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Disseminated histoplasmosis is a life-threatening condition in immunocompromised patients. The majority of healthy persons have benign disease not requiring treatment. However, in persons living with HIV, mortality is high and accurate diagnosis is paramount. We present a case of a 48-year-old HIV-positive woman who presented with haematuria and flank pain. She had a history of recurrent urinary tract infection and nephrolithiasis with obstructive hydronephrosis. During cystoscopy, a bladder lesion was found. Pathological evaluation demonstrated abundant intracellular organisms with apparent budding. Subsequent urine histoplasma antigen was negative. Given the high index of suspicion for histoplasmosis based on the surgical pathology findings and epidemiological history, the patient was started immediately on antifungal therapy. One week later, PCR results of the bladder lesion confirmed the presence of Histoplasma capsulatum. This case highlights a rare presentation of genitourinary histoplasmosis and the utility of surgical pathology evaluation and PCR for diagnosis.

BackgroundBaricitinib and dexamethasone have randomised trials supporting their use for the treatment of patients with COVID-19. We assessed the combination of baricitinib plus remdesivir versus dexamethasone plus remdesivir in preventing progression to mechanical ventilation or death in hospitalised patients with COVID-19.MethodsIn this randomised, double-blind, double placebo-controlled trial, patients were enrolled at 67 trial sites in the USA (60 sites), South Korea (two sites), Mexico (two sites), Singapore (two sites), and Japan (one site). Hospitalised adults (≥18 years) with COVID-19 who required supplemental oxygen administered by low-flow (≤15 L/min), high-flow (>15 L/min), or non-invasive mechanical ventilation modalities who met the study eligibility criteria (male or non-pregnant female adults ≥18 years old with laboratory-confirmed SARS-CoV-2 infection) were enrolled in the study. Patients were randomly assigned (1:1) to receive either baricitinib, remdesivir, and placebo, or dexamethasone, remdesivir, and placebo using a permuted block design. Randomisation was stratified by study site and baseline ordinal score at enrolment. All patients received remdesivir (≤10 days) and either baricitinib (or matching oral placebo) for a maximum of 14 days or dexamethasone (or matching intravenous placebo) for a maximum of 10 days. The primary outcome was the difference in mechanical ventilation-free survival by day 29 between the two treatment groups in the modified intention-to-treat population. Safety analyses were done in the as-treated population, comprising all participants who received one dose of the study drug. The trial is registered with ClinicalTrials.gov, NCT04640168.FindingsBetween Dec 1, 2020, and April 13, 2021, 1047 patients were assessed for eligibility. 1010 patients were enrolled and randomly assigned, 516 (51%) to baricitinib plus remdesivir plus placebo and 494 (49%) to dexamethasone plus remdesivir plus placebo. The mean age of the patients was 58·3 years (SD 14·0) and 590 (58%) of 1010 patients were male. 588 (58%) of 1010 patients were White, 188 (19%) were Black, 70 (7%) were Asian, and 18 (2%) were American Indian or Alaska Native. 347 (34%) of 1010 patients were Hispanic or Latino. Mechanical ventilation-free survival by day 29 was similar between the study groups (Kaplan-Meier estimates of 87·0% [95% CI 83·7 to 89·6] in the baricitinib plus remdesivir plus placebo group and 87·6% [84·2 to 90·3] in the dexamethasone plus remdesivir plus placebo group; risk difference 0·6 [95% CI -3·6 to 4·8]; p=0·91). The odds ratio for improved status in the dexamethasone plus remdesivir plus placebo group compared with the baricitinib plus remdesivir plus placebo group was 1·01 (95% CI 0·80 to 1·27). At least one adverse event occurred in 149 (30%) of 503 patients in the baricitinib plus remdesivir plus placebo group and 179 (37%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 7·5% [1·6 to 13·3]; p=0·014). 21 (4%) of 503 patients in the baricitinib plus remdesivir plus placebo group had at least one treatment-related adverse event versus 49 (10%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 6·0% [2·8 to 9·3]; p=0·00041). Severe or life-threatening grade 3 or 4 adverse events occurred in 143 (28%) of 503 patients in the baricitinib plus remdesivir plus placebo group and 174 (36%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 7·7% [1·8 to 13·4]; p=0·012).InterpretationIn hospitalised patients with COVID-19 requiring supplemental oxygen by low-flow, high-flow, or non-invasive ventilation, baricitinib plus remdesivir and dexamethasone plus remdesivir resulted in similar mechanical ventilation-free survival by day 29, but dexamethasone was associated with significantly more adverse events, treatment-related adverse events, and severe or life-threatening adverse events. A more individually tailored choice of immunomodulation now appears possible, where side-effect profile, ease of administration, cost, and patient comorbidities can all be considered.FundingNational Institute of Allergy and Infectious Diseases.

Background Clinical trials initiated during emerging infectious disease outbreaks must quickly enroll participants to identify treatments to reduce morbidity and mortality. This may be at odds with enrolling a representative study population especially when the population affected is undefined. Methods We evaluated the utility of CDC COVID-19-Associated Hospitalization Surveillance Network (COVID-NET), COVID-19 Case Surveillance System (CCSS), and 2020 U.S. Census data to determine demographic representation in the four stages of the Adaptive COVID-19 Treatment Trial (ACTT). We compared the cumulative proportion of participants by sex, race, ethnicity, and age enrolled at U.S. ACTT sites, with the respective 95% confidence intervals, to the reference data in forest plots. Results U.S. ACTT sites enrolled 3,509 adults hospitalized with COVID-19. When compared with COVID-NET, ACTT enrolled a similar or higher proportion of Hispanic or Latino and White participants depending on the stage, and a similar proportion of African American participants in all stages. In contrast, ACTT enrolled a higher proportion of these groups when compared with U.S. Census and CCSS. The proportion of participants ages 65 years was either similar or lower than COVID-NET and higher than CCSS and U.S. Census. The proportion of females enrolled in ACTT was lower than the proportion of females in the reference datasets. Conclusions While surveillance data of hospitalized cases may not be available early in an outbreak, it is a better comparator than U.S. Census data and surveillance of all cases, which may not reflect the population affected and at higher risk of severe disease.

We isolated a novel coronavirus from a medical team member presenting with fever and malaise after travel to Haiti. The virus showed 99.4% similarity with a recombinant canine coronavirus recently identified in a pneumonia patient in Malaysia, suggesting that infection with this virus and/or recombinant variants occurs in multiple locations.

Summary Background Functional impairment of interferon, a natural antiviral component of the immune system, is associated with the pathogenesis and severity of COVID-19. We aimed to compare the efficacy of interferon beta-1a in combination with remdesivir compared with remdesivir alone in hospitalised patients with COVID-19. Methods We did a double-blind, randomised, placebo-controlled trial at 63 hospitals across five countries (Japan, Mexico, Singapore, South Korea, and the USA). Eligible patients were hospitalised adults (aged ≥18 years) with SARS-CoV-2 infection, as confirmed by a positive RT-PCR test, and who met one of the following criteria suggestive of lower respiratory tract infection: the presence of radiographic infiltrates on imaging, a peripheral oxygen saturation on room air of 94% or less, or requiring supplemental oxygen. Patients were excluded if they had either an alanine aminotransferase or an aspartate aminotransferase concentration more than five times the upper limit of normal; had impaired renal function; were allergic to the study product; were pregnant or breast feeding; were already on mechanical ventilation; or were anticipating discharge from the hospital or transfer to another hospital within 72 h of enrolment. Patients were randomly assigned (1:1) to receive intravenous remdesivir as a 200 mg loading dose on day 1 followed by a 100 mg maintenance dose administered daily for up to 9 days and up to four doses of either 44 μg interferon beta-1a (interferon beta-1a group plus remdesivir group) or placebo (placebo plus remdesivir group) administered subcutaneously every other day. Randomisation was stratified by study site and disease severity at enrolment. Patients, investigators, and site staff were masked to interferon beta-1a and placebo treatment; remdesivir treatment was given to all patients without masking. The primary outcome was time to recovery, defined as the first day that a patient attained a category 1, 2, or 3 score on the eight-category ordinal scale within 28 days, assessed in the modified intention-to-treat population, defined as all randomised patients who were classified according to actual clinical severity. Safety was assessed in the as-treated population, defined as all patients who received at least one dose of the assigned treatment. This trial is registered with ClinicalTrials.gov , NCT04492475 . Findings Between Aug 5, 2020, and Nov 11, 2020, 969 patients were enrolled and randomly assigned to the interferon beta-1a plus remdesivir group (n=487) or to the placebo plus remdesivir group (n=482). The mean duration of symptoms before enrolment was 8·7 days (SD 4·4) in the interferon beta-1a plus remdesivir group and 8·5 days (SD 4·3) days in the placebo plus remdesivir group. Patients in both groups had a time to recovery of 5 days (95% CI not estimable) (rate ratio of interferon beta-1a plus remdesivir group vs placebo plus remdesivir 0·99 [95% CI 0·87–1·13]; p=0·88). The Kaplan-Meier estimate of mortality at 28 days was 5% (95% CI 3–7%) in the interferon beta-1a plus remdesivir group and 3% (2–6%) in the placebo plus remdesivir group (hazard ratio 1·33 [95% CI 0·69–2·55]; p=0·39). Patients who did not require high-flow oxygen at baseline were more likely to have at least one related adverse event in the interferon beta-1a plus remdesivir group (33 [7%] of 442 patients) than in the placebo plus remdesivir group (15 [3%] of 435). In patients who required high-flow oxygen at baseline, 24 (69%) of 35 had an adverse event and 21 (60%) had a serious adverse event in the interferon beta-1a plus remdesivir group compared with 13 (39%) of 33 who had an adverse event and eight (24%) who had a serious adverse event in the placebo plus remdesivir group. Interpretation Interferon beta-1a plus remdesivir was not superior to remdesivir alone in hospitalised patients with COVID-19 pneumonia. Patients who required high-flow oxygen at baseline had worse outcomes after treatment with interferon beta-1a compared with those given placebo. Funding The National Institute of Allergy and Infectious Diseases (USA).

Coronavirus disease 2019, first reported in China in late 2019, has quickly spread across the world. The outbreak was declared a pandemic by the World Health Organization on March 11, 2020. Here, we describe our initial efforts at the University of Florida Health for processing of large numbers of tests, streamlining data collection, and reporting data for optimizing testing capabilities and superior clinical management. Specifically, we discuss clinical and pathology informatics workflows and informatics instruments which we designed to meet the unique challenges of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We hope these results benefit institutions preparing to implement SARS-CoV-2 testing.

Background NDM-producing Enterobacteriaceae are a major clinical concern worldwide. We characterized NDM-positive pathogens isolated from patients and assessed the dissemination patterns of the blaNDM genes in a hospital setting. Methods Eleven NDM-positive Enterobacteriaceae (three Enterobacter hormaechei, six Klebsiella pneumoniae and two Escherichia coli) were isolated from nine patients over a 1 year period. Antimicrobial susceptibility was assessed by MICs. A combination of short- and long-read WGS was used for genome analysis. Clinical treatment history of patients was linked with genetic features of individual isolates to investigate the dissemination patterns of the blaNDM genes and NDM-positive strains. Results bla NDM in clonal K. pneumoniae were transmitted between two patients. In other instances, an identical IncC plasmid encoding NDM-1 was transmitted between E. coli and K. pneumoniae isolated from the same patient, and an IncX3 plasmid, carrying blaNDM-1 or blaNDM-5, was harboured in non-clonal E. hormaechei. Varying patterns of IS elements were identified as a critical transmission mechanism in association with blaNDM genes. Conclusions Multiple transmission patterns were identified in hospitalized patients, including dissemination of clonal bacterial strains carrying resistance genes and horizontal transfer of resistance genes among divergent bacterial strains. Controlling spread of NDM is complex: while attention to standard infection control practices is critically important, this needs to be matched by aggressive efforts to limit unnecessary antimicrobial use, to minimize the selection for and risk of transfer of ‘high mobility’ resistance genes among Enterobacteriaceae.

BackgroundThe incidence of cefepime-induced neurotoxicity (CIN) in hospitalized patients is highly variable. Although greater cefepime exposures incite neurotoxicity, data evaluating trough thresholds associated with CIN remains limited. The objectives of this study were to evaluate the incidence of CIN, assess the relationship between cefepime trough concentrations and CIN, investigate clinical factors associated with CIN, and describe electroencephalogram (EEG) abnormalities in CIN.MethodsThis was a retrospective study of adult patients who had received ≥ 5 days of cefepime with ≥ 1 trough concentration > 25 mg/L. Potential CIN cases were identified utilizing neurological symptoms, neurologist assessments, EEG findings and improvement of neurotoxicity after cefepime discontinuation.ResultsOne-hundred and forty-two patients were included. The incidence of CIN was 13% (18/142). The mean cefepime trough concentration in CIN patients was significantly greater than the non-neurotoxicity group (74.2 mg/L ± 41.1 vs. 46.6 mg/L ± 23, p=0.015). Lower renal function (creatinine clearance < 30 ml/min), greater time to therapeutic drug monitoring (TDM) (≥72 hours), and each 1 mg/mL rise in cefepime trough were independently associated with increased risk of CIN. Moderate generalized slowing of the background rhythm was the most common EEG pattern associated with CIN.ConclusionCefepime should be used cautiously in hospitalized patients due to the risk of neurotoxicity. Patients with greater renal function and those who had early cefepime TDM (≤ 72 hours) had lower risk of CIN.

Objectives: This review will briefly examine the clinical presentation and important immunology of viral pneumonia with a focus on severe acute respiratory syndrome coronavirus 2 (coronavirus disease 2019). Data Sources, Study Selection, Data Extraction, and Data Synthesis: The most relevant, original and review literature were assessed for inclusion in this review. Sources included the Centers for Disease Control and Prevention, World Health Organization, and PubMed. Conclusions: Pneumonia is a leading cause of hospitalization and death worldwide, with viral etiologies being very common. Given the rapidly emerging pandemic associated with the novel severe acute respiratory syndrome coronavirus 2 causing coronavirus disease 2019, it is important to review the clinical presentation and immunologic changes associated with viral pneumonia. Symptoms of viral pneumonia include common respiratory tract infection symptoms of cough, fever, and shortness of breath. Immunologic changes include up-regulation of airway pro-inflammatory cytokines and pathogen- and damage-associated molecular patterns contributing to cytokine and genomic changes. Coronavirus disease 2019 clinical presentation is typical of viral pneumonia with an increased prevalence of early pulmonary infiltrates and lymphopenia. Principles of early coronavirus disease 2019 management and isolation as well as potential therapeutic approaches to the emerging pandemic are discussed.

Four patients with adult-onset, disseminated mycobacterial infection had 5' UTR mutations in IKBKG without clear physical stigmata of NEMO deficiency. These mutations caused decreased levels of NEMO protein and Toll-like receptor driven cytokine production. Three patients died from disseminated disease. These mutations may be missed by whole exome sequencing.

Despite a regional decline in influenza A(H1N1)pdm09 virus infections during 2013–14, cases at a Florida hospital were more severe than those during 2009–10. Examined strains had a hemagglutinin polymorphism associated with enhanced binding to lower respiratory tract receptors. Genetic changes in this virus must be monitored to predict the effect of future pandemic viruses.

Influenza A(H3N2) strains isolated during 2014–15 in Alachua County, Florida, USA, belonged to hemagglutinin gene clade 3C.2a. High rates of influenza-like illness and confirmed influenza cases in children were associated with a decrease in estimated vaccine effectiveness. Illnesses were milder than in 2013–14; severe cases were concentrated in elderly patients with underlying diseases.

The majority of dengue fever cases reported in the United States recently have been imported. We isolated dengue virus type 2 (DENV-2) from a North-Central Florida resident with locally acquired dengue fever in May 2016. This is the first evidence of autochthonous transmission of the virus in north-central Florida.

The majority of dengue fever cases reported in the United States recently have been imported. We isolated dengue virus type 2 (DENV-2) from a North-Central Florida resident with locally acquired dengue fever in May 2016. This is the first evidence of autochthonous transmission of the virus in north-central Florida.

We measured in vitro activity of plazomicin, a next-generation aminoglycoside, and other aminoglycosides against 50 carbapenem-resistant Klebsiella pneumoniae strains from two centers and correlated the results with the presence of various aminoglycoside-modifying enzymes (AMEs). Ninety-four percent of strains were sequence type 258 (ST258) clones, which exhibited 5 ompK36 genotypes; 80% and 10% of strains produced Klebsiella pneumoniae carbapenemase 2 (KPC-2) and KPC-3, respectively. Ninety-eight percent of strains possessed AMEs, including AAC(6′)-Ib (98%), APH(3′)-Ia (56%), AAC(3)-IV (38%), and ANT(2″)-Ia (2%). Gentamicin, tobramycin, and amikacin nonsusceptibility rates were 40, 98, and 16%, respectively. Plazomicin MICs ranged from 0.25 to 1 μg/ml. Tobramycin and plazomicin MICs correlated with gentamicin MICs ( r = 0.75 and 0.57, respectively). Plazomicin exerted bactericidal activity against 17% (1× MIC) and 94% (4× MIC) of strains. All strains with AAC(6′)-Ib were tobramycin-resistant; 16% were nonsusceptible to amikacin. AAC(6′)-Ib combined with another AME was associated with higher gentamicin, tobramycin, and plazomicin MICs than AAC(6′)-Ib alone ( P = 0.01, 0.0008, and 0.046, respectively). The presence of AAC(3)-IV in a strain was also associated with higher gentamicin, tobramycin, and plazomicin MICs ( P = 0.0006, P < 0.0001, and P = 0.01, respectively). The combination of AAC(6′)-Ib and another AME, the presence of AAC(3)-IV, and the presence of APH(3′)-Ia were each associated with gentamicin resistance ( P = 0.0002, 0.003, and 0.01, respectively). In conclusion, carbapenem-resistant K. pneumoniae strains (including ST258 clones) exhibit highly diverse antimicrobial resistance genotypes and phenotypes. Plazomicin may offer a treatment option against strains resistant to other aminoglycosides. The development of molecular assays that predict antimicrobial responses among carbapenem-resistant K. pneumoniae strains should be a research priority.

Background Based on prior studies, elderly patients and those with renal dysfunction are prone to cefepime (CFP) toxicity. The toxicokinetics and toxicodynamics for CFP are not well established. Lamoth et al. reported a 50% probability of CFP neurotoxicity at a serum trough concentration of ≥22 mg/L, whereas Huwyler et al. observed CFP neurotoxicity when concentrations exceeded 35 mg/L. The objectives of this study were to quantify the incidence of CFP neurotoxicity and to assess the association between CFP concentrations and neurotoxicity. Methods We conducted a retrospective review between March 2016 and May 2018, of adult patients with serum CFP trough concentrations ≥25 mg/L. To be considered a CFP neurotoxicity case, patients were required to fulfill at least two of the NCI criteria for neurological toxicity such as, presence of new-onset confusion, delirium, or drowsiness. Following this, cases were classified as (1) high likelihood of toxicity (HLT) if they either had a neurology consult or EEG findings consistent with CFP toxicity and if their symptoms improved after discontinuation of CFP, (2) possible toxicity (PT) if neurology consult or EEG was absent or if we were unable to assess improvement after CFP was discontinued, or (3) nontoxicity (NT). Cases were independently reviewed by an ID pharmacist and physician. Additional data such as comorbidities, renal function, and use of anti-epileptics were collected. Results One hundred and forty-two patients were included in the analysis. Neurotoxicity (HLT+PT) related to CFP occurred in 18/142 (13%) patients; 67% (12/18) were considered HLT. The median age in the HLT cohort was 68 years (interquartile range [IQR], 57–74), with toxicity occurring a median of 6 days (IQR, 5–8) after starting CFP. At the time of neurotoxicity, HLT patients had diminished renal function with a median SCr of 1.6 mg/dL (IQR, 1.2–2.4) and a corresponding CrCl of 35.8 mL/minute (IQR, 19.2–50.9). The median CFP trough concentration in the HLT patients was 62 mg/L (IQR, 50–73) vs. 70mg/L (IQR, 41–115) in the PT and 42 mg/L (IQR, 31–61) in the NT groups. Conclusion Our data emphasize the need for careful dosing in older patients with renal insufficiency. Interestingly, our study reveals higher cefepime troughs (~3-fold higher) associated with neurotoxicity than previously reported. Disclosures All authors: No reported disclosures.

Campylobacter jejuni is a major cause of food-borne gastroenteritis worldwide. While mortality is low, morbidity imparted by post-infectious sequelae such as Guillain-Barré syndrome, Reiter syndrome/reactive arthritis and irritable bowel syndrome is significant. In addition, the economic cost is high due to lost productivity. Food animals, particularly poultry, are the main reservoirs of C. jejuni. The over-use of antibiotics in the human population and in animal husbandry has led to an increase in antibiotic-resistant infections, particularly with fluoroquinolones. This is problematic because C. jejuni gastroenteritis is clinically indistinguishable from that caused by other bacterial pathogens, and such illnesses are usually treated empirically with fluoroquinolones. Since C. jejuni is naturally transformable, acquisition of additional genes imparting antibiotic resistance is likely. Therefore, an understanding of the antibiotic resistance mechanisms in C. jejuni is needed to provide proper therapy both to the veterinary and human populations.

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with nosocomially acquired infections. Host cell adherence and biofilm formation of K. pneumoniae isolates is mediated by type 1 (T1P) and type 3 (MR/K) pili whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. The E. coli common pilus (ECP) is an adhesive structure produced by all E. coli pathogroups and a homolog of the ecpABCDE operon is present in the K. pneumoniae genome. In this study, we aimed to determine the prevalence of these three fimbrial genes among a collection of 69 clinical and environmental K. pneumoniae strains and to establish a correlation with fimbrial production during cell adherence and biofilm formation. The PCR-based survey demonstrated that 96% of the K. pneumoniae strains contained ecpA and 94% of these strains produced ECP during adhesion to cultured epithelial cells. Eighty percent of the strains forming biofilms on glass produced ECP, suggesting that ECP is required, at least in vitro, for expression of these phenotypes. The fim operon was found in 100% of the strains and T1P was detected in 96% of these strains. While all the strains examined contained mrkA, only 57% of them produced MR/K fimbriae, alone or together with ECP. In summary, this study highlights the ability of K. pneumoniae strains to produce ECP, which may represent a new important adhesive structure of this organism. Further, it defines the multi-fimbrial nature of the interaction of this nosocomial pathogen with host epithelial cells and inert surfaces.

Introduction: Zika virus (ZIKV) and Chikungunya virus (CHIKV) can share the same mosquito vector, and co-infections by these viruses can occur in humans. While infections with these viruses share commonalities, CHIKV is unique in causing arthritis and arthralgias that may persist for a year or more. These infections are commonly diagnosed by RT–PCR-based methods during the acute phase of infection. Even with the high specificity and sensitivity characteristic of PCR, false negatives can occur, highlighting the need for additional diagnostic methods for confirmation. Case presentation: On her return to the USA, a traveller to Colombia, South America developed an illness consistent with Zika, Chikungunya and/or Dengue. RT-PCR of her samples was positive only for ZIKV. However, arthralgias persisted for months, raising concerns about co-infection with CHIKV or Mayaro viruses. Cell cultures inoculated with her original clinical samples demonstrated two types of cytopathic effects, and both ZIKV and CHIKV were identified in the supernatants. On phylogenetic analyses, both viruses were found to be related to strains found in Colombia. Conclusion: These findings highlight the need to consider CHIKV co-infection in patients with prolonged rheumatological symptoms after diagnosis with ZIKV, and the usefulness of cell culture as an amplification step for low-viremia blood and other samples.

Zika virus and dengue virus serotype 2 were isolated from a patient with travel to Haiti who developed fever, rash, arthralgias, and conjunctivitis. The infecting Zika virus was related to Venezuelan and Brazilian strains but evolved along a lineage originating from strains isolated in 2014 in the same region of Haiti.

BACKGROUND: Synthetic cannabinoid containing products are a public health threat as reflected by a number of outbreaks of serious adverse health effects over the past 4 years. The designer drug epidemic is characterized by the rapid turnover of synthetic cannabinoid compounds on the market which creates a challenge in identifying the particular etiology of an outbreak, confirming exposure in cases, and providing current information to law enforcement. RESULTS: Between 28 May 2014 and 8 June 2014, 35 patients were evaluated and treated at the University of Florida Health Medical Center in Gainesville following reported exposure to a synthetic cannabinoid containing product obtained from a common source. Patients demonstrated acute delirium (24) and seizures (14), and five required ventilator support and ICU-level care; none died. The presence of N-[(lS)-l-(aminocarbonyl)-2-methylpropyl]-l-(cyclohexylmethyl)-lH-indazole-3-carboxamide (AB-CHMINACA), or one of its predicted metabolites was confirmed in 15 of 21 cases. A rapid public health response and aggressive public messaging prevented further morbidity, identified the source, and led to law enforcement seizure of the implicated product. DISCUSSION: The significance of this outbreak lies as much in the rapid occurrence of unpredictable, life- threatening adverse health effects from a newly identified synthetic cannabinoid compound as it does in the multidisciplinary investigation and novel partnership between local public health, the laboratory, and the chemical industry, resulting in termination of the outbreak. CONCLUSION: A coordinated response and collaboration between law enforcement, the local public health, emergency medical services and Health Center staff, were all key interventions in preventing a more substantial public health outbreak resulting from use of a novel synthetic cannabinoid compound. Real time collaborations between toxicology laboratories, suppliers of analytical standards and the public health system may be useful in the face of future novel chemical exposures.

Avibactam is a novel β-lactamase inhibitor with affinity for Klebsiella pneumoniae carbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producing K. pneumoniae (KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin gene ompK36 confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL and ompK36 mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants ( P = 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone ( P = 0.003 and P = 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.

The bactericidal/permeability increasing (BPI) and lipopolysaccharide (LPS)-binding (LBP) proteins are closely related two-domain proteins in which LPS binding is mediated by the NH(2)-terminal domain. To further define the role of the COOH-terminal domain of these proteins in delivery of LPS to specific host acceptors, we have compared interactions of LBP, BPI, LBP(N)-BPI(C) (NH(2)-terminal domain of LBP, COOH-terminal domain of BPI), and BPI(N)-LBP(C) with purified (3)H-LPS and, subsequently, with purified leukocytes and soluble (s)CD14. The COOH-terminal domain of LBP promotes delivery of LPS to CD14 on both polymorphonuclear leukocytes and monocytes resulting in cell activation. In the presence of Ca(2+) and Mg(2+), LBP and BPI each promote aggregation of LPS to protein-LPS aggregates of increased size (apparent M(r)20 x 10(6) Da), but only LPS associated with LBP and BPI(N)-LBP(C) is disaggregated in the presence of CD14. BPI and LBP(N)-BPI(C) promote apparently CD14-independent LPS association to monocytes without cell activation. These findings demonstrate that the carboxyl-terminal domain of these closely related endotoxin-binding proteins dictates the route and host responses to complexes they form with endotoxin.

Bacteria help norovius infect B cells Stomach ache, nausea, diarrhea—many people know the sort of gastrointestinal havoc norovirus can wreak. Despite this, norovirus biology remains unclear, because human norovirus cannot be grown in culture. Jones et al. now report that with the help of bacteria, human norovirus can infect cultured B cells (see the Perspective by Robinson and Pfeiffer). To infect B cells, human norovirus required the presence of gut bacteria that expressed proteins involved in determining blood type. Mouse norovirus also infected B cells, and the treatment of mice with antibiotics protected them from norovirus infection. Science , this issue p. 755 ; see also p. 700

The FilmArray blood culture identification (BCID) panel is a rapid molecular diagnostic test approved for use with positive blood culture material. We describe a fatal case of meningococcemia with central nervous system (CNS) involvement detected using the BCID test with culture-negative blood and cerebrospinal fluid.

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain ofEscherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios.

The innate immune system plays a critical role in host defense against mucosal bacteria. Campylobacter jejuni is a major cause of human gastroenteritis that usually resolves spontaneously within several days, suggesting that innate mechanisms are important to control the infection. However, the specific means by which this occurs is not well understood. While diarrheal isolates of C. jejuni usually are susceptible to human serum, we found that a systemic strain of C. jejuni, isolated from the cerebrospinal fluid of an infant with meningitis, is relatively more resistant to human serum, the Bactericidal/Permeability-Increasing Protein (BPI), an endogenous cationic antimicrobial protein, and the cationic peptide antibiotic polymyxin B. To test the hypothesis that the surface properties of this strain contributed to its ability to withstand these innate host defenses, we constructed isogenic mutants in capsule (kpsM) and lipooligosaccharide (waaF) and complemented these mutants by insertion of the complementation construct in trans into hipO, a chromosomal locus. We found that capsule expression was essential for serum resistance, whereas lipooligosaccharide played no substantial role. In contrast, the lipooligosaccharide mutant showed increased sensitivity to polymyxin B, α-defensins, cathelicidins, and BPI. These findings suggest that the polysaccharides of C. jejuni strains contribute differently to resistance against host innate immunity; whereby capsule is more important for resisting human complement and lipooligosaccharide is more important for protection against killing mediated by cationic antimicrobial peptides and proteins.

Campylobacter jejuni, a gram-negative, invasive organism, is a common cause of food-borne bacterial diarrheal disease. However, the relationship betweenC. jejuniand the innate immune system is not well described. To better characterize host defense againstC. jejuni, we investigated the ability of nitric oxide/reactive nitrogen species to kill two strains ofC. jejuni. C. jejuniviability was measured after exposure to reactive nitrogen species produced biochemically as acidified nitrite and by bone marrow-derived macrophages. We report that acidified nitrite caused a 3-log-increased kill ofC. jejuni(P< 0.05) at doses that did not affect the viability ofSalmonella entericaserovar Typhimurium. Expression ofNOS2, the gene responsible for the production of inducible nitric oxide, was increased >100-fold in murine macrophages after incubation withC. jejuni(P< 0.001). These macrophages effected a 2-log-increased kill ofC. jejuniover 24 h compared to that by NOS2−/−macrophages unable to produce nitric oxide (P< 0.05). These findings suggest that the mammalian host upregulates the production of nitric oxide in response to exposure toC. jejuniand that nitric oxide and reactive nitrogen species comprise part of the innate defense mechanisms that contribute to the resolution ofC. jejuniinfection.

Contamination of food with potentially dangerous human pathogens has been recognized since the time of Pasteur (1) and is well-documented in the modern era (2), but the development of antimicrobial agents has helped limit the consequences of such infections. Concomitantly, the widespread use of antimicrobial agents has also led to the emergence of antimicrobial drug–resistant organisms (3,4). Gupta et al. demonstrate the increasing prevalence in the United States of ciprofloxacin-resistant Campylobacter species isolated from humans and poultry from 1990 to 1997, and their studies implicate the prophylactic treatment of poultry with fluoroquinolones in this emerging problem (5). Their report indicates that the source of fluoroquinolone-resistant Campylobacter infections was consuming poultry colonized with resistant strains (Figure), rather than selection for Campylobacter resistance in the human gut after clinical fluoroquinolone use to treat the diarrheal illness (5). This work provides further evidence that fluoroquinolone use in poultry promotes the emergence of resistant Campylobacter strains that subsequently infect humans (6). That persons infected with these fluoroquinolone-resistant strains had 3 additional days of illness and were more likely to be hospitalized demonstrates the harm caused by such resistant stains (5). Figure Acquisition of fluoroquinolone (FQ)-resistant Campylobacter from poultry. Since campylobacters are normal enteric flora in many avian species, poultry represents a model system to test the hypothesis that prophylactic and growth-promoting use of antimicrobial agents in food animals selects for the emergence of antimicrobial drug–resistant organisms. In one study, chickens that were naturally colonized with fluoroquinolone-susceptible Campylobacter strains began to excrete resistant strains after 2 days of doses of enrofloxacin (7), which is commonly used for prophylaxis in the poultry industry. A single point mutation in gyrA encoding the bacterial DNA gyrase was sufficient to confer high-level resistance (7,8). This small genetic change apparently has a low "fitness cost" to the organism, as evidenced by fluoroquinolone-resistant strains' rapidly replacing susceptible Campylobacter in treated chickens (7). Developing an animal reservoir of fluoroquinolone-resistant Campylobacter has been the major factor behind transmission of quinolone resistance to humans (8,9). In contrast, among poultry treated therapeutically with enrofloxacin, no resistance was observed in the 13 C. jejuni isolates tested (9). Similarly, after the prophylactic and growth-promoting uses of macrolides in swine were banned in Denmark, the prevalence of macrolide-resistant C. coli declined (10). Thus, the major determinant of developing resistance appears to be use of subtherapeutic antimicrobial doses. The antimicrobial drug ban in Denmark did not decrease the amount of meat produced by the poultry and pig production industries, which removed a major concern (10). Evidence suggests that restricting fluoroquinolone use to therapeutic indications only in food animals could decrease rates of fluoroquinolone-resistant Campylobacter, and the Danish experience with macrolide restriction proves that such limitations need not harm the husbandry of food animals. The increased likelihood of foreign travel in persons infected with ciprofloxacin-resistant strains (5) illustrates the global threat posed by resistant strains. Appreciating such realities favors concerted efforts to limit use of fluoroquinolones (and other antimicrobial drugs) to therapy only in food animals. This view was supported by a recent (March 2004) landmark decision by Federal Drug Administration Administrative Law Judge Daniel J. Davidson, withdrawing approval for the new animal drug application to use enrofloxacin for prophylaxis or growth-promotion in poultry (11). This decision was the first occasion that a previously approved antimicrobial agent was removed from the U.S. veterinary market because of concerns about antimicrobial drug resistance. With this decision as precedent, we should follow the examples set in Europe and ban use of all antimicrobial agents in food animals, except when necessary for therapy of ill animals.