High Norovirus False Discovery Rates and Noro-1 Assay Cross-Reactivity in the BioFire FilmArray Gastrointestinal Panel

medRxiv · 2026-05-20

Abstract Molecular syndromic panels such as the BioFire FilmArray Gastrointestinal Panel (BF-GIP) have been widely adopted for gastrointestinal illness diagnosis due to their fast turnaround times and broad pathogen coverage. Recently, the BF-GIP demonstrated increased rates of norovirus false-positive detections, prompting a Class II recall of more than two million tests in February 2024. We examined the prevalence of BF-GIP norovirus false positives across four hospitals from December 2024 to June 2025. Among 185 BF-GIP norovirus-positive results confirmed with the BD MAX Enteric Viral Panel, the false discovery rate ranged from 31 to 74% across sites, with the highest rate seen at a specialized cancer care hospital. Deep sequencing of BF-GIP pouches (n=42) confirmed the Noro-1 assay as the primary source of off-target amplification, identifying 78 off-target species – predominantly commensal stool bacteria – compared to only two species for the Noro-2 assay. Off-target species amplified by the Noro-1 assay were recovered from both false-positive and true-negative pouches, suggesting no single species accounted for the false-positive results. Partial primer complementarity at off-target loci and amplicon Tm values within the acceptable range support mispriming of gut microbiota as the underlying cause. False-positive pouches exhibited significantly higher Cp values than true positives for both assays (Noro-1: 26.6 vs. 11.1, p=0.013; Noro-2: 30.0 vs. 13.1, p<0.001), consistent with low-level off-target amplification. These findings highlight the high false discovery rate of the Noro-1 assay, identify bacterial species involved in mispriming, and demonstrate the need to redesign this assay to ensure reliable testing and improved patient care. Importance Syndromic molecular panels have revolutionized gastrointestinal diagnostics. However, recent data have suggested significant norovirus false-positive results associated with the BioFire FilmArray Gastrointestinal Panel. Here, we investigate a major diagnostic failure associated with the 2024 Class II recall of this assay, revealing norovirus false discovery rates as high as 74% in certain clinical settings. By deep sequencing amplicons from FilmArray pouches, we identified widespread cross-reactivity of the Noro-1 assay with stool microbiome nucleic acid. Off-target Noro-1 amplicons were detected from 78 bacterial species across 42 clinical pouches. For 16 species with the highest read counts per pouch, amplicons mapped to discrete genomic loci with partial primer overlap, consistent with mispriming. The identification of these discrete loci, combined with the repeatedly high false discovery rate reported across multiple studies, creates a clinical imperative to redesign this assay. Our work also highlights the ongoing need for rigorous post-market surveillance and the utility of deep sequencing in troubleshooting diagnostic assay failures.

Impact of Azithromycin Administration at Hospital Discharge on Antimicrobial Resistance and Enteropathogen Carriage 3 Months Following Treatment

medRxiv · 2026-04-20

Abstract Background The Toto Bora trial tested whether a course of azithromycin reduced rates of re-hospitalization or death in the 6 months following hospitalization among Kenyan children. We hypothesized that azithromycin would reduce enteric bacteria and increase carriage of macrolide resistance in the subsequent 3 months. Methods Kenyan children (1-59 months) hospitalized and subsequently discharged for non-traumatic conditions provided fecal samples before and 3 months after randomization to a 5-day course of azithromycin or placebo. Quantitative PCR identified enteropathogens and AMR-conferring genes in fecal samples. Generalized estimating equations assessed the impact of the randomization arm on pathogen and resistance gene detection, accounting for baseline presence and site. Results Among 1,393 baseline stools, 12.4% had at least one bacterial enteropathogen, 94.7% had at least one macrolide-resistance gene, and 92.6% had at least one beta-lactamase-resistance gene identified. At month 3, children randomized to azithromycin had a 6.1% higher likelihood of carrying a macrolide resistance gene compared to placebo (adjusted prevalence ratio [aPR], 1.06; 95% CI, 1.04–1.08; P<0.001). Specifically, azithromycin randomization was associated with a higher relative prevalence of erm(B) (aPR, 1.09 [95% CI, 1.04-1.15]; P=0.001), erm(C) (aPR, 1.23 [95% CI, 1.14-1.31]; P<0.001), msr(A) (aPR, 1.14 [95% CI, 1.04-1.25]; P=0.007), and msr(D) (aPR, 1.07 [95% CI, 1.03-1.11]; P=0.001). There was no difference in overall bacterial pathogen prevalence (18.9% vs 17.3%) between randomization arms, but a slightly lower proportion of children had Shigella after randomization in the azithromycin arm (3% vs. 5%, aPR, 0.79 [95% CI, 0.62, 1.01]; P=0.063). Interpretation Azithromycin at hospital discharge was associated with higher carriage of macrolide-resistance-conferring genes in the post-discharge period compared with placebo, without significant declines in enteric pathogen carriage other than modest changes to Shigella . The potential benefits and risks of empiric azithromycin need to be considered, as children are increasingly exposed to this broad-spectrum antibiotic.

Impaired acid stress resistance in <i>Salmonella</i> Typhi Ty2

bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-10

Abstract Salmonella enterica encounters acid stress during gastrointestinal transit and within the phagosomal environment of macrophages. Acid stress resistance has been well characterized in Salmonella enterica serovar Typhimurium, but comparative studies in the human-adapted Salmonella enterica serovar Typhi are limited. We compared the growth of S. Typhimurium 14028s and S. Typhi Ty2 at pH values ranging from 3-8 and observed that Salmonella enterica serovar Typhimurium exhibits enhanced growth at pH 4.5 compared to S. Typhi. Comparative transcriptomic profiling of S. Typhimurium and S. Typhi at pH 4.5 and 7.5 identified numerous differentially expressed acid-induced genes (DEGs), including genes encoding membrane proteins (OmpC, PhoE, HydB), a transcriptional regulator (RpoS), and stress response proteins (YciG, STM14_1829, YmdF). Targeted deletion of selected genes in S. Typhimurium significantly suppressed growth at acidic pH, confirming their role in acid stress resistance. These resistance mechanisms are compromised in S. Typhi due to pseudogenization. Heterologous expression of pseudogenized genes in S. Typhi restored acid tolerance. Collectively, these findings suggest that S. Typhi has lost the ability to withstand acid stress due to genomic decay and the loss of multiple genes essential for acid survival in S. Typhimurium, reflecting divergent evolutionary paths in these two serovars. Importance Salmonella Typhimurium must adapt to acidic pH conditions in the intestinal tract and the intracellular environment to cause infection. In this study, we show that the enteric fever serovar Salmonella Typhi exhibits impaired growth at pH 4.5, in comparison to Salmonella Typhimurium. We further show that the loss of specific membrane proteins, a transcriptional regulator, and a family of stress response proteins in Salmonella Typhi are responsible for this difference. Collectively, these observations suggest that Salmonella Typhi has evolutionarily lost the ability to withstand acid stress due to differences in its interaction with the human host. This has important implications for the pathogenesis of typhoid fever.

Clusters of Invasive <i>Haemophilus influenzae</i> Type b Disease Among Adults Using Substances or Experiencing Homelessness or Housing Instability — Alaska, Oregon, and Washington, 2023─2025

MMWR Morbidity and Mortality Weekly Report · 2026-04-16

Since the introduction and widespread use of Haemophilus influenzae type b (Hib) conjugate vaccines, invasive Hib disease has become rare in the United States, and outbreaks are uncommon.During April 2023-December 2025, two genetically distinct clusters comprising 44 cases of invasive Hib disease in adults were identified: one in Alaska (14 cases) and a second spanning Washington (23) and Oregon (seven).Cases were identified through routine surveillance or notification from a hospital, and clusters were identified via whole-genome sequencing.The median patient age was 53.5 years; 43 (98%) persons had bacteremia and 42 (95%) had pneumonia.Among the 44 patients, 34 (77%) smoked one or more substances, 34 (77%) used illicit substances, and 30 (68%) were experiencing homelessness or housing instability.Overall, 40 (91%) patients did not have documentation of receipt of Hib vaccination; 35 (88%) of those would not have been eligible for routine Hib conjugate vaccination as children because they would have been older than the recommended age range for vaccination at the time the vaccine was introduced.These emerging Hib clusters reveal that adults, particularly those using substances or experiencing homelessness or housing instability, are at risk for this otherwise rare vaccine-preventable disease.Data to guide an optimal public health response to these clusters are limited.Expanding surveillance for invasive H. influenzae disease in adults could help assess the scope of this problem, identify future outbreaks, and guide the development and implementation of strategies for prevention.

Sexually transmitted enteric infections in men who have sex with men

Clinical Microbiology Reviews · 2025-09-17 · 1 citations

SUMMARY Sexual transmission of enteric infections (STEIs) among men who have sex with men (MSM) has been reported since the 1960s and is increasingly recognized since the widespread adoption of multiplex molecular diagnostics. However, the overall burden of sexually transmitted enteric infections has been difficult to ascertain, as the public health response to these infections and identification of transmission networks fall between the traditional groupings of sexually transmitted and foodborne diseases. The global emergence of extensively drug-resistant Shigella and Campylobacter infections among MSM and the potential for cross-over between different at-risk populations underscores the importance of timely diagnosis, appropriate treatment, and the need to consider community-level education and testing. Moreover, the possible impact of pre- and post-exposure prophylaxis for HIV and sexually transmitted infections on STEI is presently uncertain. This review examines our evolving understanding of STEI, discusses specific pathogens of urgent importance, and prioritizes areas for further study.

Validation of H5 influenza virus subtyping RT-qPCR assay and low prevalence of H5 detection in 2024-2025 influenza virus season

medRxiv · 2025-03-17 · 1 citations

Abstract A sustained outbreak of H5N1 influenza virus among wild fowl and domestic livestock has caused more than 70 zoonotic infections in humans in the United States, including one death. The Centers for Disease Control and Prevention has recommended rapid H5 subtyping for all hospitalized cases with influenza A virus infection to enable prompt initiation of antiviral treatment, as well as infection prevention and implementation of public health measures to control spread. To address these needs, we developed a multiplex RT-qPCR assay to subtype H5 influenza virus in nasal, nasopharyngeal, and conjunctival specimens with a limit of detection of 230 copies/mL. No cross-reactivity was observed with other common respiratory viruses, including seasonal H3N2 and H1N1 influenza A viruses. We retrospectively subtyped 590 influenza A-positive clinical specimens processed by University of Washington labs between March 2024 and February 2025, including 512 specimens collected during the 2024-2025 influenza season, and detected no H5 positives. After clinical implementation, we performed 85 clinically ordered H5 subtyping tests in February 2025 and again detected no positives. This work enhances clinical pandemic preparedness activities and highlights the exceedingly low prevalence of H5N1 influenza virus during the 2024-2025 respiratory season. Importance statement The spread of H5N1 influenza virus in the United States has led to the culling of almost 200 million birds, infected cow herds across 17 states, and resulted in 70 human infections as of March 2025. Rapid PCR subtyping of H5 influenza virus is critical to inform hospital infection prevention and public health to enable containment of viral transmission. Here, we report the design, validation, and clinical implementation of a multiplex H5-subtyping RT-qPCR assay for nasopharyngeal, nasal, and conjunctival swab specimens. Additionally, we offer the largest reported study of H5 subtyping of influenza A-positive specimens in the United States to date. No H5 infections were detected in 675 samples collected between March 2024 and February 2025 from patients with confirmed influenza A virus infection at a large academic medical center in Seattle, WA.

Multiplex Polymerase Chain Reaction Panels for Gastrointestinal Infections: Current Evidence, Regulatory Hurdles, and the Way Forward

Open Forum Infectious Diseases · 2025-07-30 · 5 citations

Syndromic multiplex polymerase chain reaction (PCR) panels have revolutionized the diagnosis of gastrointestinal infections, allowing the rapid and simultaneous detection of multiple pathogens, including rare or difficult-to-identify organisms, with superior analytic sensitivity as compared with conventional methods. Although multiplex PCR panels are costly, their costs are offset by lower health care costs resulting from improved diagnostic accuracy and more targeted therapy. However, significant barriers to reimbursement may discourage providers from ordering PCR panels or incentivize them to use smaller panels that are less comprehensive. Addressing these challenges will require a collaborative effort, including regulators, payors, and clinicians. Key steps will include updating clinical guidelines to better define appropriate utilization of gastrointestinal panels, harmonizing reimbursement criteria to align with evidence-based practice, and modernizing diagnostic codes for acute gastroenteritis to match payors' requirements. These reforms will be essential to improve access to advanced diagnostics and ensure better patient care.

Impact of Macrolide Resistance on Azithromycin for Prevention of Rehospitalization or Death Among Children Discharged From Hospitals in Western Kenya

The Journal of Infectious Diseases · 2025-04-21 · 1 citations

BACKGROUND: The Toto Bora trial tested whether a 5-day course of azithromycin reduced the risk of rehospitalization or death in the 6 months following hospitalization among Kenyan children and found no overall benefit. We hypothesized that macrolide resistance in gut microbes could modify azithromycin's effect. METHODS: From June 2016 to November 2019, Kenyan children aged 1-59 months were enrolled at hospital discharge and randomized to azithromycin or placebo. DNA from fecal samples and Escherichia coli isolates was analyzed for common macrolide resistance genes. Cox proportional hazards regression models, including interaction terms between randomization arm and individual macrolide resistance genes, were used to analyze time to rehospitalization or death, with Bonferroni correction applied to account for multiple comparisons. RESULTS: Among 1393 children tested, 94.7% had at least 1 macrolide resistance gene in their fecal DNA at hospital discharge, most commonly mph(A) (68.6% [955/1393]), followed by msr(D) (67.3% [937/1393]) and erm(B) (60.7% [846/1393]). Mef(A) (23.7% [330/1393]) was the only macrolide resistance gene that modified azithromycin's effect on rehospitalization or death (interaction P = .008). In children without the mef(A) gene, azithromycin reduced the hazard of rehospitalization or death by a third (hazard ratio [HR], 0.66 [95% confidence interval {CI}, .45-.99]) whereas among children with the mef(A) gene, there was a higher risk in those randomized to azithromycin (HR, 2.72 [95% CI, 1.21-6.09]). The effect size of azithromycin's impact on mortality and rehospitalization as separate outcomes in children with and without mef(A) were consistent but underpowered. CONCLUSIONS: Macrolide resistance in the gut microbiome may influence the efficacy of azithromycin in children discharged from the hospital. Clinical Trials Registration. NCT02414399.

Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A

PLoS Pathogens · 2025-05-02 · 3 citations

Nontyphoidal and enteric fever serovars of Salmonella enterica display distinctive interactions with serum antibodies and the complement system, which initiate the host immune response to invading microbes. This study examines the contributions of lipopolysaccharide O-antigen (O-ag) and the S. Typhi Vi polysaccharide capsule to serum resistance, complement activation and deposition, and immunoglobulin (Ig) binding in nontyphoidal S. enterica serovar Typhimurium and the enteric fever serovars S. Typhi and S. Paratyphi A. Although all three serovars are resistant to serum killing, S. Typhi and S. Paratyphi A exhibit lower levels of Ig binding, complement binding and complement activation compared to S. Typhimurium. In S. Typhimurium, WzzB-dependent long O-antigen (L O-ag) production with 16-to-35 repeating O-ag units, and FepE-dependent very long O-antigen (VL O-ag) production with over 100 repeating O-ag units, are required for serum resistance but do not prevent IgM binding or complement deposition. S. Typhi lacks VL O-ag, but its production of Vi capsule inhibits IgM binding and complement deposition, while acting in concert with L O-ag to resist serum killing. In S. Paratyphi A, L O-ag production is deficient due to a hypofunctional WzzB protein, but this is compensated by greater quantities of VL O-ag, which are required for serum resistance. Restoration of WzzB function by exchange with the S. Typhimurium or S. Typhi wzzB alleles can restore L O-ag production in S. Paratyphi A but decreases VL O-ag production, resulting in increased IgM binding. Replacement of the S. Paratyphi A O2-type polysaccharide with the S. Typhi O9 polysaccharide further increases IgM binding of S. Paratyphi A, which enhances complement activation but not complement deposition. Lastly, a gene duplication of rfbV in S. Paratyphi A is necessary for higher levels of VL O-ag and resistance to complement deposition and antibody binding. Collectively, these observations demonstrate fundamental differences between nontyphoidal and enteric fever Salmonella serovars in their interactions with innate immune effectors. Whereas nontyphoidal S. Typhimurium elicits, exploits and withstands the host acute inflammatory response, the enteric fever serovars S. Typhi and S. Paratyphi A evade it by limiting antibody recognition and complement activation and deposition.

A cytochrome <i>bd</i> repressed by a MarR family regulator confers resistance to metals, nitric oxide, sulfide, and cyanide in <i>Chromobacterium violaceum</i>

Applied and Environmental Microbiology · 2025-01-24 · 5 citations

ABSTRACT Chromobacterium violaceum is a ubiquitous environmental pathogen. Despite its remarkable adaptability, little is known about the mechanisms of stress resistance in this bacterium. Here, in a screen for iron-susceptible transposon mutants, we identified a cytochrome bd that protects C. violaceum against multiple stresses. The two subunits of this cytochrome bd (CioAB) are encoded by the cioRAB operon, which also encodes a GbsR-type MarR family transcription factor (CioR). A ∆ cioAB mutant strain was sensitive to iron and the iron-requiring antibiotic streptonigrin and showed a decrease in siderophore production. Growth curves and survival assays revealed that the ∆ cioAB strain was also sensitive to zinc, hydrogen peroxide, nitric oxide, sulfide, and cyanide. Expression analysis showed that the promoter activity of the cioRAB operon and the transcript levels of the cioAB genes were increased in a ∆ cioR mutant. CioR bound the promoter region of the cio operon in vitro , indicating that CioR is a direct repressor of its own operon. Expression of the cio operon increased at high cell density and was dependent on the quorum-sensing regulator CviR. As cyanide is also a signal for cio expression, and production of endogenous cyanide is known to be a quorum sensing-regulated trait in C. violaceum , we suggest that CioAB is a cyanide-insensitive terminal oxidase that allows respiration under cyanogenic growth conditions. Our findings indicate that the cytochrome bd CioAB protects C. violaceum against multiple stress agents that are potentially produced endogenously or during interactions with a host. IMPORTANCE The terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome bd ) to catalyze the reduction of molecular oxygen to water. Chromobacterium violaceum is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The C. violaceum genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome bd from C. violaceum , protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers C. violaceum resistance to iron, zinc, and hydrogen peroxide. This cytochrome bd is encoded by the cioRAB operon, which is under direct repression by the MarR-type regulator CioR. In addition, the cioRAB operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.