Mycoplasma genitalium is fastidious to culture, and its detection in human clinical specimens relies mainly on molecular methods. Phenotypic determination of antibiotic susceptibility for this bacterium is not a timely or feasible option for most clinical laboratories. This study sought to determine whether next-generation sequencing technologies can effectively be employed in determining genetic mutations associated with drug resistance in M. genitalium samples collected in Aptima Hologic tubes and possibly integrating them into viable workflows in public health laboratories. Following analysis by a custom-designed bioinformatics pipeline, at least one mutation/sample has been identified in 94/98 specimens in at least one of seven loci (macrolides: rrl , rplD , rplV ; fluoroquinolones: parC , parE , gyrA , gyrB ) described previously to be connected to antibiotic resistance. This method identified a total of 469 single nucleotide polymorphisms (SNPs) (452 mutations): 134 of 23S rRNA SNPs and 318 amino acid mutations: 114 substitutions and 204 synonymous; the turnaround time (sample to analyzed sequence) was typically 3 days. The assays and workflows described in this work demonstrated that the determination of a drug resistance profile for macrolides and fluoroquinolones of M. genitalium samples by using next-generation sequencing in clinical samples is a feasible approach that can be implemented in clinical laboratories, following thorough and extensive validation studies. IMPORTANCE: The mechanisms of drug resistance in Mycoplasma genitalium are complex and involve several genetic loci. The molecular methods for accurately characterizing resistance to fluoroquinolones and macrolides in this organism are often not available or approved for patient use and do not cover all genetic determinants. To this end, we propose a next-generation sequencing-based method with a turnaround time of 3 days that includes the investigation of all drug resistance loci of M. genitalium . Following adaptation, validation, and verification for routine clinical use, assays based on this method may yield molecular results that can be used to guide proper treatment regimens and for surveillance of drug resistance in the general population.

The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%–95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%–99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

Three incidents that impacted two US newborn screening (NBS) programs highlight the importance of contingency planning for the continuity of operations (COOP). Other NBS programs may benefit from the experience of these state programs for their own contingency planning efforts. Through after-action reviews conducted post-incident, crucial elements for the successful management of an incident were identified. We detailed the strengths, weaknesses, improvements needed, and future actions that will assist in preparing for other incidents as lessons learned.

Non-typhoidal Salmonella (NTS) poses a serious threat for multi-drug-resistant infections in the United States. However, most infections are not detected through traditional clinical surveillance, which severely underestimates the community disease burden. Wastewater-based epidemiology can overcome barriers in clinical surveillance and fill the gap. We performed a retrospective study to monitor disease trends and characterize viable NTS isolates from a medium-sized city’s wastewater treatment plants. Of 106 weekly raw influent samples from September 2020 to September 2021, 81 samples (76.42%) were NTS-positive via qPCR. NTS wastewater trends were a leading indicator of salmonellosis hospitalizations in the community by 1 week. The 27 unique NTS isolates (representing 11 different serotypes) recovered from wastewater revealed the biodiversity of community circulating infections. Wastewater samples showed a lower biodiversity of serotypes than environmental samples. The biodiversity of isolates collected from wastewater and clinical surveillance was similar but also represented serotypes not identified through traditional clinical surveillance approaches. Genotypic profiles of wastewater isolates revealed that many isolates have the potential for multi-drug-resistant infections and contain virulence genes associated with severe disease. This study shows that wastewater-based epidemiology is a valuable tool for monitoring disease trends and can provide early warning signs of salmonellosis in a community. We successfully enriched and cultured viable NTS isolates from raw wastewater samples frozen for over 1 year, further expanding the capabilities of retrospective studies allowing for future characterization of isolates. Overall, wastewater-based epidemiology is a powerful tool for retrospective analysis and for real-time community surveillance of NTS. IMPORTANCE: Wastewater-based epidemiology (WBE) is an emerging tool for community-level infectious disease surveillance, yet its application to bacterial pathogens remains largely underexplored. Our study demonstrated that WBE effectively tracks non-typhoidal Salmonella (NTS) trends, serving as a leading indicator of hospitalizations. We successfully recovered and characterized Salmonella isolates from wastewater, highlighting its potential for genomic surveillance. We found that NTS remains viable in frozen wastewater samples for up to 1 year, the longest reported storage time to date. Characterizing bacterial isolates allows for studying pathogen evolution, AMR dynamics, and source tracking. Expanding WBE applications to bacteria could significantly enhance our understanding of disease dynamics and inform proactive public health interventions. Further research is needed to validate WBE as an early warning system for bacterial diseases and integration into a One Health framework. Our findings highlight WBE’s growing role in infectious disease epidemiology, reinforcing its value as a powerful public health surveillance tool.