Margaret M. Williams

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.

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.

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.

Whole-genome sequencing (WGS) of microbial pathogens recovered from patients with infectious disease facilitates high-resolution strain characterization and molecular epidemiology. However, increasing reliance on culture-independent methods to diagnose infectious diseases has resulted in few isolates available for WGS. Here, we report a novel culture-independent approach to genome characterization of Bordetella pertussis , the causative agent of pertussis and a paradigm for insufficient genomic surveillance due to limited culture of clinical isolates. Sequencing libraries constructed directly from residual pertussis-positive diagnostic nasopharyngeal specimens were hybridized with biotinylated RNA “baits” targeting B. pertussis fragments within complex mixtures that contained high concentrations of host and microbial background DNA. Recovery of B. pertussis genome sequence data was evaluated with mock and pooled negative clinical specimens spiked with reducing concentrations of either purified DNA or inactivated cells. Targeted enrichment increased the yield of B. pertussis sequencing reads up to 90% while simultaneously decreasing host reads to less than 10%. Filtered sequencing reads provided sufficient genome coverage to perform characterization via whole-genome single nucleotide polymorphisms and whole-genome multilocus sequencing typing. Moreover, these data were concordant with sequenced isolates recovered from the same specimens such that phylogenetic reconstructions from either consistently clustered the same putatively linked cases. The optimized protocol is suitable for nasopharyngeal specimens with diagnostic IS 481 Ct < 35 and >10 ng DNA. Routine implementation of these methods could strengthen surveillance and study of pertussis resurgence by capturing additional cases with genomic characterization.

Health SA Gesondheid promotes communication, collaboration and teamwork between professions and disciplines within the health sciences to address problems that cross and affect disciplinary boundaries.

Multi-Locus Sequence Typing (MLST) provides allele-based characterization of bacterial pathogens in a standardized framework. However, classical MLST schemes for Bordetella pertussis, the causative agent of whooping cough, seldom reveal diversity among the small number of gene targets and thereby fail to delineate population structure. To improve discriminatory power of allele-based molecular typing of B. pertussis, we have developed a whole-genome MLST (wgMLST) scheme from 225 reference-quality genome assemblies. Iterative refinement and allele curation resulted in a scheme of 3,506 coding sequences and covering 81.4% of the B. pertussis genome. This wgMLST scheme was further evaluated with data from a convenience sample of 2,389 B. pertussis isolates sequenced on Illumina instruments, including isolates from known outbreaks and epidemics previously characterized by existing molecular assays, as well as replicates collected from individual patients. wgMLST demonstrated concordance with whole-genome single nucleotide polymorphisms (SNP) profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients during diagnostic confirmation. Additionally, a re-analysis of isolates from two statewide epidemics using wgMLST reconstructed the population structures of circulating strains with increased resolution, revealing new clusters of related cases. Comparison with an existing core-genome (cgMLST) scheme highlights the stable gene content of this bacterium and forms the initial foundation for necessary standardization. These results demonstrate the utility of wgMLST for improving B. pertussis characterization and genomic surveillance during the current pertussis disease resurgence.

Aim: The aim of this study was to evaluate a digital programme aimed to address COVID-19 related anxiety of final year undergraduate student nurses on returning to the clinical facilities during the pandemic in South Africa. Background: In South Africa, concern about the physical and psychological safety of nursing students resulted in the planning of a psychological first aid program to ensure the safe return of nursing students to clinical facilities. Design: A quantitative evaluation study was conducted in September 2020 following the August 2020 return of final year nursing students to clinical facilities. Methods: All final year nursing students were invited to participate in the study (N=196). An online survey was circulated via WhatsApp asking respondents to rate their anxiety and fear of COVID-19 before and after return to practice. Related pair analysis was done on the main outcome measures of anxiety, COVID-19 fears and concerns using paired T-Tests and Wilcoxon-ranked Tests. Results: After the programme, the respondents reported a significant reduction in anxiety (T=2.95, p=.004) and a significant reduction in COVID fear (T.3.1, p=.002), specifically related to infecting family, being exposed to COVID-19 at work, propagating infection at work and lacking access to COVID-19 information and communication. Conclusion: The implementation of a dedicated digital programme, underpinned by a sound theory base of psychological first aid, was effective in preparing nursing students for clinical practice in the pandemic.

Multi-Locus Sequence Typing (MLST) provides allele-based characterization of bacterial pathogens in a standardized framework. However, current MLST schemes for Bordetella pertussis , the causative agent of whooping cough, seldom reveal diversity among the small number of gene targets and thereby fail to delineate population structure. To improve discriminatory power of allele-based molecular typing of B. pertussis , we have developed a whole-genome MLST (wgMLST) scheme from 214 reference-quality genome assemblies. Iterative refinement and allele curation resulted in a scheme of 3,506 coding sequences and covering 81.4% of the B. pertussis genome. This wgMLST scheme was further evaluated with data from a convenience sample of 2,389 B. pertussis isolates sequenced on Illumina instruments, including isolates from known outbreaks and epidemics previously characterized by existing molecular assays, as well as replicates collected from individual patients. wgMLST demonstrated concordance with whole-genome single nucleotide polymorphisms (SNP) profiles, accurately resolved outbreak and sporadic cases in a retrospective comparison, and clustered replicate isolates collected from individual patients during diagnostic confirmation. Additionally, a re-analysis of isolates from two statewide epidemics using wgMLST reconstructed the population structures of circulating strains with increased resolution, revealing new clusters of related cases. Comparison with an existing core-genome (cgMLST) scheme highlights the genomic stability of this bacterium and forms the initial foundation for necessary standardization. These results demonstrate the utility of wgMLST for improving B. pertussis characterization and genomic surveillance during the current pertussis disease resurgence.

Respiratory diphtheria, characterized by a firmly adherent pseudomembrane, is caused by toxin-producing strains of Corynebacterium diphtheriae , with similar illness produced occasionally by toxigenic Corynebacterium ulcerans or, rarely, Corynebacterium pseudotuberculosis . While diphtheria laboratory confirmation requires culture methods to determine toxigenicity, real-time PCR (RT-PCR) provides a faster method to detect the toxin gene ( tox ).