Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru.

OBJECTIVES: Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. METHODS: Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. RESULTS: The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. CONCLUSIONS: Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.

Nosocomial infections by diverse carbapenemase-producing Aeromonas hydrophila associated with combination of plumbing issues and heat waves.

BACKGROUND: Aquatic opportunistic pathogen Aeromonas hydrophila, known to persist in low-nutrient chlorinated waters, can cause life-threatening infections. Two intensive care units experienced a cluster of Aeromonas infections following outdoor temperature spikes coinciding with recurrent plumbing issues, with fatalities due to severe underlying comorbidities co-occurring with extensively-drug resistant (XDR) Aeromonas. METHODS: We investigated this cluster using whole genome sequencing to assess genetic relatedness of isolates and identify antimicrobial resistance determinants. Three A. hydrophila were isolated from patients staying in or adjacent to rooms with plumbing issues during or immediately after periods of elevated outdoor temperatures. Sinks and faucets were swabbed for culture. RESULTS: All A. hydrophila clinical isolates exhibited carbapenem resistance but were not genetically related. Diverse resistance determinants corresponding to extensively-drug resistant were found, including co-occurring KPC-3 and VIM-2, OXA-232, and chromosomal CphA-like carbapenemase genes, contributing to major treatment challenges. All 3 patients were treated with multiple antibiotic regimens to overcome various carbapenemase classes and expired due to underlying comorbidities. Environmental culture yielded no Aeromonas. CONCLUSIONS: While the investigation revealed no singular source of contamination, it supports a possible link between plumbing issues, elevated outdoor temperatures and incidence of nosocomial Aeromonas infections. The diversity of carbapenemase genes detected in these wastewater-derived Aeromonas warrants heightened infection prevention precautions during periods of plumbing problems especially with heat waves.

Domestically Acquired NDM-1-Producing Pseudomonas aeruginosa, Southern California, USA, 2023.

We describe a case of New Delhi metallo-ß-lactamase 1-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a transplant patient with multiple hospitalizations in California, USA. Whole-genome sequencing revealed the isolate was genetically distinctive, despite ≈95% similarity to other global strains. The patient's lack of international travel suggests this CRPA was acquired domestically.

FDA trial regulation of laboratory developed tests (LDTs): An academic medical center's experience with Mpox in-house testing.

The 2022 mpox outbreak presented a familiar challenge to clinical laboratories. Accordingly, our institution was able to swiftly implement in-house mpox testing to meet the imminent diagnostic needs of the public health emergency. While the FDA authorized laboratory-developed tests (LDTs) for lesion specimens, however, it restricted the testing of rectal swabs despite mounting evidence of its clinical utility. Notably, within the short timeframe when rectal testing was available, we identified a high-risk patient without apparent lesions who tested monkeypox-positive only by our in-house rectal swab assay. In order for our institution to continue testing non-lesion samples, The FDA required a separate Emergency Use Authorization (EUA) application that demanded additional resource-costly validation studies despite utilizing the same testing platform as lesion samples. Here, we provide a brief review of the history, current status, and legal scope surrounding LDT validations, with an in-depth comparison of the technical requirements by CLIA, CAP and the FDA. Importantly, we provide our experience with the mpox EUA submission process to serve as context for the challenges that may be imposed by the new FDA regulations. We hope that our experience will offer a valuable perspective that promotes constructive discourse towards addressing the imperative to offer high-quality laboratory diagnostics without compromising on the need of the medical laboratory community to provide effective patient care.

Clinical next-generation sequencing assay combining full-length gene amplification and shotgun sequencing for the detection of CMV drug resistance mutations.

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads <500IU/mL and volumes <0.5 mL - conditions otherwise unacceptable by reference laboratories. Here, we describe the development and implementation of a robust NGS LDT that offers greater testing flexibility and sensitivity to accommodate a more diverse patient population.

Case Report: Symptomatic Herpes Simplex Virus Type 2 and Monkeypox Coinfection in an Adult Male.

A 39-year-old man presented with a history of fatigue, malaise, and rash with varied morphology on his perianal region. Polymerase chain reaction testing of the lesions confirmed coinfection with monkeypox and herpes simplex virus type 2. We emphasize the difficulty in distinguishing between monkeypox virus and herpes simplex virus type 2 based on history and examination alone.

Associations Between Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants and Risk of Coronavirus Disease 2019 (COVID-19) Hospitalization Among Confirmed Cases in Washington State: A Retrospective Cohort Study.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with 7 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. METHODS: Our study includes individuals with positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) in the Washington Disease Reporting System with available viral genome data, from 1 December 2020 to 14 January 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination. RESULTS: In total, 58 848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95% confidence interval [CI] 2.40-4.26), Beta (HR 2.85, 95% CI 1.56-5.23), Delta (HR 2.28 95% CI 1.56-3.34), or Alpha (HR 1.64, 95% CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95% CI .56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination. CONCLUSIONS: Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.

Associations between SARS-CoV-2 variants and risk of COVID-19 hospitalization among confirmed cases in Washington State: a retrospective cohort study.

BACKGROUND: The COVID-19 pandemic is dominated by variant viruses; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the hospitalization risk following infection with seven SARS-CoV-2 variants. METHODS: Our study includes individuals with positive SARS-CoV-2 RT-PCR in the Washington Disease Reporting System with available viral genome data, from December 1, 2020 to January 14, 2022. The analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for hospitalization risk following infection with a variant, adjusting for age, sex, calendar week, and vaccination. FINDINGS: 58,848 cases were sequenced through sentinel surveillance, of which 1705 (2.9%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.20, 95%CI 2.40-4.26), Beta (HR 2.85, 95%CI 1.56-5.23), Delta (HR 2.28 95%CI 1.56-3.34) or Alpha (HR 1.64, 95%CI 1.29-2.07) compared to infections with ancestral lineages; Omicron (HR 0.92, 95%CI 0.56-1.52) showed no significant difference in risk. Following Alpha, Gamma, or Delta infection, unvaccinated patients show higher hospitalization risk, while vaccinated patients show no significant difference in risk, both compared to unvaccinated, ancestral lineage cases. Hospitalization risk following Omicron infection is lower with vaccination. CONCLUSION: Infection with Alpha, Gamma, or Delta results in a higher hospitalization risk, with vaccination attenuating that risk. Our findings support hospital preparedness, vaccination, and genomic surveillance. SUMMARY: Hospitalization risk following infection with SARS-CoV-2 variant remains unclear. We find a higher hospitalization risk in cases infected with Alpha, Beta, Gamma, and Delta, but not Omicron, with vaccination lowering risk. Our findings support hospital preparedness, vaccination, and genomic surveillance.

Genomic Characterization of Campylobacter jejuni Adapted to the Guinea Pig (Cavia porcellus) Host.

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide with excessive incidence in low-and middle-income countries (LMIC). During a survey for C. jejuni from putative animal hosts in a town in the Peruvian Amazon, we were able to isolate and whole genome sequence two C. jejuni strains from domesticated guinea pigs (Cavia porcellus). The C. jejuni isolated from guinea pigs had a novel multilocus sequence type that shared some alleles with other C. jejuni collected from guinea pigs. Average nucleotide identity and phylogenetic analysis with a collection of C. jejuni subsp. jejuni and C. jejuni subsp. doylei suggest that the guinea pig isolates are distinct. Genomic comparisons demonstrated gene gain and loss that could be associated with guinea pig host specialization related to guinea pig diet, anatomy, and physiology including the deletion of genes involved with selenium metabolism, including genes encoding the selenocysteine insertion machinery and selenocysteine-containing proteins.

Contribution of Time, Taxonomy, and Selective Antimicrobials to Antibiotic and Multidrug Resistance in Wastewater Bacteria.

The use of nontherapeutic broad-spectrum antimicrobial agents triclosan (TCS) and benzalkonium chloride (BC) can contribute to bacterial resistance to clinically relevant antibiotics. Antimicrobial-resistant bacteria within wastewater may reflect the resistance burden within the human microbiome, as antibiotics and pathogens in wastewater can track with clinically relevant parameters during perturbations to the community. In this study, we monitored culturable and resistant wastewater bacteria and cross-resistance to clinically relevant antibiotics to gauge the impact of each antimicrobial and identify factors influencing cross-resistance profiles. Bacteria resistant to TCS and BC were isolated from wastewater influent over 21 months, and cross-resistance, taxonomy, and monthly changes were characterized under both antimicrobial selection regimes. Cross-resistance profiles from each antimicrobial differed within and between taxa. BC-isolated bacteria had a significantly higher prevalence of resistance to "last-resort antibiotic" colistin, while isolates resistant to TCS exhibited higher rates of multidrug resistance. Prevalence of culturable TCS-resistant bacteria decreased over time following Food and Drug Administration (FDA) TCS bans. Cross-resistance patterns varied according to sampling date, including among the most clinically important antibiotics. Correlations between strain-specific resistance profiles were largely influenced by taxonomy, with some variations associated with sampling date. The results reveal that time, taxonomy, and selection by TCS and BC impact features of cross-resistance patterns among diverse wastewater microorganisms, which could reflect the variety of factors influencing resistance patterns relevant to a community microbiome.

Microfluidic droplet application for bacterial surveillance in fresh-cut produce wash waters.

Foodborne contamination and associated illness in the United States is responsible for an estimated 48 million cases per year. Increased food demand, global commerce of perishable foods, and the growing threat of antibiotic resistance are driving factors elevating concern for food safety. Foodborne illness is often associated with fresh-cut, ready-to-eat produce commodities due to the perishable nature of the product and relatively minimal processing from farm to the consumer. The research presented here optimizes and evaluates the utility of microfluidic droplets, also termed ultra-miniaturized bioreactors, for rapid detection of viable Salmonella enterica ser. Typhimurium in a shredded lettuce wash water acquired from a major Mid-Atlantic produce processing facility (denoted as Producer) in the U.S. Using a fluorescently-labeled anti-S. Typhimurium antibody and relative fluorescence intensities, paired with in-droplet incubation, S. Typhimurium was detected and identified with 100% specificity in less than 5 h. In initial optimization experiments using S. Typhimurium-spiked sterile water, the relative fluorescence intensity of S. Typhimurium was approximately two times that of the observed relative intensities of five non-S. Typhimurium negative controls at 4-h incubation in droplets containing Rappaport-Vasiliadis (RV) broth at 37°C: relative fluorescence intensity for S. Typhimurium = 2.36 (95% CI: 2.15-2.58), Enterobacter aerogens 1.12 (95% CI: 1.09-1.16), Escherichia coli 700609 = 1.13 (95% CI: 1.09-1.17), E. coli 13706 1.13 (95% CI: 1.07-1.19), E. coli 700891 1.05 (95% CI: 1.03-1.07) and Citrobacter freundii 1.04 (95% CI: 1.03-1.05). S. Typhimurium- and E. aerogens-spiked shredded lettuce wash waters acquired from the Producer were then incubated 4 h in-droplet at 37°C with RV broth. The observed relative fluorescence of S. Typhimurium was significantly higher than that of E. aerogens, 1.56 (95% CI: 1.42-1.71) and 1.10 (95% CI: 1.08-1.12), respectively. While further optimization focusing on compatible concentration methodologies for highly-dilute produce water samples is needed, this application of droplet microfluidics shows great promise in dramatically shortening the time necessary-from days to hours-to confirm viable bacterial contamination in ready-to-eat produce wash waters used throughout the domestic and international food industry.