Search

Clostridioides difficile infection (CDI) is the fifth leading cause of death from nonmalignant gastrointestinal disease in the United States. The contribution of resistance to C. difficile-active antibiotics to the outcomes of CDI is unclear. We evaluated the antimicrobial susceptibility of C. difficile isolates in a U.S. hospital and determined associations of clinical variables and binary toxin positivity with antibiotic resistance. C. difficile spores were cultured from fecal specimens of adult patients with CDI for genotyping and antimicrobial susceptibility assay (for clindamycin [CLI], fidaxomicin [FDX], metronidazole [MTZ], moxifloxacin [MXF], tigecycline [TGC], and vancomycin [VAN]). Electronic medical records were reviewed for clinical data extraction. Ninety-seven of 130 (75%) fecal samples grew toxigenic C. difficile in culture. Most of the isolates were tcdA(+) tcdB(+) cdtB(−) (80.4%), and 18.6% and 1% were tcdA(+) tcdB(+) cdtB(+) and tcdA(−) tcdB(+) cdtB(+), respectively. Susceptibility to VAN, MTZ, FDX, TGC, MXF, and CLI was 96%, 94%, 100%, 100%, 8%, and 79%, respectively. Six isolates, all cdtB positive and belonging to the 027 ribotype, were resistant to VAN and/or MTZ. Higher MICs were found in isolates with a mutation in the VAN-related resistance gene vanR, but not vanS. In addition, cdtB(+) isolates exhibited higher MICs of VAN, MTZ, TGC, CLI, and MXF compared to cdtB(−) strains. Patients with greater intestinal inflammation or severe disease were more likely to be infected with cdtB(+) strains. Decreased susceptibility to antibiotics is not directly associated with either severe or recurrent CDI. However, antimicrobial susceptibility of C. difficile is decreased in strains positive for the binary toxin gene.

Background Implementation of the Accelerate PhenoTM Gram-negative platform (RDT) paired with antimicrobial stewardship program (ASP) intervention projects to improve time to institutional-preferred antimicrobial therapy (IPT) for Gram-negative bacilli (GNB) bloodstream infections (BSIs). However, few data describe the impact of discrepant RDT results from standard of care (SOC) methods on antimicrobial prescribing. Methods A single-center, pre-/post-intervention study of consecutive, nonduplicate blood cultures for adult inpatients with GNB BSI following combined RDT + ASP intervention was performed. The primary outcome was time to IPT. An a priori definition of IPT was utilized to limit bias and to allow for an assessment of the impact of discrepant RDT results with the SOC reference standard. Results Five hundred fourteen patients (PRE 264; POST 250) were included. Median time to antimicrobial susceptibility testing (AST) results decreased 29.4 hours (P, Implementation of a rapid phenotypic diagnostic test, plus antimicrobial stewardship intervention for Gram-negative bloodstream infections, was associated with significantly faster time to institutional preferred therapy without change in clinical outcomes. Occasional erroneous results contributed to incorrect ASP recommendations.

Wastewater-based monitoring for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the individual building level could be an efficient, passive means of early detection of new cases in congregate living settings, but this approach has not been validated. Preliminary samples were collected from a hospital and a local municipal wastewater treatment plant. Molecular diagnostic methods were compared side by side to assess feasibility, performance, and sensitivity. Refined sample collection and processing protocols were then used to monitor two occupied dormitory complexes (n = 105 and 66) over 8 weeks. Wastewater results were validated using known case counts from external clinical testing of building occupants. Results confirm that ultracentrifugation from a 24-h composite collection had a sensitivity of 96.2% and a specificity of 100%. However, the method could not distinguish new infectious cases from persistent convalescent shedding of SARS-CoV-2 RNA. If the detection of convalescent shedding is considered a false positive, then the sensitivity is 100% and specificity drops to 45%. It was determined that the proposed approach constitutes a highly sensitive wastewater surveillance method for detecting SARS-CoV-2, but it could not distinguish new infectious cases from persistent convalescent shedding. Future work must focus on approaches to distinguish new infections from convalescent shedding to fully realize the potential of building wastewater as a surveillance tool for congregate living. IMPORTANCE Some of the most severe outbreaks of COVID-19 have taken place in places where persons live together, such as nursing homes. Wastewater testing from individual buildings could be used for frequent pooled surveillance of virus from all occupants, including those who are contagious, with or without symptoms. This work provides a sensitive practical method for detecting infected individuals, as validated in two building complexes housing occupants who underwent frequent clinical testing performed by external entities. Although this sensitive method could be deployed now for pooled surveillance as an early warning system to limit outbreaks, the study shows that the approach will require further refinement to differentiate contagious, newly infected individuals from persons who have persistent viral fragments shedding in their stool outside the contagious period.

Multisystem inflammatory syndrome in children (MIS-C) is a serious, sometimes life-threatening late complication of coronavirus disease 2019 (COVID-19) with multiorgan involvement and evidence of immune activation. The pathogenesis of MIS-C is not known, nor is the pathogenesis of the severe organ damage that is the hallmark of MIS-C. Human herpesvirus 6 (HHV-6), the virus responsible for roseola, is a ubiquitous herpesvirus that causes close to universal infection by the age of 3 years. HHV-6 remains latent for life and can be activated during inflammatory states, by other viruses, and by host cell apoptosis. HHV-6 has been associated with end-organ diseases, including hepatitis, carditis, and encephalitis. In addition, ∼1% of people have inherited chromosomally integrated human herpesvirus 6 (iciHHV-6), which is HHV-6 that has been integrated into chromosomal telomeric regions and is transmitted through the germ line. iciHHV-6 can be reactivated and has been associated with altered immune responses. We report here a case of MIS-C in which an initial high HHV-6 DNA polymerase chain reaction viral load assay prompted testing for iciHHV-6, which yielded a positive result. Additional research may be warranted to determine if iciHHV-6 is commonly observed in patients with MIS-C and, if so, whether it may play a part in MIS-C pathogenesis.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, in the lung, hyaluronan synthase 1 (Has1) was the most downregulated gene and hyaluronan accumulation was decreased. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator, and indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases., Summary: IL-13 levels are elevated in patients with severe COVID-19. In a mouse model of disease, IL-13 neutralization results in reduced disease and lung hyaluronan deposition. Similarly, blockade of hyaluronan’s receptor, CD44, reduces disease, highlighting a novel mechanism for IL-13-mediated pathology.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a pandemic of the respiratory disease coronavirus disease 2019 (COVID-19). Antibody testing is essential to identify persons exposed to the virus and potentially in predicting disease immunity. 183 COVID-19 patients (68 of whom required mechanical ventilation) and 41 controls were tested for plasma IgG, IgA and IgM against the SARS-CoV-2 S1, S2, receptor binding domain (RBD) and N proteins using the MILLIPLEX® SARS-CoV-2 Antigen Panel. Plasma cytokines were concurrently measured using the MILLIPLEX® MAP Human Cytokine/Chemokine/Growth Factor Panel A. As expected the 183 COVID-19 positive patients had high levels of IgG, IgA and IgM anti-SARS-CoV-2 antibodies against each of the viral proteins. Sensitivity of anti-S1 IgG increased from 60% to 93% one week after symptom onset. S1-IgG and S1-IgA had specificities of 98% compared to the 41 COVID-19 negative patients. The 68 ventilated COVID-19 positive patients had higher antibody levels than the 115 COVID-19 positive patients who were not ventilated. IgG antibody levels against S1 protein had the strongest positive correlation to days from symptom onset. There were no statistically significant differences in IgG, IgA and IgM antibodies against S1 based on age. We found that patients with the highest levels of anti-SARS-CoV-2 antibodies had the lowest viral load in the nasopharynx. Finally there was a correlation of high plasma IL-10 with low anti-SARS-CoV-2 antibodies. Anti-SARS-CoV-2 antibody levels, as measured by a novel antigen panel, increased within days after symptom onset, achieving > 90% sensitivity and specificity within one week, and were highest in patients who required mechanical ventilation. Antibody levels were inversely associated with viral load but did not differ as a function of age. The correlation of high IL-10 with low antibody response suggests a potentially suppressive role of this cytokine in the humoral immune response in COVID-19.

Background Clostridioides difficile is the leading health care–associated pathogen, but clinicians lack a test that can reliably differentiate colonization from infection. Health care costs attributed to C. difficile are substantial, but the economic burden associated with C. difficile false positives is poorly understood. Methods A propensity score matching model for cost per hospitalization was developed to estimate the costs of both true infection and false positives. Predictors of C. difficile positivity used to estimate the propensity score were age, Charlson comorbidity index, white cell count, and creatinine. We used polymerase chain reaction (PCR) cycle threshold to identify and compare 3 groups: (1) true infection, (2) C. difficile colonization, and (3) C. difficile negative. Results A positive test was associated with $3018 higher unadjusted hospital cost. Among the 3 comparisons made with propensity-matched negative controls (all positives [+$179; P = .934], true positives [–$1892; P = .100], and colonized positives), only colonization was associated with significantly increased (+$3418; P = .012) cost. Differences in lengths of stay (all positives 0 days, P = .126; true 0 days, P = .919; colonized 1 day, P = .019) appeared to underly cost differences. Conclusions In the first C. difficile cost analysis to utilize PCR cycle threshold to differentiate colonization, we found high propensity-matched hospital costs associated with colonized but not true positives. This unexpected finding may be due to misdiagnosis of non–C. difficile diarrhea or unadjusted factors associated with colonization.

Wastewater-based monitoring for SARS-CoV-2 holds promise as tool to inform public health-decision making. Testing at individual building-level could be an efficient, passive means of preventing early detection of new cases in congregate living settings, but this approach has not been validated. Sample collection protocols were developed and refined during preliminary sampling from a hospital and a local municipal wastewater treatment plant. Molecular diagnostic methods were compared side-by-side to assess feasibility, performance and sensitivity. Optimized sample collection and processing protocols were then used to monitor two occupied dormitory complexes (n=105 and 66) over eight weeks. Wastewater results were validated using known case counts from external clinical testing of building occupants. Results confirm that ultracentrifugation from a 24 hour composite collection had a sensitivity of 95% and a specificity of 100%. However, if the detection of convalescent shedding is considered a false positive then the sensitivity would be 95.2% but the specificity would drop to 52%. We determined a highly sensitive method for detecting SARS-CoV-2 shedding in building wastewater however our methods could not distinguish new infectious cases from persistent convalescent shedding of SARS-CoV-2 RNA. Future work must focus on methods to distinguish new infections from convalescent shedding to widely deploy this promising wastewater surveillance tool.

Background Rapid diagnostics for enterococcal bloodstream infections (E-BSIs) can decrease the time to speciation and determination of vancomycin resistance but may not lead to improved antibiotic stewardship. Methods Over 3 years, the time to administration of institutionally preferred antibiotics (IPT) for patients with E-BSI was evaluated and compared between 3 intervention groups: before (baseline) and after implementation of a rapid diagnostic (BC-GP), and the use of BC-GP with an Infectious Diseases (ID) fellow–driven consultative intervention (BC-GP + ID). Results A total of 110 patients (63 baseline, 13 BC-GP, 34 BC-GP + ID) with E-BSI were evaluated. Evaluation of Enterococcus faecium BSI showed that the time IPT was significantly reduced with BC-GP + ID by 10.6 h from baseline (P = 0.02) and 5.4 h from BC-GP (P = 0.04). Conclusions An ID fellow–driven stewardship intervention was associated with a significant improvement in time to IPT for patients with E. faecium but not E. faecalis BSI.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic RNA virus characterized by high transmission rates and pathogenicity worldwide. Continued control of the COVID-19 pandemic requires the diversification of rapid, easy to use, sensitive, and portable methods for SARS-CoV-2 sample preparation and analysis. Here, we propose a method for SARS-CoV-2 viral enrichment and enzymatic extraction of RNA from clinically relevant matrices in under 10 min. This technique utilizes affinity-capture hydrogel particles to concentrate SARS-CoV-2 from solution, and leverages existing PDQeX technology for RNA isolation. Characterization of our method is accomplished with reverse transcription real-time polymerase chain reaction (RT-PCR) for relative, comparative RNA detection. In a double-blind study analyzing viral transport media (VTM) obtained from clinical nasopharyngeal swabs, our sample preparation method demonstrated both comparable results to a routinely used commercial extraction kit and 100% concordance with laboratory diagnoses. Compatibility of eluates with alternative forms of analysis was confirmed using microfluidic RT-PCR (μRT-PCR), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). The alternative methods explored here conveyed successful amplification from all RNA eluates originating from positive clinical samples. Finally, this method demonstrated high performance within a saliva matrix across a broad range of viral titers and dilutions up to 90% saliva matrix, and sets the stage for miniaturization to the microscale., Graphical abstract Image 1

Objective: Women without regular health care providers or a medical home routinely fail to complete recommended cervical cancer screening. At-home self-collection of samples to test for high-risk s...

Background We report a case of prosthetic hip joint infection in a heart transplant recipient due to Anaerobiospirillum succiniciproducens, a genus of spiral-shaped curved anaerobic gram-negative rod which colonizes the gastrointestinal tract of cats and dogs. Invasive infections in humans are rare and typically occur in immunocompromised hosts. Case presentation A 65-year-old male dog breeder with a history of rheumatoid arthritis, bilateral hip arthroplasties, and non-ischemic cardiomyopathy with a heart transplant 10 years ago presented with a three month history of progressive left hip pain and frank purulence on hip aspiration. He underwent irrigation and debridement of the left hip and one-stage revision with hardware exchange. Although gram stain and culture from synovial fluid and intraoperative cultures were initially negative, anaerobic cultures from tissue specimens later grew a spiral-shaped gram-negative rod, identified as Anaerobiospirillum spp. by 16S rRNA gene sequencing. The patient was treated with ceftriaxone 2 g daily for 6 weeks with a good response to treatment. A similar organism was unable to be isolated from culture of 2 of the patient’s dogs, however, they were thought to be the most likely source of his infection. Conclusion Anaerobiospirillum spp. should be considered in immunocompromised patients with exposure to dogs or cats who present with bacteremia, gastrointestinal infection, pyomyositis, or prosthetic joint infections, especially in cases of culture-negative or with anaerobic culture growth.

Background Molecular diagnostic panels for enteric pathogens offer increased sensitivity and reduced turnaround time. However, many pathogen detections do not change clinical management, and the cost is substantial. Methods We performed a retrospective chart review of adult outpatients with diarrhea at the University of Virginia who had samples tested by the FilmArray Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City, UT) to identify the clinical yield and to validate the clinical criteria for testing recommended in the 2017 Infectious Diseases Society of America (IDSA) guidelines. Results We analyzed 629 tests sent from adult outpatients with diarrhea between March 23, 2015, and July 18, 2016. A pathogen was detected in 127 of 629 specimens (20.2%). The most common pathogens were enteropathogenic Escherichia coli (47, 7.5%), norovirus (24, 3.8%), enteroaggregative E. coli (14, 2.2%), Campylobacter (9, 1.4%), and Salmonella (9; 1.4%). The clinical yield of testing was low, with antimicrobial treatment clearly indicated for only 18 subjects (2.9%) and any change in clinical management indicated for 33 subjects (5.2%). Following the clinical criteria for diagnostic testing from the 2017 IDSA guidelines, which suggest diagnostic testing for patients with fever, abdominal pain, blood in stool, or an immunocompromising condition, would have reduced testing by 32.3% without significantly reducing the clinical yield (sensitivity, 97.0%; 95% confidence interval [CI], 84.2%–99.9%; negative predictive value, 99.5%; 95% CI, 97.3%–100.0%). Conclusions The clinical yield of molecular diagnostic testing in this population was low. Compliance with IDSA guidelines in adult outpatients with diarrhea could reduce testing by approximately one-third.

We describe 4 children (11–17 years in age) at our institution with acute appendicitis in the setting of SARS-CoV-2 infection, suggesting a possible association. Providers should consider testing for this infection in patients with severe gastrointestinal symptoms, in order to take appropriate transmission based precautions, until more is understood.

Diagnostic stewardship is an increasingly recognized means to reduce unnecessary tests and diagnostic errors. As a leading cause of healthcare-associated infection for which accurate laboratory diagnosis remains a challenge, Clostridium difficile offers an ideal opportunity to apply the principles of diagnostic stewardship. The recently updated 2017 Infectious Diseases Society of America (IDSA)-Society for Healthcare Epidemiology of America (SHEA) Clinical Practice Guidelines for C. difficile infection now recommend separate diagnostic strategies depending on whether an institution has adopted diagnostic stewardship in test decision making. IDSA-SHEA endorsement of diagnostic stewardship for C. difficile highlights the increasing role of diagnostic stewardship in hospitals. In this opinion piece, we introduce the concept of diagnostic stewardship by discussing the new IDSA-SHEA diagnostic recommendations for laboratory diagnosis of C. difficile. We outline recent examples of diagnostic stewardship, challenges to implementation, potential downsides and propose future areas of study.

Dysregulated expression of oncogenic types of E6 and E7 is necessary for human papillomavirus (HPV)-driven carcinogenesis. An HPV E6/E7 mRNA in situ hybridization (ISH) assay covering 18 common high-risk types ("HR-RISH," aka HR-HPV RNA18 ISH) has not been extensively studied in the anogenital tract or validated on automated technology. We herein compare HR-RISH to DNA polymerase chain reaction (PCR), p16 immunohistochemistry, and a previously available HPV DNA ISH assay in HPV-related anogenital and head and neck (H&N) neoplasia. A total of 102 squamous intraepithelial lesions (16 CIN1, 25 CIN3, 3 AIN1, 12 AIN3, 9 VIN3)/invasive squamous cell carcinomas (17 cervical, 2 anal, 18 H&N) as well as 10 normal and 15 reactive cervix samples were collected. HR-RISH, DNA ISH, and p16 immunohistochemistry were performed on whole formalin-fixed, paraffin-embedded sections. RNA ISH for 6 low-risk HPV types (LR-RISH) was also performed. RNA and DNA ISH assays used automated systems. HR-HPV PCR was performed on morphology-directed formalin-fixed, paraffin-embedded punches. HR-RISH was ≥97% sensitive for PCR+ and p16+ neoplasia, as well as morphologically defined anogenital high grade squamous intraepithelial lesion/invasive squamous cell carcinoma. HR-RISH was also positive in 78% of anogenital low grade squamous intraepithelial lesion, including 81% of CIN1. Furthermore, a subset of PCR-negative/invalid and p16-negative lesions was positive for HR-RISH. Only 1 problematic reactive cervix sample and no normal cervix samples stained. These results demonstrate that HR-RISH is a robust method for the detection of HR-HPV-related neoplasia and provides insight into HPV pathobiology. Performance meets or exceeds that of existing assays in anogenital and H&N lesions and may play a role in resolving diagnostically challenging CIN1 versus reactive cases.

Educational evolution is particularly important in pathology, particularly cytopathology, due to the vast amounts of independent learning required to master this field. In this study, learning challenges faced by pathology residents were addressed through a variety of educational modalities including 24 short (∼10 minute) online tutorials (dubbed "Sound Bites") covering selected topics in cytopathology as well as other areas of anatomic and clinical pathology. Additionally, residents were provided with an annotated glass slide set covering pediatric pathology with an associated multiple choice self-assessment as well as multiheaded microscope slide review sessions. Use of these modalities was tracked and residents surveyed about their experiences using them. All 20 residents (100%) reported using Sound Bites either from work computers, home computers, or mobile devices. Residents reported that easy accessibility, brevity, and opportunities for self-assessment were important variables contributing to this use, and that Sound Bite use would make them more likely to benefit from in-person teaching through lectures and/or slide sessions. Within 12 months of the release of the first Sound Bite, individual Sound Bites were accessed a total of 1169 times (mean: 49 times per Sound Bite). In contrast, slide sets were only accessed about once a month and were only employed by 30% of residents (6 of 20) for independent study; only 20% (4 of 20) completed the accompanying multiple choice self-assessment. All residents attended multiheaded microscope slide review sessions. Whereas traditional educational methods remain valuable tools in pathology education, these data suggest that short, web-based tutorials represent a valuable adjuvant teaching tool.

Background Rapid diagnostic testing paired with ASP intervention optimizes therapy and improves outcomes but few data guide ASP response in the absence of organism identification (ID). We describe the microbiology for organisms unidentified by Accelerate Pheno™ Gram-negative platform (AXDX) in order to inform ASP-provider team communication (PTC). Methods Consecutive, non-duplicate inpatient blood cultures with Gram-negative bacilli (GNB) following AXDX implementation at a single university hospital between April 2018 and March 2019 were included. Standard of care (SOC) ID and susceptibility followed AXDX. Clinical Microbiology emailed AXDX results to the ASP in real time; results were released into the EMR paired with telephone PTC or withheld after ASP review. Bloodstream Infections (BSIs) and patient outcomes for organisms labeled no/indeterminate ID by the AXDX were characterized. Results AXDX was performed on 351 blood cultures. Among 52 (15%) labeled no/indeterminate ID, SOC methods revealed: Enterobacteriaceae (40%; 9 monomicrobial with AXDX targets), anaerobes (21%), non-lactose fermenters (NLFs) other than Pseudomonas aeruginosa (21%), and fastidious GNB (10%). Frequent organisms without AXDX targets included: Raoultella planticola (4); Bacteroides fragilis, Cupriavidus spp., Haemophilus spp., Prevotella spp., Providencia spp., non-aeruginosa Pseudomonas spp., Salmonella spp. (3 each); Pasteurella multocida, Stenotrophomonas maltophilia (2 each). BSI sources were most commonly intra-abdominal (21%), central line-associated (17%), or unknown (17%). CLABSIs were associated with immune suppression and/or substance abuse in all but 1 case. BSIs without active empiric therapy included: NDM-producing Providencia stuartii SSSI; OXA-48-producing R. planticola intraabdominal infection (IAI); Pandoraea spp. CLABSI after liver transplant; enteric fever; B. fragilis, Leptotrichia wadei, and S. maltophilia, each of unknown source. In-hospital mortality occurred in 4 of these cases. Conclusion When AXDX yields no/indeterminate ID, ASP chart review for possible anaerobic/IAI, unique environmental exposures, and travel history may assist in guiding empiric therapy. GNB with AXDX targets are not excluded. Disclosures All authors: No reported disclosures.

An outbreak of acute flaccid paralysis among children in the United States during summer 2014 was tentatively associated with enterovirus D68 infection. This syndrome in a child in fall 2014 was associated with enterovirus C105 infection. The presence of this virus strain in North America may pose a diagnostic challenge.

Whooping cough results from infection of the respiratory tract with Bordetella pertussis , and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro , ACT has not been observed or quantified in vivo , and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼10 8 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼10 8 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro , ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

The Xpert Flu Assay cartridge is a next-generation nucleic acid amplification system that provides multiplexed PCR detection of the influenza A, influenza A 2009 H1N1, and influenza B viruses in approximately 70 min with minimal hands-on time. Six laboratories participated in a clinical trial comparing the results of the new Cepheid Xpert Flu Assay to those of culture or real-time PCR with archived and prospectively collected nasal aspirate-wash (NA-W) specimens and nasopharyngeal (NP) swabs from children and adults. Discrepant results were resolved by DNA sequence analysis. After discrepant-result analysis, the sensitivities of the Xpert Flu Assay for prospective NA-W specimens containing the influenza A, influenza A 2009 H1N1, and influenza B viruses compared to those of culture were 90.0%, 100%, and 100%, respectively, while the sensitivities of the assay for prospective NP swabs compared to those of culture were 100%, 100%, and 100%, respectively. The sensitivities of the Xpert Flu Assay for archived NA-W specimens compared to those of Gen-Probe ProFlu+ PCR for the influenza A, influenza A 2009 H1N1, and influenza B viruses were 99.4%, 98.4%, and 100%, respectively, while the sensitivities of the Xpert Flu Assay for archived NP swabs compared to those of ProFlu+ were 98.1%, 100%, and 93.8%, respectively. The sensitivities of the Xpert Flu Assay with archived NP specimens compared to those of culture for the three targets were 97.5%, 100%, and 93.8%, respectively. We conclude that the Cepheid Xpert Flu Assay is an accurate and rapid method that is suitable for on-demand testing for influenza viral infection.

Candida species are common causes of bloodstream infections (BSI), with high mortality. Four species cause >90% of Candida BSI: C. albicans , C. glabrata , C. parapsilosis , and C. tropicalis . Differentiation of Candida spp. is important because of differences in virulence and antimicrobial susceptibility. Candida QuickFISH BC, a multicolor, qualitative nucleic acid hybridization assay for the identification of C. albicans (green fluorescence), C. glabrata (red fluorescence), and C. parapsilosis (yellow fluorescence), was tested on Bactec and BacT/Alert blood culture bottles which signaled positive on automated blood culture devices and were positive for yeast by Gram stain at seven study sites. The results were compared to conventional identification. A total of 419 yeast-positive blood culture bottles were studied, consisting of 258 clinical samples (89 C. glabrata , 79 C. albicans , 23 C. parapsilosis , 18 C. tropicalis , and 49 other species) and 161 contrived samples inoculated with clinical isolates (40 C. glabrata , 46 C. albicans , 36 C. parapsilosis , 19 C. tropicalis , and 20 other species). A total of 415 samples contained a single fungal species, with C. glabrata ( n = 129; 30.8%) being the most common isolate, followed by C. albicans ( n = 125; 29.8%), C. parapsilosis ( n = 59; 14.1%), C. tropicalis ( n = 37; 8.8%), and C. krusei ( n = 17; 4.1%). The overall agreement (with range for the three major Candida species) between the two methods was 99.3% (98.3 to 100%), with a sensitivity of 99.7% (98.3 to 100%) and a specificity of 98.0% (99.4 to 100%). This study showed that Candida QuickFISH BC is a rapid and accurate method for identifying C. albicans , C. glabrata , and C. parapsilosis , the three most common Candida species causing BSI, directly from blood culture bottles.

Barriers to effective diagnostic testing for human immunodeficiency virus type 1 (HIV-1) infection can be reduced with simple, reliable, and rapid detection methods. Our objective was to determine the accuracy, sensitivity, and specificity of a new rapid, lateral-flow immunochromatographic HIV-1 antibody detection device. Preclinical studies were performed using seroconversion, cross-reaction, and interference panels, archived clinical specimens, and fresh whole blood. In a multicenter, prospective clinical trial, a four-sample matrix of capillary (fingerstick) whole-blood specimens and venous whole blood, plasma, and serum was tested for HIV-1 antibodies with the Efoora HIV rapid test (Efoora Inc., Buffalo Grove, IL) and compared with an enzyme immunoassay (EIA) (Abbott Laboratories) licensed by the Food and Drug Administration. Western blot and nucleic acid test supplemental assays were employed to adjudicate discordant samples. Preclinical testing of seroconversion panels showed that antibodies were often detected earlier by the rapid test than by a reference EIA. No significant interference or cross-reactions were observed. Testing of 4,984 archived specimens yielded a sensitivity of 99.2% and a specificity of 99.7%. A prospective multicenter clinical study with 2,954 adult volunteers demonstrated sensitivity and specificity for the Efoora HIV rapid test of 99.8% (95% confidence interval [CI], 99.3 and 99.98%) and 99.0% (95% CI, 98.5 and 99.4%), respectively. Reactive rapid HIV-1 antibody detection was confirmed in 99.6% of those with a known HIV infection ( n = 939), 5.2% of those in the high-risk group ( n = 1,003), and 0.1% of those in the low-risk group ( n = 1,012). For 21 (0.71%) patients, there was discordance between the results of the rapid test and the confirmatory EIA/Western blot tests. We conclude that the Efoora HIV rapid test is a simple, rapid assay for detection of HIV-1 antibodies, with high sensitivity and specificity compared to a standardized HIV-1 EIA.

Background Molecular diagnostics for enteropathogens increase yield while reducing turnaround time. However, many pathogens do not require specific therapy, and the cost is substantial. Methods We reviewed the use of the FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, Utah) in adult outpatients at the University of Virginia and identified clinical features that could limit testing without reducing yield. We defined yield as (a) detection of a pathogen, (b) detection of a pathogen for which antimicrobial therapy is indicated, or (c) detection of a pathogen that can change management, which additionally included viral pathogens in immunocompromised patients. Results Between March 23, 2015 and February 25, 2016, we reviewed 452 tests from adult outpatients with diarrhea. A pathogen was detected in 88/452 (19.5%). The most common pathogens were: enteropathogenic E. coli (36; 8.0%), norovirus (17; 3.8%), Campylobacter (7, 1.5%), enteroaggregative E. coli (6, 1.3%), Giardia (6; 1.3%), and sapovirus (5; 1.1%). Based on clinical guidelines, antimicrobial treatment was clearly indicated for 19/452 subjects (4.2%). Limiting testing to patients with an additional enteric symptom (abdominal pain, nausea, vomiting, fecal urgency, tenesmus, or flatulence), a travel history, or an immunocompromising condition would reduce testing by 25.9%, with a treatable pathogen identified in 18/331 (5.4%) (sensitivity 94.7%, specificity 27.7%). Further modifying testing criteria to exclude subjects with vomiting, 18/288 (6.3%) had a treatable pathogen (sensitivity 94.7%, specificity 37.3%), and a pathogen which could change management was detected in 28/288 (9.7%) (sensitivity 96.6%, specificity 38.5%). Excluding immunocompromised subjects or those with a travel history, American College of Gastroenterology guidelines for testing were met by 293/348 (84.2%) with a documented duration of diarrhea, and a treatable pathogen was detected in 8/293 (2.7%) vs. 3/55 (5.5%) who did not meet testing guidelines. Conclusion Testing could be reduced by 36.3% without decreasing clinical yield by limiting testing to patients with diarrhea with an additional enteric symptom and no history of vomiting, a travel history, or an immunocompromising condition. ACG guidelines did not improve testing efficiency. Disclosures All authors: No reported disclosures.

Objective.To compare direct laboratory costs of different methods for perirectal screening for carbapenemase-producing Enterobacteriaceae (CPE) colonization.Design.Cost-benefit analysis.Setting.A university hospital and affiliated long-term acute care hospital (LTACH).Participants.Inpatients from the hospital or LTACH.Methods.Perirectal samples were collected from inpatients at risk for exposure to CPE. In 2009, we compared the accuracy of the Centers for Disease Control and Prevention (CDC)-recommended CPE screening method with similar methods incorporating a chromogenic agar (CA). We then performed a cost projection analysis using 2012 screening results for the CA method, the CDC method, and a molecular assay with wholesale pricing based on the 2009 analysis. Comparisons of turnaround and personnel time were also performed.Results.A total of 185 (2.7%) of 6,860 samples were confirmed as CPE positive during 2012. We previously found that the CDC protocol had a lower sensitivity than the CA method and predicted that the CDC protocol would have missed 92 of the CPE-positive screening results, whereas the modified protocol using CA would have missed 26, assuming similar prevalence and performance. Turnaround time was 3 days using the CDC and CA-modified protocols compared with 1 day for molecular testing. The estimated annual total program cost and total technologist's hours would be the following: CA-modified protocol, $37,441 and 376 hours; CDC protocol, $22,818 and 482 hours; and molecular testing, $224,596 and 343 hours.Conclusions.The CDC screening protocol appeared to be the least expensive perirectal screening method. However, expense must be weighed against a lower sensitivity and extra labor needed for additional work-up of non-CPE isolates. The molecular test has the shortest turnaround time but the greatest expense.

The novel IsoAmp(®) HSV Assay employs isothermal helicase-dependent nucleic acid amplification and a user-friendly disposable test device to achieve rapid (1.5h), on-demand qualitative detection of herpes simplex virus (HSV) types 1 and 2 in oral and genital lesions.To compare performance of the IsoAmp(®) HSV Assay with the ELVIS(®) HSV ID/typing (shell-vial culture and DFA) test system for clinical specimens collected from oral and genital lesions in symptomatic patients.A total of 994 specimens from male and female genital and oral lesions were obtained and evaluated at five study sites in the United States. Results from the IsoAmp(®) HSV Assay were compared to those from the ELVIS(®) system. Separate reproducibility studies were performed at 3 sites using a blinded and randomized study panel. Discrepant specimens were resolved by bidirectional sequencing analysis.After discrepant analysis, overall agreement of IsoAmp(®) with ELVIS(®) was 98.8% with 37.0% overall prevalence (all study sites). Reproducibility rates were well within expectations.The IsoAmp(®) HSV Assay showed excellent performance for clinical use for detection of HSV in genital and oral specimens. In contrast to ELVIS(®), IsoAmp(®) HSV offers excellent sensitivity plus rapid on-demand testing and simpler specimen preparation.

A simple and rapid IsoAmp HSV assay has been developed for qualitative detection of herpes simplex virus (HSV) types 1 and 2 from genital lesions. Sample preparation involved a simple dilution step and the diluted specimens were directly added to the device and amplified by isothermal helicase-dependent amplification (HDA). Amplification products were then detected by a DNA strip embedded in a disposable cassette without any instrument. The total test turn-around time is less than 1.5h from specimen processing to result reporting.To evaluate the analytical and clinical performance of the IsoAmp HSV assay as well as the robustness and reproducibility of the assay.The analytical sensitivity of the IsoAmp HSV assay was determined using both HSV-1 and HSV-2. Clinical performance was evaluated using 135 frozen specimens collected from patients with suspected HSV infection in genital area.The analytical sensitivity of the assays was 5.5 and 34.1 copies/reaction for HSV-1 and HSV-2 respectively with a 95% confidence interval. When the herpes viral culture was used as the reference standard, the clinical sensitivity and specificity of the IsoAmp HSV assay were 100.0% and 96.3% respectively. The inter-laboratory reproducibility achieved an overall 97.5% agreement by testing a total of 80 blinded HSV-1 samples among five laboratories.Adequate analytical and clinical performance of the IsoAmp HSV assay was demonstrated. This assay is simple to perform and has acceptable inter-laboratory reproducibility.

A diagnosis of pneumococcal pneumonia and empyema was established by real-time fluorescence polymerase chain reaction (PCR) performed in pleural fluid and tissue samples from a child in whom cultures were negative. This case demonstrates that the use of solid-phase nucleic acid purification technology and real-time fluorescence PCR have an application in the rapid diagnosis of S. pneumoniae pneumonia.

We report the isolation of Legionella pneumophila serogroup 4 from synovial tissue obtained from an 80-year-old female with chronic swelling of her right metacarpophalangeal joint. Synovial tissue infections caused by L. pneumophila are rare. Interestingly, this isolate was recovered from chocolate agar after 5 days of incubation.