AFLP Analysis of Trichoderma spp. from India Compared with Sequence and Morphological-based Diagnostics

Trichoderma species offer considerable potential for controlling aflatoxin contamination in groundnut and other crops. Initial classification of 48 Trichoderma isolates, derived from four different groundnut cultivation sites in India was based on alignment of 28S rDNA sequences to GenBank sequences of ex-type strains. This was found to be substantially more reliable than our routine morphological characterization, but did not provide a comprehensive diagnostic solution, as unique single nucleotide polymorphism (SNP) haplotypes could not be identified for all species. However, all the Trichoderma isolates could be readily distinguished by amplified fragment length polymorphism (AFLP) analysis, based on six primer pair combinations, which generated 234 polymorphic bands. In addition, individual AFLP bands were identified which differentiate closely related species. Similarly, AFLP bands were identified that correlated with different types of antagonism to Aspergillus flavus. The implications of these results for the development of simple polymerase chain reaction (PCR)-based diagnostic assays for antagonistic isolates of Trichoderma is discussed

Pseudomonas aeruginosa Population Structure Revisited

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS+/exoU− genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set

Risk of infection by reprocessed and resterilized virus-contaminated catheters

Aims In spite of increasing reuse of disposable catheters, there are few scientific data on potential viral transmission and infection after reuse. To determine the theoretical risk of virus transmission during reuse of catheters an in vitro study was performed using an RNA virus (echovirus-ll) and a DNA virus (adenovirus-2). Methods and Results After deliberate contamination of the catheters, reprocessing and reuse of the cleaned and glutaraldehyde sterilized catheters was simulated. The presence of residual virus was determined by cell culture and by polymerase chain reaction (PCR). After the sterilization step. infectious enterovirus was detectable in one (10%) of the samples, whereas two (20%) contained detectable enterovirus RNA. After simulated reuse, enterovirus was cultured from one (10%) of the catheters, but no less than six (60%) of the samples were enterovirus PCR positive and one (10%) contained detectable adenovirus DNA. After sonification of the catheter tips no infectious virus could be detected, but enterovirus RNA was detected in two (20%) and adenovirus DNA in three (30%) of the samples, Conclusions It has been clearly demonstrated in this in vitro study that, even after rigorous cleaning and sterilization, virus was still present in the catheter. Reuse of catheters, labelled for single-use only, is dangerous and should be prevented. (Eur Heart J 2001; 22: 378-383, doi:10,1053/cuhj.2000.2370) (C) 2001 The European Society of Cardiology

The additional value of real-time PCR in the quantitative detection of periodontal pathogens

Background and Aim: For the analysis of subgingival plaque, anaerobic bacterial culture has been the gold standard for many years. Currently, molecular microbial techniques have become available to identify and quantify target organisms with high specificity and sensitivity. The technique of real-time polymerase chain reaction (RT-PCR) provides a new tool to detect oral pathogens both in oral and non-oral human infections. The aim of this study was to compare the RT-PCR and anaerobic culture for detection and quantification of six periodontal pathogens in periodontal health and disease. Material and Methods: Subgingival plaque samples from 259 adult patients with periodontitis and 111 healthy controls were analysed with quantitative anaerobic culture and quantitative RT-PCR for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Micromonas micros and Fusobacterium spp. Results: All species were more frequently isolated from patients than controls with both culture and RT-PCR. P. gingivalis, T. forsythia and M. micros appeared significant markers for disease with both techniques. P. intermedia was significantly associated with periodontitis by RT-PCR only (OR 9.7), whereas A. actinomycetemcomitans showed a significant relationship by culture only. The critical differences between culture and RT-PCR were culture-negative/PCR-positive samples which amounted to 7% for A. actinomycetemcomitans, 3% for P. gingivalis, 7% for T. forsythia, 20% for P. intermedia, 6% for M. micros, and 0.8% for Fusobacterium spp. in periodontitis patients and 12%, 3%, 2%, 35%, 14% and 0%, respectively, in the periodontally healthy group. Furthermore, periodontitis individuals had significantly higher amount of all of the test species in the subgingival plaque samples compared with healthy subjects. Conclusion: RT-PCR provides a new rapid diagnostic tool and opens the opportunity to detect small numbers of oral pathogens in clinical specimens, which are under the detection limit by culture technique

Description of the ICTI consortium : an integrated approach to the study of Chlamydia trachomatis infection

The use of an integrated approach to the study of Chlamydia trachomatis infection of the female genital tract, presented at the mini-symposium "Chlamydia trachomatis infections" and described in the thesis of Joseph M. Lyons, has resulted in the creation of the ICTI consortium. The ICTI consortium is based on strong interaction and collaboration between basic scientists, clinicians, epidemiologists, and health care policy makers. This translational approach will help to further the valuable insight into the immunopathogenesis of this sexually transmitted infection (STI) and the development of new intervention strategies, including the vaccines and screening programs necessary to effectively diagnose, treat and prevent C. trachomatis infection. A background of the need for this integrated approach is presented and the goals and participants of the consortium are describe