Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Azole-resistant Candida albicans prosthetic joint infection treated with prolonged administration of anidulafungin and two-stage exchange with implant of a mega-prosthesis.

Fungal prosthetic joint infection (PJI) is a rare but severe complication of artroplasty. We report a case of PJI due to azole-resistant Candida albicans successfully treated with combination of prolonged administration of anidulafungin and two-stage joint exchange with insertion of a mega-prosthesis.

[Diagnosis in patients with a painful arthroplasty]. / Diagnosi differenziale in pazienti con protesi ortopedica dolorosa.

The differential diagnosis between asepting loosening or prosthetic joint infection is not always easy. Tc-99m-labelled leucocyte scans, frozen section and histology can help recognise doubtful cases. We report the experience of the Unit for Infectious Diseases and Septic Orthopaedics of the ASL-2 Liguria, Italy, with a Tc-99m-labelled leucocyte scan and intraoperative frozen section to choose the best therapeutic approach: one-stage or two-stage exchange or arthrodesis-arthroplastica. All cases underwent histology and intraoperative cultures to confirm the diagnosis, and the effectiveness of the approach was evaluated at follow up after 18 months. From January 2011 to December 2012, 36 patients were evaluated (21 hip and 15 knee arthroprosthesis). The Tc-99m-labelled leukocyte scan was positive in 31 and negative in 5 patients. Frozen section was negative in 7 patients. Five of them were patients with a negative Tc-99m-labelled leucocyte scan and were treated successfully with one-stage exchange, even if, in one of them, Enterococcus faecalis was isolated at replacement and suppressive antibiotic treatment was needed. The other 31 patients were treated with arthrodesis arthroplasty (3 patients) or a two-stage exchange. In this group the Tc-99m-labelled leucocyte scan was positive in all patients and the frozen section was positive in 29/31 cases with 6% false negative. Histology was positive in 27/31 with 13% of false negative. The sensitivity and specificity value was respectively 90% and 100% in the frozen section, 84% and 100% in histology. Cultures were positive in 23/31 cases. Patients subjected to two-stage exchange were evaluated again during prosthesis replacement but the results of the Tc-99m-labelled leucocyte scan and histology showed unclear results more frequently: the Tc-99m-labelled leucocyte scan was positive in two cases, the frozen section in three and histology in seven in spite of positive culture in three cases and one relapse in a patient with a frozen section and histology positive but negative culture. In the second step sensitivity and specificity were respectively 67% and 96% in the frozen section, 75% and 88% in histology. Finally, our experience suggests the utility of nuclear and histological tests during diagnostic work-up for differential diagnosis of aseptic loosening or prosthetic joint infection. The same tests produce more uncertain data when performed in two-stage exchange during the second step.

Prosthetic joint infections due to Mycobacterium tuberculosis: A report of 5 cases.

INTRODUCTION: Tubercular infection of prosthetic joint arthroplasty is sporadically described, but its incidence is rising. Misdiagnosis is common because of disparate clinical presentation. PRESENTATION OF CASE: We describe 1 hand, 2 hip and 2 knee prosthetic-joint infections due to Mycobacterium tuberculosis in patients without a previous history of tuberculosis. All of them were initially misdiagnosed as bacterial infections and unsuccessfully treated with antibiotic for a long period of time. Diagnosis was made by means of culture of periprosthetic tissues and histolopathological examination. Tuberculosis was cured in all patients, but two of them have had a permanent functional damage (one arthrodesis of the knee and one loss of hand function). DISCUSSION: An aggressive diagnostic approach is required to make diagnosis of periprosthetic tubercular infection. The identification of the pathogen is advisable to test drug susceptibility. CONCLUSION: The low index of suspicion of periprosthetic tubercular infection could delay a correct diagnosis with risk of permanent damage due to a late treatment. During any surgical revision of prosthetic joints with suspect infection culture for tuberculosis should be taken into consideration.