Genetic diversity and evolutionary convergence of cryptic SARS- CoV-2 lineages detected via wastewater sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitutions. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from long-term patient infections or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population.

Etiology and the challenge of diagnostic testing of community-acquired pneumonia in children and adolescents.

BACKGROUND: Pneumonia is the leading cause of mortality in pediatric population. The etiology of pneumonia in this population is variable and changes according to age and disease severity and where the study is conducted. Our aim was to determine the etiology of community-acquired pneumonia (CAP) in children aged 1 month to 17 years admitted to 13 Colombian hospitals. METHODS: Prospective cohort study. Hospitalized children with radiologically confirmed CAP and ≤ 15 days of symptoms were included and followed together with a control group. Induced sputum (IS) was submitted for stains and cultures for pyogenic bacteria and Mycobacterium tuberculosis, and multiplex PCR (mPCR) for bacteria and viruses; urinary antigens for pneumococcus and Legionella pneumophila; nasopharyngeal swabs for viruses, and paired serology for atypical bacteria and viruses. Additional cultures were taken at the discretion of primary care pediatricians. RESULTS: Among 525 children with CAP, 71.6% had non-severe pneumonia; 24.8% severe and 3.6% very severe pneumonia, and no fatal cases. At least one microorganism was identified in 84% of children and 61% were of mixed etiology; 72% had at least one respiratory virus, 28% pyogenic bacteria and 21% atypical bacteria. Respiratory syncytial virus, Parainfluenza, Rhinovirus, Influenza, Mycoplasma pneumoniae, Adenovirus and Streptococcus pneumoniae were the most common etiologies of CAP. Respiratory syncytial virus was more frequent in children under 2 years and in severe pneumonia. Tuberculosis was diagnosed in 2.3% of children. IS was the most useful specimen to identify the etiology (33.6%), and blood cultures were positive in 3.6%. The concordance between all available diagnostic tests was low. A high percentage of healthy children were colonized by S. pneumoniae and Haemophilus influenzae, or were infected by Parainfluenza, Rhinovirus, Influenza and Adenovirus. CONCLUSIONS: Respiratory viruses are the most frequent etiology of CAP in children and adolescents, in particular in those under 5 years. This study shows the challenges in making an etiologic diagnosis of CAP in pediatric population because of the poor concordance between tests and the high percentage of multiple microorganisms in healthy children. IS is useful for CAP diagnosis in pediatric population.

Early-Onset Invasive Infection Due to Corynespora cassiicola Associated with Compound Heterozygous CARD9 Mutations in a Colombian Patient.

PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

Severe Enteropathy and Hypogammaglobulinemia Complicating Refractory Mycobacterium tuberculosis Complex Disseminated Disease in a Child with IL-12Rß1 Deficiency.

PURPOSE: Mendelian susceptibility to mycobacterial disease is a rare clinical condition characterized by a predisposition to infectious diseases caused by poorly virulent mycobacteria. Other infections such as salmonellosis and candidiasis are also reported. The purpose of this article is to describe a young boy affected with various infectious diseases caused by Mycobacterium tuberculosis complex, Salmonella sp, Klebsiella pneumonie, Citrobacter sp., and Candida sp, complicated with severe enteropathy and transient hypogammaglobulinemia. METHODS: We reviewed medical records and performed flow cytometry staining for lymphocyte populations, lymphocyte proliferation in response to PHA, and intracellular IFN-γ production in T cell PHA blasts in the patient and a healthy control. Sanger sequencing was used to confirm the genetic variants in the patient and relatives. RESULTS: Genetic analysis revealed a bi-allelic mutation in IL12RB1 (C291Y) resulting in complete IL-12Rß1 deficiency. Functional analysis demonstrated the lack of intracellular production of IFN-γ in CD3+ T lymphocytes from the patient in response to rhIL-12p70. CONCLUSIONS: To our knowledge, this is the third patient with MSMD due to IL-12Rß1 deficiency complicated with enteropathy and hypogammaglobulinemia and the first case of this disease to be described in Colombia.

Standardization of the Beck Hopelessness Scale in the general population.

BACKGROUND: The Beck Hopelessness Scale (BHS) is a self-report instrument for the quantification of hopelessness in nonpsychiatric, as well as psychiatric patients. Hopelessness is a key psychological variable in suicide prediction. Until now the psychometric properties of the instrument have not been studied in a representative sample of the general population. AIMS: The objectives of the study were to generate normative data and to further investigate the construct validity and factorial structure of the BHS. METHODS: A nationally representative face-to-face household survey was conducted in Colombia in 2012 (N = 1500). RESULTS: Cronbach's alpha coefficient for the BHS was 0.81. Confirmatory factor analysis supported a three-factor model, achieving good fit indices (total sample: RMSEA = 0.043, CFI = 0.936, TLI = 0.921). Normative data for the BHS were generated for both genders and different age levels. Intercorrelations with hopelessness were highest for depression (r = 0.57), followed by anxiety (r = 0.52). CONCLUSIONS: The normative data provide a framework for the interpretation and comparisons of the BHS with other populations. Evidence supports reliability and validity of the three-factor BHS as a measure of hopelessness in the general population.

Serotype distribution, clinical characteristics, and antimicrobial resistance of pediatric invasive pneumococcal disease in Colombia during PCV10 mass vaccination (2017-2022).

Introduction: Invasive Pneumococcal Disease (IPD) causes significant morbidity and mortality in children under 5 y. Colombia introduced PCV10 vaccination in 2012, and the Neumocolombia network has been monitoring IPD in pediatric patients since 2008. Materials and methods: This study is a secondary analysis of a prospective cohort involving pediatric patients with IPD admitted to 17 hospitals in Colombia, from January 1st, 2017, to December 31st, 2022. We present data on serotypes (Spn), clinical characteristics, and resistance patterns. Results: We report 530 patients, 215 (40.5%) were younger than 24 months. Among these, 344 cases (64.7%) presented with pneumonia, 95 (17.9%) with primary bacteremia, 53 (10%) with meningitis, 6 (1.1%) had pneumonia and meningitis, and 32 (6%) had other IPD diagnosis. The median hospital stay was 12 days (RIQ 8-14 days), and 268 (50.6%) were admitted to the ICU, of whom 60 (11.3%) died. Serotyping was performed in 298 (56.1%). The most frequent serotypes were Spn19A (51.3%), Spn6C (7.7%), Spn3 (6.7%), Spn6A (3.6%), and Spn14 (3.6%). Of 495 (93%) isolates with known susceptibility, 46 (9.2%) were meningeal (M) and 449 (90.7%) non-meningeal (NM). Among M isolates, 41.3% showed resistance to penicillin, and 21.7% decreased susceptibility to ceftriaxone. For NM isolates, 28.2% had decreased susceptibility to penicilin, and 24.2% decreased susceptibility to ceftriaxone. Spn19A showed the highest resistant to penicillin at 47% and was linked to multiresistance. Conclusion: The prevalence of PCV10-included serotypes decreased, while serotypes 19A and 6C increased, with Spn19A being associated with multiresistance. These findings had played a crucial role in the decision made by Colombia to modify its immunization schedule by switching to PCV13 in July 2022.

[Effective coverage to manage domestic violence against women in Mexican municipalities: limits of metrics]. / Cobertura efectiva del manejo de la violencia contra mujeres en municipios Mexicanos: límites de la métrica.

The study estimated the effective coverage of health services in primary care for the management of domestic violence against women in three municipalities in Mexico. We estimated the prevalence and severity of violence using a validated scale, and the effective coverage proposed by Shengelia and partners with any modifications. Quality care was considered when there was a suggestion to report it to authorities. The use and quality of care was low in the three municipalities analyzed, used most frequently when there was sexual or physical violence. Effective coverage was 29.41%, 16.67% and zero in Guachochi, Jojutla and Tizimín, respectively. The effective coverage indicator had difficulties in measuring events and responses that were not based on biomedical models. Findings suggest that the indicator can be improved by incorporating other dimensions of quality.

Developing Vaccine Literacy for Urban Health Science Students, the Future Health Workforce.

The 2019 coronavirus disease pandemic underlined a shift in attitudes against vaccines and a rise in hesitancy among some members of the population, despite the overwhelming evidence that vaccinations are one of the most successful and safe health interventions. Research has shown that vaccine hesitancy is complex and can result from an intersectionality of multiple factors. Research has also shown that to tackle vaccine hesitancy in the community, health care workers play a pivotal role, as they are trusted sources who can provide reliable information and can address vaccination concerns for the public. Unfortunately, health care workers are also susceptible to vaccine hesitancy. Thus, to curb these negative attitudes and doubts against vaccinations, we propose to improve vaccine competency among health science students, who are the future health workforce. Here, we propose a comprehensive pedagogical approach that aims to improve the vaccine literacy in this student population in two urban community colleges. The approach includes the use of high-impact pedagogical interventions to achieve three main objectives: (i) to teach students the nature and process of science to have them become "competent outsiders"; (ii) to enhance students' knowledge of the complex science behind emerging infectious diseases and vaccine action, adopting a learner-centered and concept-focused instructional design, and (iii) to address the social, cultural, and historical aspects of vaccine development and the historical and present inequities that characterize this health intervention.

Experience of the use of lock therapy in pediatric patients with central venous catheter. / Experiencia del uso de terapia de sello en pacientes pediátricos con catéter venoso central.

Lock therapy is useful for preserving indwelling catheters. Few lock therapy studies have been published in Latin America. OBJECTIVE: To describe the clinical characteristics of pediatric patients using therapeutic and prophylactic lock therapy for six years in a high-complexity hospital in Colombia. PATIENTS AND METHOD: Cross-sectional descriptive study of patients aged < 18 years who received lock therapy. Collected variables included demographic data, clinical characteristics, blood test results, therapeutic interventions, frequency of admission to the pediatric critical care unit, and mortality. Descriptive analysis was performed. RESULTS: 54 patients were included in the study, most of them males, with 67 episodes of therapeutic lock therapy use. The most frequent diagnosis was hematological neoplasm (61%). Among these patients, 88% presented neutropenia while receiving lock therapy. Catheter preservation was achieved in 75% of the cases. Aminoglycosides were the most commonly used antibiotics (38%). Mortality due to catheter-related bacteremia was 6%. Catheter preservation using ethanol solution 70% was achieved in 62% of the patients with prophylactic lock therapy, all of whom had chronic gastrointestinal pathology. CONCLUSION: Catheter preservation rates were 75% and 62% in patients with therapeutic and prophylactic lock therapy, respectively, with a higher rate achieved among cancer patients with neutropenia (80%). Aminoglycosides and vancomycin were the most commonly used antibiotics.

Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

Disseminated fusariosis in children: Report of two cases in girls with leukemia.

Background and Purpose: Disseminated fusariosis is an opportunistic infection caused by the hyaline fungus Fusarium spp. and occurs mainly in patients with leukemia. Case report: Two cases of disseminated fusariosis in pediatric patients are presented. Profound and prolonged neutropenia, fever, myalgia, and skin lesions in the legs were present in two girls with leukemia undergoing chemotherapy. In the first case, infection by Fusarium spp. was confirmed by anatomopathological findings, pathogen isolation, and polymerase chain reaction. In the second case, Fusarium solani infection was confirmed by mass spectrometry using blood cultures and skin lesion samples. Conclusion: It is important to consider disseminated fusariosis in high-risk patients who present with profound and prolonged neutropenia and persistent fever that does not resolve after broad-spectrum antibiotics to initiate antifungal therapy in a timely manner.

Acute Intrathoracic Tuberculosis in Children and Adolescents with Community-Acquired Pneumonia in an Area with an Intermediate Disease Burden.

Tuberculosis (TB) in the pediatric population is a major challenge. Our objective was to describe the clinical and microbiological characteristics, radiological patterns, and treatment outcomes of children and adolescents (from 1 month to 17 years) with community-acquired pneumonia (CAP) caused by TB. We performed a prospective cohort study of a pediatric population between 1 month and 17 years of age and hospitalized in Medellín, Colombia, with the diagnosis of radiologically confirmed CAP that had ≤ 15 days of symptoms. The mycobacterial culture of induced sputum was used for the bacteriological confirmation; the history of TB contact, a tuberculin skin test, and clinical improvement with treatment were used to identify microbiologically negative TB cases. Among 499 children with CAP, TB was diagnosed in 12 (2.4%), of which 10 had less than 8 days of a cough, 10 had alveolar opacities, 9 were younger than 5 years old, and 2 had close contact with a TB patient. Among the TB cases, 50% (6) had microbiological confirmation, 8 had viral and/or bacterial confirmation, one patient had multidrug-resistant TB, and 10/12 had non-severe pneumonia. In countries with an intermediate TB burden, Mycobacterium tuberculosis should be included in the etiological differential diagnosis (as a cause or coinfection) of both pneumonia and severe CAP in the pediatric population.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing.

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author Summary: During the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

Induced sputum as an adequate clinical specimen for the etiological diagnosis of community-acquired pneumonia (CAP) in children and adolescents.

OBJECTIVES: This study aimed to evaluate the utility of induced sputum (IS) for the diagnosis of community-acquired pneumonia (CAP) in pediatric population. METHODS: This cross-sectional study included pediatric population aged between 1 month and 17 years who were hospitalized with a diagnosis of CAP in 13 hospitals in Colombia, in whom an IS sample was obtained. Gram staining, aerobic bacterial and mycobacterial culture tests, and polymerase chain reaction (PCR) for 6 atypical bacteria and 15 respiratory viruses were performed. We evaluated the quality of IS samples. RESULTS: IS samples were collected in 516 of 525 children included in this study. The median age was 32 months, 38.6% were younger than 2 years, and 40.9% were between 2 and 5 years. Two patients had transient hypoxemia during the procedure. The quality of the IS obtained was good in 48.4% and intermediate in 24.5%. Identification of a respiratory pathogen was achieved with an IS sample (with Gram staining, culture test, and PCR) in 372 of 516 children with CAP. CONCLUSION: Our study shows that IS is an adequate sample for the diagnosis of CAP in pediatric population that required hospitalization. The procedure was safe, well tolerated, and with better diagnostic yields compared with the rest of the samples obtained.

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

Changes in the incidence of acute bacterial meningitis caused by Streptococcus pneumoniae and the implications of serotype replacement in children in Colombia after mass vaccination with PCV10.

Introduction: Acute bacterial meningitis (ABM) is a public health problem. The disease has reemerged after the introduction of pneumococcal conjugate vaccines (PCVs) due to an increase in serotypes that are not covered. The objective was to determine the changes in the disease incidence before and after the introduction of the 10-valent vaccine (PCV10) in Colombia. Methods: This multicenter study was conducted in 17 hospitals in Colombia. Data were collected from January 2008 to December 2019 in 10 hospitals in Bogotá and from January 2017 to December 2019 in seven hospitals in Cali, Medellín and Cartagena. The data were grouped into three periods: 2008-2011, 2012-2015, and 2016-2019. Results: Of the 706 cases of invasive pneumococcal disease, 81 (11.4%) corresponded to meningitis. The relative incidence in Bogotá in the first period was 0.6 per 100,000 patients ≤ 5 years, decreased to 0.4 per 100,000 patients ≤ 5 years in the second period and increased in the third period to 0.7 per 100,000 patients ≤ 5 years. Serotypes covered by PCV10 decreased from 75 to 9.1%, with Spn19A (31.8%) and Spn34 (13.6%) emerging in the third period. Increased resistance to penicillin (13 to 37%) and to ceftriaxone (5.9 to 16%) was due to the emergence of multidrug-resistant Spn19A. The total mortality rate was 23.5% and increased from 12 to 33%. Conclusions: ABM due to pneumococcus has high morbidity and mortality rates. Reemergence of the disease has been observed due to the inclusion of polymerase chain reaction (PCR) for diagnosis and replacement of circulating serotypes after the introduction of PCV10, with an increase in Spn19A, which causes death and exhibits antimicrobial resistance. Continued surveillance is needed.

17-Hydroxycyclooctatin, a fused 5-8-5 ring diterpene, from Streptomyces sp. MTE4a.

A new diterpene with a fused 5-8-5 ring system was isolated from the fermentation broth of a soil actinomycete. The stereochemistry at C-15 was determined in an unusual manner using a decomposition product.

Why Do Antibiotics Exist?

In the struggle with antibiotic resistance, we are losing. There is now a serious threat of moving into a postantibiotic world. High levels of resistance, in terms of both frequency and strength, have evolved against all clinically approved antibiotics worldwide. The usable life span of new clinically approved antibiotics is typically less than a decade before resistance reaches frequencies so high as to require only guarded usage. However, microbes have produced antibiotics for millennia without resistance becoming an existential issue. If resistance is the inevitable consequence of antibiotic usage, as has been the human experience, why has it not become an issue for microbes as well, especially since resistance genes are as prevalent in nature as the genes responsible for antibiotic production? Here, we ask how antibiotics can exist given the almost ubiquitous presence of resistance genes in the very microbes that have produced and used antibiotics since before humans walked the planet. We find that the context of both production and usage of antibiotics by microbes may be key to understanding how resistance is managed over time, with antibiotic synthesis and resistance existing in a paired relationship, much like a cipher and key, that impacts microbial community assembly. Finally, we put forward the cohesive, ecologically based "secret society" hypothesis to explain the longevity of antibiotics in nature.

Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater.

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.