Crykey: Rapid Identification of SARS-CoV-2 Cryptic Mutations in Wastewater.

We present Crykey, a computational tool for rapidly identifying cryptic mutations of SARS-CoV-2. Specifically, we identify co-occurring single nucleotide mutations on the same sequencing read, called linked-read mutations, that are rare or entirely missing in existing databases, and have the potential to represent novel cryptic lineages found in wastewater. While previous approaches exist for identifying cryptic linked-read mutations from specific regions of the SARS-CoV-2 genome, there is a need for computational tools capable of efficiently tracking cryptic mutations across the entire genome and for tens of thousands of samples and with increased scrutiny, given their potential to represent either artifacts or hidden SARS-CoV-2 lineages. Crykey fills this gap by identifying rare linked-read mutations that pass stringent computational filters to limit the potential for artifacts. We evaluate the utility of Crykey on >3,000 wastewater and >22,000 clinical samples; our findings are three-fold: i) we identify hundreds of cryptic mutations that cover the entire SARS-CoV-2 genome, ii) we track the presence of these cryptic mutations across multiple wastewater treatment plants and over a three years of sampling in Houston, and iii) we find a handful of cryptic mutations in wastewater mirror cryptic mutations in clinical samples and investigate their potential to represent real cryptic lineages. In summary, Crykey enables large-scale detection of cryptic mutations representing potential cryptic lineages in wastewater.

Somatic evolution of marine transmissible leukemias in the common cockle, Cerastoderma edule.

Transmissible cancers are malignant cell lineages that spread clonally between individuals. Several such cancers, termed bivalve transmissible neoplasia (BTN), induce leukemia-like disease in marine bivalves. This is the case of BTN lineages affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and northwest Africa. To investigate the evolution of cockle BTN, we collected 6,854 cockles, diagnosed 390 BTN tumors, generated a reference genome and assessed genomic variation across 61 tumors. Our analyses confirmed the existence of two BTN lineages with hemocytic origins. Mitochondrial variation revealed mitochondrial capture and host co-infection events. Mutational analyses identified lineage-specific signatures, one of which likely reflects DNA alkylation. Cytogenetic and copy number analyses uncovered pervasive genomic instability, with whole-genome duplication, oncogene amplification and alkylation-repair suppression as likely drivers. Satellite DNA distributions suggested ancient clonal origins. Our study illuminates long-term cancer evolution under the sea and reveals tolerance of extreme instability in neoplastic genomes.

Wastewater early warning system for SARS-CoV-2 outbreaks and variants in a Coruña, Spain.

Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 103 and 106 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time.

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer.

The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. Here, we contrasted the genomic profiles of CTC pools recovered from metastatic colorectal cancer (mCRC) patients using different enrichment strategies (CellSearch, Parsortix, and FACS). Mutations inferred in the CTC pools differed depending on the enrichment strategy and, in all cases, represented a subset of the mutations detected in the matched primary tumor samples. However, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures, and drug-suitability scores remarkably close to those found in matching primary tumor samples. In addition, FACS CTC pools were enriched in apparent sequencing artifacts, leading to much higher genomic diversity estimates. Our results highlight the utility of CTCs to assess the genomic heterogeneity of individual tumors and help clinicians prioritize drugs in mCRC.

Clonality and timing of relapsing colorectal cancer metastasis revealed through whole-genome single-cell sequencing.

Recurrence of tumor cells following local and systemic therapy is a significant hurdle in cancer. Most patients with metastatic colorectal cancer (mCRC) will relapse, despite resection of the metastatic lesions. A better understanding of the evolutionary history of recurrent lesions is required to identify the spatial and temporal patterns of metastatic progression and expose the genetic and evolutionary determinants of therapeutic resistance. With this goal in mind, here we leveraged a unique single-cell whole-genome sequencing dataset from recurrent hepatic lesions of an mCRC patient. Our phylogenetic analysis confirms that the treatment induced a severe demographic bottleneck in the liver metastasis but also that a previously diverged lineage survived this surgery, possibly after migration to a different site in the liver. This lineage evolved very slowly for two years under adjuvant drug therapy and diversified again in a very short period. We identified several non-silent mutations specific to this lineage and inferred a substantial contribution of chemotherapy to the overall, genome-wide mutational burden. All in all, our study suggests that mCRC subclones can migrate locally and evade resection, keep evolving despite rounds of chemotherapy, and re-expand explosively.

Malignant transformation and genetic alterations are uncoupled in early colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) development is generally accepted as a sequential process, with genetic mutations determining phenotypic tumor progression. However, matching genetic profiles with histological transition requires the analyses of temporal samples from the same patient at key stages of progression. RESULTS: Here, we compared the genetic profiles of 34 early carcinomas with their respective adenomatous precursors to assess timing and heterogeneity of driver alterations accompanying the switch from benign adenoma to malignant carcinoma. In almost half of the cases, driver mutations specific to the carcinoma stage were not observed. In samples where carcinoma-specific alterations were present, TP53 mutations and chromosome 20 copy gains commonly accompanied the switch from adenomatous tissue to carcinoma. Remarkably, 40% and 50% of high-grade adenomas shared TP53 mutations and chromosome 20 gains, respectively, with their matched carcinomas. In addition, multi-regional analyses revealed greater heterogeneity of driver mutations in adenomas compared to their matched carcinomas. CONCLUSION: Genetic alterations in TP53 and chromosome 20 occur at the earliest histological stage in colorectal carcinomas (pTis and pT1). However, high-grade adenomas can share these alterations despite their histological distinction. Based on the well-defined sequence of CRC development, we suggest that the timing of genetic changes during neoplastic progression is frequently uncoupled from histological progression.

Differential development of large-cell neuroendocrine or small-cell lung carcinoma upon inactivation of 4 tumor suppressor genes.

High-grade neuroendocrine lung malignancies (large-cell neuroendocrine cell carcinoma, LCNEC, and small-cell lung carcinoma, SCLC) are among the most deadly lung cancer conditions with no optimal clinical management. The biological relationships between SCLC and LCNEC are still largely unknown and a current matter of debate as growing molecular data reveal high heterogeneity with potential therapeutic consequences. Here we describe murine models of high-grade neuroendocrine lung carcinomas generated by the loss of 4 tumor suppressors. In an Rbl1-null background, deletion of Rb1, Pten, and Trp53 floxed alleles after Ad-CMVcre infection in a wide variety of lung epithelial cells produces LCNEC. Meanwhile, inactivation of these genes using Ad-K5cre in basal cells leads to the development of SCLC, thus differentially influencing the lung cancer type developed. So far, a defined model of LCNEC has not been reported. Molecular and transcriptomic analyses of both models revealed strong similarities to their human counterparts. In addition, a 68Ga-DOTATOC-based molecular-imaging method provides a tool for detection and monitoring the progression of the cancer. These data offer insight into the biology of SCLC and LCNEC, providing a useful framework for development of compounds and preclinical investigations in accurate immunocompetent models.

Duration of Antibiotic Treatment in Community-Acquired Pneumonia: A Multicenter Randomized Clinical Trial.

IMPORTANCE: The optimal duration of antibiotic treatment for community-acquired pneumonia (CAP) has not been well established. OBJECTIVE: To validate Infectious Diseases Society of America/American Thoracic Society guidelines for duration of antibiotic treatment in hospitalized patients with CAP. DESIGN, SETTING, AND PARTICIPANTS: This study was a multicenter, noninferiority randomized clinical trial performed at 4 teaching hospitals in Spain from January 1, 2012, through August 31, 2013. A total of 312 hospitalized patients diagnosed as having CAP were studied. Data analysis was performed from January 1, 2014, through February 28, 2015. INTERVENTIONS: Patients were randomized at day 5 to an intervention or control group. Those in the intervention group were treated with antibiotics for a minimum of 5 days, and the antibiotic treatment was stopped at this point if their body temperature was 37.8°C or less for 48 hours and they had no more than 1 CAP-associated sign of clinical instability. Duration of antibiotic treatment in the control group was determined by physicians. MAIN OUTCOMES AND MEASURES: Clinical success rate at days 10 and 30 since admission and CAP-related symptoms at days 5 and 10 measured with the 18-item CAP symptom questionnaire score range, 0-90; higher scores indicate more severe symptoms. RESULTS: Of the 312 patients included, 150 and 162 were randomized to the control and intervention groups, respectively. The mean (SD) age of the patients was 66.2 (17.9) years and 64.7 (18.7) years in the control and intervention groups, respectively. There were 95 men (63.3%) and 55 women (36.7%) in the control group and 101 men (62.3%) and 61 women (37.7%) in the intervention group. In the intent-to-treat analysis, clinical success was 48.6% (71 of 150) in the control group and 56.3% (90 of 162) in the intervention group at day 10 (P = .18) and 88.6% (132 of 150) in the control group and 91.9% (147 of 162) in the intervention group at day 30 (P = .33). The mean (SD) CAP symptom questionnaire scores were 24.7 (11.4) vs 27.2 (12.5) at day 5 (P = .10) and 18.6 (9.0) vs 17.9 (7.6) at day 10 (P = .69). In the per-protocol analysis, clinical success was 50.4% (67 of 137) in the control group and 59.7% (86 of 146) in the intervention group at day 10 (P = .12) and 92.7% (126 of 137) in the control group and 94.4% (136 of 146) in the intervention group at day 30 (P = .54). The mean (SD) CAP symptom questionnaire scores were 24.3 (11.4) vs 26.6 (12.1) at day 5 (P = .16) and 18.1 (8.5) vs 17.6 (7.4) at day 10 (P = .81). CONCLUSIONS AND RELEVANCE: The Infectious Diseases Society of America/American Thoracic Society recommendations for duration of antibiotic treatment based on clinical stability criteria can be safely implemented in hospitalized patients with CAP. TRIAL REGISTRATION: clinicaltrialsregister.eu Identifier: 2011-001067-51.

Neumomediastino y daño alveolar difuso. Afección pulmonar severa por dermatomiositis / Pneumomediastinum and diffuse alveolar pain. Severe interstitial pneumopathy due to dermatomyositis

Presentamos el caso de un varón de 36 años con dermatomiositis de reciente comienzo, que desarrolló neumomediastino y enfisema subcutáneo masivo al inicio de una neumopatía intersticial progresiva y severa. En el momento del diagnóstico no había imágenes parenquimatosas quísticas evidentes; sin embargo, la broncoscopia permitió evidenciar una lesión endobronquial de aspecto necrótico que se consideró como probable origen de la fuga aérea. El paciente se trató con corticoides a dosis altas, ciclofosfamida y ciclosporina A, con resolución del neumomediastino y del enfisema subcutáneo. Con el tratamiento se observó una mejoría progresiva de la afección parenquimatosa pulmonar y de la insuficiencia respiratoria, lo que permitió la disminución progresiva de los corticoides (AU)

We have recently observed the case of a 36-year-old man with dermatomyositis of recent onset, who developed masive pneumomediastinum and subcutaneous emphysema at the onset of a progresive and severe pulmonary disease. Although there were no sign of parenchymal cysts, after the bronchoscopy it was possible to observe endobronchial necrotic injury which was considered as the likely source of the air leak. He was treated with high dose of corticosteroids, cyclophosphamide and cyclosporin A which resulted in the disappearance of the pneumomediastinum and subcutaneous emphysema and the progressive improvement of both parenchymal lung disease and respiratory insufficiency, enabled us to progressively taper the dose of corticosteroids (AU)

[Pneumomediastinum and diffuse alveolar pain. Severe interstitial pneumopathy due to dermatomyositis]. / Neumomediastino y daño alveolar difuso. Afección pulmonar severa por dermatomiositis.

We have recently observed the case of a 36-year-old man with dermatomyositis of recent onset, who developed masive pneumomediastinum and subcutaneous emphysema at the onset of a progresive and severe pulmonary disease. Although there were no sign of parenchymal cysts, after the bronchoscopy it was possible to observe endobronchial necrotic injury which was considered as the likely source of the air leak. He was treated with high dose of corticosteroids, cyclophosphamide and cyclosporin A which resulted in the disappearance of the pneumomediastinum and subcutaneous emphysema and the progressive improvement of both parenchymal lung disease and respiratory insufficiency, enabled us to progressively taper the dose of corticosteroids.