Re-evaluation of Streptococcus pneumoniae carriage in Portuguese elderly by qPCR increases carriage estimates and unveils an expanded pool of serotypes.

Streptococcus pneumoniae (pneumococcus) is a leading cause of infections worldwide. Disease is preceded by asymptomatic colonization of the upper respiratory tract. Classical culture-based methods (CCBM) suggest that colonization in the elderly is <5%. Recently, use of qPCR has challenged these observations. We estimated pneumococcal carriage prevalence and serotypes among Portuguese elderly using qPCR and compared results with those obtained by CCBM. Nasopharyngeal and oropharyngeal paired samples (599 each) of individuals over 60 years living in nursing (n = 299) or family (n = 300) homes were screened for the presence of pneumococci by qPCR targeting lytA and piaB. Positive samples were molecular serotyped. Use of qPCR improved detection of pneumococci in oropharyngeal samples compared to CCBM: from 0.7% to 10.4% (p < 0.001) in the nursing home collection, and from 0.3% to 5.0% (p < 0.001) in the family home collection. No significant differences were observed between both methods in nasopharyngeal samples (5.4% vs. 5.4% in the nursing homes; and 4.3% vs. 4.7% in the family homes). Twenty-one serotypes/serogroups were detected by qPCR compared to 14 by CCBM. In conclusion, use of qPCR suggests that pneumococcal carriage in Portuguese elderly is approximately 10%, and unveiled a large pool of serotypes. These results are important to understand progression to disease and impact of pneumococcal vaccines in the elderly.

Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants.

BACKGROUND: Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type). RESULTS: Somatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV. CONCLUSIONS: Cut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in some of the regenerated plants showed that both viruses are seed-transmitted. The regeneration of true-to-type virus-free plants from all infected varieties indicates that this methodology may represent an alternative procedure for virus cleaning in grapevine.

In vitro propagation of Vitis vinifera L. cv. 'Monastrell'

Background: A protocol for the micropropagation of the grape (Vitis vinifera L.) cultivar 'Monastrell' was developed. Initial plant material was obtained from the sanitary selection of grapevine plants performed by real-time RT-PCR to confirm the absence of Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3, and Grapevine fleck virus. Results: The effects of the salt composition (comparing Lloyd and McCown woody plant medium and Murashige and Skoog medium 1/2 macronutrients) and the growth regulator benzylaminopurine (BAP), at 0 and 8.9 µM, on plant propagation were evaluated using nodes as explants. The most efficient procedure consisted of bud induction in the medium with Lloyd and McCown woody plant salts and 8.9 µM BAP for 30 d along with elongation in cytokinin-free medium for 60 d, which gave 22 nodes/explant (174 plants/initial plant). A second cycle of propagation in a medium without BAP for another 60 d could give approximately 10,000 nodes, which can be obtained after an additional 2 months of culture. All plants acclimatized after the second cycle of multiplication were successfully transferred to soil. Conclusion: We developed an optimal protocol for V. vinifera cv. 'Monastrell' micropropagation, the first described for this cultivar.