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In this retrospective analysis, we describe weekly croup and corresponding viral prevalence patterns in a pediatric quaternary care system in metropolitan Atlanta. We characterize a series of 24 patients with croup associated with SARS-CoV-2 infection and show that this clinical presentation increased substantially in frequency during the period of high Omicron vs Delta transmission.

Background Easy identification of immunocompromised hosts (ICHs) would allow for stratification of culture results based on host type. Methods We utilized antimicrobial stewardship program (ASP) team notes written during handshake stewardship rounds in the pediatric intensive care unit (PICU) as the gold standard for host status; clinical notes from the primary team, medication orders during the encounter, problem list, and billing diagnoses documented prior to the ASP documentation were extracted to develop models that predict host status. We calculated performance for three models based on diagnoses/medications, with and without natural language processing from clinical notes. The susceptibility of pathogens causing bacteremia to commonly used empiric antibiotic regimens was then stratified by host status. Results We identified 844 antimicrobial episodes from 666 unique patients; 160 (18.9%) were identified as ICHs. We randomly selected 675 initiations (80%) for model training and 169 initiations (20%) for testing. A rule-based model using diagnoses and medications alone yielded a sensitivity of 0.87 (08.6–0.88), specificity of 0.93 (0.92–0.93), and positive predictive value (PPV) of 0.74 (0.73–0.75). Adding clinical notes into XGBoost model led to improved specificity of 0.98 (0.98–0.98) and PPV of 0.9 (0.88–0.91), but with decreased sensitivity 0.77 (0.76–0.79). There were 77 bacteremia episodes during the study period identified and a host-specific visualization was created. Conclusions An electronic health record–based phenotype based on notes, diagnoses, and medications identifies ICH in the PICU with high specificity.

Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. Objective.— To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. Design.— We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription–polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. Results.— Concurrently collected saliva and mid-turbinate swabs had greater than 95% positive agreement with NP swabs when obtained within 10 days of symptom onset. Positive agreement of saliva and mid-turbinate samples collected from children with symptom onset >10 days prior, or without symptoms, was 82% compared to NP swab samples. Cycle threshold (Ct) values for mid-turbinate nasal samples more closely correlated with Ct values from NP samples than from saliva samples. Conclusions.— These findings suggest that all 3 sample types from children are useful for SARS-CoV-2 diagnostic testing by RT-PCR, and that concordance is greatest when the child has had symptoms of COVID-19 within the past 10 days. This study provides scientific justification for using sample types other than the NP swab for SARS-CoV-2 testing in pediatric populations.

The optimal care of septic patients depends on the successful recovery of clinically relevant microorganisms from blood cultures and the timely reporting of organism identification and antimicrobial susceptibility testing (AST) results. Many preanalytic factors play a critical role in culturing microorganisms, and advancements in blood culture instrument technology have reduced the time to positive results. Additionally, rapid organism identification and AST results directly from positive blood culture broth via new methods help to further shorten the time from empiric to targeted treatment. This article summarizes the current state of blood culture methods, including preanalytic, analytical, and postanalytic factors that are available to clinical microbiology laboratories.

Group A Streptococcus (GAS) (Streptococcus pyogenes) is the most common bacterial cause of acute pharyngitis. Diagnosed cases of GAS pharyngitis should be treated with antibiotics to prevent primary and secondary complications. Commonly, testing for GAS pharyngitis can be done rapidly (in minutes) using rapid antigen detection tests (RADTs), which generally have relatively high specificity (>90%) but lower sensitivity (70 to 90%). The low sensitivity of GAS RADTs requires that culture be performed to maximize sensitivity, which delays the final result by 24 to 48 hours. New molecular GAS assays are on the market and offer relatively rapid (i.e., minutes to tens of minutes) and highly sensitive results so that culture is not required for negative results. We describe our experience with moving from RADT and culture testing for GAS to solely molecular testing, providing quicker results for improved patient care.

BACKGROUND Urinary tract infections (UTI) are the most common bacterial infections among infants and young children with fever without a source. Extended-spectrum β-lactamases (ESBLs) have emerged as emerging cause of UTI globally; however, data about risk factors and clinical features of children with ESBL-UTI have been scarce. OBJECTIVE To describe the predisposing risk factors, clinical and microbiologic features associated with pediatric UTIs caused by ESBL-producing bacteria (ESBL-PB). METHODS Our nested case-control study ran from January 1, 2012 to December 31, 2016. Pediatric patients with ESBL-PB UTI were compared with patients with non-ESBL-PB UTI matched for age and year of diagnosis. RESULTS A total of 720 children were enrolled (240 cases and 480 controls). Patients with ESBL-PB UTI were more likely to have a history of prior intensive care unit (ICU) admission (22.5% vs. 12.3%, P < 0.001), at least one underlying comorbidity (19.2% vs. 5.8%, P < 0.001), prior hospitalization (47.1% vs. 32.9%, P < 0.001), exposure to a cephalosporin antibiotic within 30 days before culture (7.5% vs. 4.2%, P = 0.035), and to have cystostomy (7.9% vs. 1.5%, P < 0.001) compared with those with non-ESBL-PB UTI. Patients with ESBL-PB UTI were more likely to present with hypothermia (48.8% vs. 38.5%, P = 0.009); had significantly longer average hospital stays {8.7 days [95% confidence interval (CI): 3.2-14.3] vs. 4.0 days (95% CI: 2.5-5.5)} and were more likely to be admitted to the ICU [odds ratio (OR) 1.8; 95% CI: 1.1-2.9). Multivariate analysis determined that only having cystostomy (OR 3.7; 95% CI: 1.4-9.4] and at least one underlying comorbidity (OR 2.4; 95% CI: 1.3-4.3) were the independent risk factors for ESBL-PB UTI. All ESBL-PB isolates tested against meropenem were susceptible, majority were resistant to multiple non-beta-lactam antibiotics. CONCLUSIONS Children with underlying comorbidities and cystostomy are at higher risk for ESBL-PB UTI, but majority of ESBL cases were patients without any known risk factors. Clinical signs/symptoms and commonly used biochemical markers were unreliable to differentiate cases caused by ESBL-PB from those caused by non-ESBL-PB. Further research is needed to elucidate the conditions most associated with ESBL-PB UTIs among children to properly guide empirical therapy in patients at-risk for these infections, to improve the outcomes, and finally, to determine strategies for rational antimicrobial use.

The impact of repeated sample collection on COVID-19 test performance is unknown. The FDA and CDC currently recommend the primary collection of diagnostic samples to minimize the perceived risk of false-negative findings. We therefore evaluated the association between repeated sample collection and test performance among 325 symptomatic patients undergoing COVID-19 testing in Atlanta, GA. High concordance was found between consecutively collected mid-turbinate samples with both molecular (n = 74, 100% concordance) and antigen-based (n = 147, 97% concordance, kappa = 0.95, CI = 0.88–1.00) diagnostic assays. Repeated sample collection does not decrease COVID-19 test performance, demonstrating that multiple samples can be collected for assay validation and clinical diagnosis.

While there has been significant progress in the development of rapid COVID-19 diagnostics, as the pandemic unfolds, new challenges have emerged, including whether these technologies can reliably detect the more infectious variants of concern and be viably deployed in non-clinical settings as “self-tests”. Multidisciplinary evaluation of the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW, a widely used rapid antigen test, included limit of detection, variant detection, test performance across different age-groups, and usability with self/caregiver-administration. While BinaxNOW detected the highly infectious variants, B.1.1.7 (Alpha) first identified in the UK, B.1.351 (Beta) first identified in South Africa, P.1 (Gamma) first identified in Brazil, B.1.617.2 (Delta) first identified in India and B.1.2, a non-VOC, test sensitivity decreased with decreasing viral loads. Moreover, BinaxNOW sensitivity trended lower when devices were performed by patients/caregivers themselves compared to trained clinical staff, despite universally high usability assessments following self/caregiver-administration among different age groups. Overall, these data indicate that while BinaxNOW accurately detects the new viral variants, as rapid COVID-19 tests enter the home, their already lower sensitivities compared to RT-PCR may decrease even more due to user error.

Noroviruses are a leading cause of acute gastroenteritis (AGE) among adults and children worldwide. NoroSurv is a global network for norovirus strain surveillance among children

Rapid diagnostic tests (RDTs) for bloodstream infections (BSIs) decrease the time to organism identification and resistance detection. RDTs are associated with early deescalation of therapy for Gram-positive BSIs. However, it is less clear how RDTs influence antibiotic management for Gram-negative BSIs and whether RDT results are acted on during off-hours. We performed a single-center, retrospective review of children with BSI and Verigene (VG) testing at a children’s hospital. Of the 301 positive cultures included in the study (196 Gram-positive, 44 Gram-negative, 32 polymicrobial, and 29 non-VG targets), the VG result had potential to impact antibiotic selection in 171 cases; among these, antibiotic changes occurred in 119 (70%) cases. For Gram-negative cultures, the Verigene result correlated with unnecessary antibiotic escalation and exposure to broader-spectrum antibiotics than needed. In contrast, for Gram-positive cultures, the VG results correlated with appropriate antibiotic selection. VG results permitted early deescalation for methicillin-susceptible Staphylococcus aureus (MSSA) (19/24 [79%]) and avoidance of antibiotics for skin contaminants (30/85 [35%]). Antibiotic changes occurred more quickly during the day than at night (4.6 versus 11.7 h, respectively; P

We evaluated six commercial molecular tests targeting Mycoplasma pneumoniae, namely, the BioFire FilmArray respiratory panel (RP), the Meridian Alethia Mycoplasma Direct, the GenMark ePlex respiratory pathogen panel (RPP), the Luminex NxTAG RPP, the ELITech ELITe InGenius Mycoplasma MGB research use only (RUO) PCR, and the SpeeDx Resistance Plus MP assays. Laboratory-developed PCR assays at the University of Alabama at Birmingham and the Centers for Disease Control and Prevention were used as reference standards. Among 428 specimens, 212 were designated confirmed positives for M. pneumoniae. The highest clinical sensitivities were found with the InGenius PCR (99.5%) and the FilmArray RP (98.1%). The Resistance Plus MP identified 93.3% of the confirmed-positive specimens, whereas 83.6, 64.6, and 55.7% were identified by the ePlex RPP, NxTAG RPP, and Mycoplasma Direct assays, respectively. There was no significant difference between the sensitivity of the reference methods and that of the FilmArray RP and InGenius assays, but the remaining four assays detected significantly fewer positive specimens (P

In clinical microbiology laboratories, advancements in the methods used for routine organism identification have facilitated more accurate species level resolution. This, along with increasing knowledge regarding “new” pathogens, has provided new insights into biology and has revealed clinical associations not previously known. Aerococcus urinae, Alloscardovia omnicolens, and Actinotignum schaalii are Gram-positive bacteria associated with urinary tract infections but can also be members of the urinary tract microbiota. In addition, invasive infections have been reported for the three bacteria, usually with a urinary tract source. Importantly, the use of routine biochemical testing methods for identification of these bacteria can result in misidentification or the incorrect determination that they are not clinically significant. Of these bacteria, only A. urinae has antimicrobial susceptibility testing criteria and interpretative breakpoints. Microbiologists and clinicians need to be aware of these urinary tract pathogens and how to correctly identify them.

Background: Discontinuation of contact precautions for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have failed to show an increase in associated transmission or infections in adult healthcare settings. Pediatric experience is limited. Objective: We evaluated the impact of discontinuing contact precautions for MRSA, VRE, and extended-spectrum β-lactamase–producing gram-negative bacilli (ESBLs) on device-associated healthcare-associated infections (HAIs). Methods: In October 2018, contact precautions were discontinued for children with MRSA, VRE, and ESBLs in a large, tertiary-care pediatric healthcare system comprising 2 hospitals and 620 beds. Coincident interventions that potentially reduced HAIs included blood culture diagnostic stewardship (June 2018), a hand hygiene education initiative (July 2018), a handshake antibiotic stewardship program (December 2018) and multidisciplinary infection prevention rounding in the intensive care units (November 2018). Compliance with hand hygiene and HAI prevention bundles were monitored. Device-associated HAIs were identified using standard definitions. Annotated run charts were used to track the impact of interventions on changes in device-associated HAIs over time. Results: Average hand hygiene compliance was 91%. Compliance with HAI prevention bundles was 81% for ventilator-associated pneumonias, 90% for catheter-associated urinary tract infections, and 97% for central-line–associated bloodstream infections. Overall, device-associated HAIs decreased from 6.04 per 10,000 patient days to 3.25 per 10,000 patient days after October 2018 (Fig. 1). Prior to October 2018, MRSA, VRE and ESBLs accounted for 10% of device-associated HAIs. This rate decreased to 5% after October 2018. The decrease in HAIs was likely related to interventions such as infection prevention rounds and handshake stewardship. Conclusions: Discontinuation of contact precautions for children with MRSA, VRE, and ESBLs were not associated with increased device-associated HAIs, and such discontinuation is likely safe in the setting of robust infection prevention and antibiotic stewardship programs.Funding: NoneDisclosures: None

We report a cluster of pediatric cryptosporidiosis infections among solid organ transplant recipients at a summer camp in Georgia, USA. A retrospective cohort study was conducted to investigate the risk factors for infection. A total of 118 campers attended the camp during July 23-28, 2017. The overall attack rate among campers during the outbreak was 11% (13/118). Sanger-based amplicon sequencing of stool specimens from 7 (80%) campers identified Cryptosporidium hominis as the suspected etiologic agent. All infected campers were heart or kidney transplant recipients receiving immunosuppressive therapy. The median reported symptom duration was 12 days (range 6-18 days) and 9 (69.2%) were hospitalized for at least one night (median length of stay 5 days, range 2-16 days). There were no deaths or acute rejection events attributed to infection. The results of the epidemiologic and environmental investigation suggest a recreational pool as the presumed source, although there was no direct evidence to support this. Many long-term interventions were implemented, and there have been no further outbreaks at the camp in the following two years. This outbreak demonstrates that cryptosporidiosis may be associated with notable burden in pediatric transplant recipients, and illustrates the challenges associated with source identification and containment.

Non-Staphylococcus aureus staphylococcal species (non-SASS) are important pathogens in both animal and human populations. The development of β-lactam resistance in non-SASS through acquisition and expression of penicillin-binding protein 2a (PBP2a) represents a significant clinical and public health threat. Here, we evaluated the diagnostic performance of two versions of a PBP2a immunochromatographic assay with non-SASS. Our data show that the revised version of the assay, the PBP2a SA culture colony test, has superior diagnostic sensitivity compared to the previous version of the assay, the PBP2a culture colony test, 100% (95% confidence interval [CI], 93.3 to 100%) versus 67.9% (95% CI, 53.7 to 80.1%), respectively, while both assays display a specificity of 100% (95% CI, 92.5 to 100%). Therefore, the PBP2a SA culture colony test offers a rapid, accurate, and relatively inexpensive method for detecting PBP2a-mediated β-lactam resistance in clinically relevant non-SASS for the management of infections due to these organisms and for antimicrobial stewardship.

Background: SARS-CoV-2 infection in children does not seem to follow the same pattern as in adults. Limited information is published on the level of antibody production and the duration of antibody response in children with COVID-19. Moreover, it is unknown if all children have a similar immune response to the infection, or if there are age dependent differences. In these data, we look at the IgM and IgG levels and duration of two age groups infected by the SARS-CoV-2 virus. Methods: Residual laboratory specimens from pediatric patients positive for SARS-CoV-2 infection were tested for IgM and IgG against SARS-CoV-2 using an automated Abbott ARCHITECT i1000. We tested 181 specimens from 41 patients with a positive molecular result. Data was grouped either as time after nucleic acid amplification test (NAAT) or time after symptom onset. Patient samples were divided into 2 age groups: 0 to 11 years old and 12 to 19 years old. The assays detect IgM against the spike protein and IgG against the nucleocapsid protein.

Objectives Group A Streptococcus (GAS) is the most common bacterial cause of pediatric acute pharyngitis, and its quick identification is important for subsequent treatment. We sought to determine whether molecular GAS-based testing can successfully replace GAS antigen testing and subsequent culture in a pediatric urgent care center. Methods We tested 160 patient oropharyngeal samples by a rapid antigen GAS test, the Alere i Strep A test, and throat culture in a pediatric urgent care setting and calculated basic statistical metrics. Results The sensitivity and specificity of the molecular test were 98% and 100%, respectively, compared with culture. There was a 9% false-positive rate with the rapid antigen-based testing. Conclusions The Alere test is sufficiently sensitive and specific for definitive GAS testing in a pediatric urgent care setting. This implementation has enabled us to provide definitive patient results at the time of each patient encounter.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

Background The reservoir of pathogenic ciprofloxacin-resistant Escherichia coli remains unknown. Methods We conducted a prospective cohort study of 80 healthy twins and their mothers to determine the frequency of excretion of ciprofloxacin-resistant, potentially pathogenic E. coli. Stool specimens were cultured selectively for ciprofloxacin-resistant gram-negative bacteria. Isolates were categorized on the basis of additional resistance and virulence profiles. We also prospectively collected clinical metadata. Results Fifteen children (19%) and 8 mothers (20%) excreted ciprofloxacin-resistant E. coli at least once. Overall, 33% of 40 families had at least 1 member whose stool specimen yielded ciprofloxacin-resistant E. coli on culture. Fifty-seven submitted stool specimens (2.8%) contained such organisms; clones ST131-H30 and ST405 accounted for 52 and 5 of the positive specimens, respectively. Length of hospital stay after birth (P = .002) and maternal colonization (P = .0001) were associated with subsequent childhood carriage of ciprofloxacin-resistant E. coli; antibiotic use, acid suppression, sex, mode of delivery, and maternal perinatal antibiotic use were not. Ciprofloxacin-resistant E. coli were usually resistant to additional antibiotic classes, and all had virulence genotypes typical of extraintestinal pathogenic E. coli. Conclusions Healthy children and their mothers commonly harbor ciprofloxacin-resistant E. coli with pathogenic potential.

Clinical laboratory workers encounter a variety of occupational hazards, including exposure to infectious agents. The routes of pathogen exposure associated with laboratory work include ingestion, inhalation, direct inoculation, and contamination of skin and mucous membranes (1). The accidental inoculation of infectious materials (i.e., via contaminated needles, broken glass, or other sharps) is the leading cause of laboratory-associated infections (1). In the 1980s, the emergence of the HIV epidemic created an appreciation for biosafety and laboratory-acquired infections, ultimately leading to both practice guidelines and legislation to reduce the risk of exposure of laboratory workers to bloodborne pathogens; this was implemented by the adoption of Universal Precautions in 1987, and later, in 1996, these became a component of Standard Precautions (2). The actual incidence and risk of laboratory-acquired infections is very difficult to quantify in the absence of standardized reporting systems, as well as the challenge of attributing a specific exposure to acquisition of infection. The most comprehensive studies to date attempting to estimate the incidence and epidemiology of laboratory-acquired infections were completed prior to 1980, and it is difficult to generalize them to today's laboratory environment, for a variety of reasons—standards for personal protective equipment have changed, the availability of vaccines, the epidemiology of bloodborne infections has changed, and the way laboratory testing is performed has changed (with a shift away from primarily manual methods to more automated methods). That said, since 1999, only 1 confirmed case of laboratory-acquired HIV infection has been reported in the US; this case was secondary to a needle stick injury sustained in an individual working with a live HIV culture (2). The fear associated with the recent Ebola virus (EV)3 epidemic triggered a renewed interest in occupationally acquired infections in healthcare workers in the US, including the safety of laboratory workers in …

The advent of new diagnostics assays for Group A Streptococcus, influenza, and respiratory syncytial virus now provide rapid results with increased sensitivity and specificity. Molecular testing is no longer confined to the walls of the laboratory, but moving to the patient in the form of point-of-care tests. In addition, multiplex syndromic panels are allowing broad testing of pathogens associated with a single clinical presentation. This article focuses specifically on rapid diagnostic tests for pathogens most affecting children. Rapid and accurate pathogen detection in children may result in decreased time to optimal antimicrobial treatment and improved patient outcomes.

Sepsis is a global problem and laboratory methods must be optimized to effect rapid, positive patient outcomes. The proverbial reference standard method for diagnosis, the actual culture of blood, suffers from a variety of preanalytical issues such as sufficient blood volume, prior antimicrobial treatment, time from sampling to incubation, and analytical issues of prolonged turnaround time until final identification and availability of antimicrobial susceptibility testing results. Advancements have occurred in molecular methods to provide rapid microorganism identification and resistance gene detection (e.g., mecA in Staphylococcus spp.) from positive blood culture broths. In a meta-analysis of these new methods, a significant reduction in time to targeted treatment was observed ( 1 ). However, these methods are dependent on having a positive blood culture, which is subject to the preanalytical issues described above. The ability to directly identify pathogens from blood specimens would greatly reduce the time to identification and time to optimization of therapy, as well as potentially contributing to avoiding unnecessary antimicrobials and/or discontinuation of treatment in patients with negative testing results. Direct detection methods for viruses from patient specimens have benefited from advances in molecular methods, which is largely because of the high viral loads present in those specimens. In contrast, the majority of septic patients have a paucity of bacteria or fungi per milliliter of blood, necessitating the need for larger blood volumes to increase blood culture sensitivity ( 2 – 4 ).

Background Bordetella holmesii is a respiratory pathogen, known to cause bacteremia predominantly among patients with functional or anatomical asplenia. Currently, there is no consensus on optimal treatment for B. holmesii infection nor are there established interpretative criteria. This study aims to describe treatment of pediatric patients diagnosed with B. holmesii bacteremia, and treatment outcomes, in order to help establish an optimal therapeutic strategy. Methods We conducted a retrospective chart review of pediatric patients with microbiologically confirmed B. holmesii bacteremia at Children’s Healthcare of Atlanta, 2011–2018. We extracted demographic and clinical information of the identified patients from the medical record, and evaluated antimicrobial choice, hospital days, and treatment outcomes. Results Seven patients were identified; all had sickle cell disease and five had moderate to severe asthma requiring controller medications. They presented to the emergency department with mild respiratory illness with fevers, but had hemodynamic stability. Peripheral blood cultures were obtained and intravenous ceftriaxone was administered as the empiric antibiotic therapy. Six patients were discharged home after evaluation, and one patient was admitted for treatment for acute chest syndrome with venoocclusive crisis (see figure). When the blood cultures grew B. holmesii, previously discharged patients were called back for follow-up; three were admitted, and only one patient had a subsequent blood culture growing B. holmesii. Hospitalization days ranged from 3 to 5 days, and two patients went home with oral ciprofloxacin at the time of discharge. Total antibiotic days ranged from 1 to 15 days among the seven patients. No one required an intensive level care, and all were asymptomatic without recurrence of B. holmesii infections at the post-discharge follow-up. Conclusion In our pediatric patients with B. holmesii bacteremia, clinical recovery was favorable with no severe illness, despite widely different treatment regimens and length of therapy. The questions still remain regarding pathogenicity of B. holmesii infection and efficacy of antibiotic use. Disclosures All authors: No reported disclosures.

Necrotizing fasciitis is a life-threatening, rapidly progressing infection of fascia and subcutaneous cellular tissue typically caused by mixed aerobic and anaerobic bacteria. We present a case report of an immunocompromised 4-year-old female with necrotizing fasciitis from a rare fungal organism, Mucor indicus. The patient underwent multiple debridements and was treated for 10 months, first on liposomal amphotericin B (2 months) then posaconazole (8 months). Mucor indicus is a rarely described pathogen with only nine other cases described. Identification of this organism remains a challenge, and the need for further understanding of risk factors and organism susceptibility testing to help guide treatment is crucial.

We performed a retrospective analysis of a simple modification to MALDI-TOF MS for microorganism identification to accurately improve the turnaround time (TAT) for identification of Enterobacteriaceae recovered in blood cultures. Relative to standard MALDI-TOF MS procedures, we reduced TAT from 28.3 (n=90) to 21.2h (n=107).

Infectious diarrhea is a major cause of morbidity. A rapid and inexpensive assay for the diagnosis of infectious gastroenteritis would expedite appropriate therapy and prevent unnecessary and potentially invasive testing. This article summarizes assays for the diagnosis of infectious gastroenteritis based on the host response to bacterial, viral, or parasitic infection. This includes both systemic biomarkers (such as C-reactive protein, erythrocyte sedimentation rate, and serum cytokines) and fecal biomarkers (such as lactoferrin, fecal leukocyte analysis, and calprotectin). Although some of these assays have value as adjunct diagnostics, they lack sensitivity and specificity as stand-alone tests in this setting.

Hemophagocytic lymphohistiocytosis (HLH) is a rare potentially fatal syndrome characterized by an uncontrolled hyperinflammatory response. The secondary form of HLH is usually triggered by a causative agent. Ehrlichia chaffeensis is a rare trigger of secondary HLH. We present a case series of five adolescents and adults diagnosed with Ehrlichia-induced HLH and we discuss their clinical and laboratory findings. We also review the literature for similar cases. Between October 2003 and June 2014, we identified 76 cases of HLH in adolescents and adults, 5 of which were induced by Ehrlichia. All 5 patients had fever, cytopenias, hypertriglyceridemia, and high ferritin. Hyperferritinemia was striking with a median admission ferritin of 47,290 μg/L (range: 2,863-85,517). In addition to the positive Ehrlichia PCR testing on peripheral blood of all patients, two patients with neurologic symptoms tested positive for E. chaffeensis in CSF specimens. Early treatment with doxycycline was effective. After a median follow up of 7.3 months, all patients were alive and none had recurrence of HLH. Clinicians should consider E. chaffeensis as a potential trigger for HLH especially in areas with tick activity. Prompt diagnosis and treatment with doxycycline are required for a better outcome.

In order to divide, the bacterium Escherichia coli must assemble a set of at least 10 essential proteins at the nascent division site. These proteins localize to midcell according to a linear hierarchy, suggesting that cell division proteins are added to the nascent divisome in strict sequence. We previously described a method, 'premature targeting', which allows us to target a protein directly to the division site independently of other cell division proteins normally required for its localization at midcell. By systematically applying this method to probe the recruitment of and associations among late cell division proteins, we show that this linear assembly model is likely incorrect. Rather, we find that the assembly of most of the late proteins can occur independently of 'upstream' proteins. Further, most late proteins, when prematurely targeted to midcell, can back-recruit upstream proteins in the reverse of the predicted pathway. Together these observations indicate that the late proteins, with the notable exception of the last protein in the pathway, FtsN, are associated in a hierarchical set of protein complexes. Based on these observations we present a revised model for assembly of the E. coli division apparatus.

We report two cases of bacteremia with Actinobaculum schaalii, a rarely reported, anaerobic, Gram-positive bacterium. The first case was a patient with renal cancer who developed pyelonephritis after cryoablation, and the second was a patient who developed sepsis after a urogenital procedure. Bacteremia resolved after administration of empiric antibiotic therapy.

Sulfate-reducing bacteria (SRB) colonize the guts of ∼50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing, RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger , the most common SRB in a surveyed cohort of healthy US adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types of carbohydrates and sulfur sources. Diet was a major determinant of functions expressed by this artificial nine-member community and of the genes that impact D. piger fitness; the latter includes high- and low-affinity systems for using ammonia, a limiting resource for D. piger in mice consuming a polysaccharide-rich diet. Although genes involved in hydrogen consumption and sulfate reduction are necessary for its colonization, varying dietary-free sulfate levels did not significantly alter levels of D. piger , which can obtain sulfate from the host in part via cross-feeding mediated by Bacteroides -encoded sulfatases. Chondroitin sulfate, a common dietary supplement, increased D. piger and H 2 S levels without compromising gut barrier integrity. A chondroitin sulfate-supplemented diet together with D. piger impacted the assemblage’s substrate utilization preferences, allowing consumption of more reduced carbon sources and increasing the abundance of the H 2 -producing Actinobacterium, Collinsella aerofaciens . Our findings provide genetic and metabolic details of how this H 2 -consuming SRB shapes the responses of a microbiota to diet ingredients and a framework for examining how individuals lacking D. piger differ from those who harbor it.

Cell division in bacteria requires the coordinated action of a set of proteins, the divisome, for proper constriction of the cell envelope. Multiple protein-protein interactions are required for assembly of a stable divisome. Within the Escherichia coli divisome is a conserved subcomplex of inner membrane proteins, the FtsB/FtsL/FtsQ complex, which is necessary for linking the upstream division proteins, which are predominantly cytoplasmic, with the downstream division proteins, which are predominantly periplasmic. FtsB and FtsL are small bitopic membrane proteins with predicted coiled-coil motifs, which themselves form a stable subcomplex that can recruit downstream division proteins independently of FtsQ; however, the details of how FtsB and FtsL interact together and with other proteins remain to be characterized. Despite the small size of FtsB, we identified separate interaction domains of FtsB that are required for interaction with FtsL and FtsQ. The N-terminal half of FtsB is necessary for interaction with FtsL and sufficient, when in complex with FtsL, for recruitment of downstream division proteins, while a portion of the FtsB C terminus is necessary for interaction with FtsQ. These properties of FtsB support the proposal that its main function is as part of a molecular scaffold to allow for proper formation of the divisome.

In order to divide, the bacterium Escherichia coli must assemble a set of at least 10 essential proteins at the nascent division site. These proteins localize to midcell according to a linear hierarchy, suggesting that cell division proteins are added to the nascent divisome in strict sequence. We previously described a method, 'premature targeting', which allows us to target a protein directly to the division site independently of other cell division proteins normally required for its localization at midcell. By systematically applying this method to probe the recruitment of and associations among late cell division proteins, we show that this linear assembly model is likely incorrect. Rather, we find that the assembly of most of the late proteins can occur independently of 'upstream' proteins. Further, most late proteins, when prematurely targeted to midcell, can back-recruit upstream proteins in the reverse of the predicted pathway. Together these observations indicate that the late proteins, with the notable exception of the last protein in the pathway, FtsN, are associated in a hierarchical set of protein complexes. Based on these observations we present a revised model for assembly of the E. coli division apparatus.

In this study, we investigated the requirement of theListeria monocytogenesbroad-range phospholipase C (PC-PLC) during infection of human epithelial cells.L. monocytogenesis a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell,L. monocytogenesis initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies withL. monocytogeneshave been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However,L. monocytogenescan escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative ofL. monocytogenesstrain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression inL. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.