Florence Whitehill

A pictographic representation of the text abstract showing wastewater and epidemiologic data flow and data integration. To understand the utility of healthcare facility-level wastewater surveillance (WWS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is important to correlate wastewater SARS-CoV-2 RNA detection with the number of clinical infections. WWS for SARS-CoV-2 was performed at three skilled nursing facilities (SNFs) over 25 weeks. Electronegative membrane filtration (enMF) and Nanotrap® Magnetic Virus Particles (NP) virus concentration methods were compared. Extracts were tested by droplet digital polymerase chain reaction. Spearman's correlations (ρ) between wastewater virus RNA concentrations and infection counts were calculated. From split wastewater samples, enMF recovered higher SARS-CoV-2 RNA concentrations than NP. Combining data from all facilities, the median concentrations were 53.0 versus 38.6 gc/100 mL for enMF and NP, respectively (p = 0.001). Using enMF, correlations were moderate to strong at SNF A (ρ ranged 0.67–0.86, all p-values <0.001). Weak to moderate correlations may reflect the sampled manhole not representing the entire facility (SNF B, ρ ranged 0.47–0.72, p-values ranged <0.001–0.12) and longitudinal data gaps from summer heat and equipment maintenance (SNF C, ρ ranged 0.14–0.59, p-values ranged<0.01 to 0.52). WWS can be a valuable tool for tracking dynamics of SARS-CoV-2 infections in healthcare facilities.

To understand the utility of healthcare facility-level wastewater surveillance (WWS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is important to correlate wastewater SARS-CoV-2 RNA detection with the number of clinical infections. WWS for SARS-CoV-2 was performed at three skilled nursing facilities (SNFs) over 25 weeks. Electronegative membrane filtration (enMF) and Nanotrap® Magnetic Virus Particles (NP) virus concentration methods were compared. Extracts were tested by droplet digital polymerase chain reaction. Spearman’s correlations (ρ) between wastewater virus RNA concentrations and infection counts were calculated. From split wastewater samples, enMF recovered higher SARS-CoV-2 RNA concentrations than NP. Combining data from all facilities, the median concentrations were 53.0 versus 38.6 gc/100 mL for enMF and NP, respectively (p=0.001). Using enMF, correlations were moderate to strong at SNF A (ρ ranged 0.67 to 0.86, all p-values <0.001). Weak to moderate correlations can be explained by the sampled manhole not representing the entire facility (SNF B, ρ ranged 0.47 to 0.72, p-values ranged <0.001 to 0.12) and longitudinal data gaps from summer heat and equipment maintenance (SNF C, ρ ranged 0.14 to 0.59, p-values ranged 0.52 to <0.01). WWS can be a valuable tool for tracking dynamics of SARS-CoV-2 infections in healthcare facilities. Highlights: Healthcare facility-level wastewater surveillance may be useful as a complementary surveillance method for infectious disease targets, including SARS-CoV-2. Two virus concentration methods were compared, electronegative membrane filtration (enMF) and Nanotrap® Magnetic Virus Particles (NP). Spearman’s correlations (ρ) between wastewater virus RNA concentrations and infection counts were calculated among several subpopulations at three skilled nursing facilities. Graphical

Wastewater surveillance (WWS) at healthcare facilities is a nascent field with knowledge gaps in the feasibility of conducting such surveillance at this specialized facility type, and for how to best implement and interpret wastewater (WW) data. WWS was piloted at skilled nursing facilities, including conducting tracer studies, optimization of a low-flow strainer for autosampler WW collection, and preliminary testing of a WW access survey. An expanded WW access survey with collaboration from additional partners was distributed to 16 post-acute/long-term care facilities. The lessons learned obtained through the pilot ‘use cases’ demonstrated minimal clogging and consistent collection of WW using a low-flow strainer (±0.36 L) and tracer studies highlighted the importance to confirm facility effluent source with an average visual dye detection between 1.5 and 2.5 min from the toilet flush. The expanded WW access survey assessed the feasibility of WWS regarding physical onsite manhole access, safety aspects, effluent flow, and other factors, where 75% (12 of 16) of surveyed facilities demonstrated feasibility. Healthcare facility-level WWS includes specialized methodological approaches prior to implementation to achieve the intended public health impact. These considerations support the continued overall goal of detecting emerging biological public health threats at healthcare facilities using WWS.

Carbapenem resistance is an urgent public health threat. Wastewater surveillance could support antimicrobial resistance monitoring at long-term care facilities (LTCFs). Feasibility of wastewater sampling (via composite or passive sampler, and sewer biofilm swabs) for carbapenemase genes ( bla _KPC , bla _VIM , bla _OXA-48-like , bla _NDM , and bla _IMP ) detected by qPCR or GeneXpert® Carba-R from a LTCF was assessed over 16 months and compared to clinical infections. bla _KPC , bla _OXA-48-like , and bla _VIM were routinely detected in composite wastewater samples (6.4±0.8 log ₁₀ gene copy (GC)/ml (100% of 61 samples), 5.5±0.8 (98%), and 5.9±1.3 (34%), respectively), passive samples (8.2±0.5 log ₁₀ GC/g (31% of 55), 6.6±0.5 (96%), and 6.6±0.7 (31%)) and sewer biofilm (6.0±0.7 log ₁₀ GC/cm ² of pipe (100% of 17), 5.0±0.7 (100%), and 5.8±2.7 (24%)). Resistomes of wastewater and sewer biofilms differed, but both contained bla _KPC , bla _VIM , and bla _IMP . Passive sampling may be a suitable alternative to composite sampling. Wastewater surveillance is a promising addition to carbapenemase monitoring.

Background: Wastewater surveillance at healthcare facilities for carbapenem-resistant (CR) bacteria harboring carbapenemase genes could result in earlier detection of circulating or emerging antimicrobial resistance (AR) threats. However, knowledge gaps exist regarding the CR bacterial community and carbapenemase genes present in facility wastewater and plumbing, which need to be addressed. Methods: Wastewater effluent samples from three skilled nursing facilities (SNFs) (Georgia, USA) were collected weekly over six months. Selective chromogenic media were examined for their performance in screening the samples for targeted CR bacteria. Culture-dependent and culture-independent methods (ie, PCR) were applied to enriched wastewater samples (n = 73) to screen isolates and pooled samples for the most common carbapenemase genes circulating in the United States (blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP), and method sensitivity was compared. Whole genome sequencing (WGS) was used to identify all AR genes present in isolates. Results: Bacteria (n = 209 isolates) were identified using MALDI-TOF and included >20 different genera. Clinically relevant (Acinetobacter baumannii complex, Enterobacter cloacae complex, Enterococcus faecium, Stenotrophomonas spp.) and environmental/opportunistic (Comamonas spp., Pandoraea spp., Pseudomonas putida group) CR bacteria were identified in each of the three SNFs. In Facility A and C, only blaKPC was detected in 9 (26.5%) of 34 and 14 (11.4%) of 123 recovered isolates, respectively. There were no PCR-positive results in Facility B. WGS confirmed the presence or absence of all genes screened by PCR (100% concordance). Conclusions: These data provide a proof-of-concept and insights into CR bacteria present in healthcare facility wastewater using culture, PCR, and sequencing methods.

Background: Surveillance of wastewater from healthcare facilities has the potential to identify the emergence of multidrug-resistance (MDR) genes of public health importance. Specifically, wastewater surveillance (WWS) can provide sentinel surveillance of novel MDR genes or organisms in healthcare facilities, helping to direct targeted prevention efforts and monitor longitudinal effects. Several knowledge gaps need to be addressed before WWS can be used routinely for MDR surveillance, including determining optimal approaches to sampling, processing, and testing wastewater. Methods: To this end, we evaluated multiple methods for wastewater collection (passive, composite, and grab), concentration (nanoparticles, filtration, and centrifugation), and PCR quantification (real-time quantitative PCR vs. digital PCR) for Candida auris and 5 carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like) twice weekly for 6 months at a long-term acute care hospital in Chicago, IL. We also tested the effects of different transport and sample storage conditions on PCR quantification. Results: All genes were detected in facility wastewater, with blaKPC being the most consistently abundant. Experiments were done in triplicate with gene copy, variance, and number of detections between triplicates used to determine method efficacy. We found that passive samples processed immediately by centrifugation followed by bead-beating and dPCR provided the most reliable results for detecting MDR genes and C. auris. We also present the tradeoffs of different approaches and use culture and metagenomics to elucidate clinical relevance. Conclusions: This study establishes a practical approach for WWS as a potential tool for public health monitoring of MDR burden in healthcare facilities.

Background: Borealpox virus (previously known as Alaskapox virus) is an Orthopoxvirus species first identified in a patient living near Fairbanks, Alaska, in 2015; the source of the patient's infection was unknown. Six additional borealpox cases have been identified through 2023. Methods: We conducted interviews to ascertain travel history and potential exposures for the six patients, trapped small mammals for orthopoxvirus testing, and performed a phylogenetic analysis of viral DNA sequences. Results: Five patients had similar clinical illness, consisting of at least one lesion and, in most cases, lymphadenopathy. One patient with underlying immunosuppression experienced severe illness, hospitalization, and died despite receiving investigational orthopoxvirus therapeutics. One of the patients had a self-reported prior history of smallpox vaccination. None had traveled outside of Alaska, and there was no evidence of person-to-person transmission. Borealpox virus DNA and serologic evidence for past orthopoxvirus infection were detected in multiple small-mammal species. Genetic distance and phylogenetic analyses are compatible with multiple spillover events from animals to humans. Conclusions: Epidemiologic, ecologic, and phylogenetic evidence indicate that borealpox is a zoonotic disease, and current data suggest the reservoir is likely small mammals. Although most infections are mild and self-limiting, immunosuppressed persons may be at risk for severe disease. Further studies are needed to determine the geographic range of borealpox virus in small mammals throughout the Circumpolar North.

Farmed mink are one of few animals in which infection with SARS-CoV-2 has resulted in sustained transmission among a population and spillback from mink to people. In September 2020, mink on a Michigan farm exhibited increased morbidity and mortality rates due to confirmed SARS-CoV-2 infection. We conducted an epidemiologic investigation to identify the source of initial mink exposure, assess the degree of spread within the facility’s overall mink population, and evaluate the risk of further viral spread on the farm and in surrounding wildlife habitats. Three farm employees reported symptoms consistent with COVID-19 the same day that increased mortality rates were observed among the mink herd. One of these individuals, and another asymptomatic employee, tested positive for SARS-CoV-2 by real-time reverse transcription PCR (RT-qPCR) 9 days later. All but one mink sampled on the farm were positive for SARS-CoV-2 based on nucleic acid detection from at least one oral, nasal, or rectal swab tested by RT-qPCR (99%). Sequence analysis showed high degrees of similarity between sequences from mink and the two positive farm employees. Epidemiologic and genomic data, including the presence of F486L and N501T mutations believed to arise through mink adaptation, support the hypothesis that the two employees with SARS-CoV-2 nucleic acid detection contracted COVID-19 from mink. However, the specific source of virus introduction onto the farm was not identified. Three companion animals living with mink farm employees and 31 wild animals of six species sampled in the surrounding area were negative for SARS-CoV-2 by RT-qPCR. Results from this investigation support the necessity of a One Health approach to manage the zoonotic spread of SARS-CoV-2 and underscores the critical need for multifaceted public health approaches to prevent the introduction and spread of respiratory viruses on mink farms.

This report describes a case of rabies in a dog imported from Azerbaijan to the United States.