable region-7 (HVR-7) in the hexon gene.

ABSTRACTEleven reference and 25 clinical isolates of Fusariumwere subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1a); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusariumspecies and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella(Fusarium) fujikuroispecies complex was the commonest (40%) and was followed by the Fusarium solanispecies complex (FSSC) (36%) and the F. incarnatum-F. equisetispecies complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1a, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 µg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria.

The identification of fastidious aerobic Actinomycètes such as Gordonia, Rhodococcus,and Tsukamurellahas remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5’-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus,and Tsukamurella,and defined potential reference sequences for species from each of these genera.

ABSTRACTSequence analysis of the Nocardiaessential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia(16 species) was studied in comparison with 5'-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1sequences for the species, including 29 Nocardia cyriacigeorgica(96.6 to 98.9% similarity) and 4 Nocardia veterana(91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia novastrains (identified as various Nocardiaspecies by secA1sequencing) and 1 isolate each of Nocardia abscessus(identified as Nocardia asiatica), Nocardia elegans(Nocardia africana), and Nocardia transvalensis(Nocardia blacklockiae); both secA1gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova(14 secA1sequence types), followed by Nocardia farcinicaand N. veterana(n= 7 each); there was only a single sequence type among eight Nocardia paucivoransstrains. The secA1locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.

Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5'-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.

ABSTRACTStaphylococcal cassette chromosome mec(SCCmec) is a large mobile genetic element which is used frequently for subtyping of methicillin-resistant Staphylococcus aureus(MRSA) strains. MRSA SCCmectype IV not only predominates among community-acquired MRSA (CA-MRSA) strains but also is associated with several genetic lineages of hospital-acquired MRSA (HA-MRSA) and with other species. The objective of this study was to investigate the diversity of MRSA strains classified as SCCmectype IV by using a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay as well as spatyping and pulsed-field gel electrophoresis (PFGE). Sixty-two primer pairs and 63 probes were designed to interrogate each open reading frame (ORF) of SCCmectype IV sequences. A set of 131 MRSA SCCmectype IV isolates were classified into 79 subtypes by this method. There was considerable concordance between SCCmectype IV subtyping, spatyping, and PFGE patterns for clinical isolates, and the stability of SCCmectype IV subtyping was comparable to that of the other two methods. Using an in-house computer program, we showed that a subset of 20 genetic markers could achieve the same level of discrimination between isolates as the full set of 62, with a Simpson's index of diversity of 0.975. SCCmectype IV has a much higher level of diversity than previously suggested. The application of the mPCR/RLB hybridization assay to MRSA SCCmectype IV subtyping can improve the discriminatory power and throughput of MRSA typing and has the potential to enhance rapid infection control surveillance and outbreak detection.

ABSTRACTThe relatively high-level clonality of methicillin-resistant Staphylococcus aureus(MRSA) and its frequent high-level endemicity in nosocomial settings complicate the development of methods for rapid subtyping of MRSA strains that are capable of identifying person-to-person transmission in hospitals. Phage-derived open reading frame (PDORF) typing is an MRSA typing method targeting mobile genetic elements that was recently described and evaluated using a geographically restricted set of isolates. The objective of this study was to develop a multiplex PCR-reverse line blot (mPCR/RLB) assay for PDORF typing and to test its applicability on a broad range of isolates and in an environment where MRSA is highly endemic. The 16 targets were identified using a 23-primer-pair mPCR/RLB assay with two probes for each target. The method was evaluated using 42 MRSA reference strains, including those representing major international clones, and 35 isolates from episodes of suspected nosocomial transmission. In vivostability was explored using 81 isolate pairs. Pulsed-field gel electrophoresis (PFGE) and spatyping were performed for comparison. Among the 42 reference strains, there were 33 PFGE subtypes, 30 PDORF types, and 22 spatypes. Simpson's index of diversity was 0.987, 0.971, and 0.926 for PFGE subtyping, PDORF typing, and spatyping, respectively. Typing of clinical isolates by PDORF typing and PFGE demonstrated concordant results. mPCR/RLB-based PDORF typing has similar discriminatory power to that of PFGE, can assist in tracking MRSA transmission events in a setting of high-level endemicity, and has the advantage of being a high-throughput technique.

ABSTRACTConventional approaches to characterizing human enteroviruses (HEVs) are based on viral isolation and neutralization. Molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) and nucleotide sequencing of the entire or partial VP1 gene. A modified RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient way to characterize common and nonserotypeable (by neutralization) HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at a reference laboratory from 1979 to 2007; these common serotypes were identified using one sense and three antisense primers and a set of 80 serotype-specific probes in VP1 (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). In this study, one HEV-specific primer pair, two probes in the 5' untranslated region (UTR), and a new set of 80 serotype-specific probes in VP1 were designed. First, we successfully applied the modified RT-PCR-RLB (using two HEV-specific probes and two sets of serotype-specific probes) to synchronously detect the 5' UTR and VP1 regions of 131/132 isolates previously studied (F. Zhou et al., J. Clin. Microbiol. 47:2737-2743, 2009). Then, this method was used to identify 73/92 nonserotypeable HEV isolates; 19 nonserotypeable isolates were hybridized only with HEV-specific probes. The VP1 region of 92 nonserotypeable HEV isolates was sequenced; 73 sequences corresponded with one or both RLB results and 19 (not belonging to the 20 most common genotypes) were identified only by sequencing. Two sets of serotype-specific probes can capture the majority of strains belonging to the 20 most common serotypes/genotypes simultaneously or complementarily. Synchronous detection of the 5' UTR and VP1 region by RT-PCR-RLB will facilitate the identification of HEVs, especially nonserotypeable isolates.

ABSTRACTMacrolide resistance in Streptococcus pneumoniaehas emerged as an important clinical problem worldwide over the past decade. The aim of this study was to analyze the phenotypes (serotype and antibiotic susceptibility), genotypes (multilocus sequence type [MLST] and antibiotic resistance gene/transposon profiles) among the 31% (102/328) of invasive isolates from children in New South Wales, Australia, in 2005 that were resistant to erythromycin. Three serotypes—19F (47 isolates [46%]), 14 (27 isolates [26%]), and 6B (12 isolates [12%])—accounted for 86 (84%) of these 102 isolates. Seventy four (73%) isolates had the macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype and carried Tn916transposons (most commonly Tn6002); of these, 73 (99%) contained the erythromycin ribosomal methylase gene [erm(B)], 34 (47%) also carried the macrolide efflux gene [mef(E)], and 41 (55%) belonged to serotype 19F. Of 28 (27%) isolates with the M phenotype, 22 (79%) carried mef(A), including 16 (57%) belonging to serotype 14, and only six (19%) carried Tn916transposons. Most (84%) isolates which contained mefalso contained one of the msr(A) homologues, melor msr(D); 38 of 40 (95%) isolates with mef(E) (on mega) carried mel, and of 28 (39%) isolates with mef(A), 10 (39%) carried meland another 11(39%) carried msr(D), on Tn1207.1. Two predominant macrolide-resistant S. pneumoniaeclonal clusters (CCs) were identified in this population. CC-271 contained 44% of isolates, most of which belonged to serotype 19F, had the MLSBphenotype, were multidrug resistant, and carried transposons of the Tn916family; CC-15 contained 23% of isolates, most of which were serotype 14, had the M phenotype, and carried mef(A) on Tn1207.1.Erythromycin resistance among S. pneumoniaeisolates in New South Wales is mainly due to the dissemination of multidrug-resistant S. pneumoniaestrains or horizontal spread of the Tn916family of transposons.

ABSTRACTAlthough 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardiaspecies, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined NocardiaPCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardiaisolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivoransstrains (n= 10 strains) and a Nocardia asteroidesisolate with a novel 16S rRNA gene sequence. Nocardia farcinicaand Nocardia cyriacigeorgicastrains were identified by the sequential use of an N. farcinica-“negative” probe and a combined N. farcinica/N. cyriacigeorgicaprobe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensisprobe with the Nocardia thailandicaDNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivoransstrains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinicastains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardiaspecies and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended.

ABSTRACTWith molecular sequencing as a gold standard, the Vitek MS, Bruker Biotyper MS, and Vitek-2 Compact systems correctly identified 92.7%, 97.0%, and 15.2% of 164 Candida guillermondiiisolates, respectively, and none of 8 C. fermentatiisolates. All of the isolates showed high susceptibility to echinocandins, but some C. guilliermondiiisolates showed low azole susceptibility.

Several virulent clones of group B streptococcus (GBS) are known to be associated with certain serotypes and molecular epidemiological markers. It is unclear, however, whether the clinical significance of GBS can be predicted based solely on such molecular markers. The aim of this study was to test the hypothesis that GBS virulence can be predicted by using the molecular epidemiology markers.

ABSTRACTThe more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3' half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.

ABSTRACTIn this study, we developed a simple, reliable, serotype-specific PCR method to differentiate Streptococcus pneumoniaeserotypes 6A, 6B, and 6C. It was more efficient and practical than the assays currently being used to identify serotypes 6A, 6B, and 6C. Of 120 selected serogroup 6 isolates from subjects with invasive (n= 101) and noninvasive (n= 19) pneumococcal disease, most of which were collected after 2003 in New South Wales, 45 had been identified as 6A and 75 had been identified as 6B by the Quellung reaction. PCR analysis confirmed the results for serotype 6B isolates and identified two different subtypes. Fourteen of 45 isolates that had been identified as serotype 6A actually belonged to serotype 6C.

ABSTRACTSerotypes of group B streptococcus (GBS) that cause urinary tract infection (UTI) are poorly characterized. We conducted a prospective study of GBS UTI in adults to define the clinical and microbiological characteristics of these infections, including which serotypes cause disease. Patients who had GBS cultured from urine over a 1-year period were grouped according to symptoms, bacteriuria, and urinalysis. Demographic data were obtained by reviewing medical records. Isolates were serotyped by latex agglutination and multiplex PCR-reverse line blotting (mPCR/RLB). Antibiotic susceptibilities were determined by disc diffusion. GBS was cultured from 387/34,367 consecutive urine samples (1.1%): 62 patients had bacteriuria of >107CFU/liter and at least one UTI symptom; of these patients, 31 had urinary leukocyte esterase and pyuria (others not tested), 50 (81%) had symptoms consistent with cystitis, and 12 (19%) had symptoms of pyelonephritis. Compared with controls (who had GBS isolated without symptoms), a prior history of UTI was an independent risk factor for disease. Increased age was also significantly associated with acute infection. Serotyping results were consistent between latex agglutination and mPCR/RLB for 331/387 (85.5%) isolates; 22 (5.7%) and 7 (1.8%) isolates were nontypeable with antisera and by mPCR/RLB, respectively; and 45/56 (80.4%) isolates with discrepant results were typed by mPCR/RLB as belonging to serotype V. Serotypes V, Ia, and III caused the most UTIs; serotypes II, Ib, and IV were less common. Nontypeable GBS was not associated with UTI. Erythromycin (39.5%) and clindamycin (26.4%) resistance was common. We conclude that a more diverse spectrum of GBS serotypes causes UTI than previously recognized, with the exception of nontypeable GBS.

ABSTRACTThe aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis, Mycoplasma hominis, M. genitalium, and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 × 10-1to 7.0 × 10-11ng/µl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.

ABSTRACTAmong 1,762 isolates of Streptococcus agalactiae(group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning cpsHto cpsM). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the cpsE-cpsF-cpsGregion of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the cpsE-cpsF-cspGregion of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516; P< 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.

ABSTRACTA sensitive rolling-circle amplification (RCA)-based method utilizing species-specific padlock probes targeted to the internal transcribed spacer 2 region of the fungal ribosomal DNA gene complex was developed. The assay was rapid (2 hours) and specific. Of 28 fungal isolates (16 of Candida, six of Aspergillus, and six of Scedosporiumspp.), all were all identified correctly.

ABSTRACTDNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophytonspp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophytonspp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanensebut not T. rubrum. Signature sets of SNPs between T. rubrumand T. soudanense(4-bp difference) and T. violaceumand T. soudanense(3-bp difference) were identified. The RCA assay correctly identified five Trichophytonspecies. Although the use of two “group-specific” probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophytonspp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophytonspp.

ABSTRACTWe compared three novel methicillin-resistant Staphylococcus aureus(MRSA) genotyping methods with multilocus sequence typing (MLST) and spatyping to assess their utility for routine strain typing. The new methods were femAand nucsequence typing and toxin gene profiling (TGP), using a multiplex-PCR-based reverse line blot assay to detect 13 pyrogenic superantigen and exfoliative toxin genes. Forty-two well-characterized MRSA strains, representing 15 MLSTs or 9 clonal clusters (CCs), were genotyped by all methods. Twenty-two spa, nine femA, and seven nucsequence types were identified. The femAsequence types correlated exactly with CCs; nucsequences types were less discriminatory but generally correlated well with femAtypes and CCs. Ten isolates contained none of 13 toxin genes; TGPs of the remainder comprised 1 to 5 toxin genes. The combination of spatyping and TGPs identified 26 genotypes among the 42 strains studied. A combination of two or three rapid, inexpensive genotyping methods could potentially provide rapid MRSA strain typing as well as useful information about clonal origin and virulence.

ABSTRACTWe developed a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify 50 uncommon pneumococcal serotypes. In combination with a previously described mPCR/RLB assay (3), all 90 pneumococcal serotypes can be identified individually (32 serotypes) or, because of predictable cross-reactions, to within small groups of two to five related serotypes (58 serotypes), which can be distinguished using serotype-specific antisera.