Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time ...PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.
A loss of physical functioning (i.e., a low physical capacity and/or a low physical activity) is a common feature in patients with chronic obstructive pulmonary disease (COPD). To date, the primary ...care physiotherapy and specialized pulmonary rehabilitation are clearly underused, and limited to patients with a moderate to very severe degree of airflow limitation (GOLD stage 2 or higher). However, improved referral rates are a necessity to lower the burden for patients with COPD and for society. Therefore, a multidisciplinary group of healthcare professionals and scientists proposes a new model for referral of patients with COPD to the right type of exercise-based care, irrespective of the degree of airflow limitation. Indeed, disease instability (recent hospitalization, yes/no), the burden of disease (no/low, mild/moderate or high), physical capacity (low or preserved) and physical activity (low or preserved) need to be used to allocate patients to one of the six distinct patient profiles. Patients with profile 1 or 2 will not be referred for physiotherapy; patients with profiles 3–5 will be referred for primary care physiotherapy; and patients with profile 6 will be referred for screening for specialized pulmonary rehabilitation. The proposed Dutch model has the intention to get the right patient with COPD allocated to the right type of exercise-based care and at the right moment.
The population structure of Staphylococcus aureus carried by healthy humans was determined using a large strain collection of nonclinical origin (n = 829). High-throughput amplified fragment length ...polymorphism (AFLP) analysis revealed 3 major and 2 minor genetic clusters of S. aureus, which were corroborated by multilocus sequence typing. Major AFLP cluster I comprised 44.4% of the carriage isolates and showed additional heterogeneity whereas major AFLP groups II and III presented 2 homogeneous clusters, including 47.3% of all carriage isolates. Coanalysis of invasive S. aureus strains and epidemic methicillin-resistant S. aureus (MRSA) revealed that all major clusters contained invasive and multiresistant isolates. However, clusters and subclusters with overrepresentation of invasive isolates were also identified. Bacteremia in elderly adults, for instance, was caused by a IVa cluster-derived strain significantly more often than by strains from other AFLP clusters. Furthermore, expansion of multiresistant clones or clones associated with skin disease (impetigo) was detected, which suggests that epidemic potential is present in pathogenic strains of S. aureus. In addition, the virulence gene encoding Panton-Valentine leukocidin was significantly enriched in S. aureus strains causing abscesses and arthritis in comparison with the carriage group. We provide evidence that essentially any S. aureus genotype carried by humans can transform into a life-threatening human pathogen but that certain clones are more virulent than others.
Mutation-induced recessive alleles
(mlo) of the barley
Mlo locus confer a leaf lesion phenotype and broad spectrum resistance to the fungal pathogen, Erysiphe graminis f. sp. hordei. The gene has ...been isolated using a positional cloning approach. Analysis of 11 mutagen-induced
mlo alleles revealed mutations leading in each case to alterations of the deduced Mlo wild-type amino acid sequence. Susceptible intragenic recombinants, isolated from
mlo heteroallelic crosses, show restored
Mlo wild-type sequences. The deduced 60 kDa protein is predicted to be membrane-anchored by at least six membrane-spanning helices. The findings are compatible with a dual negative control function of the Mlo protein in leaf cell death and in the onset of pathogen defense; absence of Mlo primes the responsiveness for the onset of multiple defense functions.
1 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
2 Department of Pediatrics, ...Erasmus MC, University Medical Center Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands
3 Department of Microbial Genomics, Keygene NV, Agro Business Park 90, 6708 PW Wageningen, The Netherlands
4 Department of Bioinformatics, Erasmus MC, University Medical Center Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands
5 PathoFinder BV, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands
6 Laboratory of Pediatric Infectious Diseases, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands
Correspondence Damian C. Melles d.melles{at}erasmusmc.nl
Bacterial interference between Staphylococcus aureus and Streptococcus pneumoniae in the nasopharynx has been observed during colonization, which might have important clinical implications for the widespread use of pneumococcal conjugate vaccine in young children. This study aimed to determine whether the capacity of Staph. aureus to compete with Strep. pneumoniae is dependent on bacterial genotype. Demographic and microbiological determinants of carriage of specific genotypes of Staph. aureus in children were also studied. Children ( n =3198) were sampled in the nasopharynx to detect carriage of Staph. aureus , Strep. pneumoniae and Neisseria meningitidis . Staph. aureus genotypes and pneumococcal sero- and genotypes were determined. Age, gender, zip code, active smoking and co-colonization with N. meningitidis or Strep. pneumoniae , both vaccine- and non-vaccine types, were not associated with colonization by specific Staph. aureus genotypes. Based on the whole-genome typing data obtained, there was no obvious correlation between staphylococcal and pneumococcal genotypes during co-colonization. Passive smoking showed a significant association ( P =0.003) with carriage of a specific Staph. aureus cluster. This study suggests that there are no major differences between Staph. aureus clones (with different disease-invoking potential) in their capacity to compete with Strep. pneumoniae subtypes. Further studies should demonstrate whether differences in bacterial interference are due to more subtle genetic changes.
Abbreviations: AFLP, amplified fragment length polymorphism; ht-AFLP, high-throughput amplified fragment length polymorphism; PCA, principal component analysis; PCV7, 7-valent pneumococcal-conjugate vaccine; RFEL, restriction fragment end labelling
Resistance of barley (Hordeum vulgare) to the powdery mildew fungus Erysiphe graminis f.sp. hordei is conferred by several dominant genes, but also by recessive alleles of the Mlo locus mapping on ...the long arm of chromosome 4. In addition, this single-factor-mediated resistance is active against all known physiological races of the parasite. Thus the mechanism underlying mlo-mediated resistance should differ substantially from that mediated by the dominant genes. A positional cloning strategy to isolate the Mlo gene from the barley genome, the size of which is almost double the size of the human genome, has been designed. The AFLP technique was employed to identify markers tightly linked to the Mlo locus and to produce a local high-resolution genetic map. The use of this high-volume marker technology allowed the rapid screening of approximately 250,000 loci for linkage to Mlo. A large number of Mlo-linked AFLP markers were identified, one of which cosegregated with Mlo on the basis of more than 4000 meiotic events. A four-genome-equivalent barley YAC library (average insert size 480 kb) was constructed and screened with this cosegregating marker. Four YACs containing this marker were isolated and subsequent characterization by AFLP-based physical mapping allowed the physical delimitation of the Mlo locus to a DNA segment of 30 kb.
The minor capsid proteins C and D from phage M13 have been characterized by differential amino acid labeling and amino-terminal sequence analysis. We demonstrate that D protein (Mr12,260) is the ...product of gene VI, whereas the C component is composed of the products of both gene VII (Mr3580) and gene IX (Mr3650). Our data further show that the proteins of genes VI, VII, and IX are not subject to proteolytic processing but are packaged into mature virions as their primary translational products. On the basis of incorporation of specific amino acids, the copy numbers of these proteins in M13 virions could be estimated relative to the number of A protein molecules. The M13 phage contains on the average 5 molecules of A protein, 5 molecules of VI protein and 3-4 molecules of both VII protein and IX protein. These copy numbers remained unchanged in M13 recombinant phages of up to two times the length of wild-type phages, a fact that indicates that these minor capsid proteins are located at either one or both ends of the phage filament.
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