A tollgate for typhoid.

Humans are uniquely susceptible to typhoid fever caused by infection with Salmonella enterica serovar Typhi. Mathur et al. now report that mice lacking Toll-like receptor 11, which is absent in humans, can be lethally infected with S. Typhi, a breakthrough that promises to speed the development of better vaccines.

Cluster of Nontoxigenic Corynebacterium diphtheriae Infective Endocarditis and Rising Background C. diphtheriae Cases-Seattle, Washington, 2020-2023.

BACKGROUND: Nontoxigenic Corynebacterium diphtheriae, often associated with wounds, can rarely cause infective endocarditis (IE). Five patients with C. diphtheriae IE were identified within 12 months at a Seattle-based hospital system. We reviewed prior C. diphtheriae-positive cultures to determine if detections had increased over time and evaluated epidemiologic trends. METHODS: We conducted a formal electronic health record search to identify all patients aged ≥18 years with C. diphtheriae detected in a clinical specimen (ie, wound, blood, sputum) between 1 September 2020 and 1 April 2023. We collected patient demographics, housing status, comorbidities, substance-use history, and level of medical care required at detection. We extracted laboratory data on susceptibilities of C. diphtheriae isolates and on other pathogens detected at the time of C. diphtheriae identification. RESULTS: Between 1 September 2020 and 1 April 2023, 44 patients (median age, 44 years) had a C. diphtheriae-positive clinical culture, with most detections occurring after March 2022. Patients were predominantly male (75%), White (66%), unstably housed (77%), and had a lifetime history of injecting drugs (75%). Most C. diphtheriae-positive cultures were polymicrobial, including wound cultures from 36 (82%) patients and blood cultures from 6 (14%) patients, not mutually exclusive. Thirty-four patients (77%), including all 5 patients with C. diphtheriae IE, required hospital admission for C. diphtheriae or a related condition. Of the 5 patients with IE, 3 died of IE and 1 from COVID-19. CONCLUSIONS: Findings suggest a high-morbidity outbreak disproportionately affecting patients who use substances and are unstably housed.

Epidemiology and Economic Burden of Acute Infectious Gastroenteritis Among Adults Treated in Outpatient Settings in US Health Systems.

INTRODUCTION: Acute infectious gastroenteritis (AGE) is a common reason for outpatient visits and hospitalizations in the United States. This study aimed to understand the demographic and clinical characteristics, common pathogens detected, health care resource utilization (HRU), and cost among adult outpatients with AGE visiting US health systems. METHODS: A retrospective cohort study was conducted using one of the largest hospital discharge databases (PINC AI Healthcare Database) in the United States. Adult patients (aged ≥18 years) with a principal diagnosis of AGE during an outpatient visit between January 1, 2016, and June 30, 2021, were included. Pathogen detection analysis was performed in those with microbiology data available. RESULTS: Among 248,896 patients, the mean age was 44.3 years (range 18-89+ years), 62.9% were female, and 68.5% were White. More than half (62.0%) of the patients did not have any preexisting comorbidity, and only 18.3% underwent stool workup at the hospital. Most patients (84.7%) were seen in the emergency department, and most (96.4%) were discharged home. Within 30 days of discharge, 1.0% were hospitalized, and 2.8% had another outpatient visit due to AGE. The mean cost of the index visit plus 30-day AGE-related follow-up was $1,338 per patient, amounting to $333,060,182 for the total study population. Among patients with microbiology data available (n = 12,469), common pathogens detected were Clostridioides difficile (32.2%), norovirus (6.3%), and Campylobacter spp. (4.0%). DISCUSSION: AGE is a common and costly disease affecting adults of all ages and more females than males, including individuals with or without baseline conditions in a hospital-based outpatient setting. C. difficile was the most common pathogen detected.

Relationship between Diagnostic Method and Pathogen Detection, Healthcare Resource Use, and Cost in U.S. Adult Outpatients Treated for Acute Infectious Gastroenteritis.

A retrospective observational study was performed to assess the relationship between diagnostic method (traditional work-up [TW], multiplex PCR panel with < 12 target pathogens [PCR < 12], or multiplex PCR panel with ≥ 12 target pathogens [PCR12]), and diagnostic yield, health care resource use (HRU), and cost in adult outpatients visiting U.S. hospitals for acute infectious gastroenteritis (AGE). Using data from PINC AI Healthcare Database during January 1, 2016-June 30, 2021, we analyzed adult patients with an AGE diagnosis and stool testing performed during an outpatient visit. Detection rates for different pathogens were analyzed for those with microbiology data available. Among 36,787 patients, TW was most often performed (57.0%). PCR12 testing was more frequent in patients from large, urban, and teaching hospitals, compared to TW (all P < 0.01). PCR12 was associated with a higher mean index visit cost (by $97) but lower mean 30-day AGE-related follow-up cost (by $117) than TW. Patients with PCR12 had a lower 30-day AGE-related hospitalization risk than TW (1.7% versus 2.7% P < 0.01). Among the 8,451 patients with microbiology data, PCR12 was associated with fewer stool tests per patient (mean 1.61 versus 1.26), faster turnaround time (mean 6.3 versus 25.7 h) and lower likelihood of receiving in-hospital antibiotics (39.4% versus 47.1%, all P < 0.01) than TW. A higher percentage of patients with PCR12 had a target pathogen detected (73.1%) compared to PCR < 12 (63.6%) or TW (45.4%, P < 0.01). Thus, we found that large multiplex PCR panels were associated with lower 30-day AGE-related follow-up cost and risk of AGE-related hospitalization, and increased diagnostic yield compared to TW.

Cyclopropane Fatty Acids Are Important for Salmonella enterica Serovar Typhimurium Virulence.

A variety of eubacteria, plants, and protozoa can modify membrane lipids by cyclopropanation, which is reported to modulate membrane permeability and fluidity. The ability to cyclopropanate membrane lipids has been associated with resistance to oxidative stress in Mycobacterium tuberculosis, organic solvent stress in Escherichia coli, and acid stress in E. coli and Salmonella. In bacteria, the cfa gene encoding cyclopropane fatty acid (CFA) synthase is induced during the stationary phase of growth. In the present study, we constructed a cfa mutant of Salmonella enterica serovar Typhimurium 14028s (S. Typhimurium) and determined the contribution of CFA-modified lipids to stress resistance and virulence in mice. Cyclopropane fatty acid content was quantified in wild-type and cfa mutant S. Typhimurium. CFA levels in the cfa mutant were greatly reduced compared to CFA levels in the wild type, indicating that CFA synthase is the major enzyme responsible for cyclopropane modification of lipids in Salmonella. S. Typhimurium cfa mutants were more sensitive to extreme acid pH, the protonophore CCCP, and hydrogen peroxide compared to the wild type. In addition, cfa mutants exhibited reduced viability in murine macrophages and could be rescued by the addition of the NADPH phagocyte oxidase inhibitor diphenyleneiodonium (DPI) chloride. S. Typhimurium lacking cfa was also attenuated for virulence in mice. These observations indicate that CFA modification of lipids makes an important contribution to Salmonella virulence.

Availability of Ferritin-Bound Iron to Enterobacteriaceae.

The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.

Elevated White Blood Cell Count Does Not Predict Clostridium difficile Nucleic Acid Testing Results.

BACKGROUND: An elevated white blood cell count (WBC; >15 000/µL) is an established prognostic marker in patients with Clostridium difficile infection (CDI). Small observational studies have suggested that a markedly elevated WBC should prompt consideration of CDI. However, there is limited evidence correlating WBC elevation with the results of C. difficile nucleic acid amplification testing (NAAT). METHODS: Retrospective review of laboratory testing, outcomes, and treatment of 16 568 consecutive patients presenting to 4 hospitals over 4 years with NAAT and WBC testing on the same day. RESULTS: No significant relationship between C. difficile NAAT results and concurrent WBC in the inpatient setting was observed. Although an elevated WBC did predict NAAT results in the outpatient and emergency department populations (P < .001), accuracy was poor, with receiver-operator areas under the curve of 0.59 and 0.56, respectively. An elevated WBC (>15 000/µL) in CDI was associated with a longer median hospital length of stay (15.5 vs 11.0 days; P < .01), consistent with leukocytosis as a prognostic marker in CDI. NAAT-positive inpatients with elevated WBC were more likely to be treated with metronidazole and/or vancomycin (relative ratio, 1.2; 95% confidence interval [CI], 1.1-1.3) and die in the hospital (relative ratio, 2.9; 95% CI, 2.0-4.3). CONCLUSIONS: Although WBC is an important prognostic indicator in patients with CDI, an isolated WBC elevation has low sensitivity and specificity as a predictor of fecal C. difficile NAAT positivity in the inpatient setting. A high or rising WBC in isolation is not a sufficient indication for CDI testing.

COVID-19-Lessons Learned and Questions Remaining.

In this article, the editors of Clinical Infectious Diseases review some of the most important lessons they have learned about the epidemiology, clinical features, diagnosis, treatment and prevention of SARS-CoV-2 infection and identify essential questions about COVID-19 that remain to be answered.

The intracellular pathogen concept.

The intracellular pathogen concept classifies pathogenic microbes on the basis of their site of replication and dependence on host cells. This concept played a fundamental role in establishing the field of cellular microbiology, founded in part by Dr. Pascale Cossart, whose seminal contributions are honored in this issue of Molecular Microbiology. The recognition that microbes can access and replicate in privileged compartments within host cells has led to many new and fruitful lines of investigation into the biology of the cell and mechanisms of cell-mediated immunity. However, like any scientific concept, the intracellular pathogen concept can become a dogma that constrains thinking and oversimplifies complex and dynamic host-pathogen interactions. Growing evidence has blurred the distinction between "intracellular" and "extracellular" pathogens and demonstrated that many pathogens can exist both within and outside of cells. Although the intracellular pathogen concept remains useful, it should not be viewed as a rigid classification of pathogenic microbes, which exhibit remarkable variation and complexity in their behavior in the host.

The Laboratory Diagnosis of Coronavirus Disease 2019- Frequently Asked Questions.

Diagnostic testing has played and will continue to play a major role in the coronavirus disease 2019 (COVID-19) pandemic. The ability to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory secretions is essential to determine when an individual is infected and potentially infectious to others. Viral detection is used for the identification, management, and isolation of individual patients. Viral detection is also used to determine when the virus has entered a community and how rapidly it is spreading. As communities attempt to reopen following periods of shutdown, the detection of both SARS-CoV-2 and specific antibodies that recognize the virus will become increasingly important as a means to assess infection and immunity in individuals and communities. Here, we discuss questions commonly asked by clinicians about COVID-19 diagnostic testing.

Gastroenteritis in Men Who Have Sex With Men in Seattle, Washington, 2017-2018.

BACKGROUND: Men who have sex with men (MSM) are at risk for sexual transmission of enteric pathogens. The microbiology of gastroenteritis in MSM has not been examined since the advent of antiretroviral therapy and molecular diagnostics. Our objective was to assess the causes of gastroenteritis among MSM living with and without human immunodeficiency virus (HIV) coinfection in Seattle, Washington. METHODS: We conducted a retrospective cohort study of 235 MSM who underwent multiplex stool polymerase chain reaction (PCR) testing between 1 January 2017 and 1 June 2018. We abstracted clinical and laboratory data from electronic medical records. Parallel or reflexive culture and susceptibility testing were performed when PCR detected cultivable pathogens. RESULTS: Among 235 MSM tested (268 episodes), 131 had 151 episodes with positive test results. 148 (63.0%) individuals were living with HIV. Among positive tests, 88.7% detected a bacterial pathogen, 26% a virus, and 40% a parasite. Diarrheagenic Escherichia coli (enteroaggretative, enteropathogenic), Shigella, and Campylobacter were the most commonly detected bacteria (33.1%, 30.5%, and 17.2% of positive samples, respectively). Forty-three percent of positive specimens had ≥2 pathogens. Etiologies and clinical presentations were similar between men living with and without HIV. Cultured Shigella and Campylobacter isolates were frequently resistant to multiple antibiotics. CONCLUSIONS: MSM present with gastroenteritis from varied pathogens, including some not detected by conventional stool culture. High levels of antibiotic resistance are consistent with frequent antibiotic exposure in this population and the transmission of multiresistant strains. New approaches are needed to detect, treat, and prevent enteric infections in MSM.

International Spread of Multidrug-Resistant Campylobacter coli in Men Who Have Sex With Men in Washington State and Québec, 2015-2018.

BACKGROUND: Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species. METHODS: This was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS). RESULTS: We report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information's Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate. CONCLUSIONS: As previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data.

Streptococcus pyogenes pbp2x Mutation Confers Reduced Susceptibility to ß-Lactam Antibiotics.

Two near-identical clinical Streptococcus pyogenes isolates of emm subtype emm43.4 with a pbp2x missense mutation (T553K) were detected. Minimum inhibitory concentrations (MICs) for ampicillin and amoxicillin were 8-fold higher, and the MIC for cefotaxime was 3-fold higher than for near-isogenic control isolates, consistent with a first step in developing ß-lactam resistance.

Modulation of H-NS transcriptional silencing by magnesium.

The bacterial histone-like protein H-NS silences AT-rich DNA, binding DNA as 'stiffened' filaments or 'bridged' intrastrand loops. The switch between these modes has been suggested to depend on the concentration of divalent cations, in particular Mg2+, with elevated Mg2+ concentrations associated with a transition to bridging. Here we demonstrate that the observed binding mode is a function of the local concentration of H-NS and its cognate binding sites, as well as the affinity of the interactions between them. Mg2+ does not control a binary switch between these two modes but rather modulates the affinity of this interaction, inhibiting the DNA-binding and silencing activity of H-NS in a continuous linear fashion. The direct relationship between conditions that favor stiffening and transcriptional silencing activity suggests that although both modes can occur in the cell, stiffening is the predominant mode of binding at silenced genes.

Rapid and Extensive Expansion in the United States of a New Multidrug-resistant Escherichia coli Clonal Group, Sequence Type 1193.

We describe the rapid and ongoing emergence across multiple US cities of a new multidrug-resistant Escherichia coli clone-sequence type (ST) 1193-resistant to fluoroquinolones (100%), trimethoprim-sulfamethoxazole (55%), and tetracycline (53%). ST1193 is associated with younger adults (age <40 years) and currently comprises a quarter of fluoroquinolone-resistant clinical E. coli urine isolates.

Long-Term Carriage of Medicopsis romeroi, an Agent of Black-Grain Mycetoma, Presenting as Phaeohyphomycosis in a Renal Transplant Patient.

Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a key feature of coelomycetes. Immunocompromised patients are at risk of these infections, even after remote exposure, and typically present with phaeohyphomycoses without dissemination. We present the case of a renal transplant recipient 6.5 years post-transplant who developed a slowly progressive soft tissue infection mimicking a synovial cyst. A cultured isolate was identified as Medicopsis romeroi by sequencing of multiple ribosomal loci. The patient responded well to debridement and posaconazole therapy. Solid-organ transplant patients are at risk of opportunistic fungal infection long after transplant, and molecular methods are often required for definitive identification.

Changes in molecular epidemiology and antimicrobial resistance profiles of Clostridioides (Clostridium) difficile strains in the United States between 2011 and 2017.

PCR ribotyping and antimicrobial susceptibility testing were used to characterize 940 Clostridioides (Clostridium) difficile isolates collected from 26 U S. hospitals over three time periods from 2011 to 2017. The proportion of ribotype (RT) 027 isolated during the three surveys decreased significantly over time from 31% in 2011-2012, to 22% in 2013-2014, and to 14% in 2015-2017 (p < 0.001 and p = 0.010, respectively), while we observed an increase in prevalence of RT106, that rose from 7% in our first survey to 19% of isolates in our last survey (p < 0.001). In addition, both RT056 and RT002 rose from 3% to 10% (p < 0.001). The proportions of all other ribotypes remained steady over time, and RT014/020 was the third most common strain type in our convenience sample in the final survey. Overall, resistance to moxifloxacin, rifampin, and vancomycin decreased during our studies, mainly due to the decline in RT027 isolates. A decrease in moxifloxacin resistance and an increase in tetracycline resistance were found among RT027 strains isolated in the last survey. Although the proportion of RT027 isolates declined, multidrug resistance among this ribotype continues to be common.

Testing Hypotheses on Risk Factors for Scientific Misconduct via Matched-Control Analysis of Papers Containing Problematic Image Duplications.

It is commonly hypothesized that scientists are more likely to engage in data falsification and fabrication when they are subject to pressures to publish, when they are not restrained by forms of social control, when they work in countries lacking policies to tackle scientific misconduct, and when they are male. Evidence to test these hypotheses, however, is inconclusive due to the difficulties of obtaining unbiased data. Here we report a pre-registered test of these four hypotheses, conducted on papers that were identified in a previous study as containing problematic image duplications through a systematic screening of the journal PLoS ONE. Image duplications were classified into three categories based on their complexity, with category 1 being most likely to reflect unintentional error and category 3 being most likely to reflect intentional fabrication. We tested multiple parameters connected to the hypotheses above with a matched-control paradigm, by collecting two controls for each paper containing duplications. Category 1 duplications were mostly not associated with any of the parameters tested, as was predicted based on the assumption that these duplications were mostly not due to misconduct. Categories 2 and 3, however, exhibited numerous statistically significant associations. Results of univariable and multivariable analyses support the hypotheses that academic culture, peer control, cash-based publication incentives and national misconduct policies might affect scientific integrity. No clear support was found for the "pressures to publish" hypothesis. Female authors were found to be equally likely to publish duplicated images compared to males. Country-level parameters generally exhibited stronger effects than individual-level parameters, because developing countries were significantly more likely to produce problematic image duplications. This suggests that promoting good research practices in all countries should be a priority for the international research integrity agenda.

Diagnostic Stewardship: Opportunity for a Laboratory-Infectious Diseases Partnership.

Recent advances in microbial diagnostics are providing clinicians with information about microbes causing infections and their resistance to antimicrobial agents more rapidly than ever before. Diagnostic stewardship refers to the appropriate use of laboratory testing to guide patient management, including treatment, in order to optimize clinical outcomes and limit the spread of antimicrobial resistance. Fulfilling the promise of diagnostic stewardship requires a seamless partnership between clinical laboratories, pharmacists, and infectious diseases clinicians, so that appropriate tests are ordered and diagnostic information is translated into appropriate management in real time.

Clinical Impact of a Multiplex Gastrointestinal Polymerase Chain Reaction Panel in Patients With Acute Gastroenteritis.

Background: Molecular syndromic diagnostic panels can enhance pathogen identification in the approximately 2-4 billion episodes of acute gastroenteritis that occur annually worldwide. However, the clinical utility of these panels has not been established. Methods: We conducted a prospective, multi-center study to investigate the impact of the BioFire FilmArray Gastrointestinal polymerase chain reaction panel on clinical diagnosis and decision-making, and compared the clinical acuity of patients with positive results obtained exclusively with the FilmArray with those detected by conventional stool culture. A total of 1887 consecutive fecal specimens were tested in parallel by FilmArray and stool culture. Laboratory and medical records were reviewed to determine rates of detection, turnaround times, clinical features, and the nature and timing of clinical decisions. Results: FilmArray detected pathogens in 35.3% of specimens, compared to 6.0% for culture. Median time from collection to result was 18 hours for FilmArray and 47 hours for culture. Median time from collection to initiation of antimicrobial therapy was 22 hours for FilmArray and 72 hours for culture. Patients diagnosed by FilmArray were more likely to receive targeted rather than empirical therapy, compared to those diagnosed by culture (P = .0148). Positive Shiga-like toxin-producing E. coli results were reported 47 hours faster with FilmArray and facilitated discontinuation of empirical antimicrobials. Patients diagnosed exclusively by FilmArray had clinical characteristics similar to those identified by culture. Conclusions: FilmArray markedly improved clinical sensitivity in patients with acute diarrhea, identified cases with clinical acuity comparable to those identified by culture, and enabled clinicians to make more timely and targeted therapeutic decisions.