RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

Nuclear compartmentalization as a mechanism of quantitative control of gene expression.

Gene regulation requires the dynamic coordination of hundreds of regulatory factors at precise genomic and RNA targets. Although many regulatory factors have specific affinity for their nucleic acid targets, molecular diffusion and affinity models alone cannot explain many of the quantitative features of gene regulation in the nucleus. One emerging explanation for these quantitative properties is that DNA, RNA and proteins organize within precise, 3D compartments in the nucleus to concentrate groups of functionally related molecules. Recently, nucleic acids and proteins involved in many important nuclear processes have been shown to engage in cooperative interactions, which lead to the formation of condensates that partition the nucleus. In this Review, we discuss an emerging perspective of gene regulation, which moves away from classic models of stoichiometric interactions towards an understanding of how spatial compartmentalization can lead to non-stoichiometric molecular interactions and non-linear regulatory behaviours. We describe key mechanisms of nuclear compartment formation, including emerging roles for non-coding RNAs in facilitating their formation, and discuss the functional role of nuclear compartments in transcription regulation, co-transcriptional and post-transcriptional RNA processing, and higher-order chromatin regulation. More generally, we discuss how compartmentalization may explain important quantitative aspects of gene regulation.

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus.

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.

Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

India Field Epidemiology Training Program Response to COVID-19 Pandemic, 2020-2021.

The India Field Epidemiology Training Program (FETP) has played a critical role in India's response to the ongoing COVID-19 pandemic. During March 2020-June 2021, a total of 123 FETP officers from across 3 training hubs were deployed in support of India's efforts to combat COVID-19. FETP officers have successfully mitigated the effect of COVID-19 on persons in India by conducting cluster outbreak investigations, performing surveillance system evaluations, and developing infection prevention and control tools and guidelines. This report discusses the successes of select COVID-19 pandemic response activities undertaken by current India FETP officers and proposes a pathway to augmenting India's pandemic preparedness and response efforts through expansion of this network and a strengthened frontline public health workforce.

Comprehensive immune cell profiling depicts an early immune response associated with severe coronavirus disease 2019 in cancer patients.

Recent studies have highlighted multiple immune perturbations related to severe acute respiratory syndrome coronavirus 2 infection-associated respiratory disease [coronavirus disease 2019 (COVID-19)]. Some of them were associated with immunopathogenesis of severe COVID-19. However, reports on immunological indicators of severe COVID-19 in the early phase of infection in patients with comorbidities such as cancer are scarce. We prospectively studied about 200 immune response parameters, including a comprehensive immune-cell profile, inflammatory cytokines and other parameters, in 95 patients with COVID-19 (37 cancer patients without active disease and intensive chemo/immunotherapy, 58 patients without cancer) and 21 healthy donors. Of 95 patients, 41 had severe disease, and the remaining 54 were categorized as having a nonsevere disease. We evaluated the association of immune response parameters with severe COVID-19. By principal component analysis, three immune signatures defining characteristic immune responses in COVID-19 patients were found. Immune cell perturbations, in particular, decreased levels of circulating dendritic cells (DCs) along with reduced levels of CD4 T-cell subsets such as regulatory T cells (Tregs ), type 1 T helper (Th1) and Th9; additionally, relative expansion of effector natural killer (NK) cells were significantly associated with severe COVID-19. Compared with patients without cancer, the levels of terminal effector CD4 T cells, Tregs , Th9, effector NK cells, B cells, intermediate-type monocytes and myeloid DCs were significantly lower in cancer patients with mild and severe COVID-19. We concluded that severely depleted circulating myeloid DCs and helper T subsets in the initial phase of infection were strongly associated with severe COVID-19 independent of age, type of comorbidity and other parameters. Thus, our study describes the early immune response associated with severe COVID-19 in cancer patients without intensive chemo/immunotherapy.

Genomic Analysis of AZD1222 (ChAdOx1) Vaccine Breakthrough Infections in the City of Mumbai.

Background: This manuscript describes the genetic features of SARS-CoV-2 mutations, prevalent phylogenetic lineages, and the disease severity amongst COVID-19-vaccinated individuals in a tertiary cancer hospital during the second wave of the pandemic in Mumbai, India. Methods: This observational study included 159 COVID-19 patients during the second wave of the pandemic from 17th March to 1st June 2021 at a tertiary cancer care centre in Mumbai. The cohort comprised of healthcare workers, staff relatives, cancer patients, and patient relatives. For comparison, 700 SARS-CoV-2 genomes sequenced during the first wave (23rd April to 25th September 2020) at the same centre were also analysed. Patients were assigned to nonvaccinated (no vaccination or <14 days from the 1st dose, n = 92), dose 1(≥14 days from the 1st dose to <14 days from the 2nd dose, n = 29), and dose 2 (≥14 days from the 2nd dose, n = 38) groups. Primary measure was the prevalence of SARS-CoV-2 genomic lineages among different groups. In addition, severity of COVID-19 was assessed according to clinical and genomic variables. Results: Kappa B.1.1671.1 and delta B.1.617.2 variants contributed to an overwhelming majority of sequenced genomes (unvaccinated: 40/92, 43.5% kappa, 46/92, 50% delta; dose 1: 14/29, 48.3% kappa, 15/29, 51.7% delta; and dose 2: 23/38, 60.5% kappa, 14/38 36.8% delta). The proportion of the kappa and delta variants did not differ significantly across the unvaccinated, dose 1, and dose 2 groups (p = 0.27). There was no occurrence of severe COVID-19 in the dose 2 group (0/38, 0% vs. 14/121, 11.6%; p = 0.02). SARS-CoV-2 genomes from all three severe COVID-19 patients in the vaccinated group belonged to the delta lineage (3/28, 10.7% vs. 0/39, 0.0%, p = 0.04). Conclusions: Sequencing analysis of SARS-COV-2 genomes from Mumbai during the second wave of COVID-19 suggests the prevalence of the kappa B.1.617.1 and the delta B.1.627.2 variants among both vaccinated and unvaccinated individuals. Continued evaluation of genomic sequencing data from breakthrough COVID-19 is necessary for monitoring the properties of evolving variants of concern and formulating appropriate immune response boosting and therapeutic strategies.

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Isoniazid Preventive Therapy among Children Living with Tuberculosis Patients: Is It Working? A Mixed-Method Study from Bhopal, India.

Objective: We assessed uptake of isoniazid preventive therapy (IPT) among child contacts of smear-positive tuberculosis (TB) patients and its implementation challenges from healthcare providers' and parents' perspectives in Bhopal, India. Methods: A mixed-method study design: quantitative phase (review of programme records and house-to-house survey of smear-positive TB patients) followed by qualitative phase (interviews of healthcare providers and parents). Results: Of 59 child contacts (<6 years) of 129 index patients, 51 were contacted. Among them, 19 of 51 (37%) were screened for TB and one had TB. Only 11 of 50 (22%) children were started and 10 of 50 (20%) completed IPT. Content analysis of interviews revealed lack of awareness, risk perception among parents, cumbersome screening process, isoniazid stock-outs, inadequate knowledge among healthcare providers and poor programmatic monitoring as main barriers to IPT implementation. Conclusion: National TB programme should counsel parents, train healthcare providers, simplify screening procedures, ensure regular drug supply and introduce an indicator to strengthen monitoring and uptake of IPT.

A first country-wide review of Diabetes Mellitus care in Bhutan: time to do better.

BACKGROUND: There is an increasing trend of non-communicable diseases in Bhutan including Diabetes Mellitus (DM). To address this problem, a National Diabetes Control Programme was launched in 1996. There is anecdotal evidence that many patients do not visit the DM clinics regularly, but owing to lack of cohort monitoring, the magnitude of such attrition from care is unknown. Knowledge of the extent of this problem will provide a realistic assessment of the situation on the ground and would be helpful to initiate corrective actions. In this first country-wide audit, we thus aimed to determine among type 2 DM patients registered for care the i) pre-treatment attrition ii) one-year programme outcomes including retention in care, died and Lost-to-follow-up (LTFU, defined as not having visited the clinic at least once within a year of registration) iii) factors associated with attrition from care (death + LTFU) and iv) quality of follow-up care, measured by adherence to recommended patient-monitoring protocols including glycaemic control. METHODS: A retrospective cohort study involving a review of records routinely maintained under the National Diabetes Control Programme. All type 2 DM patients registered between 1st January and 31st December 2012 in 18 district hospitals of Bhutan were included. Glycaemic control was defined as glycosylated haemoglobin of <7% or [Fasting Blood Sugar of <130 mg/dl and, Post-prandial Blood Sugar of <180 mg/dl]. RESULTS: Of 350 registered DM patients (52% female, median age 55 years), 63(18%) were LTFU before treatment initiation (pre-treatment attrition). Of the remaining 287 individuals who started treatment, 226(79%) were retained in care while 61(21%) either died or were LTFU. Glycaemic control was achieved in 85(38%) patients retained in care. Between 7 and 98% of monitoring parameters had missing data. CONCLUSION: Nearly one-third of DM patients were LTFU and there were short comings in monitoring. Qualitative research is urgently needed to find out the reasons for high attrition. Given the high political commitment by the Royal Government of Bhutan, the findings provide ample grounds for instituting corrective measures and propelling DM care further. It is time to do better!

3D genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized such that factors involved in transcription and processing of distinct classes of RNA are organized within specific nuclear bodies. One such nuclear body is the nuclear speckle, which is defined by high concentrations of protein and non-coding RNA regulators of pre-mRNA splicing. What functional role, if any, speckles might play in the process of mRNA splicing remains unknown. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs, and higher co-transcriptional splicing levels relative to genes that are located farther from nuclear speckles. We show that directed recruitment of a pre-mRNA to nuclear speckles is sufficient to drive increased mRNA splicing levels. Finally, we show that gene organization around nuclear speckles is highly dynamic with differential localization between cell types corresponding to differences in Pol II occupancy. Together, our results integrate the longstanding observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a critical role for dynamic 3D spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

The Potential of CRISPR-Guided Therapies in the Dermatology Clinic.

Over the past decade, CRISPR has rapidly made its way from the bench to the bedside, providing a newfound therapeutic avenue to not only treat genetic diseases but also permanently cure them. Although there are several clinical trials in early stages, there are so far no CRISPR-based clinical trials for cutaneous disease. In this review, we describe multiple cutaneous diseases that represent ideal targets for CRISPR-based therapeutics owing to known single gene‒causing mutations. We also explore the potential of CRISPR nucleases to treat inflammatory disorders such as eczema and psoriasis, which are not classically categorized as genodermatoses. We describe the therapeutic solutions for these diseases that are guided by various CRISPR-associated (Cas) effector proteins, for example, using Cas9 to permanently edit the DNA of somatic cells, Cas3 to target foreign DNA to combat viral/bacterial skin infections, and Cas13 to edit mutated RNA transcripts in diseases where permanent DNA editing is untenable. Furthermore, we discuss various drug delivery modalities for CRISPR therapeutics, including transdermal patches and microneedles, which are uniquely suited for dermatological diseases. In summary, we highlight the potential of CRISPR-based therapeutics to revolutionize the treatment of cutaneous disease with a goal of being accessible to the practicing dermatologist.

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

SPRITE: a genome-wide method for mapping higher-order 3D interactions in the nucleus using combinatorial split-and-pool barcoding.

A fundamental question in gene regulation is how cell-type-specific gene expression is influenced by the packaging of DNA within the nucleus of each cell. We recently developed Split-Pool Recognition of Interactions by Tag Extension (SPRITE), which enables mapping of higher-order interactions within the nucleus. SPRITE works by cross-linking interacting DNA, RNA and protein molecules and then mapping DNA-DNA spatial arrangements through an iterative split-and-pool barcoding method. All DNA molecules within a cross-linked complex are barcoded by repeatedly splitting complexes across a 96-well plate, ligating molecules with a unique tag sequence, and pooling all complexes into a single well before repeating the tagging. Because all molecules in a cross-linked complex are covalently attached, they will sort together throughout each round of split-and-pool and will obtain the same series of SPRITE tags, which we refer to as a barcode. The DNA fragments and their associated barcodes are sequenced, and all reads sharing identical barcodes are matched to reconstruct interactions. SPRITE accurately maps pairwise DNA interactions within the nucleus and measures higher-order spatial contacts occurring among up to thousands of simultaneously interacting molecules. Here, we provide a detailed protocol for the experimental steps of SPRITE, including a video ( https://youtu.be/6SdWkBxQGlg ). Furthermore, we provide an automated computational pipeline available on GitHub that allows experimenters to seamlessly generate SPRITE interaction matrices starting with raw fastq files. The protocol takes ~5 d from cell cross-linking to high-throughput sequencing for the experimental steps and 1 d for data processing.

Sudden deaths due to accidental leakage of Lindane from a storage tank in a village, Sitapur, Uttar Pradesh, India, 2020: A field epidemiological investigation.

Background: Chemical leakages cause devastating health effects on humans. On 6 February 2020, seven deaths were reported following a hazardous chemical leakage in a village in Uttar Pradesh, India. We investigated the event to identify the cause and propose recommendations. Methods: We defined a case as sudden onset of breathlessness, headache, or death in the village, 6-7 February 2020. We conducted a house-to-house case search and calculated attack rate (AR) and case-fatality rate (CFR) by age and gender. We conducted an environmental investigation at the leakage site and sent the chemicals for forensic analysis. We obtained the cause of death through autopsy reports. Results: Out of 2,942 residents, we identified 23 cases (AR = 8/1,000) and seven deaths (CFR = 30%). The median age of the case was 42 years (range, 2-64 years). The AR was higher among males (14/1,000 [19/1,402]). All the 23 case-patients who were sleeping at the chemical leakage site or visited to witness the event developed symptoms, and all seven cases who were sleeping within 150 meters of the leakage site died. The environmental investigation revealed leakage of hazardous substances from the storage tank. Toxicology analysis confirmed the leaked chemical as Lindane (gamma-hexachlorocyclohexane), and autopsy reports confirmed the cause of death as asphyxia. Conclusions: Asphyxia following the leakage of Lindane from the storage tank possibly led to sudden deaths. We recommend using leak-proof tanks to ensure safe storage and disposal, law enforcement, and regulations to prevent people from staying close to chemical storage sites.

Interplay Between Duration of Androgen Deprivation Therapy and External Beam Radiotherapy With or Without a Brachytherapy Boost for Optimal Treatment of High-risk Prostate Cancer: A Patient-Level Data Analysis of 3 Cohorts.

IMPORTANCE: Radiotherapy combined with androgen deprivation therapy (ADT) is a standard of care for high-risk prostate cancer. However, the interplay between radiotherapy dose and the required minimum duration of ADT is uncertain. OBJECTIVE: To determine the specific ADT duration threshold that provides a distant metastasis-free survival (DMFS) benefit in patients with high-risk prostate cancer receiving external beam radiotherapy (EBRT) or EBRT with a brachytherapy boost (EBRT+BT). DESIGN, SETTINGS, AND PARTICIPANTS: This was a cohort study of 3 cohorts assembled from a multicenter retrospective study (2000-2013); a post hoc analysis of the Randomized Androgen Deprivation and Radiotherapy 03/04 (RADAR; 2003-2007) randomized clinical trial (RCT); and a cross-trial comparison of the RADAR vs the Deprivación Androgénica y Radio Terapía (Androgen Deprivation and Radiation Therapy; DART) 01/05 RCT (2005-2010). In all, the study analyzed 1827 patients treated with EBRT and 1108 patients treated with EBRT+BT from the retrospective cohort; 181 treated with EBRT and 203 with EBRT+BT from RADAR; and 91 patients treated with EBRT from DART. The study was conducted from October 15, 2020, to July 1, 2021, and the data analyses, from January 5 to June 15, 2021. EXPOSURES: High-dose EBRT or EBRT+BT for an ADT duration determined by patient-physician choice (retrospective) or by randomization (RCTs). MAIN OUTCOMES AND MEASURES: The primary outcome was DMFS; secondary outcome was overall survival (OS). Natural cubic spline analysis identified minimum thresholds (months). RESULTS: This cohort study of 3 studies totaling 3410 men (mean age [SD], 68 [62-74] years; race and ethnicity not collected) with high-risk prostate cancer found a significant interaction between the treatment type (EBRT vs EBRT+BT) and ADT duration (binned to <6, 6 to <18, and ≥18 months). Natural cubic spline analysis identified minimum duration thresholds of 26.3 months (95% CI, 25.4-36.0 months) for EBRT and 12 months (95% CI, 4.9-36.0 months) for EBRT+BT for optimal effect on DMFS. In RADAR, the prolongation of ADT for patients receiving only EBRT was not associated with significant improvements in DMFS (hazard ratio [HR], 1.01; 95% CI, 0.65-1.57); however, for patients receiving EBRT+BT, a longer duration was associated with improved DMFS (DMFS HR, 0.56; 95% CI, 0.36-0.87; P = .01). For patients receiving EBRT alone (DART), 28 months of ADT was associated with improved DMFS compared with 18 months (RADAR HR, 0.37; 95% CI, 0.17-0.80; P = .01). CONCLUSIONS AND RELEVANCE: These cohort study findings suggest that the optimal minimum ADT duration for treatment with high-dose EBRT alone is more than 18 months; and for EBRT+BT, it is 18 months or possibly less. Additional studies are needed to determine more precise minimum durations.

Kyasanur Forest Disease, is our surveillance system healthy to prevent a larger outbreak? A mixed-method study, Shivamogga, Karnataka, India: 2019.

BACKGROUND: Kyasanur Forest disease (KFD) is a tick-borne zoonosis that is endemic in Karnataka. Against the backdrop of the recent geographical expansion of KFD, indicating the inadequacy of policy and surveillance systems, the present study was performed to evaluate the KFD surveillance system in Shivamogga. METHODS: US Centers for Disease Control and Prevention guidelines for surveillance system evaluation were followed. Nine attributes of the system towards its objectives were evaluated in a mixed study in Shivamogga. RESULTS: Two of three medical officers and four of six health staff at the institutions visited were found to be untrained in KFD surveillance. Integrated disease surveillance formats did not capture KFD data. Surveillance (tick, monkey, human) was mostly driven by the Health Department. Some of the critical findings of the evaluations were the absence of an animal and entomological surveillance line list, non-standardized reporting formats for human suspects, varying delays in the time-to-test across laboratories (2-16 days), and a lack of systematic data-sharing practices. Significant issues that emerged in the interview were deforestation with a change in ecosystem dynamics, limited diagnostic capacity, non-availability of point-of-care tests, outdated surveillance guidelines, a confusing surveillance perimeter (5 km), non-existing co-ownership among stakeholders, limited vaccine production capacity, and inadequate operational research. CONCLUSIONS: The system should consider integrating a One Health approach with defined ownership of activities among stakeholders. Revision of the guidelines is mandatory.

Performance of a Prostate-Specific Membrane Antigen Positron Emission Tomography/Computed Tomography-Derived Risk-Stratification Tool for High-risk and Very High-risk Prostate Cancer.

Importance: Prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography (PET/CT) can detect low-volume, nonlocalized (ie, regional or metastatic) prostate cancer that was occult on conventional imaging. However, the long-term clinical implications of PSMA PET/CT upstaging remain unclear. Objectives: To evaluate the prognostic significance of a nomogram that models an individual's risk of nonlocalized upstaging on PSMA PET/CT and to compare its performance with existing risk-stratification tools. Design, Setting, and Participants: This cohort study included patients diagnosed with high-risk or very high-risk prostate cancer (ie, prostate-specific antigen [PSA] level >20 ng/mL, Gleason score 8-10, and/or clinical stage T3-T4, without evidence of nodal or metastatic disease by conventional workup) from April 1995 to August 2018. This multinational study was conducted at 15 centers. Data were analyzed from December 2020 to March 2021. Exposures: Curative-intent radical prostatectomy (RP), external beam radiotherapy (EBRT), or EBRT plus brachytherapy (BT), with or without androgen deprivation therapy. Main Outcomes and Measures: PSMA upstage probability was calculated from a nomogram using the biopsy Gleason score, percentage positive systematic biopsy cores, clinical T category, and PSA level. Biochemical recurrence (BCR), distant metastasis (DM), prostate cancer-specific mortality (PCSM), and overall survival (OS) were analyzed using Fine-Gray and Cox regressions. Model performance was quantified with the concordance (C) index. Results: Of 5275 patients, the median (IQR) age was 66 (60-72) years; 2883 (55%) were treated with RP, 1669 (32%) with EBRT, and 723 (14%) with EBRT plus BT; median (IQR) PSA level was 10.5 (5.9-23.2) ng/mL; 3987 (76%) had Gleason grade 8 to 10 disease; and 750 (14%) had stage T3 to T4 disease. Median (IQR) follow-up was 5.1 (3.1-7.9) years; 1221 (23%) were followed up for at least 8 years. Overall, 1895 (36%) had BCR, 851 (16%) developed DM, and 242 (5%) died of prostate cancer. PSMA upstage probability was significantly prognostic of all clinical end points, with 8-year C indices of 0.63 (95% CI, 0.61-0.65) for BCR, 0.69 (95% CI, 0.66-0.71) for DM, 0.71 (95% CI, 0.67-0.75) for PCSM, and 0.60 (95% CI, 0.57-0.62) for PCSM (P < .001). The PSMA nomogram outperformed existing risk-stratification tools, except for similar performance to Staging Collaboration for Cancer of the Prostate (STAR-CAP) for PCSM (eg, DM: PSMA, 0.69 [95% CI, 0.66-0.71] vs STAR-CAP, 0.65 [95% CI, 0.62-0.68]; P < .001; Memorial Sloan Kettering Cancer Center nomogram, 0.57 [95% CI, 0.54-0.60]; P < .001; Cancer of the Prostate Risk Assessment groups, 0.53 [95% CI, 0.51-0.56]; P < .001). Results were validated in secondary cohorts from the Surveillance, Epidemiology, and End Results database and the National Cancer Database. Conclusions and Relevance: These findings suggest that PSMA upstage probability is associated with long-term, clinically meaningful end points. Furthermore, PSMA upstaging had superior risk discrimination compared with existing tools. Formerly occult, PSMA PET/CT-detectable nonlocalized disease may be the main driver of outcomes in high-risk patients.