Amanda K. Lyons

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.

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.

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. Wastewater effluent samples from three skilled nursing facilities (SNFs) (Georgia, USA) were collected weekly over six months each. Selective chromogenic media were examined for their performance in screening the samples for targeted CR bacteria. Culture-dependent and culture-independent methods (i.e., 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 ( bla _KPC , bla _NDM , bla _VIM , bla _OXA-48-like , and bla _IMP ), and method sensitivity was compared. Whole genome sequencing (WGS) was used to identify all AR genes present in isolates. 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 bla _KPC 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 for by PCR (100% concordance). These data provide a proof of concept and insights into CR bacteria present in healthcare facility wastewater using culture, PCR, and sequencing methods.

The purpose of this study was to determine the feasibility of facility-level wastewater surveillance in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in skilled nursing facility (SNF) wastewater using three concentration methods, as well as a proof-of-concept for antimicrobial resistance (AR) genes/organisms detection. Wastewater effluent samples were collected from an SNF over an 8-week period. Wastewater was concentrated using electronegative membrane filtration (enMF), polyethylene glycol precipitation, and Nanotrap® magnetic virus particles (NP). Quantification of the genome copy concentration from SARS-CoV-2 and bovine respiratory syncytial virus (BRSV), a SARS-CoV-2 surrogate spiked into all samples, was performed with droplet digital polymerase chain reaction (ddPCR). Wastewater sample aliquots were also enriched in microbiological culture media and screened for organisms with AR phenotypes on selective and differential agars. Multiplex real-time PCR was used to detect a broad array of carbapenem resistance genes. SARS-CoV-2 was detected and quantified from a single enMF-concentrated wastewater sample. The highest concentration of BRSV came from enMF-concentrated samples. Klebsiella, Enterobacter, Citrobacter, and Escherichia coli exhibiting AR phenotypes were successfully detected using culture-dependent approaches. Culture-independent, multiplex PCR indicated that blaKPC was the main carbapenemase gene detected in wastewater samples. Facility-level wastewater surveillance could be a useful strategy for SNFs.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

Results from sampling healthcare surfaces for pathogens are difficult to interpret without understanding the factors that influence pathogen detection. We investigated the recovery of four healthcare-associated pathogens from three common surface materials, and how a body fluid simulant (artificial test soil, ATS), deposition method, and contamination levels influence the percent of organisms recovered (%R). Known quantities of carbapenemase-producing KPC+ Klebsiella pneumoniae (KPC), Acinetobacter baumannii, vancomycin-resistant Enterococcus faecalis, and Clostridioides difficile spores (CD) were suspended in Butterfield’s buffer or ATS, deposited on 323cm² steel, plastic, and laminate surfaces, allowed to dry 1h, then sampled with a cellulose sponge wipe. Bacteria were eluted, cultured, CFU counted and %R determined relative to the inoculum. The %R varied by organism, from 1 log₁₀ within the 60 min drying time. For all organisms, the %R was significantly greater if suspended in ATS than if suspended in Butterfield’s buffer (p<0.05), and for most organisms the %R was not significantly different when sampled from any of the three surfaces. Organisms deposited in multiple droplets were recovered at equal or higher %R than if spread evenly on the surface. This work assists in interpreting data collected while investigating a healthcare infection outbreak or while conducting infection intervention studies.

Key Points: Patients with ESKD on dialysis had persistent and intermittently positive RT-PCR tests for severe acute respiratory syndrome coronavirus 2. Evidence for presence of infectious virus was lacking in most cases. A symptom-based approach, instead of a test-based approach, should be used to decide when to discontinue transmission-based precautions. Background: Patients with ESKD on maintenance dialysis receive dialysis in common spaces with other patients and have a higher risk of severe SARS-CoV-2 infections. They may have persistently or intermittently positive SARS-CoV-2 RT-PCR tests after infection. We describe the clinical course of SARS-CoV-2 infection and the serologic response in a convenience sample of patients with ESKD to understand the duration of infectivity. Methods: From August to November 2020, we enrolled patients on maintenance dialysis with SARS-CoV-2 infections from outpatient dialysis facilities in Atlanta, Georgia. We followed participants for approximately 42 days. We assessed COVID-19 symptoms and collected specimens. Oropharyngeal (OP), anterior nasal (AN), and saliva (SA) specimens were tested for the presence of SARS-CoV-2 RNA, using RT-PCR, and sent for viral culture. Serology, including neutralizing antibodies, was measured in blood specimens. Results: Fifteen participants, with a median age of 58 (range, 37‒77) years, were enrolled. Median duration of RT-PCR positivity from diagnosis was 18 days (interquartile range [IQR], 8‒24 days). Ten participants had at least one, for a total of 41, positive RT-PCR specimens ≥10 days after symptoms onset. Of these 41 specimens, 21 underwent viral culture; one (5%) was positive 14 days after symptom onset. Thirteen participants developed SARS-CoV-2–specific antibodies, 11 of which included neutralizing antibodies. RT-PCRs remained positive after seroconversion in eight participants and after detection of neutralizing antibodies in four participants; however, all of these samples were culture negative. Conclusions: Patients with ESKD on maintenance dialysis remained persistently and intermittently SARS-CoV-2–RT-PCR positive. However, of the 15 participants, only one had infectious virus, on day 14 after symptom onset. Most participants mounted an antibody response, including neutralizing antibodies. Participants continued having RT-PCR–positive results in the presence of SARS-CoV-2–specific antibodies, but without replication-competent virus detected.