Multicenter clinical evaluation of the Luminex Aries Flu A/B & RSV assay for pediatric and adult respiratory tract specimens

ABSTRACT Influenza A and B viruses and respiratory syncytial virus (RSV) are three common viruses implicated in seasonal respiratory tract infections and are a major cause of morbidity and mortality in adults and children worldwide. In recent years, an increasing number of commercial molecular tests have become available to diagnose respiratory viral infections. The Luminex Aries Flu A/B &amp; RSV assay is a fully automated sample-to-answer molecular diagnostic assay for the detection of influenza A, influenza B, and RSV. The clinical performance of the Aries Flu A/B &amp; RSV assay was prospectively evaluated in comparison to that of the Luminex xTAG respiratory viral panel (RVP) at four North American clinical institutions over a 2-year period. Of the 2,479 eligible nasopharyngeal swab specimens included in the prospective study, 2,371 gave concordant results between the assays. One hundred eight specimens generated results that were discordant with those from the xTAG RVP and were further analyzed by bidirectional sequencing. Final clinical sensitivity values of the Aries Flu A/B &amp; RSV assay were 98.1% for influenza A virus, 98.0% for influenza B virus, and 97.7% for RSV. Final clinical specificities for all three pathogens ranged from 98.6% to 99.8%. Due to the low prevalence of influenza B, an additional 40 banked influenza B-positive specimens were tested at the participating clinical laboratories and were all accurately detected by the Aries Flu A/B &amp; RSV assay. This study demonstrates that the Aries Flu A/B &amp; RSV assay is a suitable method for rapid and accurate identification of these causative pathogens in respiratory infections.</jats:p

Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification

The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-manolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes

Comparison of the Panther Fusion and BD MAX GBS Assays for Detection of Group B Streptococcus in Prenatal Screening Specimens.

Streptococcus agalactiae, or Group B Streptococcus (GBS) is the cause of early and late-onset GBS disease in neonates and can present as septicemia, meningitis, and pneumonia. Our objective was to compare the performance of two FDA-approved nucleic acid amplification tests (NAATs), the Panther Fusion and BD MAX™ systems, for detection of group B Streptococcus (GBS) in vaginal-rectal screening specimens. A total of 510 vaginal-rectal prepartum specimens were tested simultaneously in both NAATs following broth enrichment. Assay agreement was calculated using the kappa statistics. Overall agreement between assays was 99.0% (505/510; 95% CI: 0.951 to 0.997; kappa = 0.974). Discordant results were re-tested with both assays and by standard culture. The assays were also compared for workflow characteristics, including time to first results (TFR), total turnaround time (TAT), number of return visits to load additional specimens, and hands-on time (HoT).Using a standard run size of 60 specimens/day, the Panther Fusion assay had a longer time to TFR (2.4 vs. 2.0 hours), but showed a shorter overall TAT for all 60 samples (3.98 vs. 7.18 hours) due to an increased initial sample loading capacity, required less labor (35.0 vs. 71.3 sec/sample) and fewer return visits for loading additional specimens (0 vs. 2). The Panther Fusion system also had a larger sample loading capacity (120 vs. 24 samples) and greater 8-hour throughput (335 vs. 96 samples). In summary, the Panther Fusion GBS assay has comparable clinical performance to the BD MAX GBS assay, but provides a faster TAT, less HoT, and higher throughput

Automated real-time collection of pathogen-specific diagnostic data: Syndromic infectious disease epidemiology

© Lindsay Meyers, Christine C Ginocchio, Aimie N Faucett, Frederick S Nolte, Per H Gesteland, Amy Leber, Diane Janowiak,. Background: Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. Objective: The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. Methods: We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. Results: The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. Conclusions: Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks

Identification and quantification of microbial populations in activated sludge and anaerobic digestion processes

Eight different phenotypes were studied in an activated sludge process (AeR) and anaerobic digester AnD) in a full-scale wastewater treatment plant by means of fluorescent in situ hybridization (FISH) and automated FISH quantification software. The phenotypes were ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, denitrifying bacteria, phosphate-accumulating organisms (PAO), glycogen-accumulating organisms (GAO), sulphate-reducing bacteria (SRB), methanotrophic bacteria and methanogenic archaea. Some findings were unexpected: (a) Presence of PAO, GAO and denitrifiers in the AeR possibly due to unexpected environmental conditions caused by oxygen deficiencies or its ability to survive aerobically; (b) presence of SRB in the AeR due to high sulphate content of wastewater intake and possibly also due to digested sludge being recycled back into the primary clarifier; (c) presence of methanogenic archaea in the AeR, which can be explained by the recirculation of digested sludge and its ability to survive periods of high oxygen levels; (d) presence of denitrifying bacteria in the AnD which cannot be fully explained because the nitrate level in the AnD was not measured. However, other authors reported the existence of denitrifiers in environments where nitrate or oxygen was not present suggesting that denitrifiers can survive in nitrate-free anaerobic environments by carrying out low-level fermentation; (e) the results of this paper are relevant because of the focus on the identification of nearly all the significant bacterial and archaeal groups of microorganisms with a known phenotype involved in the biological wastewater treatment.This study was realized with support from the Universitat Politecnica de Valencia, Universitat de Valencia and CONACYT (National Council of Science and Technology of Mexico).Reyes Sosa, MB.; Borrás Falomir, L.; Seco Torrecillas, A.; Ferrer, J. (2015). Identification and quantification of microbial populations in activated sludge and anaerobic digestion processes. Environmental Technology. 36(1):45-53. doi:10.1080/09593330.2014.934745S455336

NmcA Carbapenem-hydrolyzing Enzyme in Enterobacter cloacae in North America1

An imipenem-resistant Enterobacter cloacae isolate was recovered from the blood of a patient with a hematologic malignancy. Analytical isoelectric focusing, inhibitor studies, hydrolysis, induction assays, and molecular sequencing methods confirmed the presence of a NmcA carbapenem-hydrolyzing enzyme. This first report of NmcA detected in North America warrants further investigation into its distribution and clinical impact

Use of cultivation-dependent and -independent techniques to assess contamination of central venous catheters: a pilot study

<p>Abstract</p> <p>Background</p> <p>Catheters are the most common cause of nosocomial infections and are associated with increased risk of mortality, length of hospital stay and cost. Prevention of infections and fast and correct diagnosis is highly important.</p> <p>Methods</p> <p>In this study traditional semiquantitative culture-dependent methods for diagnosis of bacteria involved in central venous catheter-related infections as described by Maki were compared with the following culture-independent molecular biological methods: Clone libraries, denaturant gradient gel electrophoresis, phylogeny and fluorescence in situ hybridization.</p> <p>Results</p> <p>In accordance with previous studies, the cultivation of central venous catheters from 18 patients revealed that <it>S. epidermidis </it>and other coagulase-negative staphylococci were most abundant and that a few other microorganisms such as <it>P. aeruginosa </it>and <it>K. pneumoniae </it>occasionally were found on the catheters. The molecular analysis using clone libraries and sequencing, denaturant gradient gel electrophoresis and sequencing provided several important results. The species found by cultivation were confirmed by molecular methods. However, many other bacteria belonging to the phyla <it>Proteobacteria, Firmicutes, Actinobacteria </it>and <it>Bacteroidetes </it>were also found, stressing that only a minor portion of the species present were found by cultivation. Some of these bacteria are known to be pathogens, some have not before been described in relation to human health, and some were not closely related to known pathogens and may represent new pathogenic species. Furthermore, there was a clear difference between the bacterial species found in biofilm on the external (exluminal) and internal (luminal) side of the central venous catheter, which can not be detected by Maki's method. Polymicrobial biofilms were observed on most of the catheters and were much more common than the cultivation-dependent methods indicated.</p> <p>Conclusion</p> <p>The results show that diagnosis based on molecular methods improves the detection of microorganisms involved in central catheter-related infections. The importance of these microorganisms needs to be investigated further, also in relation to contamination risk from improper catheter handling, as only in vivo contaminants are of interest. This information can be used for development of fast and more reliable diagnostic tools, which can be used in combination with traditional methods.</p

Microbial community structure elucidates performance of Glyceria maxima plant microbial fuel cell

The plant microbial fuel cell (PMFC) is a technology in which living plant roots provide electron donor, via rhizodeposition, to a mixed microbial community to generate electricity in a microbial fuel cell. Analysis and localisation of the microbial community is necessary for gaining insight into the competition for electron donor in a PMFC. This paper characterises the anode–rhizosphere bacterial community of a Glyceria maxima (reed mannagrass) PMFC. Electrochemically active bacteria (EAB) were located on the root surfaces, but they were more abundant colonising the graphite granular electrode. Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, indicating that the current was probably generated via the hydrolysis of cellulose. Due to the presence of oxygen and nitrate, short-chain fatty acid-utilising denitrifiers were the major competitors for the electron donor. Acetate-utilising methanogens played a minor role in the competition for electron donor, probably due to the availability of graphite granules as electron acceptors

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB

Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators

BACKGROUND: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. METHODOLOGY/PRINCIPAL FINDINGS: Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and alpha-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. CONCLUSIONS/SIGNIFICANCE: This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health