A Randomized Clinical Trial of Bayesian-Guided Beta-Lactam Infusion Strategy and Associated Bacterial Resistance and Clinical Outcomes in Patients With Severe Pneumonia.

BACKGROUND: Antimicrobial resistance is a growing health concern worldwide. The objective of this study was to evaluate the effect of beta-lactam infusion on the emergence of bacterial resistance in patients with severe pneumonia in the intensive care unit. METHODS: Adult intensive care patients receiving cefepime, meropenem, or piperacillin-tazobactam for severe pneumonia caused by Gram-negative bacteria were randomized to receive beta-lactams as an intermittent (30 minutes) or continuous (24 hours) infusion. Respiratory samples for culture and susceptibility testing, with minimum inhibitory concentrations (MIC), were collected once a week for up to 4 weeks. Beta-lactam plasma concentrations were measured and therapeutic drug monitoring was performed using Bayesian software as the standard of care. RESULTS: The study was terminated early owing to slow enrollment. Thirty-five patients were enrolled in this study. Cefepime (n = 22) was the most commonly prescribed drug at randomization, followed by piperacillin (n = 8) and meropenem (n = 5). Nineteen patients were randomized into the continuous infusion arm and 16 into the intermittent infusion arm. Pseudomonas aeruginosa was the most common respiratory isolate (n = 19). Eighteen patients were included in the final analyses. No differences in bacterial resistance were observed between arms ( P = 0.67). No significant differences in superinfection ( P = 1), microbiological cure ( P = 0.85), clinical cure at day 7 ( P = 0.1), clinical cure at end of therapy ( P = 0.56), mortality ( P = 1), intensive care unit length of stay ( P = 0.37), or hospital length of stay ( P = 0.83) were observed. Achieving 100% ƒT > MIC ( P = 0.04) and ƒT > 4 × MIC ( P = 0.02) increased likelihood of clinical cure at day 7 of therapy. CONCLUSIONS: No differences in the emergence of bacterial resistance or clinical outcomes were observed between intermittent and continuous infusions. Pharmacokinetic/pharmacodynamic target attainment may be associated with a clinical cure on day 7.

Dynamics of Anti-influenza Mucosal IgA Over a Season in a Cohort of Individuals Living or Working in a Long-term Care Facility.

BACKGROUND: Serological surveys are used to ascertain influenza infection and immunity, but evidence for the utility of mucosal immunoglobulin A (IgA) as a correlate of infection or protection is limited. METHODS: We performed influenza-like illness (ILI) surveillance on 220 individuals living or working in a retirement community in Gainesville, Florida from January to May 2018, and took pre- and postseason nasal samples of 11 individuals with polymerase chain reaction (PCR)-confirmed influenza infection and 60 randomly selected controls. Mucosal IgA against 10 strains of influenza was measured from nasal samples. RESULTS: Overall, 28.2% and 11.3% of individuals experienced a 2-fold and 4-fold rise, respectively, in mucosal IgA to at least 1 influenza strain. Individuals with PCR-confirmed influenza A had significantly lower levels of preseason IgA to influenza A. Influenza-associated respiratory illness was associated with a higher rise in mucosal IgA to influenza strains of the same subtype, and H3N2-associated respiratory illness was associated with a higher rise in mucosal IgA to other influenza A strains. CONCLUSIONS: By comparing individuals with and without influenza illness, we demonstrated that mucosal IgA is a correlate of influenza infection. There was evidence for cross-reactivity in mucosal IgA across influenza A subtypes.

A Gastrointestinal PCR Panel Improves Clinical Management and Lowers Health Care Costs.

Conventional methods for the identification of gastrointestinal pathogens are time-consuming and expensive and have limited sensitivity. The aim of this study was to determine the clinical impact of a comprehensive molecular test, the BioFire FilmArray gastrointestinal (GI) panel, which tests for many of the most common agents of infectious diarrhea in approximately 1 h. Patients with stool cultures submitted were tested on the GI panel (n = 241) and were compared with control patients (n = 594) from the year prior. The most common organisms detected by the GI panel were enteropathogenic Escherichia coli (EPEC, n = 21), norovirus (n = 21), rotavirus (n = 15), sapovirus (n = 9), and Salmonella (n = 8). Patients tested on the GI panel had an average of 0.58 other infectious stool tests compared with 3.02 in the control group (P = 0.0001). The numbers of days on antibiotic(s) per patient were 1.73 in the cases and 2.12 in the controls (P = 0.06). Patients with the GI panel had 0.18 abdomen and/or pelvic imaging studies per patient compared with 0.39 (P = 0.0002) in the controls. The average length of time from stool culture collection to discharge was 3.4 days in the GI panel group versus 3.9 days in the controls (P = 0.04). The overall health care cost could have decreased by $293.61 per patient tested. The GI panel improved patient care by rapidly identifying a broad range of pathogens which may not have otherwise been detected, reducing the need for other diagnostic tests, reducing unnecessary use of antibiotics, and leading to a reduction in hospital length of stay.

Semi-quantitative Influenza A population averages from a multiplex respiratory viral panel (RVP): potential for reflecting target sequence changes affecting the assay.

BACKGROUND: Yearly influenza virus mutations potentially affect the performance of molecular assays, if nucleic acid changes involve the sequences in the assay. Because individual patient viral loads depend on variables such as duration of illness, specimen type, age, and immunosuppression, we examined seasonal population averages of positive tests to smooth inherent variability. METHODS: We studied the population seasonal averages of the semi-quantitative nAMPs for the influenza matrix and hemagglutinin genes in the GenMark (Carlsbad, CA) Respiratory Viral Panel assay between 3 institutions over 3 Influenza seasons. RESULTS: Population average nAMPs were strikingly consistent between separate institutions, but differed substantially between H3N2 and H1N1 seasons. In the 2012-2013 and 2014-2015 influenza seasons, matrix gene H3N2 nAMP averages were 50-70% less than those of the same assay in the 2013-2014 H1N1 season. Influenza strains representative of these seasons were grown in tissue culture and when the supernatant virus was adjusted to the same copy number using a TaqMan assay, the same relative differences were reproduced in the RVP assay. Because the sequences for the PCR and PCR product detection in the GenMark assay are proprietary, the manufacturer provided single stranded DNA matching the capture probe for the representative H3N2 (3 mismatches) and H1N1 strains (2 different mismatches). Equimolar concentrations of these synthetic DNA sequences gave average nAMP values that closely correlated with the average nAMPS of the representative strains and their respective seasonal averages. CONCLUSIONS: Seasonal averages of semi-quantitative data may provide a means to follow assay performance as a reflection of the effects of molecular drift.

Hemagglutinin Gene Clade 3C.2a Influenza A(H3N2) Viruses, Alachua County, Florida, USA, 2014-15.

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.

Severity of influenza A(H1N1) illness and emergence of D225G variant, 2013-14 influenza season, Florida, USA.

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.

Detection of Neisseria meningitidis from negative blood cultures and cerebrospinal fluid with the FilmArray blood culture identification panel.

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.

Time to positivity of blood cultures supports antibiotic de-escalation at 48 hours.

BACKGROUND: Appropriate de-escalation of empirical antimicrobial therapy is a fundamental component of antimicrobial stewardship. Concern for the late detection of bloodstream pathogens may undermine early streamlining efforts and subject patients to protracted courses of nonessential therapy. OBJECTIVE: To quantify the prevalence of bacterial bloodstream infection (BSI) detection after more than 48 hours of culture incubation. We also assessed the impact of antimicrobial therapy delivered prior to blood sample collection. METHODS: We retrospectively evaluated time to blood culture positivity (TTP) in adult patients at an academic tertiary care hospital. Microbiology reports were reviewed to identify the TTP for the first positive blood culture bottle for each episode of BSI occurring from February 1, 2011, to July 31, 2011. Isolates were classified as true pathogens or contaminants. Blood culture results after 48 hours of incubation were compared with results after 120 hours of incubation. RESULTS: The median TTP of 416 monomicrobial BSIs and 210 contamination episodes was 13.7 and 24.4 hours, respectively (P < .001). The median TTPs in those who received and did not receive prior antibiotics were 17.0 and 12.8 hours, respectively (P < .001). By 48 hours, 98% of aerobic Gram-positive and Gram-negative BSIs were detected. Culture results at 48 hours were 97% sensitive and had a negative predictive value of 99.8%. CONCLUSION: Few true BSIs are detected after more than 48 hours of culture incubation. Clinicians may adjust empirical antibiotic coverage at this time with little risk for subsequent bacterial pathogen detection.

Universal intravenous access cleaning device fails to sterilize stopcocks.

BACKGROUND: Contamination of a central venous catheter may occur through use of conventional open-lumen stopcock devices (COLDs), or disinfectable, needleless, closed connectors (DNCCs). We investigated the effectiveness of a new universal IV access cleaning device (Site-Scrub) compared with 70% isopropyl alcohol prep pads for sanitizing COLDs or DNCCs inoculated with common catheter-associated pathogens. METHODS: Site-Scrub was compared with 70% alcohol prep pads for sanitizing contaminated female Luer lock COLD or DNCC filled with sterile saline or propofol and 2 common bacterial central venous catheter contaminants (Staphylococcus epidermidis or Pseudomonas aeruginosa). Devices were contaminated using a glove touch (COLD and DNCC) or syringe tip (COLD). The primary end point of the study was colony-forming units (CFU) after 24 hours. RESULTS: The use of glove touch contamination, the contaminants, S epidermidis and P aeruginosa, produced CFU in saline-filled COLDs treated with the Site-Scrub, but not in those treated with alcohol pads (P < 0.001). Similar results were observed with propofol-filled COLDs (P < 0.001). For DNCCs filled with saline or propofol, both alcohol and Site-Scrub effectively reduced CFU growth compared with contaminated controls (P < 0.001). When COLDs were contaminated by treated syringe tips, there was no significant evidence of reduction in CFU growth by using either alcohol pads or Site-Scrub compared with contaminated controls. CONCLUSIONS: These data suggest that when the inner surface of the COLD is contaminated, both alcohol pads and Site-Scrub were not significantly effective in decontaminating the COLD. When the COLD rim is contaminated, however, alcohol pads outperform Site-Scrub. DNCCs were uniformly decontaminated with either treatment. Future work should focus on better access systems because current COLDs are difficult to decontaminate.

Intestinal dysbiosis and depletion of butyrogenic bacteria in Clostridium difficile infection and nosocomial diarrhea.

Clostridium difficile infection (CDI) causes nearly half a million cases of diarrhea and colitis in the United States each year. Although the importance of the gut microbiota in C. difficile pathogenesis is well recognized, components of the human gut flora critical for colonization resistance are not known. Culture-independent high-density Roche 454 pyrosequencing was used to survey the distal gut microbiota for 39 individuals with CDI, 36 subjects with C. difficile-negative nosocomial diarrhea (CDN), and 40 healthy control subjects. A total of 526,071 partial 16S rRNA sequence reads of the V1 to V3 regions were aligned with 16S databases, identifying 3,531 bacterial phylotypes from 115 fecal samples. Genomic analysis revealed significant alterations of organism lineages in both the CDI and CDN groups, which were accompanied by marked decreases in microbial diversity and species richness driven primarily by a paucity of phylotypes within the Firmicutes phylum. Normally abundant gut commensal organisms, including the Ruminococcaceae and Lachnospiraceae families and butyrate-producing C2 to C4 anaerobic fermenters, were significantly depleted in the CDI and CDN groups. These data demonstrate associations between the depletion of Ruminococcaceae, Lachnospiraceae, and butyrogenic bacteria in the gut microbiota and nosocomial diarrhea, including C. difficile infection. Mechanistic studies focusing on the functional roles of these organisms in diarrheal diseases and resistance against C. difficile colonization are warranted.

Is Kawasaki Disease Caused by a Respiratory Virus?

BACKGROUND: Kawasaki disease is characterized by high fever, rash, cervical lymphadenopathy, conjunctival injection, oral mucous membrane changes and swelling of the extremities followed by skin sloughing. Despite >50 years of study, no bacterial, viral or other infectious agent has been consistently associated with the illness. The lockdown and social distancing for COVID-19 in March 2020 led to a marked decrease in respiratory virus circulation. This provided an "experiment of nature" to determine whether Kawasaki disease would decline in parallel. METHODS: Discharge ICD-10 diagnosis codes were obtained from the Vizient Clinical Data Base for Kawasaki disease and respiratory viruses, and analyzed for the age group < 5 years. Weekly respiratory virus positivity data were also obtained from BioFire Diagnostics. RESULTS: Common enveloped respiratory viruses declined precipitously from April 2020 through March 2021 to levels at or below historical seasonal minimum levels. Kawasaki Disease declined about 40% compared with 2018-2019, which is distinctly different from the pattern seen for the enveloped respiratory viruses. Strong seasonality was seen for Kawasaki disease as far back as 2010, and correlated most closely with respiratory syncytial virus, human metapneumovirus and less so with influenza virus suggesting there is a baseline level of Kawasaki disease activity that is heightened during yearly respiratory virus activity but that remains at a certain level even in the near total absence of respiratory viruses. CONCLUSIONS: The striking decrease in enveloped respiratory viruses after lockdown and social distancing was not paralleled by a comparable decrease in Kawasaki disease incidence, suggesting a different epidemiology.

Ultrastructural, immunofluorescence, and RNA evidence support the hypothesis of a "new" virus associated with Kawasaki disease.

BACKGROUND: Intracytoplasmic inclusion bodies (ICI) have been identified in ciliated bronchial epithelium of Kawasaki disease (KD) patients using a synthetic antibody derived from acute KD arterial IgA plasma cells; ICI may derive from the KD etiologic agent. METHODS: Acute KD bronchial epithelium was subjected to immunofluorescence for ICI and cytokeratin, high-throughput sequencing, and transmission electron microscopy (TEM). Interferon pathway gene expression profiling was performed on KD lung. RESULTS: An intermediate filament cytokeratin "cage" was not observed around KD ICI, making it unlikely that ICI are overproduced or misfolded human protein aggregates. Many interferon-stimulated genes were detected in the bronchial epithelium, and significant modulation of the interferon response pathway was observed in the lung tissue of KD patients. No known virus was identified by sequencing. Aggregates of virus-like particles (VLP) were detected by TEM in all 3 acute KD patients from whom nonembedded formalin-fixed lung tissue was available. CONCLUSIONS: KD ICI are most likely virus induced; bronchial cells with ICI contain VLP that share morphologic features among several different RNA viral families. Expedited autopsies and tissue fixation from acute KD fatalities are urgently needed to more clearly ascertain the VLP. These findings are compatible with the hypothesis that the infectious etiologic agent of KD may be a "new" RNA virus.

Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP.

We compared the FilmArray RP (Idaho Technology, Inc., Salt Lake City, UT) and the xTAG RVP (Luminex Corporation, Toronto, Canada) multiplex respiratory virus PCR methods for the detection of respiratory viruses in a set of 200 patient specimens frozen at -70 °C after standard viral culture and antigen detection methods were done. Both systems detected between 40 to 50% more viruses than traditional methods, primarily rhinoviruses and human metapneumovirus. The FilmArray RP detected significantly more total viruses either alone or as part of mixed infections than the xTAG RVP, as well as an additional 21.6% more respiratory syncytial viruses. The xTAG RVP requires 5 to 6 h with 2.5 to 3 h of hands-on time, while the FilmArray RP takes about an hour with 3 to 5 min of hands-on time, making it much easier to perform.

Evaluation of a rapid and completely automated real-time reverse transcriptase PCR assay for diagnosis of enteroviral meningitis.

Nucleic acid amplification tests (NAATs) for enterovirus RNA in cerebrospinal fluid (CSF) have emerged as the new gold standard for diagnosis of enteroviral meningitis, and their use can improve the management and decrease the costs for caring for children with enteroviral meningitis. The Xpert EV assay (Cepheid, Sunnyvale, CA) is a rapid, fully automated real-time PCR test for the detection of enterovirus RNA that was approved by the U.S. Food and Drug Administration for in vitro diagnostic use in March 2007. In this multicenter trial we established the clinical performance characteristics of the Xpert EV assay in patients presenting with meningitis symptoms relative to clinical truth. Clinical truth for enteroviral meningitis was defined as clinical evidence of meningitis, the absence of another detectable pathogen in CSF, and detection of enterovirus in CSF either by two reference NAATs or by viral culture. A total of 199 prospectively and 235 retrospectively collected specimens were eligible for inclusion in this study. The overall prevalence of enteroviral meningitis was 26.04%. The Xpert EV assay had a sensitivity of 94.69% (90% confidence interval [CI] = 89.79 to 97.66%), specificity of 100% (90% CI = 99.07 to 100%), positive predictive value of 100%, negative predictive value of 98.17, and an accuracy of 98.62% relative to clinical truth. The Xpert EV assay demonstrated a high degree of accuracy for diagnosis of enteroviral meningitis. The simplicity and on-demand capability of the Xpert EV assay should prove to be a valuable adjunct to the evaluation of suspected meningitis cases.

Target capture sequencing of SARS-CoV-2 genomes using the ONETest Coronaviruses Plus.

We introduce a target capture next-generation sequencing methodology, the ONETest Coronaviruses Plus, to sequence the SARS-CoV-2 genome and select loci of other respiratory viruses. We applied the ONETest on 70 respiratory samples (collected in Florida, USA between May and July, 2020), in which SARS-CoV-2 had been detected by a PCR assay. For 48 of the samples, we also applied the ARTIC protocol. Of the 70 ONETest libraries, 45 (64%) had a (near-)complete sequence (>29,000 bases and >90% covered by >9 reads). Of the 48 ARTIC libraries, 25 (52%) had a (near-)complete sequence. In 19 out of 25 (76%) samples in which both the ONETest and ARTIC yielded (near-)complete sequences, the lineages assigned were identical. As a target capture approach, the ONETest is less prone to loss of sequence coverage than amplicon approaches, and thus can provide complete genomic information more often to track and monitor SARS-CoV-2 variants.

Performance of a Semiquantitative Multiplex Bacterial and Viral PCR Panel Compared With Standard Microbiological Laboratory Results: 396 Patients Studied With the BioFire Pneumonia Panel.

BACKGROUND: Microbiologic results are critical to optimal management of patients with lower respiratory tract infection, but standard methods may take several days. The multiplex polymerase chain reaction BioFire Pneumonia (PN) panel detects 15 common bacterial species semiquantitatively as copy number/mL, 8 viral species, and 7 resistance genes in about an hour within the clinical laboratory. METHODS: We tested 396 unique endotracheal or bronchoalveolar lavage specimens with the BioFire Pneumonia panel and compared the bacterial detections to conventional gram stain and culture results. RESULTS: Of the 396 patients, 138 grew at least 1 bacterium that had a target on the PN panel, and 136/138 (98.6%) were detected by the panel. A total of 177 isolates were recovered in culture and the PN panel detected 174/177 (98.3%). A further 20% of patients had additional targets detected that were not found on standard culture (specificity 69%, positive predictive value 63%, and negative predictive value 98.9%). Copy number was strongly related to standard semiquantitative growth on plates reported by the laboratory (eg, 1+, 2+, 3+ growths) and was significantly higher in those specimens that grew a potential pathogen. Both higher copy number and bacterial detections found by the PN panel, but not found in culture, were strongly positively related to the level of white blood cells reported in the initial gram stain. CONCLUSIONS: Higher copy number and bacterial detections by the PN panel are related to the host respiratory tract inflammatory response. If laboratories can achieve a rapid turnaround time, the PN panel should have a significant impact both on patient management and on antibiotic stewardship.

Dissemination mechanisms of NDM genes in hospitalized patients.

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 bla NDM 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 bla NDM 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 bla NDM-1 or bla NDM-5, was harboured in non-clonal E. hormaechei. Varying patterns of IS elements were identified as a critical transmission mechanism in association with bla NDM 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.

Impact of a Point-of-care Respiratory PCR Panel in a Pediatric Clinic on Postvisit Communication and Follow-up Visits.

While the FilmArray Respiratory Panel EZ has been proven to reduce inappropriate antibiotic use in the outpatient pediatric setting, it is unclear whether its implementation will also reduce downstream health costs such as provider visits and telephone calls. This analysis will help pediatricians make more informed decisions on the implementation and judicious use of the Respiratory Panel EZ in their clinical practice.

Isolation gowns as a potential work hazard.

OBJECTIVES: Isolation gowns are used as a barrier to bacterial transmission from patient to provider and vice versa. If an isolation gown is ineffective, the patient and provider have a potential breach of safety and increased infection risk. This study compared the bacterial permeability of differently rated, commonly uses isolation gowns to assess their effectiveness in preventing simulated bacterial transmittance, and thus contamination, from patient to provider. METHODS: Serial dilutions of Staphylococcus epidermidis in sterile saline were applied to a simulated skin surface. Unrated and Levels 1 through 4 non-sterile isolation gowns contacted the solution, simulating patient contact. Both sides of the contaminated gowns were then cultured on blood agar by rolling a sterile swab across the gown and evenly inoculating the culture plate. Colony counts from inside and outside of the gowns were compared. Separately, S. epidermidis was placed on a sample of each gown and scanning electron microscopy was used to visualize the contaminated gowns' physical structure. RESULTS: Mean bacterial transmittance from outside of the gown (i.e. patient contact side) to inside of the gowns (i.e. provider clothing or skin side) based on gown rating was as follows: unrated: 50.4% (SD 9.0%); Level 1: 39.7% (SD 11.2%); Level 2: 16.3% (SD 10.3%); Level 3: 0.3% (SD 0.8%); Level 4: 0.0% (SD 0.0%). Scanning electron microscope imaging of unrated, Level 1, and Level 2 gowns revealed gown pore sizes much larger than the bacteria. The Welch one-way analysis of variance statistic showed significant difference dependent on gown-level rating. CONCLUSIONS: Unrated, Level 1, and Level 2 isolation gowns do not provide effective bacterial isolation barriers when bacteria like S. epidermidis make contact with one side of the gown material. Not studied, but implied, is that unrated and lower rated isolation gowns would be as or even more physically permeable to virus particles, which are much smaller than bacteria.