Rosemary C. She

Characterization of monospecies biofilm formation by Helicobacter pylori.

As all bacteria studied to date, the gastric pathogen Helicobacter pylori has an alternate lifestyle as a biofilm. H. pylori forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral H. pylori to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed H. pylori arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the H. pylori life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic H. pylori while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach, H. pylori planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene, luxS, and the cagE type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by H. pylori and the selective enhancement of planktonic growth in the presence of gastric mucin.

Invasive Haemophilus influenzae Disease in Utah Children: An 11-Year Population-Based Study in the Era of Conjugate Vaccine

BACKGROUND The incidence of invasive Haemophilus influenzae infection decreased dramatically since the introduction of the H. influenzae serotype b (Hib) conjugate vaccine. H. influenzae invasive disease continues to occur and cause significant morbidity and mortality in children aged <5 years. We aimed to report the epidemiology and serotypes of invasive H. influenzae disease in children from Utah in the post-Hib vaccine era. METHODS We identified all cases of invasive H. influenzae disease, defined as H. influenzae isolated from a sterile site, during the period 1998-2008 among children aged <18 years who were living in Utah. RESULTS We identified 91 cases of invasive H. influenzae disease in children. Children aged <5 years accounted for 78 cases (86%). H. influenzae serotype a (Hia) was the most common serotype (22 cases), representing 28% of all cases of invasive disease among children aged <5 years. The majority (15 cases [93%]) of Hib disease cases occurred among children aged <5 years and accounted for 18% of all cases of H. influenzae invasive disease in this age group. The mean incidence of Hia disease increased from 0.8 cases per 100,000 child-years in 1998 to 2.6 cases per 100,000 child-years in 2008. The incidence of Hib disease among children aged <5 years remained steady at 0.5 cases per 100,000 child-years. Bacteremia accounted for 61% of all cases of invasive disease. One-half (13 of 26) of cases of H. influenzae meningitis were due to Hia. CONCLUSIONS H. influenzae continues to cause invasive disease in Utah children. Hia is the primary cause of the overall increased incidence of invasive H. influenzae disease and leads to disease similar to Hib. Isolated cases of Hib disease demonstrate a continued reservoir. The success of the Hib conjugate vaccine may therefore be vulnerable to vaccine shortages and refusal of vaccination.

Limited Utility of Culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae for Diagnosis of Respiratory Tract Infections

ABSTRACT We assessed the utility of culture for Mycoplasma pneumoniae and Chlamydophila pneumoniae to diagnose respiratory tract infections. Compared to PCR and IgM serology, culture was less sensitive and had extremely low yield. Culture is not recommended for these pathogens, and this method should be eliminated from routine practice.

Respiratory syncytial virus outbreak in a long-term care facility detected using reverse transcriptase polymerase chain reaction: an argument for real-time detection methods.

OBJECTIVES: To report an outbreak of respiratory synctyial virus (RSV) in a long‐term care facility (LTCF) during ongoing routine respiratory illness surveillance.

Clinical Impact of Laboratory Implementation of Verigene BC-GN Microarray-Based Assay for Detection of Gram-Negative Bacteria in Positive Blood Cultures

ABSTRACT Gram-negative bacteremia is highly fatal, and hospitalizations due to sepsis have been increasing worldwide. Molecular tests that supplement Gram stain results from positive blood cultures provide specific organism information to potentially guide therapy, but more clinical data on their real-world impact are still needed. We retrospectively reviewed cases of Gram-negative bacteremia in hospitalized patients over a 6-month period before (n = 98) and over a 6-month period after (n = 97) the implementation of a microarray-based early identification and resistance marker detection system (Verigene BC-GN; Nanosphere) while antimicrobial stewardship practices remained constant. Patient demographics, time to organism identification, time to effective antimicrobial therapy, and other key clinical parameters were compared. The two groups did not differ statistically with regard to comorbid conditions, sources of bacteremia, or numbers of intensive care unit (ICU) admissions, active use of immunosuppressive therapy, neutropenia, or bacteremia due to multidrug-resistant organisms. The BC-GN panel yielded an identification in 87% of Gram-negative cultures and was accurate in 95/97 (98%) of the cases compared to results using conventional culture. Organism identifications were achieved more quickly post-microarray implementation (mean, 10.9 h versus 37.9 h; P < 0.001). Length of ICU stay, 30-day mortality, and mortality associated with multidrug-resistant organisms were significantly lower in the postintervention group (P < 0.05). More rapid implementation of effective therapy was statistically significant for postintervention cases of extended-spectrum beta-lactamase-producing organisms (P = 0.049) but not overall (P = 0.12). The Verigene BC-GN assay is a valuable addition for the early identification of Gram-negative organisms that cause bloodstream infections and can significantly impact patient care, particularly when resistance markers are detected.

Evaluation of enzyme immunoassays to detect Clostridium difficile toxin from anaerobic stool culture.

Stool culture for Clostridium difficile, while necessary for strain typing and antimicrobial surveillance, cannot determine toxin production. We prospectively tested in triplicate 91 C difficile cultured isolates for toxin production by 2 enzyme immunoassays (EIAs) (Meridian Premier Toxins A&B, Meridian Bioscience, Cincinnati, OH; and TechLab Tox A/B II, TechLab, Blacksburg, VA) and cytotoxin neutralization bioassay (CTN). By CTN, 88% (80/91) were toxigenic. Reproducibility was 93% (85/91) for CTN, 80% (73/91) for Meridian EIA, and 79% (72/91) for TechLab EIA. Compared with CTN, sensitivities were 87.1% and 89.2% for the Meridian and TechLab EIAs, respectively. In an additional 115 stool specimens, CTN detected toxin more frequently from cultured isolates (96/115) than stool (84/115). For C difficile toxin detection from isolates, EIA was less reproducible than CTN. EIA methods can be falsely negative in 10% to 12% of isolates, and these should be tested by CTN or polymerase chain reaction. When positive, EIA is fast and reliable for detecting C difficile toxin from culture.

Evaluation of Helicobacter pylori immunoglobulin G (IgG), IgA, and IgM serologic testing compared to stool antigen testing.

ABSTRACT The utility of Helicobacter pylori serology was evaluated in 4,722 specimens and compared to stool antigen detection. Immunoglobulin M (IgM) sensitivity (6.8%) was unacceptably low. Key performance differences were observed in IgG specificity, IgA sensitivity, and specificity between adults and children that may warrant differentiating optimal serologic cutoff values by age.

Development of a multiplex real-time RT-PCR assay for detection of influenza A, influenza B, RSV and typing of the 2009-H1N1 influenza virus

A high-throughput real-time RT-PCR assay was developed to amplify and detect a conserved region of the hemagglutinin gene of the 2009-H1N1 influenza A virus using a minor groove binder-conjugated hybridization probe. The assay was paired with a separate triplex real-time assay that detects influenza A via the matrix gene, influenza B and RSV in a multiplex format and compared with the Centers for Disease Control and Prevention (CDC) rRT-PCR assay using 143 samples. The 2009-H1N1 portion of the multiplex assay had 100% correlation with the CDC assay, while the triplex assay had a 99% agreement. An additional 105 samples collected from October to November 2009 were also tested using both the individual 2009-H1N1 and triplex assays. Of these 105 samples, eight were positive for the hemagglutinin target in the H1N1 assay and negative for the matrix target in the triplex assay. Discrepant analysis revealed single nucleotide polymorphisms within the matrix gene of 2009-H1N1 virus-positive samples. The limit of detection for the 2009-H1N1 assay was between 750 and 1,500 copies/reaction and no cross-reactivity with other respiratory pathogens was observed. Overall, this multiplexed format proved to be sensitive, robust and easy to use and serves as a useful tool for pandemic testing.

Performance of diagnostic tests to detect respiratory viruses in older adults

Abstract The performance of 4 laboratory methods for diagnosis of viral respiratory tract infections (RTI) in older adults was evaluated. Seventy-four nasopharyngeal (NP) swab specimens were obtained from 60 patients with RTI at a long-term care facility over 2 respiratory seasons. Sixteen specimens were positive for a respiratory virus by at least 1 method. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) by the Luminex xTAG® Respiratory Viral Panel (RVP) detected 16 (100%) of the positive specimens, RVP of 24-h culture supernatant detected 8 (50%), direct fluorescent antibody testing detected 4 (25%), rapid culture detected 2 (12.5%), and rapid antigen testing detected none. For a comparison group, RVP was performed on NP swabs from 20 outpatient children with RTI. The mean fluorescence intensity by RVP was significantly lower for positive adult patients than pediatric patients (P = 0.0373). Our data suggest that older adult patients shed lower titers of viruses, necessitating a highly sensitive assay such as RT-PCR to reliably detect respiratory viral pathogens.

Carcinosarcoma of the liver: a case report and review of the literature.

No more than 11 cases of carcinosarcoma of the liver have been reported in the past 40 years that fulfill the definition of hepatocellular carcinoma combined with differentiated sarcomatous elements. Most cases consist of hepatocellular carcinoma with 1 to 2 heterologous elements. We report a case of a 51-year-old woman with liver carcinosarcoma consisting of 3 carcinomatous components and 4 sarcomatous components. Hepatocellular carcinoma, fibrolamellar type, was accompanied by neuroendocrine carcinoma (neuron-specific enolase and synaptophysin positive) and adenocarcinoma (cytokeratin 7 and 20 positive). The sarcomatous elements consisted of poorly differentiated spindle cell neoplasm (vimentin positive), leiomyosarcoma (smooth muscle actin positive), rhabdomyosarcoma (desmin positive), and osteosarcoma. To our knowledge, this is the first case of liver carcinosarcoma with this many differentiated heterologous features. There are differing views on the pathogenesis of this tumor. Findings in this case support the view that metaplasia of carcinomatous cells gives rise to the sarcomatous elements.