Environmental drivers of phytoplankton community dynamics in an agriculturally-influenced tributary in the lower Great Lakes.

Phytoplankton community composition in tributaries differs from that in their receiving waters, due to light limitation from suspended particles and other factors such as nutrient availability and temperature. This study was designed to manipulate light levels in early, mid, and late summer to determine the combined effects of light attenuation and naturally varying nutrient availability on phytoplankton community composition in an agriculturally-influenced tributary of the lower Great Lakes. In all trials, in situ microcosm experiments show that phytoplankton abundance increased under three light attenuation treatments (60 %, 75 %, and 85 % attenuation) relative to time-zero, but higher light attenuation reduced total phytoplankton abundance relative to controls. Highest phytoplankton diversity in terms of richness and evenness occurred in September (late summer), and across all three trials was lowest under the highest light attenuation treatments (85 %). Phytoplankton community composition followed a normal seasonal shift from diatoms dominating in June (early summer), followed by cyanobacteria dominating in mid to late summer. In general, lower light levels (especially 85 % attenuation) corresponded with an increased dominance of cyanobacteria. These findings support the hypothesis that phytoplankton abundance and diversity vary with light and nutrient availability and that light attenuation promotes the shift from buoyant cyanobacteria to other taxa more tolerant of low light levels.

Low-dose naltrexone for post-COVID fatigue syndrome: a study protocol for a double-blind, randomised trial in British Columbia.

INTRODUCTION: A significant proportion of individuals suffering from post COVID-19 condition (PCC, also known as long COVID) can present with persistent, disabling fatigue similar to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and post-viral fatigue syndromes. There remains no clear pharmacological therapy for patients with this subtype of PCC, which can be referred to as post-COVID fatigue syndrome (PCFS). A low dose of the opioid antagonist naltrexone (ie, low-dose naltrexone (LDN)) has emerged as an off-label treatment for treating fatigue and other symptoms in PCC. However, only small, non-controlled studies have assessed LDN in PCC, so randomised trials are urgently required. METHODS AND ANALYSIS: A prospective, randomised, double-blind, parallel arm, placebo-controlled phase II trial will be performed to assess the efficacy of LDN for improving fatigue in PCFS. The trial will be decentralised and open to eligible individuals throughout the Canadian province of British Columbia (BC). Participants will be recruited through the province-wide Post-COVID-19 Interdisciplinary Clinical Care Network (PC-ICCN) and research volunteer platform (REACH BC). Eligible participants will be 19-69 years old, have had a confirmed or physician-suspected SARS-CoV-2 infection at least 3 months prior and meet clinical criteria for PCFS adapted from the Institute of Medicine ME/CFS criteria. Individuals who are taking opioid medications, have a history of ME/CFS prior to COVID-19 or history of significant liver disease will be excluded. Participants will be randomised to an LDN intervention arm (n=80) or placebo arm (n=80). Participants in each arm will be prescribed identical capsules starting at 1 mg daily and follow a prespecified schedule for up-titration to 4.5 mg daily or the maximum tolerated dose. The trial will be conducted over 16 weeks, with assessments at baseline, 6, 12 and 16 weeks. The primary outcome will be fatigue severity at 16 weeks evaluated by the Fatigue Severity Scale. Secondary outcomes will include pain Visual Analogue Scale score, overall symptom severity as measured by the Patient Phenotyping Questionnaire Short Form, 7-day step count and health-related quality of life measured by the EuroQol 5-Dimension questionnaire. ETHICS AND DISSEMINATION: The trial has been authorised by Health Canada and approved by The University of British Columbia/Children's and Women's Health Centre of British Columbia Research Ethics Board. On completion, findings will be disseminated to patients, caregivers and clinicians through engagement activities within existing PCC and ME/CFS networks. Results will be published in academic journals and presented at conferences. TRIAL REGISTRATION NUMBER: NCT05430152.

Evaluating and optimizing Acid-pH and Direct Lysis RNA extraction for SARS-CoV-2 RNA detection in whole saliva.

COVID-19 has been a global public health and economic challenge. Screening for the SARS-CoV-2 virus has been a key part of disease mitigation while the world continues to move forward, and lessons learned will benefit disease detection beyond COVID-19. Saliva specimen collection offers a less invasive, time- and cost-effective alternative to standard nasopharyngeal swabs. We optimized two different methods of saliva sample processing for RT-qPCR testing. Two methods were optimized to provide two cost-efficient ways to do testing for a minimum of four samples by pooling in a 2.0 mL tube and decrease the need for more highly trained personnel. Acid-pH-based RNA extraction method can be done without the need for expensive kits. Direct Lysis is a quick one-step reaction that can be applied quickly. Our optimized Acid-pH and Direct Lysis protocols are reliable and reproducible, detecting the beta-2 microglobulin (B2M) mRNA in saliva as an internal control from 97 to 96.7% of samples, respectively. The cycle threshold (Ct) values for B2M were significantly higher in the Direct Lysis protocol than in the Acid-pH protocol. The limit of detection for N1 gene was higher in Direct Lysis at ≤ 5 copies/µL than Acid-pH. Saliva samples collected over the course of several days from two COVID-positive individuals demonstrated Ct values for N1 that were consistently higher from Direct Lysis compared to Acid-pH. Collectively, this work supports that each of these techniques can be used to screen for SARS-CoV-2 in saliva for a cost-effective screening platform.

Lake Erie ice is a repository of organisms.

Organism abundance and diversity were assessed in Lake Erie ice samples using sequences derived from a combined metagenomic and metatranscriptomic analysis. The 68,417 unique sequences were from Bacteria (77.5%), Eukarya (22.3%), and Archaea (0.2%) and indicated diverse species of organisms from 32 bacterial, 8 eukaryotic, and 2 archaeal taxonomic groups.

Declines in ice cover are accompanied by light limitation responses and community change in freshwater diatoms.

The rediscovery of diatom blooms embedded within and beneath the Lake Erie ice cover (2007-2012) ignited interest in psychrophilic adaptations and winter limnology. Subsequent studies determined the vital role ice plays in winter diatom ecophysiology as diatoms partition to the underside of ice, thereby fixing their location within the photic zone. Yet, climate change has led to widespread ice decline across the Great Lakes, with Lake Erie presenting a nearly "ice-free" state in several recent winters. It has been hypothesized that the resultant turbid, isothermal water column induces light limitation amongst winter diatoms and thus serves as a competitive disadvantage. To investigate this hypothesis, we conducted a physiochemical and metatranscriptomic survey that spanned spatial, temporal, and climatic gradients of the winter Lake Erie water column (2019-2020). Our results suggest that ice-free conditions decreased planktonic diatom bloom magnitude and altered diatom community composition. Diatoms increased their expression of various photosynthetic genes and iron transporters, which suggests that the diatoms are attempting to increase their quantity of photosystems and light-harvesting components (a well-defined indicator of light limitation). We identified two gene families which serve to increase diatom fitness in the turbid ice-free water column: proton-pumping rhodopsins (a potential second means of light-driven energy acquisition) and fasciclins (a means to "raft" together to increase buoyancy and co-locate to the surface to optimize light acquisition). With large-scale climatic changes already underway, our observations provide insight into how diatoms respond to the dynamic ice conditions of today and shed light on how they will fare in a climatically altered tomorrow.

Increasing mercury bioaccumulation and biomagnification rates of Nile perch (Lates niloticus L.) in Winam Gulf, Lake Victoria, Kenya.

The Nile perch (Lates niloticus L.) commercial fishery for Lake Victoria in East Africa is an important source of revenue and employment. We focused on shifts in food web structure and total mercury (THg) bioaccumulation and biomagnification in Nile perch, and lower food web items collected from Winam Gulf (Kenya) sampled 24 years apart (1998 and 2022). Stable isotope carbon (δ13C) values were higher in all species from 2022 compared to 1998. Stable nitrogen isotope (δ15N) values in baseline organisms were lower in 2022 compared to 1998. In Nile perch, δ15N values were correlated with total length, but the δ15N-length regressions were steeper in 1998 compared to 2022 except for one large (158 cm) Nile perch from 1998 with an uncharacteristically low δ15N value. Total Hg concentrations were lower in lower trophic species from 2022 compared to 1998. However, the THg bioaccumulation rate (as a function of fish length) in Nile perch was greater in 2022 compared to 1998 resulting in 24.2 % to 42.4 % higher wet weight dorsal THg concentrations in 2022 Nile perch for market slot size (50 to 85 cm) fish. The contrasting observations of increased THg bioaccumulation with size in 2022 against decreases in the rate of trophic increase with size and lower THg concentrations of lower food web items imply reduced fish growth and potential bioenergetic stressors on Winam Gulf Nile perch. All samples except 1 large Nile perch (139 cm total length collected in 2022) had THg concentrations below the European Union trade limit (500 ng/g wet weight). However, for more vulnerable individuals (women, children and frequent fish eaters), we recommend a decrease in maximum monthly meal consumption for 55-75 cm Nile perch from 16 meals per month calculated for 1998 to a limit of 8 meals per month calculated for 2022.

Microcystin aids in cold temperature acclimation: Differences between a toxic Microcystis wildtype and non-toxic mutant.

For Microcystis aeruginosa PCC 7806, temperature decreases from 26 °C to 19 °C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, reactive oxygen species damage, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19 °C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

Rapidly developed, optimized, and applied wastewater surveillance system for real-time monitoring of low-incidence, high-impact MPOX outbreak.

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Cool temperature acclimation in toxigenic Microcystis aeruginosa PCC 7806 and its non-toxigenic mutant.

For Microcystis aeruginosa PCC 7806, temperature decreases from 26° C to 19° C double the microcystin quota per cell during growth in continuous culture. Here we tested whether this increase in microcystin provided M. aeruginosa PCC 7806 with a fitness advantage during colder-temperature growth by comparing cell concentration, cellular physiology, and the transcriptomics-inferred metabolism to a non-toxigenic mutant strain M. aeruginosa PCC 7806 ΔmcyB. Photo-physiological data combined with transcriptomic data revealed metabolic changes in the mutant strain during growth at 19° C, which included increased electron sinks and non-photochemical quenching. Increased gene expression was observed for a glutathione-dependent peroxiredoxin during cold treatment, suggesting compensatory mechanisms to defend against reactive oxygen species are employed in the absence of microcystin in the mutant. Our observations highlight the potential selective advantages of a longer-term defensive strategy in management of oxidative stress (i.e., making microcystin) vs the shorter-term proactive strategy of producing cellular components to actively dissipate or degrade oxidative stress agents.

A combined microscopy and single-cell sequencing approach reveals the ecology, morphology, and phylogeny of uncultured lineages of zoosporic fungi.

Environmental DNA analyses of fungal communities typically reveal a much larger diversity than can be ascribed to known species. Much of this hidden diversity lies within undescribed fungal lineages, especially the early diverging fungi (EDF). Although these EDF often represent new lineages even at the phylum level, they have never been cultured, making their morphology and ecology uncertain. One of the methods to characterize these uncultured fungi is a single-cell DNA sequencing approach. In this study, we established a large data set of single-cell sequences of EDF by manually isolating and photographing parasitic fungi on various hosts such as algae, protists, and micro-invertebrates, combined with subsequent long-read sequencing of the ribosomal DNA locus (rDNA). We successfully obtained rDNA sequences of 127 parasitic fungal cells, which clustered into 71 phylogenetic lineages belonging to seven phylum-level clades of EDF: Blastocladiomycota, Chytridiomycota, Aphelidiomycota, Rozellomycota, and three unknown phylum-level clades. Most of our single cells yielded novel sequences distinguished from both described taxa and existing metabarcoding data, indicating an expansive and hidden diversity of parasitic taxa of EDF. We also revealed an unexpected diversity of endobiotic Olpidium-like chytrids and hyper-parasitic lineages. Overall, by combining photographs of parasitic fungi with phylogenetic analyses, we were able to better understand the ecological function and morphology of many of the branches on the fungal tree of life known only from DNA sequences. IMPORTANCE Much of the diversity of microbes from natural habitats, such as soil and freshwater, comprise species and lineages that have never been isolated into pure culture. In part, this stems from a bias of culturing in favor of saprotrophic microbes over the myriad symbiotic ones that include parasitic and mutualistic relationships with other taxa. In the present study, we aimed to shed light on the ecological function and morphology of the many undescribed lineages of aquatic fungi by individually isolating and sequencing molecular barcodes from 127 cells of host-associated fungi using single-cell sequencing. By adding these sequences and their photographs into the fungal tree, we were able to understand the morphology of reproductive and vegetative structures of these novel fungi and to provide a hypothesized ecological function for them. These individual host-fungal cells revealed themselves to be complex environments despite their small size; numerous samples were hyper-parasitized with other zoosporic fungal lineages such as Rozellomycota.

Fragmentomic analysis of circulating tumor DNA-targeted cancer panels.

BACKGROUND: The isolation of cell-free DNA (cfDNA) from the bloodstream can be used to detect and analyze somatic alterations in circulating tumor DNA (ctDNA), and multiple cfDNA-targeted sequencing panels are now commercially available for Food and Drug Administration (FDA)-approved biomarker indications to guide treatment. More recently, cfDNA fragmentation patterns have emerged as a tool to infer epigenomic and transcriptomic information. However, most of these analyses used whole-genome sequencing, which is insufficient to identify FDA-approved biomarker indications in a cost-effective manner. PATIENTS AND METHODS: We used machine learning models of fragmentation patterns at the first coding exon in standard targeted cancer gene cfDNA sequencing panels to distinguish between cancer and non-cancer patients, as well as the specific tumor type and subtype. We assessed this approach in two independent cohorts: a published cohort from GRAIL (breast, lung, and prostate cancers, non-cancer, n = 198) and an institutional cohort from the University of Wisconsin (UW; breast, lung, prostate, bladder cancers, n = 320). Each cohort was split 70%/30% into training and validation sets. RESULTS: In the UW cohort, training cross-validated accuracy was 82.1%, and accuracy in the independent validation cohort was 86.6% despite a median ctDNA fraction of only 0.06. In the GRAIL cohort, to assess how this approach performs in very low ctDNA fractions, training and independent validation were split based on ctDNA fraction. Training cross-validated accuracy was 80.6%, and accuracy in the independent validation cohort was 76.3%. In the validation cohort where the ctDNA fractions were all <0.05 and as low as 0.0003, the cancer versus non-cancer area under the curve was 0.99. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that sequencing from targeted cfDNA panels can be utilized to analyze fragmentation patterns to classify cancer types, dramatically expanding the potential capabilities of existing clinically used panels at minimal additional cost.

Actionable wastewater surveillance: application to a university residence hall during the transition between Delta and Omicron resurgences of COVID-19.

Wastewater surveillance has gained traction during the COVID-19 pandemic as an effective and non-biased means to track community infection. While most surveillance relies on samples collected at municipal wastewater treatment plants, surveillance is more actionable when samples are collected "upstream" where mitigation of transmission is tractable. This report describes the results of wastewater surveillance for SARS-CoV-2 at residence halls on a university campus aimed at preventing outbreak escalation by mitigating community spread. Another goal was to estimate fecal shedding rates of SARS-CoV-2 in a non-clinical setting. Passive sampling devices were deployed in sewer laterals originating from residence halls at a frequency of twice weekly during fall 2021 as the Delta variant of concern continued to circulate across North America. A positive detection as part of routine sampling in late November 2021 triggered daily monitoring and further isolated the signal to a single wing of one residence hall. Detection of SARS-CoV-2 within the wastewater over a period of 3 consecutive days led to a coordinated rapid antigen testing campaign targeting the residence hall occupants and the identification and isolation of infected individuals. With knowledge of the number of individuals testing positive for COVID-19, fecal shedding rates were estimated to range from 3.70 log10 gc ‧ g feces-1 to 5.94 log10 gc ‧ g feces-1. These results reinforce the efficacy of wastewater surveillance as an early indicator of infection in congregate living settings. Detections can trigger public health measures ranging from enhanced communications to targeted coordinated testing and quarantine.

Interactions between chytrids cause variable infection strategies on harmful algal bloom forming species.

Cyanobacteria have a great diversity of natural enemies, such as herbivores and pathogens, including fungal pathogens within the Chytridiomycota (chytrids). While these pathogens have been previously described on a select number of cyanobacterial hosts and are suspected to play a significant ecological role, little is understood about species interactions and how competition between parasites can affect epidemic development and bloom formation. Here, three Planktothrix agardhii isolates from Sandusky Bay, Lake Erie (OH, USA) were challenged in monoculture and polyculture against infection by three isolates (C1, C2, C10) of their obligate chytrid fungal pathogen, Rhizophydiales sp. The chytrid isolates were inoculated as single isolates or a mixture of up to three different isolates. In monoculture, host isolates were characterized as highly susceptible (P. agardhii 1030), moderately susceptible (P. agardhii 1808) or mostly resistant (P. agardhii 1801). Co-infection of chytrid isolates on the highly susceptible host isolate had an additive effect on chytrid prevalence, leading to a culture crash where 2 or 3 chytrid isolates were present. Co-infection of chytrid isolates on the moderately susceptible and mostly resistant isolates had no effect on chytrid infection outcome or prevalence compared to infection with a single isolate. In polyculture, the effect on host growth was most significant in the single chytrid isolate treatment, which was attenuated with the addition of mixed chytrid treatments. Genetic analysis of the resulting population after the experimental period showed a tendency for the chytrid isolate C1 and P. agardhii 1801 to dominate in mixed population samples. Two different interspecific interactions seem to be in play; varied parasite infection strategies allow for the amplification of infection prevalence due to mixed chytrids in a susceptible monoculture, or competition allows for the dominance of a single chytrid isolate in monoculture and the reduction of infection prevalence in a host polyculture. This work thus highlights how interactions between chytrid infections can change the course of epidemic development and harmful algal bloom formation.

Evaluating fatigue in patients recovering from COVID-19: validation of the fatigue severity scale and single item screening questions.

BACKGROUND: Fatigue is a common symptom in hospitalized and non-hospitalized patients recovering from COVID-19, but no fatigue measurement scales or questions have been validated in these populations. The objective of this study was to perform validity assessments of the fatigue severity scale (FSS) and two single-item screening questions (SISQs) for fatigue in patients recovering from COVID-19. METHODS: We examined patients ≥ 28 days after their first SARS-CoV-2 infection who were hospitalized for their acute illness, as well as non-hospitalized patients referred for persistent symptoms. Patients completed questionnaires through 1 of 4 Post COVID-19 Recovery Clinics in British Columbia, Canada. Construct validity was assessed by comparing FSS scores to quality of life and depression measures. Two SISQs were evaluated based on the ability to classify fatigue (FSS score ≥ 4). RESULTS: Questionnaires were returned in 548 hospitalized and 546 non-hospitalized patients, with scores computable in 96.4% and 98.2% of patients respectively. Cronbach's alpha was 0.96 in both groups. The mean ± SD FSS score was 4.4 ± 1.8 in the hospitalized and 5.2 ± 1.6 in the non-hospitalized group, with 62.5% hospitalized and 78.9% non-hospitalized patients classified as fatigued. Ceiling effects were 7.6% in the hospitalized and 16.1% in non-hospitalized patients. FSS scores negatively correlated with EQ-5D scores in both groups (Spearman's rho - 0.6 in both hospitalized and non-hospitalized; p < 0.001) and were higher among patients with a positive PHQ-2 depression screen (5.4 vs. 4.0 in hospitalized and 5.9 vs. 4.9 in non-hospitalized; p < 0.001). An SISQ asking whether there was "fatigue present" had a sensitivity of 70.6% in hospitalized and 83.2% in non-hospitalized patients; the "always feeling tired" SISQ, had a sensitivity of 70.5% and 89.6% respectively. CONCLUSIONS: Fatigue was common and severe in patients referred for post COVID-19 assessment. Overall, the FSS is suitable for measuring fatigue in these patients, as there was excellent data quality, strong internal consistency, and construct validity. However, ceiling effects may be a limitation in the non-hospitalized group. SISQs had good sensitivity for identifying clinically relevant fatigue in non-hospitalized patients but only moderate sensitivity in the hospitalized group, indicating that there were more false negatives.

Fusing Higher-Order Features in Graph Neural Networks for Skeleton-Based Action Recognition.

Skeleton sequences are lightweight and compact and thus are ideal candidates for action recognition on edge devices. Recent skeleton-based action recognition methods extract features from 3-D joint coordinates as spatial-temporal cues, using these representations in a graph neural network for feature fusion to boost recognition performance. The use of first-and second-order features, that is, joint and bone representations, has led to high accuracy. Nonetheless, many models are still confused by actions that have similar motion trajectories. To address these issues, we propose fusing higher-order features in the form of angular encoding (AGE) into modern architectures to robustly capture the relationships between joints and body parts. This simple fusion with popular spatial-temporal graph neural networks achieves new state-of-the-art accuracy in two large benchmarks, including NTU60 and NTU120, while employing fewer parameters and reduced run time. Our source code is publicly available at: https://github.com/ZhenyueQin/Angular-Skeleton-Encoding.

Genomic comparison of Planktothrix agardhii isolates from a Lake Erie embayment.

Planktothrix agardhii is a filamentous cyanobacterial species that dominates harmful algal blooms in Sandusky Bay, Lake Erie and other freshwater basins across the world. P. agardhii isolates were obtained from early (June) blooms via single filament isolation; eight have been characterized from 2016, and 12 additional isolates have been characterized from 2018 for a total of 20 new cultures. These novel isolates were processed for genomic sequencing, where reads were used to generate scaffolds and contigs which were annotated with DIAMOND BLAST hit, Pfam, and GO. Analyses include whole genome alignment to generate phylogenetic trees and comparison of genetic rearrangements between isolates. Nitrogen acquisition and metabolism was compared across isolates. Secondary metabolite production was genetically explored including microcystins, two types of aeruginosin clusters, anabaenopeptins, cyanopeptolins, microviridins, and prenylagaramides. Two common and 4 unique CRISPR-cas islands were analyzed for similar sequences across all isolates and against the known Planktothrix-specific cyanophage, PaV-LD. Overall, the uniqueness of each genome from Planktothrix blooms sampled from the same site and at similar times belies the unexplored diversity of this genus.

Metatranscriptomic Sequencing of Winter and Spring Planktonic Communities from Lake Erie, a Laurentian Great Lake.

Previous reports suggest planktonic and under-ice winter microbial communities in Lake Erie are dominated by diatoms. Here, we report the assembled metatranscriptomes of 79 Lake Erie surface water microbial communities spanning both the winter (28 samples) and spring (51 samples) months over spatial, temporal, and climatic gradients in 2019 through 2020.

Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.