Morphological and Physiological Development of Pyricularia oryzae Isolates from North-western Region of Sarawak on Different Media under Laboratory Conditions Laboratory Conditions

Rice blast (causal agent: Pyricularia oryzae) is an important disease of rice in Sarawak. Understanding the pathogen’s morphological characteristics, genetic diversity and pathogenicity is important. Having a suitable medium for culturing and maintaining P. oryzae is important to ensure the availability of inoculum or materials under laboratory conditions. Oatmeal agar (OMA) and potato dextrose agar (PDA) are common media used for growing P. oryzae. OMA allows better mycelial growth and better sporulation as compared to PDA. There are also other alternatives such as fresh rice leaf agar and rice straw agar. Although OMA seems to be the best medium, unfortunately the opaqueness of the medium causes difficulty in observing the morphology and growth of mycelia. In addition, it is known that different isolates of P. oryzae will respond differently to different media. This study aims to identify the best media for culturing and maintaining P. oryzae isolates from Sarawak. A total of 14 P. oryzae isolates were characterised for their morphological characteristics, growth rate and sporulation rate using seven growing media. These 14 isolates included seven newly identified isolates in this study and seven isolates from a previous study, which were verified using internal transcribed spacer DNA sequence. The colony surface of the 14 P. oryzae isolates varied on different growing media. The pigmentation of colony surface varied from different shades of grey, translucent light brown, white and colourless. Pyricularia oryzae isolates grew better on OMA and PDA, while OMA was the best for sporulation. These two media can be recommended for culturing and maintaining different P. oryzae isolates under laboratory conditions

New variants of AvrPiz-t identified in Pyricularia oryzae from Malaysia

Rice blast disease (causal agent: Pyricularia oryzae) is an important rice disease worldwide as it can cause significant yield loss. Resistance genes in rice can recognise the corresponding avirulence genes (Avr-gene) of P. oryzae and suppress the attack of the pathogen as proposed in ‘gene-for-gene’ interaction. This interaction is highly specific. The resistance will be rapidly breakdown owing to the plasticity of Avr-gene. The breaking down of disease resistance can cause problem in managing the disease as well as sustaining a resistant rice variety. This paper aimed to study and analyse the sequence variation of AvrPiz-t gene from P. oryzae isolates in Malaysia (Sarawak) together with AvrPiz-t sequences available from National Centre for Biotechnology Information (NCBI). This study compared the AvrPiz-t haplotype found with previously reported haplotypes. A total of 20 Malaysian P. oryzae isolates were obtained from six divisions of Sarawak and were verified using internal transcribed spacer. Together with seven Malaysian P. oryzae isolates from a previous study, a total of 27 AvrPiz-t gene sequences from Malaysian isolates were successfully amplified and verified. The AvrPiz-t open reading frame (ORF) of 27 Malaysian isolates were aligned with 123 AvrPiz-t ORF from other countries that were available in NCBI database and 100 AvrPiz-t ORF from a previous study (Total = 250 sequences). Unique sequences were identified and translated into amino sequence. The amino acid sequences were aligned with haplotype sequences from previous studies. In total, there were 19 AvrPiz-t haplotypes, with eight newly identified haplotypes. The AvrPiz-t haplotypes were distributed across 14 countries with haplotype H9 as the dominant haplotype. Neutrality tests were performed for the 250 AvrPiz-t ORF sequences. The 250 isolates of P. oryzae were undergoing directional natural selection.. Detached leaf inoculation using filter paper method of selected Malaysia (Sarawak) P. oryzae isolates carrying one of the new AvrPiz-t haplotypes showed different level of pathogenicity towards Sarawak rice landraces. The data obtained serves as baseline information for disease management in Malaysia, especially for Sarawak state. Additionally, the information is also a reference for future rice breeding program in Sarawak

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

A cross-sectional study of burnout and its associations with learning environment and learner factors among psychiatry residents within a National Psychiatry Residency Programme

10.1136/bmjopen-2019-030619BMJ OPEN9

Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer

Introduction Circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together. Methods Concurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients. Results Higher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression. Conclusions A standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality