Over 60 countries have integrated wastewater-based epidemiology (WBE) in their COVID-19 surveillance programs, focusing on wastewater treatment plants (WWTP). In this paper, we piloted the assessment of SARS-CoV-2 WBE as a complementary public health surveillance method in susceptible communities in a highly urbanized city without WWTP in the Philippines by exploring the extraction and detection methods, evaluating the contribution of physico-chemical–anthropogenic factors, and attempting whole-genome sequencing (WGS). Weekly wastewater samples were collected from sewer pipes or creeks in six communities with moderate-to-high risk of COVID-19 transmission, as categorized by the City Government of Davao from November to December 2020. Physico-chemical properties of the wastewater and anthropogenic conditions of the sites were noted. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. A subset of nine samples were subjected to WGS using the Minion sequencing platform. SARS-CoV-2 RNA was detected in twenty-two samples (91.7%) regardless of the presence of new cases. Cycle threshold values correlated with RNA concentration and attack rate. The lack of a sewershed map in the sampled areas highlights the need to integrate this in the WBE planning. A combined analysis of wastewater physico-chemical parameters such as flow rate, surface water temperature, salinity, dissolved oxygen, and total dissolved solids provided insights on the ideal sampling location, time, and method for WBE, and their impact on RNA recovery. The contribution of fecal matter in the wastewater may also be assessed through the coliform count and in the context of anthropogenic conditions in the area. Finally, our attempt on WGS detected single-nucleotide polymorphisms (SNPs) in wastewater which included clinically reported and newly identified mutations in the Philippines. This exploratory report provides a contextualized framework for applying WBE surveillance in low-sanitation areas.

The population of the endemic and critically endangered Philippine eagle ( Pithecophaga jefferyi Ogilvie‐Grant 1896) has dramatically declined, which may lead to reduced genetic diversity and increased inbreeding and extinction risk. To address the concern, there is an urgent need for genetic data to guide conservation breeding, preserve the genetic diversity of the species, and improve reintroduction outcomes. In this study, we performed mitochondrial genome sequencing and assessed the genetic diversity of Philippine eagles. Analysis of partial mitogenomes (12 protein‐coding genes, 17 tRNAs, and 16S rRNA) revealed a very low overall nucleotide diversity ( π = 0.00054 ± 0.00030) among 32 eagles from the Greater Mindanao Faunal Region. A higher number of haplotypes were observed in the eagles bred in captivity at the Philippine Eagle Center ( h = 17) compared to the wild captives ( h = 13), but both population subsets had comparable nucleotide and haplotype diversity. At least two distinct Philippine eagle subpopulations based on maternal ancestry were identified, although there is generally low genetic differentiation based on F _ST analysis. Furthermore, isolation by distance analysis (geographic isolation) showed a weak positive correlation between the genetic distance and the geographic distance of the eagle population. This suggests possible historical connectivity or limited influence of geographic separation on the genetic differentiation in Philippine eagles. Nevertheless, three genetically distant haplotypes and several eagles from KBA dyads with moderate levels of genetic differentiation were identified, representing the existence of genetically useful breeding stocks that can be prioritized in the current conservation program. Finally, phylogenetic analysis of Philippine eagles and other members of the family Accipitridae corroborates previous findings on the inclusion of the species in the subfamily Circaetinae and its monophyletic status. Altogether, we demonstrate the utility of mitogenomes in assessing genetic diversity and evolutionary relationships for species conservation.

Background Over 50 countries have used Wastewater-Based Epidemiology (WBE) and Whole-Genome Sequencing (WGS) of SARS-CoV-2 for monitoring COVID-19 cases. COVID-19 surveillance in the Philippines relies on clinical monitoring and contact tracing, with both having limited use in early detection or prediction of community outbreaks. Thus, complementary public health surveillance methods that can provide community-level infection data faster and using lesser resources must be explored. Objectives This study piloted and assessed WBE and WGS as approaches for COVID-19 surveillance in low-resource and low-sanitation communities in Davao City, Philippines. Methods Weekly wastewater samples were collected from six barangay community sewer pipes or creeks from November to December 2020. Samples were concentrated using a PEG-NaCl precipitation method and analyzed by RT-PCR to detect the SARS-CoV-2 N, RdRP, and E genes. In addition, SARS-CoV-2 RNA-positive samples were subjected to WGS for genomic mutation surveillance. Public data from clinical surveillance were also reviewed to interpret WBE data. Results Twenty-two of the 24 samples (91.7%) obtained from the six barangays tested positive for SARS-CoV-2 RNA. The cycle threshold (Ct) values were correlated with RNA concentration and attack rate. Thirty-two SARS-CoV-2 mutations were detected in WGS, including novel non-synonymous mutations or indels in seven SARS-CoV-2 genes and ten mutations previously reported in the Philippines. Discussion SARS-CoV-2 RNA was detected in community wastewater from the six barangays of Davao City, even when the barangays were classified as having a low risk of COVID-19 transmission and no new cases were reported. Despite the fragmented genome sequences analyzed, our genomic surveillance in wastewater confirmed the presence of previously reported mutations while identifying mutations not yet registered in clinical surveillance. The local context of a community must be considered when planning to adopt WBE and WGS as complementary COVID-19 surveillance methodologies, especially in low-sanitation and low-resource settings.

The technical limitations of capillary sequencing in providing insights on phylogeny have been greatly aided in recent years by the implementation of next generation sequencing platforms which can generate whole mitochondrial genome (mitogenome) sequences. In this study, enriched mitochondrial DNA ofCynopterus brachyotisfrom Mindanao, Philippines was sequenced using the Illumina MiSeq platform. A total of 653,967 clean paired-end reads was assembled using a MIRA-MITObim pipeline, resulting in a consensus mitogenome sequence length of 17,382 bases and a GC content of 41.48%, which is consistent with other published mitogenomes in fruit bats. The assembledC. brachyotismitogenome was annotated using the MITOS online server and was able to resolve all mitochondrial genes, except for one transfer RNA gene (trnT) which may be further resolved by additional capillary sequencing of the region. Sequence analysis showed that the PhilippineC. brachyotisis only 90%-91% homologous with otherCynopterusspp., based on its full mitogenome sequence. Phylogenetic analysis of fruit bat mitogenomes, deposited in online repositories, revealed that the PhilippineC. brachyotisin this study has diverged from AsianCynopterus, namelyCynopterus brachyotisandCynopterus sphinxfrom other parts of Asia (100% bootstrap support) with the latter two forming a separate clade. This divergence at the species level was consistent with phylogentic inference using cytochrome oxidase 1 (CO1) and cytochrome B (cytb) gene markers. Our results strengthen the previously reported hypothesis that the Cynopterus cf. brachyotis in the Philippines is distinct from its Asian counterparts and should be, therefore, treated as a new species.