Amy Leber

Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis

ABSTRACT The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx 1 and stx 2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.

Multicenter Evaluation of BioFire FilmArray Meningitis/Encephalitis Panel for Detection of Bacteria, Viruses, and Yeast in Cerebrospinal Fluid Specimens

ABSTRACT Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis. The FilmArray Meningitis/Encephalitis (ME) Panel is a multiplexed in vitro diagnostic test for the simultaneous, rapid (∼1-h) detection of 14 pathogens directly from CSF specimens: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, and Cryptococcus neoformans/Cryptococcus gattii. We describe a multicenter evaluation of 1,560 prospectively collected CSF specimens with performance compared to culture (bacterial analytes) and PCR (all other analytes). The FilmArray ME Panel demonstrated a sensitivity or positive percentage of agreement of 100% for 9 of 14 analytes. Enterovirus and human herpesvirus type 6 had agreements of 95.7% and 85.7%, and L. monocytogenes and N. meningitidis were not observed in the study. For S. agalactiae, there was a single false-positive and false-negative result each, for a sensitivity and specificity of 0 and 99.9%, respectively. The specificity or negative percentage of agreement was 99.2% or greater for all other analytes. The FilmArray ME Panel is a sensitive and specific test to aid in diagnosis of ME. With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship are anticipated.

Validation of an in-house assay for cytomegalovirus immunoglobulin G (CMV IgG) avidity and relationship of avidity to CMV IgM levels.

ABSTRACT Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of >60%. Thus, low avidity in the in-house ELISA was defined as an AI of ⩽50%, whereas high avidity was defined as an AI of ⩾60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of ⩾3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (n = 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of ⩾3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.

Human Adenovirus Infection in Kawasaki Disease: A Confounding Bystander?

BACKGROUND Human adenovirus (HAdV) infection mimics Kawasaki disease (KD) but can also be detected in KD patients. Evidence suggests that HAdV-C species can persist in pediatric adenoids and/or tonsils. We sought to determine (1) the frequency of HAdV detection by real-time polymerase chain reaction in KD patients, (2) the differences in HAdV semiquantitative nasopharyngeal viral loads between KD patients with detectable HAdV vs those with HAdV disease, and (3) whether nasopharyngeal HAdV-C shedding is occurring in KD. METHODS From August 2009 through April 2011, HAdV-positive patients were identified in 1 of the following groups: group I, complete or incomplete KD as defined by the American Heart Association (AHA); group II, treated for incomplete KD but not fulfilling AHA criteria; and group III, otherwise healthy children with some KD-like features ultimately diagnosed with HAdV disease. RESULTS Among 77 KD patients diagnosed, 8.8% (5/57) of group I and 25% (5/20) of group II KD patients had HAdV detected. Viral loads were significantly lower in group I (n = 5) vs group III (n = 26; P = .034). Of the 13 specimens available for HAdV typing, 7 of 7 group III and 1 of 3 group II specimens were determined to be HAdV-B using viral culture. The remaining 5 KD samples were unable to be cultured and molecular typing showed either HAdV-C (n = 3) or were nontypeable (n = 2). CONCLUSIONS In KD, molecular-based HAdV detection is not uncommon, may represent persistence of HAdV-C, and should be interpreted with caution. Together, quantitative polymerase chain reaction and HAdV typing may aid in distinguishing HAdV disease mimicking KD from KD with concomitant HAdV detection.

Comparison of Polyurethane Foam to Nylon Flocked Swabs for Collection of Secretions from the Anterior Nares in Performance of a Rapid Influenza Virus Antigen Test in a Pediatric Emergency Department

ABSTRACT Rapid antigen testing of upper respiratory secretions collected with various swab types is often utilized for laboratory diagnoses of influenza virus infection. There are limited data on the effects of swab composition on test performance. This study compared the performance of the Quidel QuickVue Influenza A+B test on secretions from the anterior nares when a polyurethane foam swab was used for collection to that when a nylon flocked swab was used for collection. One hundred subjects who presented to a pediatric emergency department with symptoms suggestive of an influenza virus infection were recruited for the study. Foam and flocked swabs of the anterior nares were obtained from separate nares of each subject before a posterior nasopharyngeal swab was collected and placed into viral transport medium. The QuickVue test was performed directly on each swab type, and the results were compared to the results of reverse transcription-PCR (RT-PCR), direct fluorescent antibody (DFA) test, and viral culture performed on the transport medium. RT-PCR alone and DFA combined with culture were utilized as separate gold standards. There were 56 cases of influenza detected by RT-PCR; the QuickVue test was positive for 40 foam and 30 flocked swabs, for sensitivities of 71% and 54%, respectively (P = 0.01). Similarly, there were 49 influenza cases detected by DFA and/or culture; the QuickVue test was positive for 38 foam and 30 flocked swabs, for sensitivities of 78% and 61%, respectively (P = 0.13). This study suggests that polyurethane foam swabs perform better than nylon flocked swabs for the collection of secretions from anterior nares in the Quidel QuickVue Influenza A+B test.

Comparison of Complement Fixation and Hemagglutination Inhibition Assays for Detecting Antibody Responses following Influenza Virus Vaccination

ABSTRACT Complement fixation (CF) was compared to hemagglutination inhibition (HI) as a method for identifying antibody responses to influenza virus vaccination. CF assays were performed at two different laboratories using paired (pre- and postvaccination) sera from 38 vaccinated laboratory employees; HI assays were performed at a third laboratory. As expected, most vaccinees (31/38 = 82%) responded to at least one of three influenza virus antigens as measured by HI. In contrast, only 21% (8/38) of vaccinees showed a response by CF at laboratory 1, and only 29% (11/38) showed a response by CF at laboratory 2. These findings indicate that due to low sensitivity, CF assays should not be used to assess the antibody response to influenza virus vaccination.

Occurrence of 3 Bordetella species during an outbreak of cough illness in Ohio: epidemiology, clinical features, laboratory findings and antimicrobial susceptibility.

Background: An increase in laboratory diagnosis of pertussis was noted in central Ohio during 2010. Diagnosis was made using a polymerase chain reaction assay targeting the multicopy insertion sequence IS481, which is found in both Bordetella pertussis (Bp) and Bordetella holmesii (Bh). An increase in specimens testing positive for Bordetella parapertussis (Bpp) using insertion sequence IS1001 was also noted. Methods: Nasopharyngeal swab specimens submitted April 1, 2010, to March 31, 2011, were tested using a multiplex polymerase chain reaction assay for Bp/Bh (IS481) and Bpp followed by singleplex assays for Bp and Bh. A subgroup of specimens was also cultured for Bordetella species, and antimicrobial susceptibility testing was performed on recovered organisms. Demographic and clinical features were compared for patients with Bp, Bh and Bpp. Results: Of 520 IS481-positive specimens, 214 (41.1%) were positive for Bp, 79 (15.2%) were positive for Bh and 5 (1.0%) were positive for both Bp and Bh; 222 (42.7%) were negative for both targets. An additional 220 specimens were positive for Bpp. Among a sample of 155 IS481-positive specimens, 40, 15 and 0 were culture positive for Bp, Bh and Bpp, respectively. Among a sample of 55 BparaIS1001-positive (Bpp) specimens, 22, 0 and 0 were culture positive for Bpp, Bp and Bh, respectively. All Bordetella species were susceptible to macrolide antibiotics. Patients with Bh were older than patients with Bp, who were older than those positive for Bpp (mean ages: 12.0, 8.0 and 4.2 years, respectively; P < 0.001). One or more classic signs of pertussis (ie, paroxysmal cough, whoop, post-tussive emesis) were seen in 55.9% of 263 patients (59 Bp, 24 Bh, 80 Bpp and 100 negative for Bordetella species), but did not differ statistically among the groups (&khgr;2 = 5.1, P = 0.17). Conclusions: All 3 Bordetella species, Bp, Bh and Bpp, were detected during on outbreak of pertussis-like cough illness. There were noted differences in age and seasonality, but clinical features at the time of presentation did not allow clear differentiation of these infections. All Bordetella species recovered from culture and tested were susceptible in vitro to macrolide antibiotics. Additional study is necessary to further characterize epidemiologic and clinical characteristics of Bh-associated cough illness and to determine potential co-occurrence of Bordetella species with other bacterial and viral respiratory tract pathogens.

A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

Successful Treatment of Bloodstream Infection Due to Metallo-β-Lactamase-Producing Stenotrophomonas maltophilia in a Renal Transplant Patient

ABSTRACT Stenotrophomonas maltophilia is an emerging multidrug-resistant (MDR) opportunistic pathogen for which new antibiotic options are urgently needed. We report our clinical experience treating a 19-year-old renal transplant recipient who developed prolonged bacteremia due to metallo-β-lactamase-producing S. maltophilia refractory to conventional treatment. The infection recurred despite a prolonged course of colistimethate sodium (colistin) but resolved with the use of a novel drug combination with clinical efficacy against the patients S. maltophilia isolate.

Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction–dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses

The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.