A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

Double-stranded RNAs purified from the V2356 (‘Successa’) sour cherry source of the Shirofugen stunt disease (SSD) were sequenced using a 454 pyrosequencing multiplex approach. The 15,646 reads obtained were assembled into 279 contigs, 5 of which, totaling almost 16.9 kbp and 5,332 reads (34% of sample reads), showed high Blast scores and homology to Little cherry virus 1 (LChV1). The five contigs were further assembled manually into three supercontigs spanning the full LChV1 genome with only two small gaps (17 and 55 bases). Completion of the sequencing of the viral genome was performed using targeted polymerase chain reaction and primers designed from the contigs. No evidence for the presence of other viral agents in the V2356 source could be obtained in the remaining contigs or singletons. The V2356 LChV1 isolate is only ≈76% identical with the reference complete LChV1 sequences and, in particular, with the ITMAR isolate associated with the Kwanzan stunting syndrome. However, it is highly homologous (97 to 100% identity) in two short genome regions with divergent LChV1 from North America, providing the first complete sequence for such divergent isolates. Although not providing a definite proof, the failure to detect any other viral agent in the V2356 SSD source and the identification of LChV1 in a second, independent, source of the disease suggests that LChV1 isolates could be responsible for the SSD syndrome.

BACKGROUND: The increasing use of macrolides especially in the treatment of Helicobacter pylori infection has led to an increase in resistant strains. The resistance of H pylori to macrolides, especially clarithromycin, is one of the major causes of eradication failure. In H pylori , clarithromycin resistance is due to point mutations localised in domain V of 23S rRNA. AIM: To develop a molecular technique based on amplification of a relevant fragment of the 23S rRNA and colorimetric hybridisation in liquid phase to detect directly in biopsy specimens the type of mutation associated with resistance of H pylori to clarithromycin. METHODS: Gastric biopsy samples from 61 patients were submitted to this test. The results were compared with standard methods (determination of minimal inhibition concentration, polymerase chain reaction/restriction fragment length polymorphism, and/or DNA sequencing) in order to evaluate the test and to define the cut off values, specificity, and sensitivity. RESULTS: The 14 biopsy samples in which H pylori was not detected did not give a positive result in any assay, and the 14 samples harbouring strains susceptible to clarithromycin gave a positive result with the wild type probe as expected. The 33 biopsy specimens containing resistant strains always gave a positive signal with one of the probes detecting resistant organisms, but in eight cases they also reacted with the wild type probe, indicating that a mixture of resistant and susceptible organisms was present. CONCLUSION: The importance of this new assay is that it allows the detection of multiple genotypes corresponding to either heterogeneous genotypes or mixed infections. Moreover, it allows in a single step not only the detection of H pylori but also the determination of its susceptibility to clarithromycin directly in biopsy specimens without the need for culture.

The differences in eradication rates reported in clinical trials aiming to cure Helicobacter pylori infection cannot be entirely explained by the type of regimen, bacterial resistance, or lack of compliance. Using data from a clinical trial, a logistic regression model was constructed to determine whether cagA status, assessed by PCR, affects the outcome of eradication. Resistance to clarithromycin (10% of the strains) predicted failure perfectly. In the model ( n = 156), a cagA -lacking strain (odds ratio [OR] = 2.2; 95% confidence interval [CI], (1.1 to 4.7), tobacco smoking OR = 3.1; 95% CI, 1.3 to 7.0), and a double dose of proton pump inhibitor in the treatment regimen (OR = 0.3; 95% CI, 0.2 to 0.7) were associated with the treatment outcome. The exact role of cagA in the outcome of H. pylori eradication therapy has not been explored. However, the type of histological lesions which it causes in the gastric mucosa may be implicated. Regardless of the mechanism involved, cagA status is a good predictive marker of eradication outcome.