Combining variant detection and fragment length analysis improves detection of minimal residual disease in postsurgery circulating tumour DNA of stage II-IIIA NSCLC patients.

Stage II-IIIA nonsmall cell lung cancer (NSCLC) patients receive adjuvant chemotherapy after surgery as standard-of-care treatment, even though only approximately 5.8% of patients will benefit. Identifying patients with minimal residual disease (MRD) after surgery using tissue-informed testing of postoperative plasma circulating cell-free tumour DNA (ctDNA) may allow adjuvant therapy to be withheld from patients without MRD. However, the detection of MRD in the postoperative setting is challenging, and more sensitive methods are urgently needed. We developed a method that combines variant calling and a novel ctDNA fragment length analysis using hybrid capture sequencing data. Among 36 stage II-IIIA NSCLC patients, this method distinguished patients with and without recurrence of disease in a 20 times repeated 10-fold cross validation with 75% accuracy (P = 0.0029). In contrast, using only variant calling or only fragment length analysis, no signification distinction between patients was shown (P = 0.24 and P = 0.074 respectively). In addition, a variant-level fragmentation score was developed that was able to classify variants detected in plasma cfDNA into tumour-derived or white-blood-cell-derived variants with 84% accuracy. The findings in this study may help drive the integration of various types of information from the same data, eventually leading to cheaper and more sensitive techniques to be used in this challenging clinical setting.

An Automated Correction Algorithm (ALPACA) for ddPCR Data Using Adaptive Limit of Blank and Correction of False Positive Events Improves Specificity of Mutation Detection.

BACKGROUND: Bio-Rad droplet-digital PCR is a highly sensitive method that can be used to detect tumor mutations in circulating cell-free DNA (cfDNA) of patients with cancer. Correct interpretation of ddPCR results is important for optimal sensitivity and specificity. Despite its widespread use, no standardized method to interpret ddPCR data is available, nor have technical artifacts affecting ddPCR results been widely studied. METHODS: False positive rates were determined for 6 ddPCR assays at variable amounts of input DNA, revealing polymerase induced false positive events (PIFs) and other false positives. An in silico correction algorithm, known as the adaptive LoB and PIFs: an automated correction algorithm (ALPACA), was developed to remove PIFs and apply an adaptive limit of blank (LoB) to individual samples. Performance of ALPACA was compared to a standard strategy (no PIF correction and static LoB = 3) using data from commercial reference DNA, healthy volunteer cfDNA, and cfDNA from a real-life cohort of 209 patients with stage IV nonsmall cell lung cancer (NSCLC) whose tumor and cfDNA had been molecularly profiled. RESULTS: Applying ALPACA reduced false positive results in healthy cfDNA compared to the standard strategy (specificity 98 vs 88%, P = 10-5) and stage IV NSCLC patient cfDNA (99 vs 93%, P = 10-11), while not affecting sensitivity in commercial reference DNA (70 vs 68% P = 0.77) or patient cfDNA (82 vs 88%, P = 0.13). Overall accuracy in patient samples was improved (98 vs 92%, P = 10-7). CONCLUSIONS: Correction of PIFs and application of an adaptive LoB increases specificity without a loss of sensitivity in ddPCR, leading to a higher accuracy in a real-life cohort of patients with stage IV NSCLC.

FIMAR: A new Fluoride Index to mitigate geogenic contamination by Managed Aquifer Recharge.

The objective of this study is to develop a Fluoride Index for mitigation of geogenic contamination by Managed Aquifer Recharge (FIMAR). This index was tested by applying to the Pambar river basin in southern India. About 40% of the study area had fluoride >1.5 mg/L, contributed from the dissolution of fluorite, fluorapatite, biotite and hornblende. The relationship between groundwater level and fluoride concentration exhibited two types of relationship. In shallow wells, groundwater recharge during monsoon increases the groundwater level and dilutes the fluoride concentration. During summer, evaporation decreases the groundwater levels and increases the fluoride content. In deep wells, the fluoride-rich salts that is deposited in the unsaturated zone due to evaporation in the pre-monsoon season is flushed during groundwater recharge and thus, the fluoride concentration increases with increase in groundwater level. This relationship was used as a key layer in FIMAR. Overlay analysis including the well type derived from the groundwater level and fluoride fluctuation, average fluoride concentration, geology, geomorphology, soil, drainage density, land use, lineament density and thickness of the weathered zone indicated 30% of the area was suitable for MAR to mitigate fluoride contamination. Since the construction of new MAR structures is cost-intensive, it is proposed to rejuvenate the existing ponds in the areas identified by FIMAR to induce recharge and dilute fluoride concentration. The positive impact was confirmed using numerical and geochemical modelling. This proves that FIMAR can be used as a planning tool in fluoride endemic areas and that the future MAR methods will be beneficial to mitigate geogenic fluoride contamination.

Role-play as an educational tool in medication communication skills: Students' perspectives.

OBJECTIVES: Medication communication skills are vital aspects of patient care that may influence treatment outcomes. However, traditional pharmacology curriculum deals with imparting factual information, with little emphasis on patient communication. The current study aims to explore students' perceptions of role-play as an educational tool in acquiring communication skills and to ascertain the need of role-play for their future clinical practice. MATERIALS AND METHODS: This questionnaire-based study was done in 2nd professional MBBS students. A consolidated concept of six training cases, focusing on major communication issues related to medication prescription in pharmacology, were developed for peer-role-play sessions for 2nd professional MBBS (n = 122) students. Structured scripts with specific emphasis on prescription medication communication and checklists for feedback were developed. Prevalidated questionnaires measured the quantitative aspects of role-plays in relation to their relevance as teaching-learning tool, perceived benefits of sessions, and their importance for future use. STATISTICAL ANALYSIS: Data analysis was performed using descriptive statistics. RESULTS: The role-play concept was well appreciated and considered an effective means for acquiring medication communication skills. The structured feedback by peers and faculty was well received by many. Over 90% of the students reported immense confidence in communicating therapy details, namely, drug name, purpose, mechanism, dosing details, and precautions. Majority reported a better retention of pharmacology concepts and preferred more such sessions. CONCLUSIONS: Most students consider peer-role-play as an indispensable tool to acquire effective communication skills regarding drug therapy. By virtue of providing experiential learning opportunities and its feasibility of implementation, role-play sessions justify inclusion in undergraduate medical curricula.

Fluoride in weathered rock aquifers of southern India: Managed Aquifer Recharge for mitigation.

Climatic condition, geology, and geochemical processes in an area play a major role on groundwater quality. Impact of these on the fluoride content of groundwater was studied in three regions-part of Nalgonda district in Telangana, Pambar River basin, and Vaniyar River basin in Tamil Nadu, southern India, which experience semi-arid climate and are predominantly made of Precambrian rocks. High concentration of fluoride in groundwater above 4 mg/l was recorded. Human exposure dose for fluoride through groundwater was higher in Nalgonda than the other areas. With evaporation and rainfall being one of the major contributors for high fluoride apart from the weathering of fluoride rich minerals from rocks, the effect of increase in groundwater level on fluoride concentration was studied. This study reveals that groundwater in shallow environment of all three regions shows dilution effect due to rainfall recharge. Suitable managed aquifer recharge (MAR) methods can be adopted to dilute the fluoride rich groundwater in such regions which is explained with two case studies. However, in deep groundwater, increase in fluoride concentration with increase in groundwater level due to leaching of fluoride rich salts from the unsaturated zone was observed. Occurrence of fluoride above 1.5 mg/l was more in areas with deeper groundwater environment. Hence, practicing MAR in these regions will increase the fluoride content in groundwater and so physical or chemical treatment has to be adopted. This study brought out the fact that MAR cannot be practiced in all regions for dilution of ions in groundwater and that it is essential to analyze the fluctuation in groundwater level and the fluoride content before suggesting it as a suitable solution. Also, this study emphasizes that long-term monitoring of these factors is an important criterion for choosing the recharge areas.