Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Perceived wellbeing of patients one year post stroke in general practice - <Emphasis Type="Italic">recommendations for quality aftercare</Emphasis>

Background  

Annually, 41,000 people in the Netherlands have strokes. This has multiple physical and psychosocial consequences. Most patients return home after discharge from hospital. Quality aftercare by general practitioners is important to support patients at home. The purpose of this study is to examine the wellbeing of patients who returned home immediately after discharge from hospital, one year post stroke, in comparison with the general Dutch population of the same age and to determine factors that could influence wellbeing.     

Highly Pathogenic H5N1 Influenza Viruses Carry Virulence Determinants beyond the Polybasic Hemagglutinin Cleavage Site

Highly pathogenic avian influenza viruses (HPAIV) originate from avirulent precursors but differ from all other influenza viruses by the presence of a polybasic cleavage site in their hemagglutinins (HA) of subtype H5 or H7. In this study, we investigated the ability of a low-pathogenic avian H5N1 strain to transform into an HPAIV. Using reverse genetics, we replaced the monobasic HA cleavage site of the low-pathogenic strain A/Teal/Germany/Wv632/2005 (H5N1) (TG05) by a polybasic motif from an HPAIV (TG05_poly). To elucidate the virulence potential of all viral genes of HPAIV, we generated two reassortants carrying the HA from the HPAIV A/Swan/Germany/R65/06 (H5N1) (R65) plus the remaining genes from TG05 (TG05-HA_R65) or in reversed composition the mutated TG05 HA plus the R65 genes (R65-HA_TG05poly). In vitro, TG05_poly and both reassortants were able to replicate without the addition of trypsin, which is characteristic for HPAIV. Moreover, in contrast to avirulent TG05, the variants TG05_poly, TG05-HA_R65, and R65-HA_TG05poly are pathogenic in chicken to an increasing degree. Whereas the HA cleavage site mutant TG05_poly led to temporary non-lethal disease in all animals, the reassortant TG05-HA_R65 caused death in 3 of 10 animals. Furthermore, the reassortant R65-HA_TG05poly displayed the highest lethality as 8 of 10 chickens died, resembling “natural” HPAIV strains. Taken together, acquisition of a polybasic HA cleavage site is only one necessary step for evolution of low-pathogenic H5N1 strains into HPAIV. However, these low-pathogenic strains may already have cryptic virulence potential. Moreover, besides the polybasic cleavage site, the additional virulence determinants of H5N1 HPAIV are located within the HA itself and in other viral proteins.     

A microsatellite linkage map of Drosophila mojavensis

Background

Drosophila mojavensishas been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives.

Results

We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis.

Conclusions

The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.     

Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography-tandem mass spectrometry

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.     

First evidence of natural reproduction of the Allis shad Alosa alosa in the River Rhine following re‐introduction measures

After stocking with larvae from the Gironde–Garonne–Dordogne population, in 2013, three young‐of‐the‐year Allis shad Alosa alosa, probably originating from natural reproduction, were documented for the first time in a period of nearly 100 years in the River Rhine. In 2014, a further increase was observed when 57 juveniles and eight adults were caught; seven of these eight adults were derived from stocking, indicating the success of stocking measures within the framework of the EU‐LIFE project.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.