Evaluation of two commercial methods for the susceptibility testing of Candida species: Vitek 2® and Sensititre YeastOne®

Background

An increased incidence of fungal infections caused by Candida species, especially Candida glabrata and Candida krusei, which are less susceptible to azoles, has been observed. Standardized susceptibility testing is essential for clinical management and for monitoring the epidemiology of resistance.

Evaluation of the Vitek-MS™ system in the identification of Candida isolates from bloodstream infections

Background

Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) represents a revolution in the identification of microorganisms of clinical interest. Many studies have confirmed the accuracy and fastness of this tool with routine strains.

A new amplification target for PCR–RFLP detection and identification of Chlamydiaceae species

The family Chlamydiaceae contains nine species pathogenic to humans and animals, but their routine identification is hampered by inadequate detection methods. In an attempt to find a new region for PCR detection and discrimination of the Chlamydiaceae species, the 3 end of the omp2 gene of Chlamydiaceae has been examined. Since sequence data for this part of the genes of Chlamydophila felis and Chlamydia suis had not been available, the near full length of the omp2 genes of these species were cloned and sequenced. Consensus primers enabling amplification of a previously untargeted region spanning 1,030 bp at the 3 end of the gene were designed. Discrimination of all nine Chlamydiaceae species was achieved via RFLP analysis of the amplicons with RsaI and HinfI or RsaI and TaqI endonucleases or via electrophoretic mobility analysis of the RsaI restriction fragments in agarose gel with bisbenzimide-PEG. Intraspecies uniformity of the RFLP patterns was evaluated by the typing of reference strains, isolates of human and animal origin from culture collections, and clinical specimens, and by computer analysis of GenBank sequences. The 3 end of the omp2 gene was shown to be an appropriate marker region suitable for rapid identification of Chlamydiaceae species and can be used for characterization of collection strains and new isolates in taxonomic, epidemiological, and clinical purposes.     

Comparison of the Kato-Katz and Helmintex methods for the diagnosis of schistosomiasis in a low-intensity transmission focus in Bandeirantes, Paraná, southern Brazil

The diagnosis of schistosomiasis is problematic in low-intensity transmission areas because parasitological methods lack sensitivity and molecular methods are neither widely available nor extensively validated. Helmintex is a method for isolating eggs from large faecal samples. We report preliminary results of a comparative evaluation of the Helmintex and Kato-Katz (KK) methods for the diagnosis of schistosomiasis in a low-intensity transmission area in Bandeirantes, Paraná, southern Brazil. Eggs were detected by both methods in seven patients, whereas only Helmintex yielded positive results in four individuals. The results confirm the previously demonstrated higher sensitivity of the Helmintex method compared with the KK method.     

Comparison of ‘ignition’ and ‘extraction’ methods for the determination of organic phosphate in rocks and soils

Summary Ignition markedly increased the solubility in dilute acid of iron- and aluminium-bound inorganic phosphate in samples of weathered New Zealand greywacke rock. This observation supports the view that ignition methods may sometimes overestimate the total organic phosphate of soils.     

Comparison between Generalized-Born and Poisson–Boltzmann methods in physics-based scoring functions for protein structure prediction

Continuum solvent models such as Generalized-Born and Poisson–Boltzmann methods hold the promise to treat solvation effect efficiently and to enable rapid scoring of protein structures when they are combined with physics-based energy functions. Yet, direct comparison of these two approaches on large protein data set is lacking. Building on our previous work with a scoring function based on a Generalized-Born (GB) solvation model, and short molecular-dynamics simulations, we further extended the scoring function to compare with the MM-PBSA method to treat the solvent effect. We benchmarked this scoring function against seven publicly available decoy sets. We found that, somewhat surprisingly, the results of MM-PBSA approach are comparable to the previous GB-based scoring function. We also discussed the effect to the scoring function accuracy due to presence of large ligands and ions in some native structures of the decoy sets.     

Synergistic antibacterial effect between silybin and N,N′-dicyclohexylcarbodiimide in clinical Pseudomonas aeruginosa isolates

Silybin is a composition of the silymarin group as a hepatoprotective agent, and it exhibits various biological activities, including an antibacterial activity. In this study, the effects of a combination of silybin with N,N'-dicyclohexylcarbodiimide (DCCD) against clinical isolates of Pseudomonas aeruginosa were investigated. In the results of susceptibility assay, silybin showed more potent antibacterial activity in methicilin-resistant Staphylococcus aureus (MRSA) than in P. aeruginosa, but DCCD significantly increased the antibacterial activity of silybin in P. aeruginosa. The antibacterial activity of silybin was affected by the strong action of multidrug-resistant pumps rather than by a permeable disruption of lipopolysaccharide and silybin showed a remarkable synergistic activity in combination with some antibiotic agents against drug-resistant bacteria. Therefore, silybin has a potential as a combination therapeutic agent for treatment of infectious diseases by multidrug-resistant bacteria.     

Do the phenology and functional stem attributes of woody species allow for the identification of functional groups in the semiarid region of Brazil?

The phenology of tree species in environments that are subject to strong climatic seasonality is mainly determined by water availability, which may vary as a function of wood density. The relationship among phenology, water potential, wood density and the capacity of water storage in the stem were determined for woody species of caatinga vegetation (dry forest) in the semiarid region of NE Brazil. Leaf flush and fall, flowering and fruiting events were recorded over a 31-month period, and the water potential was measured over a two-year period. These data were related to precipitation, water availability in the soil and photoperiod. Seven deciduous species exhibited low wood density (DLWD,?<0.5?g?cm^?3), high capacity of water storage in the stem (until 250?% of the dry weight) and high water potential during the year, as opposed to 15 deciduous species that showed high wood density (DHWD,?≥0.5?g?cm^?3). Leaf flush, flowering and the fruiting of DHWD species were related to precipitation, whereas these phenological events occurred at the end of the dry season and/or the beginning of the rainy season for DLWD species and were related to the photoperiod. The two evergreen species showed variations of water potential that were intermediate between those of DHWD and DLWD deciduous species, leaf flush during the dry season and flowering at the end of dry season. These results suggest the existence of three functional groups: evergreen species, DHWD deciduous species and DLWD deciduous species.     

CODEHOP-mediated PCR – A powerful technique for the identification and characterization of viral genomes

Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) PCR primers derived from amino acid sequence motifs which are highly conserved between members of a protein family have proven to be highly effective in the identification and characterization of distantly related family members. Here, the use of the CODEHOP strategy to identify novel viruses and obtain sequence information for phylogenetic characterization, gene structure determination and genome analysis is reviewed. While this review describes techniques for the identification of members of the herpesvirus family of DNA viruses, the same methodology and approach is applicable to other virus families.     

Development of Capsicum EST–SSR markers for species identification and in silico mapping onto the tomato genome sequence

Capsicum spp. are widely cultivated for use as vegetables and spices. The Kihara Institute for Biological Research, Yokohama City University, Japan, has stocks of approximately 800 lines of Capsicum spp. collected from various regions of Central and South America, the regions of origin for Capsicum spp. In this study, 5,751 primer pairs for simple sequence repeat markers, based on 118,060 publicly available sequences of expressed sequence tags of Capsicum annuum, were designed and subjected to a similarity search against the genomic sequence of tomato, a model Solanaceae species. Nucleotide sequences spanning 2,245 C. annuum markers were successfully mapped onto the tomato genome, and 96 of these, which spanned the entire tomato genome, were selected for further analysis. In genotyping analysis, 60 out of the 77 markers that produced specific DNA amplicons showed polymorphism among the Capsicum lines examined. On the basis of the resulting data, the 192 tested lines were grouped into five main clusters. The additional sequencing analysis of the plastid genes, matK and rbcL, divided the resources into three groups. As a result, 19 marker loci exhibited genotypes specific to species and cluster, suggesting that the DNA markers are useful for species identification. Information on the DNA markers will contribute to Capsicum genetics, genomics, and breeding.     

Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan^® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector—leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890 bp. From this amplified product, specifically a target of 69 bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9?×?10⁵ g of tissue) followed by roots (7.4?×?10⁵ g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

     

Is PCR the Next Reference Standard for the Diagnosis of Schistosoma in Stool? A Comparison with Microscopy in Senegal and Kenya

Background

The current reference test for the detection of S. mansoni in endemic areas is stool microscopy based on one or more Kato-Katz stool smears. However, stool microscopy has several shortcomings that greatly affect the efficacy of current schistosomiasis control programs. A highly specific multiplex real-time polymerase chain reaction (PCR) targeting the Schistosoma internal transcriber-spacer-2 sequence (ITS2) was developed by our group a few years ago, but so far this PCR has been applied mostly on urine samples. Here, we performed more in-depth evaluation of the ITS2 PCR as an alternative method to standard microscopy for the detection and quantification of Schistosoma spp. in stool samples.

Methodology/Principal findings

Microscopy and PCR were performed in a Senegalese community (n = 197) in an area with high S. mansoni transmission and co-occurrence of S. haematobium, and in Kenyan schoolchildren (n = 760) from an area with comparatively low S. mansoni transmission. Despite the differences in Schistosoma endemicity the PCR performed very similarly in both areas; 13–15% more infections were detected by PCR when comparing to microscopy of a single stool sample. Even when 2–3 stool samples were used for microscopy, PCR on one stool sample detected more infections, especially in people with light-intensity infections and in children from low-risk schools. The low prevalence of soil-transmitted helminthiasis in both populations was confirmed by an additional multiplex PCR.

Conclusions/Significance

The ITS2-based PCR was more sensitive than standard microscopy in detecting Schistosoma spp. This would be particularly useful for S. mansoni detection in low transmission areas, and post-control settings, and as such improve schistosomiasis control programs, epidemiological research, and quality control of microscopy. Moreover, it can be complemented with other (multiplex real-time) PCRs to detect a wider range of helminths and thus enhance effectiveness of current integrated control and elimination strategies for neglected tropical diseases.     

Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip®arrays

Background

The availability of both mouse and human draft genomes has marked the beginning of a new era of comparative mammalian genomics. The two available mouse genome assemblies, from the public mouse genome sequencing consortium and Celera Genomics, were obtained using different clone libraries and different assembly methods.

Results

We present here a critical comparison of the two latest mouse genome assemblies. The utility of the combined genomes is further demonstrated by comparing them with the human 'golden path' and through a subsequent analysis of a resulting conserved sequence element (CSE) database, which allows us to identify over 6,000 potential novel genes and to derive independent estimates of the number of human protein-coding genes.

Conclusion

The Celera and public mouse assemblies differ in about 10% of the mouse genome. Each assembly has advantages over the other: Celera has higher accuracy in base-pairs and overall higher coverage of the genome; the public assembly, however, has higher sequence quality in some newly finished bacterial artifical chromosome clone (BAC) regions and the data are freely accessible. Perhaps most important, by combining both assemblies, we can get a better annotation of the human genome; in particular, we can obtain the most complete set of CSEs, one third of which are related to known genes and some others are related to other functional genomic regions. More than half the CSEs are of unknown function. From the CSEs, we estimate the total number of human protein-coding genes to be about 40,000. This searchable publicly available online CSEdb will expedite new discoveries through comparative genomics.     

A new method for the identification of SCE's per cell cycle in BrdUrd — Substituted chromosomes

If cells are allowed to replicate in the presence of different concentrations of BrdUrd throughout three consecutive cell cycles, FPG stained third division chromosomes show three different levels of staining. This fact allows identification of four different types of SCE's, corresponding to the first-, second-and third-cycle exchanges. Finally, the relationship between the degree of BrdUrd-substitution and the frequency of SCE's per cell cycle supports that most of the SCE's detected in BrdUrd-substituted chromosomes are induced by this thymidine analogue incorporated into DNA.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.