April N. Abbott

Identification of Potential Virulence Determinants by Himar1 Transposition of Infectious Borrelia burgdorferi B31

ABSTRACT Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

Reflexive Culture in Adolescents and Adults With Group A Streptococcal Pharyngitis

BACKGROUND Guidelines currently provide conflicting recommendations regarding the diagnosis of group A streptococcal (GAS) pharyngitis in adults. Clinical guidelines state that negative rapid antigen detection tests (RADTs) do not require confirmation by a backup method in adults, whereas laboratory-based guidelines mandate confirmation of a negative RADT in patients of all ages. The objective of this study was to assess the utility of reflexive culture following a negative RADT in adolescents and adults with suspected GAS pharyngitis. METHODS A retrospective analysis of 726 patients, aged ≥13 years, with negative RADTs and positive GAS throat cultures, was performed between 1 January 2000 and 31 December 2011 at 2 academic medical centers in Seattle, Washington. Complication rates, treatment, modified Centor score, and bacterial burden in patients with negative RADTs and positive GAS throat cultures were assessed. RESULTS Modified Centor scores ≥2 were observed in 55% of patients with a negative RADT and positive GAS culture. Of these, 77% of patients had a moderate or heavy bacterial burden (≥2+). RADTs failed to detect some patients who presented with serious complications of GAS pharyngitis: 29 (4.0%) had peritonsillar abscesses and 2 (0.28%) were diagnosed with acute rheumatic fever. Providers found culture results to be useful for initiating antibiotic therapy or confirming a clinical diagnosis. Antibiotic treatment was prescribed in 68.7% of patients, with culture-directed initiation of therapy documented in 43.5%. CONCLUSIONS Reflexive GAS culture is clinically useful when RADTs are negative. RADTs fail to detect a substantial number of adult patients with clinically significant pharyngitis who can benefit from treatment.

Predictors of Relapse of Methicillin-Resistant Staphylococcus aureus Bacteremia after Treatment with Vancomycin

ABSTRACT The risk factors for relapse of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin treatment are unknown. Diversilab typing was used to classify recurrent bacteremia as relapse or reinfection. Bacteremia for >7 days and staphylococcal cassette chromosome mec element (SCCmec) type II were independently associated with relapse of MRSA bacteremia after vancomycin treatment.

Comparison of the Verigene Clostridium difficile, Simplexa C. difficile Universal Direct, BD MAX Cdiff, and Xpert C. difficile assays for the detection of toxigenic C. difficile

We compared the Verigene Clostridium difficile test (Nanosphere, Northbrook, IL, USA), the Simplexa C. difficile Universal Direct (Focus Diagnostics, Cypress, CA, USA), the BD MAX Cdiff (Becton Dickinson, Franklin Lakes, NJ, USA), and the Xpert C. difficile (Cepheid, Sunnyvale, CA, USA) assays for the detection of toxigenic C. difficile. One hundred and ninety deidentified, remnant diarrheal specimens were included in this study. After resolution of discordant results by toxigenic culture, the Xpert C. difficile assay displayed the highest sensitivity (100%), with a specificity of 98.8%. The sensitivity and specificity were 95.2% and 99.4% and 87% and 100% for the Verigene CDF and Simplexa Universal Direct assays, respectively. Finally, the BD MAX assay showed a sensitivity of 87% and a specificity of 98.8%. Despite differences in the overall performance of these assays, these results support the routine use of these platforms for the detection of toxigenic C. difficile in the clinical laboratory.

11β-Hydroxysteroid Dehydrogenases Are Regulated during the Pulmonary Granulomatous Response to the Mycobacterial Glycolipid Trehalose-6,6′-Dimycolate

Objective: Tuberculosis has a staggering influence on world health, resulting in nearly 2 million deaths per year. The influence of glucocorticoids during Mycobacterium tuberculosis infection has been under investigation for decades; however, the identity of mycobacterial factors and the mechanism by which glucocorticoids are tissue specifically regulated to influence immune function during acute granuloma formation are unknown. Methods: One factor implicated in initiating immunopathology during M. tuberculosis infection is trehalose-6,6′-dimycolate (TDM), a glycolipid component of the mycobacterial cell wall. Intravenous administration of TDM causes inflammatory responses in lungs of mice similar to M. tuberculosis infection and has been used as a successful model to examine proinflammatory regulation and early events involved in the manifestation of pathology. Results and Conclusion: IL-6, IL-1α and TNF-α mRNA and protein peaked during the initiation of granuloma formation. Pulmonary corticosterone levels were elevated when the proinflammatory response was greatest, dropping to half of that upon the establishment of granuloma pathology on day 7. It is hypothesized that once corticosterone reaches the site of inflammation, the enzymes 11β-hydroxysteroid dehydrogenases (11βHSDs) can influence bioavailability by interconverting corticosterone and the inert metabolite 11-dehydrocorticosterone. RT-PCR demonstrated that pulmonary 11βHSD type 1 mRNA decreased 4-fold and 11βHSD type 2 (11βHSD2) mRNA expression increased 2.5-fold on day 3 after injection, suggesting that corticosterone regulation in the lung, specifically the reduction of active corticosterone by 11βHSD2, may influence the progression of granuloma formation in response to the mycobacterial glycolipid.

Accidental Exposure to Burkholderia pseudomallei in the Laboratory in the Era of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Burkholderia pseudomallei is the causative agent of melioidosis, a disease that can vary in severity from fulminant sepsis to chronic infection with fatality rates of up to 42% ([1][1], [2][2]). As world travel has increased, so have melioidosis rates in travelers to countries where melioidosis is

Coinfection of Fusobacterium nucleatum and Actinomyces israelii in Mastoiditis Diagnosed by Next-Generation DNA Sequencing

ABSTRACT Some bacterial infections involve potentially complex mixtures of species that can now be distinguished using next-generation DNA sequencing. We present a case of mastoiditis where Gram stain, culture, and molecular diagnosis were nondiagnostic or discrepant. Next-generation sequencing implicated coinfection of Fusobacterium nucleatum and Actinomyces israelii, resolving these diagnostic discrepancies.

Human Diphyllobothrium nihonkaiense Infection in Washington State

ABSTRACT A patient in Washington State harbored a fish tapeworm most likely acquired from eating raw salmon. Diphyllobothrium nihonkaiense was identified by cox1 sequence analysis. Although this is the first documented human D. nihonkaiense infection in the United States, the parasite may have been present earlier but misidentified as Diphyllobothrium latum.

Direct-from-Blood-Culture Disk Diffusion To Determine Antimicrobial Susceptibility of Gram-Negative Bacteria: Preliminary Report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group

ABSTRACT The performance of a disk diffusion test using broth from positive blood cultures as inoculum (direct disk diffusion [dDD]) was evaluated for a collection of 20 challenge isolates of Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Isolates seeded into human blood were inoculated into Bactec Plus Aerobic/F, VersaTREK Redox 1, and BacT/Alert FA Plus bottles and incubated in the respective automated blood culture systems. Disk diffusion results were compared to reference disk diffusion results. Categorical agreement (CA) values for dDD, after removal of random errors due to natural MIC variation, were 87.8%, 88.4%, and 92.2% for the BacT/Alert, Bactec, and VersaTREK systems, respectively. No very major errors (VME) were observed, and major error (ME) rates were 3.0%, 2.3%, and 1.7%, respectively. Incubation of the dDD test samples for 6 h compared to incubation for 16 to 18 h resulted in 19.9% of tests having too light of growth to allow reading of zones of inhibition. Among the evaluable dDD tests, CA values were 58.9%, 76.6%, and 73.2% for the isolates seeded into the BacT/Alert, Bactec, and VersaTREK systems, respectively. VME rates for isolates seeded into these systems were 2.2%, 1.8%, and 3.0%, respectively, and ME rates were 25.4%, 6.1%, and 2.8%, respectively, at the 6-h reading. The best performance of dDD was found for blood cultures with bacterial concentrations in the range of 7.6 × 107 to 5.0 × 108 CFU/ml; CA values ranged from 94.7 to 96.2% for these concentrations after 18 h of incubation and from 76.9 to 84.1% after 6 h of incubation. These preliminary data demonstrate the potential accuracy of dDD testing by the clinical laboratory.

Performance of a New Rapid Immunoassay Test Kit for Point-of-Care Diagnosis of Significant Bacteriuria

ABSTRACT Urinary tract infections (UTIs) are frequently encountered in clinical practice and most commonly caused by Escherichia coli and other Gram-negative uropathogens. We tested RapidBac, a rapid immunoassay for bacteriuria developed by Silver Lake Research Corporation (SLRC), compared with standard bacterial culture using 966 clean-catch urine specimens submitted to a clinical microbiology laboratory in an urban academic medical center. RapidBac was performed in accordance with instructions, providing a positive or negative result in 20 min. RapidBac identified as positive 245/285 (sensitivity 86%) samples with significant bacteriuria, defined as the presence of a Gram-negative uropathogen or Staphylococcus saprophyticus at ≥103 CFU/ml. The sensitivities for Gram-negative bacteriuria at ≥104 CFU/ml and ≥105 CFU/ml were 96% and 99%, respectively. The specificity of the test, detecting the absence of significant bacteriuria, was 94%. The sensitivity and specificity of RapidBac were similar on samples from inpatient and outpatient settings, from male and female patients, and across age groups from 18 to 89 years old, although specificity was higher in men (100%) compared with that in women (92%). The RapidBac test for bacteriuria may be effective as an aid in the point-of-care diagnosis of UTIs especially in emergency and primary care settings.