Applied and Environmental Microbiology

Background: Privacy curtains represent a uniquely difficult infection control surface because they are frequently touched by patients, visitors, and healthcare workers, yet are difficult to disinfect using routine environmental cleaning protocols. We sought to quantify the performance of a novel automated aerosolized hydrogen peroxide system against pathogens on privacy curtains. Methods: This study was performed in an enclosed office space ({approx} 65 m^3), approximating a patient exam room. Four Bacillus atrophaeus (ATCC 9372) biological indicators (BIs) were affixed to a standard polyester privacy curtain. A single one-minute fogging phase was followed by a ten-minute contact/aeration phase (total = 11 min). Four corresponding chemical indicators measured peak airborne aerosolized hydrogen peroxide concentration, and two control BIs were placed outside of the room. Results: All four BIs demonstrated complete inactivation (no growth after seven-day incubation), representing [≥] 4-log10 spore reduction. Peak dosages reached 42 ppm nearest the aerosolized hydrogen peroxide jet outlet, with the lowest value at the near point on the back side of the curtain reaching 27 ppm (exceeding the target dosage of 25 ppm). Control exposures failed to achieve sterilization. Conclusions: A next-generation aerosolized hydrogen peroxide dry-fogging disinfection system achieved full inactivation of resistant spores on both sides of privacy curtains within a short 11-minute automated cycle.

Wastewater-based epidemiology uses both targeted and non-targeted bioanalytical approaches to detect and track disease-causing microbes in wastewater. Targeted approaches such as quantitative polymerase chain reaction (qPCR) remain widely used while sequencing-based approaches have slowly gained popularity. However, concordance between established targeted molecular assays and sequencing approaches remains incompletely characterized. To assess the validity of sequencing as an alternative to qPCR, we compared outcomes of both methods targeting five respiratory viruses in wastewater samples collected across 16 Texas cities over 4 years (May 2022-July 2026) of continuous sampling. We found positive correlations between qPCR and sequencing measurements, with both methods independently correlating with statewide clinical emergency department visits. qPCR positivity also increased with genomic sequencing coverage, and sequencing demonstrated greater potential than qPCR as an early respiratory virus outbreak detection method. Our findings support metagenomic sequencing as a sensitive and highly specific alternative to targeted qPCR for wastewater surveillance, and reinforce its utility towards long-term longitudinal monitoring of respiratory viruses.

Tick-borne disease risk reflects the combined influence of vector abundance, pathogen prevalence, human exposure, and seasonal activity, yet these components are rarely integrated at the county level. We developed species-specific ecological risk scores for three medically important tick species (Acari: Ixodidae): Amblyomma americanum (L.), Dermacentor variabilis (Say), and Ixodes scapularis Say, across all 102 Illinois counties using data from 2019 to 2022. County-level predicted tick abundance and pathogen prevalence were scaled and combined using equal weights to generate primary vector-level risk scores. Robustness to the equal-weight assumption was evaluated by systematically varying the relative weights assigned to abundance and pathogen prevalence, while a separate sensitivity analysis incorporated reported tick-bite incidence where available. Associations with human disease were evaluated using negative binomial regression of tick-borne disease case counts on the corresponding risk scores, with county population included as an offset. Monthly phenology indicators were constructed by integrating human disease cases, reported tick bites, and sampled tick abundance and were evaluated in relation to temperature and precipitation. Seasonal risk was estimated by combining county-level vector risk with species-specific monthly phenology, generating monthly risk surfaces that were deployed through an interactive R Shiny application. Higher risk scores were significantly associated with greater disease case rates for each tick species. County rankings were highly stable under moderate changes in abundance and prevalence weighting. Seasonal activity differed among species, with A. americanum and D. variabilis showing concentrated warm-season risk and I. scapularis exhibiting a broader seasonal profile. These county-level seasonal risk maps identify where and when relative tick-borne disease risk is elevated in Illinois, supporting targeted prevention, surveillance, and clinical awareness.

Vegetation plays a critical role in shaping fine-scale tick density by structuring local microclimates, habitat resources, and host interactions that largely determine tick survival and activity, with implications for tick-borne disease transmission. Tick burden in the United States continues to increase, with annual reported tick-borne disease cases more than doubling from 2004 through 2019. These trends call for a broader scope of tick-mitigation strategies that go beyond traditional prevention measures that focus on personal protection; and there is an opportunity to inform habitat-level interventions. Here, we quantify fine-scale vegetation structure across 48 subplots at tick surveillance sites in Western Massachusetts to assess local habitat features associated with Ixodes scapularis density and potential for human-tick contact risk. In assessing understory and overstory vegetation attributes, this study identified life stage-specific differences in predicted tick density, with higher adult densities in areas with deeper leaf litter and presence of invasive plant species and nymphs associated with high presence of ferns. Both adult and nymphal Ix. scapularis were predicted at higher densities in relatively low basal area forests (18.9 to 26.9 m2/hectare). This study design provides a simple, low-cost framework for organizations looking to incorporate tick-informed forest measurement ecology into their public health tick surveillance protocols with minimal training.

Wolbachia-based interventions were less effective in achieving introgression in Rio de Janeiro than other settings. Reduced introgression occurred in locations where dengue was historically high, raising concerns of inequity. We examined factors affecting Wolbachia introgression in Rio through trajectory modeling, spatiotemporal Bayesian models, and Ae. aegypti abundance estimation. During the intervention (2017-2020), Wolbachia introgression was 26% (median; IQR,14-44%), with four distinct trajectories. Despite receiving more releases, densely populated areas with low socioeconomic status had lower introgression. In spatiotemporally explicit multivariable regressions, higher Ae. aegypti abundance, temperature, population density and lower socioeconomic status were associated with reduced introgression. Improved characterization of Ae. aegypti abundance and other structural/environmental predictors of introgression could be used to calibrate release doses to ensure equitable benefits from Wolbachia interventions.

Background: Commercial spaceflight now carries civilians on short missions, yet their stress-axis physiology is poorly described. Earlier shuttle data suggest that sympathetic responses predominate after short flights and glucocorticoid responses after long ones. The neutrophil-to-lymphocyte ratio (NLR) is a low-cost readout of both. Methods: Preregistered secondary analysis of open clinical laboratory data (NASA Open Science Data Repository OSD-569 and OSD-575) from the four Inspiration4 crew members, each sampled seven times: three draws before launch, one the day after return (R+1) and three during recovery (R+45 to R+194). Log-transformed NLR was modelled with a linear mixed model with a random intercept per crew member and Kenward-Roger inference. Lymphocyte and eosinophil counts and C-reactive protein were secondary outcomes with false discovery rate correction. Results: All 28 draws were analysed. NLR at R+1 did not differ from pre-flight (fold change 1.04; 95% CI 0.81 to 1.34; p = 0.73); two crew members increased (+25% and +46%) and two decreased (-27% and -11%). During recovery NLR was 23% lower than pre-flight (fold change 0.77; 95% CI 0.64 to 0.92; p = 0.005). No secondary outcome changed after correction; lymphocytes were 18% lower at R+1 (95% CI -35% to +4%; q = 0.31). Between-person differences explained 58% of the variance. An ordinary regression ignoring repeated measures missed the recovery effect (p = 0.08). Conclusions: A 3-day orbital flight produced no consistent leukocyte stress signature one day after return. The lower NLR during recovery suggests that pre-flight values, not recovery values, deviate from the resting state, which has implications for how baselines are defined in short-duration spaceflight studies.

Importance: Coccidioidomycosis, also known as Valley fever, is a serious fungal disease caused by inhaling Coccidioides spores aerosolized from the soil. Despite rising public health concern, the distribution and incidence of coccidioidomycosis in the US remain poorly characterized as not all states are required to report cases, and case under-ascertainment is presumed to be high, particularly outside of the endemic region. Objective: To estimate monthly, county-level coccidioidomycosis incidence across the US while accounting for widespread under-ascertainment. Design, Setting, and Participants: We integrated multisource data, including human cases diagnosed in electronic health records, serological testing of dogs, and ecological and sociodemographic predictors, in a machine learning model estimating monthly, county-level coccidioidomycosis incidence. The model was trained on surveillance data from Arizona and California from 2013 to 2023, then applied to predict incidence in states across the contiguous US over the same time period. Main Outcomes and Measures: Incidence of coccidioidomycosis at the monthly county level. Results: The model predicted coccidioidomycosis incidence from 2013 to 2023 for 3,108 counties from all states in the contiguous US. The model identified 60 counties in non-reporting states where predicted incidence was [≥]2 cases per 100,000 population (considered moderate to high incidence). In states with mandatory reporting outside of Arizona and California, the model predicted county-level incidence rates that were 28% higher (95% bootstrap interval, 0.1%-69.8%) than those captured in national surveillance, equating to a total of 608 potentially missed cases per year (95% bootstrap interval, 340-964). Conclusions and Relevance: In this cross-sectional study, the model quantified case under-ascertainment for coccidioidomycosis relative to Arizona and California, and highlighted the potential for alternative data sources to fill in gaps in mandatory reporting. Estimating incidence while accounting for under-ascertainment and non-reporting may help efforts to improve public and physician awareness and to detect emergence in new regions.

Importance: Clinicians often look for a dental source in Staphylococcus aureus bacteremia (SAB), but how often dental disease is found, compared with other bacteremias, is unclear. Objective: To compare documented dental disease in adults with SAB, Escherichia coli bacteremia, and a general population cohort. Design, Setting, and Participants: Retrospective cohort study in the Protege database of 48 975 adults with bacteremia from January 1, 2015, through December 31, 2023, assigned by earliest in-window organism, and 81 945 general population adults. Exposures: SAB, with E coli bacteremia as the primary comparator and the general population as the secondary comparator. Main Outcomes and Measures: Documented dental disease from 7 days before through 30 days after index, defined by a qualifying dental procedure or disease-specific note text and adjusted for age, sex, race, and documentation intensity. Results: Dental disease occurred in 528 of 16 435 patients with SAB (3.21%) and 179 of 24 942 patients with E coli bacteremia (0.72%). The adjusted odds ratio was 3.86 (95% CI, 3.23 to 4.63). By the pathology-only definition, disease occurred in 517 patients with SAB (3.15%) and 399 general population patients (0.49%). The adjusted odds ratio was 3.71 (95% CI, 3.08 to 4.46). Within SAB, rates were highest with prior infective endocarditis (9.8%), opioid use disorder (6.2%), injection drug use (5.3%), and prosthetic valve (5.0%). Prevalence ranged from 0.49% in the general population to 7.98% in other streptococcal bacteremia. Of 437 patients with SAB and an assignable first finding date, 333 (76.2%) had the finding after the index date. Conclusions and Relevance: Dental disease was documented more often in SAB than in E coli bacteremia after adjustment. Most findings were first recorded after bacteremia was recognized, during SAB evaluation. These data do not show that dental disease caused the bacteremia.

Introduction: Pesticide exposure has been associated with poorer mental health, yet little is known about whether seasonal fluctuations in agricultural pesticide use are associated with cyclical changes in mood among people living in agricultural communities. We evaluated associations between 10 peak and off-peak pesticide spray seasons and ecological momentary assessment (EMA)-derived mood in a floricultural region in Ecuador. Methods: 496 participants completed repeated EMA surveys across 10 assessment administrations over 2.5 years (2022-2024), including one single-assessment administration and nine 10-day bursts (Nobservations=14,332). Mood outcomes included negative affect (sadness, anxiety, stress, anger, tiredness, difficulty concentrating; overall mean=2.79, Standard Deviation [SD]=1.32), positive affect (happiness, motivation; overall mean=4.21, SD=1.60), and behavioral dysregulation (aggression and impulsivity, overall mean=1.86, SD=1.29). Generalized linear mixed models estimated pooled, between-person and within-person associations between peak pesticide spray seasons and mood, adjusting for demographic and longitudinal factors Effect modification by sex, floricultural occupation, and cohabitation with a floricultural worker was assessed. Results: Peak spray seasons were associated with higher negative affect ({beta}=0.11 [0.06, 0.16]) and behavioral dysregulation ({beta}=0.10 [0.05, 0.15]) but not positive affect ({beta}=0.01 [-0.05, 0.07]). Associations were driven by within-person changes. Sex modified associations with negative affect, which were stronger in males than females. Floricultural occupation modified associations, with poorer mood, greater behavioral dysregulation and lower positive affect among floricultural workers than non-workers. Discussion: Peak pesticide spray seasons were associated with worsening of negative affect and behavioral dysregulation. EMA may provide a sensitive approach for detecting short-term neurobehavioral changes associated with seasonal pesticide exposure.

Introduction: Acute gastroenteritis (AGE) is a cause of childhood morbidity and may be associated with human adenovirus (HAdV) infection. This study aimed to investigate the occurrence of HAdV in fecal samples from children with AGE in Salvador, Bahia, Brazil, as well as to molecularly characterize the identified strains and evaluate their cytopathic effects in cell culture. Methods: A total of 131 samples were analyzed using the nested polymerase chain reaction (Nested-PCR) technique. Molecular characterization was performed through Sanger sequencing and phylogenetic analysis, while Vero cell cultures were used to investigate the occurrence of cytopathic effects. Results: Of the samples analyzed, 13 (9.9%) tested positive for HAdV, while 118 (90.1%) were negative. The highest detection frequency was observed in children aged three years or younger, particularly those under one year of age. Phylogenetic analysis identified variants related to HAdV-F41 and HAdV-B3, revealing genetic differences and distinct clustering patterns compared with reference sequences. In cell culture, cytopathic effects were observed four days after inoculation, although further analyses are required to confirm productive viral replication. Conclusion: These findings demonstrate the presence of HAdV in the samples studied and contribute to understanding its genetic diversity and circulation in Salvador, highlighting the importance of epidemiological surveillance and further studies on its role in the etiology of childhood AGE.

Background. Nigeria's reported Lassa fever case fatality ratio (CFR) rose from 18.9% at week 23 of 2025 to 25.0% in 2026, prompting concerns over failing clinical care. Because surveillance artefacts can also alter CFRs, candidate mechanisms that published data can resolve were identified and evaluated. Methods. This ecological study extracted weekly state-level counts from 474 Nigeria Centre for Disease Control and Prevention situation reports (2017-2026). Per a pre-specified plan, years were compared at phase-matched points, year-on-year CFR changes were decomposed into within-state and between-state components, and tipping-point ascertainment shifts calculated. Weekly incident and cumulative published totals were compared post hoc. Findings. Of 11 candidate mechanisms, 2 could be evaluated fully and 5 in part. On weekly incident counts, 2026 did not differ from 2025 at the phase-matched midpoint (+0.3 percentage points, 95% CI -5.0 to 7.6); within-state (-1.1, 95% CI -4.7 to 3.0) and between-state (+0.5, -2.0 to 2.9) components spanned zero. Conversely, the published cumulative CFR was 5.4 to 6.2 percentage points higher because 70 deaths were added retrospectively by week 36 outside weekly reports. Reconciling cumulative figures would require 279 missed survivors (6% of suspected cases not confirmed). Interpretation. The 2026 CFR rise came from deaths added to cumulative totals outside the weekly counts, and published data cannot attribute it to care. Endemic zoonotic CFRs cannot serve as clinical outcome measures unless testing volumes, deaths added late, and post-mortem sampling are published alongside case counts. Funding. US NSF-NIH-NIFA EEID; UK BBSRC; Birgitta Sintring Fellowship (Uppsala University).

Introduction: Acute gastroenteritis (AGE) is a global public health problem and a major cause of mortality in low- and lower-middle-income countries. Human bocavirus (HBoV) is among the viral agents associated with this condition. Therefore, investigating the presence of HBoV in cases of AGE in Salvador, Bahia, as well as its prevalence and circulating strains, is important. Objective: To investigate the occurrence of HBoV infection during an AGE outbreak in Salvador. Methods: A total of 178 samples were analyzed for HBoV using nested polymerase chain reaction (nested PCR), and the amplification products were visualized by agarose gel electrophoresis. HBoV was detected in 5.7% of the samples analyzed. Conclusion: This study contributes to the understanding of HBoV epidemiology as a secondary agent associated with acute gastroenteritis during a norovirus outbreak in Salvador. Continued investigation and identification of the etiological agents involved in outbreaks are essential for evaluating the causes of AGE. These findings also highlight the need for broader diagnostic approaches and the importance of phylogenetic analysis for monitoring and controlling viral infections.

Abstract Background: Cyprus has transitioned into a high-alert zone for mosquito-borne viruses. This necessitates a proactive assessment of its future physicians. Following the 2022 re-establishment of Aedes aegypti in Larnaca and Aedes albopictus in Limassol, Cyprus, the island is now biologically primed for local outbreaks of dengue, Zika, and chikungunya, alongside Culex-borne West Nile virus. The study assessed medical students' knowledge and risk perception. Methods: A cross-sectional online survey administered the validated MosquitoWise Health Belief Model instrument to undergraduate-entry and graduate-entry students at a university in Cyprus in mid-2026. Results: Of 1023 students, 119 responded (11.6%), with pre-clinical students overrepresented (p<0.001). Mean knowledge was 5.43/9, stronger on mosquito biology (72.5%) than virus knowledge (54.2%). Median severity was 6.00/7 against susceptibility 4.25/7. Knowledge differed by study stage (p<0.001), correlated with severity (rs = 0.399) but not susceptibility. Conclusions: The study establishes a critical baseline for pre-clinical medical students in Cyprus. Overall, participants performed better on mosquito biology when compared to mosquito-borne viruses and how they spread. Our findings highlight the value of integrating theoretical microbiology with clinical practice in medical education to enhance physicians' preparedness for the changing landscape of newly emerging infections in Cyprus and across Europe as a whole. Key Words: Arboviruses, Cross-Sectional Studies, Cyprus, Health Belief Model, Medical Students, Risk Perception.

Community-led urban greening may contribute to neighborhood safety, but its relationship with crime remains uncertain. We examined whether Keep Indianapolis Beautiful Project GreenSpaces in Indianapolis were associated with changes in violence and total crime. We used a matched-pair difference-in-differences design comparing funded sites with matched unfunded applicant sites. The primary analysis included 29 matched pairs with two years of pre- and post-grant follow-up. Monthly Part I violent offenses and total crime within 600-m buffers were modeled using Poisson pseudo-maximum likelihood. In the first post-grant year, the violent-crime estimate was lower but imprecise (IRR, 0.903; 95% CI, 0.805-1.014). In the second post-grant year, the relative rate was lower (IRR, 0.830; 95% CI, 0.718-0.961). Total crime estimates showed no clear post-grant change. Additional analyses indicated some sensitivity to spatial scale, but no sustained divergence in pre-grant crime patterns. Analyses of short-term crime dependence showed clear month-to-month persistence in violent crime within sites but limited evidence of stable spatial dependence across nearby sites. Therefore, within the two-year post-grant period examined, Project GreenSpaces was associated most consistently with lower relative violent-crime rates in the second year, while no corresponding decrease was observed for total recorded crime. Longer follow-up is needed to determine whether these patterns persist or change as projects mature.

Brazil has one of the highest dengue burdens globally, with transmission influenced by climate events. Yet, the effects of compound climate extremes on dengue risk and prediction have not been quantified to date. Using a Bayesian spatiotemporal model of monthly dengue cases across 435 health regions from 2010 to 2025, we estimated relative risk associated with interacting long-lag and short-lag drought indicators and temperature anomalies across nine Koppen-Geiger climatic zones, adjusting for absolute temperature and El Nino-Southern Oscillation. Long-lag dry, mid-lag warm and short-lag wet conditions were associated with the highest relative risk in five climatic zones, with temperature anomalies modifying hydrometeorological dynamics more strongly in temperate than tropical areas. Climate covariates improved the prediction of case counts and outbreaks compared to baseline models at lead times of 1, 3, 6 and 12 months, and for the upcoming season 5 months ahead. Improvements were greatest in the Northeast, Southeast, and Midwest. Recent dengue incidence provided the greatest improvement at the 1-month lead, whereas combining incidence with climate information performed best at 3- and 6-month leads. We extended our framework to weekly state-level predictions of the season ahead to align with national public health planning. We proposed a multi-staged dengue forecasting approach to enhance early warning and response across Brazil.

Total serum perfluorooctane sulfonate (PFOS) concentration and its isomer composition describe different aspects of internal exposure, but their respective associations with depressive symptoms remain uncertain. We examined adults aged 18-25 years in NHANES August 2021-August 2023 using survey-weighted models of the Patient Health Questionnaire-9 (PHQ-9). Total measured PFOS was the sum of linear PFOS and pooled monomethyl-branched PFOS; composition was their log-ratio. The analytic cohort included 169 participants, with 147 complete cases for core adjustment. Median total PFOS was 1.70 ng/mL, the median linear fraction was 72.2%, and the weighted prevalence of PHQ-9 [&ge;]10 was 22.6%. In the joint adjusted model, a doubling of total PFOS at fixed composition corresponded to -0.49 PHQ-9 points (95% CI, -1.65 to 0.68); a doubling of the linear-to-branched ratio at fixed total corresponded to 0.21 points (-1.94 to 2.36). Algebraic rescaling across the reported interquartile ranges yielded differences of -0.58 points (-1.95 to 0.81) for total PFOS and 0.13 points (-1.16 to 1.41) for composition. The inverse continuous-score association weakened with adjustment, whereas the screening odds ratio primarily lost precision. Excluding three observations with an isomer below detection shifted both joint coefficients. The data therefore do not establish an independent composition association or equivalent isomer effects. Experimental evidence supports candidate oxidative, inflammatory, and synaptic pathways, but differential protein binding and elimination prevent a serum composition ratio from being interpreted directly as brain dose or toxicity. Larger longitudinal analyses with isomer-resolved exposure and toxicokinetic measurements are needed to test these pathways.

Population genomic studies have revealed strong regional structure in Plasmodium knowlesi across Southeast Asia. To facilitate routine genomic surveillance, we developed a targeted amplicon sequencing panel that preserves the large- and small-scale population genetic structure. We screened published genomes for short, highly polymorphic targets and a 30-SNP barcode. We validated multiplexed amplification and sequencing using a portable Oxford Nanopore Technologies MinION sequencer on 28 clinical infections from four southern Thai provinces collected between 2018-2023. Twenty-seven loci yielded sequence data and 23 infections with data from at least eight loci were retained for analysis. Across the 23 Thai infections and 104 published Malaysian genomes, 586 diverse SNPs were observed. The amplicon panel and 30-SNP barcode retained the major population structure observed in genome-wide data, including a clear distinction between Bornean and Peninsular Malaysian infections and two clusters in Borneo. Across newly sequenced Thai infections, 25 loci were diverse and showed substantial diversity (mean expected heterozygosity = 0.69). All infections were effectively monoclonal. Pairwise identity-by-descent revealed substantial recent relatedness, with 46 of 253 pairs at or above 0.5. Two Thai genetic groups (n = 12 and n = 11) were observed, mirroring previously observed structure in peninsular Malaysia. Groups were not significantly associated with province or collection year. In conclusion, targeted sequencing can reveal geographically informative population structure across scales and thus support surveillance.

Background Community-led interventions for female sex workers (FSW) can improve HIV prevention and treatment outcomes by lowering self-stigma and strengthening social cohesion to improve service uptake. In Zimbabwe, the AMETHIST intervention trial integrated peer-led microplanning with self-help groups for FSW and demonstrated improved HIV treatment outcomes. We investigated the role of social cohesion and self-stigma in explaining these findings. Method We conducted a mixed-method analysis of data collected during AMETHIST. We re-analysed qualitative data from interviews and group discussions to identify narratives linking self-stigma, engagement with microplanners and/or self-help groups and HIV service use. We analysed quantitative data from endline respondent-driven sampling surveys for differences in self-reported measures of social cohesion and self-stigma between trial arms, and between those who did and did not participate in intervention activities or access services. Results In both arms, FSW self-reported engaging with peer supporters, participating in self-help groups and accessing HIV services. Qualitative narratives suggested peer support and group participation could reduce self-stigma and support service uptake. Lower levels of self-stigma were seen in the intervention arm (58.6% vs 63.2%; aOR 0.77; 95% CI 0.61-0.86). Social cohesion exceeded 50% across the whole sample, but was not associated with study arm, nor with engagement in intervention-related activities. Discussion Although qualitative data suggested some FSW felt their uptake of services was influenced by intervention components, quantitative analyses did not suggest either variable was on the causal pathway to the outcomes of the intervention. A limitation may have been the measurement of these complex constructs and the high levels of social cohesion across both study arms.

Introduction Despite the significant burden of Shigellosis, routine surveillance systems in resource-limited settings lack predictive capabilities to guide proactive public health responses. Consequently, we developed and evaluated the accuracy, uncertainty and computational cost of multi-horizon forecasting models for Shigella incidence among children <5 years in rural western Kenya using 16-year longitudinal incidence time-series. Methods We estimated monthly Shigella incidence using data from three pediatric enteric studies (2008-2024), supplemented with Kenya Health Information System during inter-study gaps. Incidence estimates incorporated sampling, healthcare-seeking, stool-testing, and pathogen-specific weights, with bootstrap-derived 95% confidence intervals. Predictors included Google Trends health-related indicators, climate, temporal trends, and seasonality. Forecasts at 1-, 3-, and 6-month horizons were evaluated using rolling-origin validation. Following horizon-specific predictor selection using Boruta, 14 state-space, machine-learning, and hybrid models were compared using point-forecast and probabilistic metrics, bootstrap confidence intervals, and Model Confidence Sets. Adaptive conformal prediction recalibrated prediction intervals, while Shapley Additive exPlanations assessed predictor contributions. Results Mean monthly Shigella incidence was 2.51 per 100 child-years (standard deviation=1.85). Shigella dynamics in Kenya can be forecast beyond seasonal persistence alone for up to six months reducing absolute errors by ~ 33%. However, optimal model architectures and predictor sets shift systematically across forecasting horizons. Temporal trend and recent incidence were the dominant predictors, while predictor importance shifted with forecast horizon toward syndromic, pharmaceutical, seasonal, and lagged environmental signals. Extreme Gradient Boosting performed best at 1 month (Root mean squared error [RMSE]=1.86), remained competitive at 3 months (RMSE=1.89), whereas exponential smoothing (ETS) performed best at 6 months (RMSE=2.05). Forecast accuracy declined with increasing horizon. ETS and TBATS provided the best-calibrated prediction intervals, whereas machine-learning models generally under-covered. Model Confidence Sets demonstrated horizon-dependent model distinguishability. Conclusion Horizon-specific model selection balancing predictive accuracy, uncertainty calibration, and computational feasibility could strengthen operational early warning systems for shigellosis.

Clinical metagenomic sequencing detects hundreds of organisms whose relevance to infection requires clinicians' manual interpretation. We retrospectively reviewed 4,855 specimens from 19 sampling sites, linked 1.33 million detected organisms to clinical culprit-pathogen determinations, and fine-tuned a large language model on these labels. The model recovered 92.44% of reference culprit calls in the held-out test set and 84.49% in a later cohort. In a blinded re-adjudication of 215 cases, the model matched 90.5% of culprit calls on which both expert reviewers agreed. Adjudication accuracy varied with organism identity and specimen types. mNGS-derived metrics such as read counts or genome coverage alone were insufficient to identify culprit pathogens, and organisms classified as common colonizers could still be judged causative. Certain organisms were frequently co-reported as causative in the same specimen. These findings support further evaluation of human and AI-assisted report review, particularly regarding missed pathogens, excess calls, and differences across specimen types.