Multicenter Diagnostic Accuracy Evaluation of the Luminex Aries Real-Time PCR Assay for Group B Streptococcus Detection in Lim Broth-Enriched Samples.

The vertical transmission of group B Streptococcus (GBS) strains causing neonatal sepsis is one of the leading reasons for neonatal mortality worldwide. The gold standard for GBS detection is enriched culture with or without the aid of chromogenic agars. Given the high risk for morbidity and mortality in this population, high assay sensitivity is required to prevent the personal and economic costs of GBS disease. Nucleic acid amplification tests (NAATs) allow for objective determination of GBS colonization with a sensitivity and a specificity higher than those of traditional culture methods. In this study, we determined the analytical and clinical performance of the Aries GBS assay compared to those of the enrichment culture method, biochemical identification, and the NAATs used at the study sites. Remnant Lim broth samples were used to perform the Aries assay and reference testing. Upon first testing using enriched culture as the reference standard, the Aries GBS assay identified GBS with a 96.1% sensitivity (95% confidence interval [CI], 91.2 to 98.7%) and a 91.4% specificity (95% CI, 88.8 to 93.6%). The test performed with 100% positive agreement (95% CI, 83.2 to 100%) compared to the results of the BD Max GBS assay and 98.0% positive agreement (95% CI, 89.2 to 99.9%) compared to the results of the Cepheid Xpert GBS LB test. Repeatability and reproducibility were maintained in intra- and interlaboratory testing, regardless of the instrument, module, or user who performed the test. The Aries GBS assay can be set up in less than 5 min and produces results in 2 h. The easy setup, with minimal hands-on time, and high assay sensitivity and specificity make this a useful testing option for GBS screening in prepartum women.

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria.

AIMS: Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer. METHODS AND RESULTS: Nine 16S rRNA amplicons were tested on an Ion Torrent PGM to identify 41 strains of clinical importance. The V1-V2 region identified 40 of 41 isolates to the species level. Three data analysis methods were tested, finding that the Ribosomal Database Project's SequenceMatch outperformed BLAST and the Ion Reporter Metagenomics analysis pipeline. Lastly, 16S rRNA gene sequencing mixtures of four species through a six log range of dilution showed species were identifiable even when present as 0·1% of the mixture. CONCLUSIONS: Sequencing the V1-V2 16S rRNA gene region, made possible by the increased read length Ion Torrent PGM sequencer's 400 base pair chemistry, may be a better choice over other commonly used regions for identifying clinically important bacteria. In addition, the SequenceMatch algorithm, freely available from the Ribosomal Database Project, is a good choice for matching filtered reads to organisms. Lastly, 16S rRNA gene sequencing's sensitivity to the presence of a bacterial species at 0·1% of a mixture suggests it has sufficient sensitivity for samples in which important bacteria may be rare. SIGNIFICANCE AND IMPACT OF THE STUDY: We have validated 16S rRNA gene sequencing on a benchtop sequencer including simple mixtures of organisms; however, our results highlight deficits for clinical application in place of current identification methods.

Multicenter evaluation of the Sepsityper™ extraction kit and MALDI-TOF MS for direct identification of positive blood culture isolates using the BD BACTEC™ FX and VersaTREK(®) diagnostic blood culture systems.

AIMS: (i) Evaluation of delayed time to blood culture extraction by the Sepsityper kit and impact of shipping pellets off-site for MALDI-TOF MS analysis. (ii) Comparison of Sepsityper and laboratory-developed extraction methods from a literature review. METHODS AND RESULTS: Using two blood culture systems (BD BACTEC and VersaTREK), we extracted 411 positive blood cultures using the Sepsityper kit to mimic a potential protocol for institutions without a MALDI-TOF MS. Extracted pellets were shipped and analysed on the Bruker UltraflexIII. Successful extraction of 358 (87·1%) samples was determined by the presence of detectable proteins. MALDI-TOF MS correctly identified 332 (80·8%) samples. CONCLUSIONS: Delayed time to extraction did not affect Sepsityper extraction or MALDI-TOF MS accuracy. The extracted pellets remain stable and provide accurate results by MALDI-TOF MS when shipped at room temperature to off-site reference laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show that institutions without a MALDI-TOF MS can take advantage of this innovative technology by shipping a volume of blood to an off-site laboratory for extraction and MALDI-TOF MS analysis. We also performed a literature review to compare various extraction methods.

Detection of respiratory viruses on air filters from aircraft.

AIMS: To evaluate the feasibility of identifying viruses from aircraft cabin air, we evaluated whether respiratory viruses trapped by commercial aircraft air filters can be extracted and detected using a multiplex PCR, bead-based assay. METHODS AND RESULTS: The ResPlex II assay was first tested for its ability to detect inactivated viruses applied to new filter material; all 18 applications of virus at a high concentration were detected. The ResPlex II assay was then used to test for 18 respiratory viruses on 48 used air filter samples from commercial aircraft. Three samples tested positive for viruses, and three viruses were detected: rhinovirus, influenza A and influenza B. For 33 of 48 samples, internal PCR controls performed suboptimally, suggesting sample matrix effect. CONCLUSION: In some cases, influenza and rhinovirus RNA can be detected on aircraft air filters, even more than 10 days after the filters were removed from aircraft. SIGNIFICANCE AND IMPACT OF THE STUDY: With protocol modifications to overcome PCR inhibition, air filter sampling and the ResPlex II assay could be used to characterize viruses in aircraft cabin air. Information about viruses in aircraft could support public health measures to reduce disease transmission within aircraft and between cities.

Comparison of MRSASelect Agar, CHROMagar Methicillin-Resistant Staphylococcus aureus (MRSA) Medium, and Xpert MRSA PCR for detection of MRSA in Nares: diagnostic accuracy for surveillance samples with various bacterial densities.

Rapid laboratory methods provide optimal support for active surveillance efforts to screen for methicillin-resistant Staphylococcus aureus (MRSA). Most laboratories struggle to determine the optimal use of resources, considering options to balance cost, speed, and diagnostic accuracy. To assess the performance of common methods, the first comparison of MRSASelect agar (MS) and CHROMagar MRSA (CA), with and without broth enrichment followed by a 24-h subculture to MS, was performed. Results were compared to those of the Xpert MRSA assay. For direct culture methods, the agreement between MS and CA was 98.8%. At 18 h, direct MS identified 93% of all positive samples from direct culture and 84% of those identified by the Xpert MRSA. For Trypticase soy broth-enriched MS culture, incubated overnight and then subcultured for an additional 24 h, the agreement with Xpert MRSA was 96%. The agreement between direct MS and Xpert MRSA was 100% when semiquantitative culture revealed a bacterial density of 2+ or greater; however, discrepancies between culture and Xpert MRSA arose for MRSA bacterial densities of 1+ or less, indicating low density as a common cause of false-negative culture results. Since 1+ or less was established as the most common MRSA carrier state, broth enrichment or PCR may be critical for the identification of all MRSA carriers who may be reservoirs for transmission. In this active-surveillance convenience sample, the use of broth enrichment followed by subculture to MS offered a low-cost but sensitive method for MRSA screening, with performance similar to that of Xpert MRSA PCR.

Rapid detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays.

A multicenter preclinical evaluation was conducted to evaluate the performance of two Cepheid Xpert assays for detection of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus. Sensitivity was 97.1% and 98.3% for MRSA in wound and blood culture specimens, respectively. Sensitivity was 100% for S. aureus from both specimen types.

Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares.

The first U.S. multicenter clinical trial to assess the performance of the Cepheid Xpert MRSA assay (Xpert MRSA) was conducted. The assay is a qualitative test designed for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from nares swabs. This novel test combines integrated nucleic acid extraction and automated real-time PCR for the detection of a MRSA-specific signature sequence. A total of 1,077 nares specimens were collected from seven geographically distinct health care sites across the United States with prevalence rates ranging from 5.2% to 44%. Nares specimens were tested by (i) the Xpert MRSA assay, (ii) direct culture on CHROMagar MRSA medium (direct CM culture), and (iii) broth-enriched culture (Trypticase soy broth with 6.5% sodium chloride) followed by plating onto CHROMagar MRSA medium (broth-enriched CM culture). When direct CM culture was designated the reference method, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA assay were 94.3%, 93.2%, 73.0%, and 98.8%, respectively. When broth-enriched CM culture was used as the reference method, the clinical sensitivity, specificity, PPV, and NPV of the Xpert MRSA assay were 86.3%, 94.9%, 80.5%, and 96.6%, respectively. The BD GeneOhm MRSA (BDGO) assay was performed as a comparative molecular method. No statistical performance differences were observed between the Xpert MRSA and BDGO assays when they were compared to culture methods. From this large-scale, multicenter clinical comparison, we conclude that the Xpert MRSA assay is a simple, rapid, and accurate method for performing active surveillance for MRSA in a variety of health care populations.

In situ hybridization for the differentiation of Aspergillus, Fusarium, and Pseudallescheria species in tissue section.

Identification of fungi in tissue sections can be difficult. In particular, species of Aspergillus, Fusarium, and Pseudallescheria all appear as septate, branched hyphae. However, their differentiation can have significant clinical implications, as the latter two groups are often resistant to commonly used antifungal agents. In situ hybridization may assist in rapidly distinguishing these organisms in the absence of available culture. Oligonucleotide DNA probes were directed against the 5S, 18S, or 28S rRNA sequences of three groups of fungi with a high degree of specificity for each. Probes were tested on 26 formalin-fixed, paraffin-embedded tissue specimens, each with culture-proven involvement by one of these organisms: Fusarium species, n = 12; Pseudallescheria boydii, n = 5; Aspergillus species, n = 9 ( probe set validated in an earlier study). Accuracy of both ISH and morphology was compared with culture. Morphologic examination (GMS and PAS) showed a greater sensitivity in detecting fungi (100%) as compared with in situ hybridization (84.6%). When detected, however, DNA probes allowed definitive identification of organisms. While there was no ability to distinguish between the three groups of organisms by morphologic features, ISH probes showed 100% positive predictive value (PPV, 19/19 organisms identified correctly). No cross-reactivity was observed when the probes were tested against other genera (100% specificity). Furthermore, the use of ISH allowed the detection of mixed fungal infections involving multiple organism types in two cases, demonstrating another advantage over morphology. In situ hybridization, directed against rRNA sequences, provides a rapid and accurate technique for distinguishing commonly encountered, nonpigmented filamentous fungi in histologic sections. While less sensitive than morphology, ISH is highly accurate and may help to distinguish between organisms that have similar or identical morphologic features by light microscopy.

Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens.

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10(2) to 10(4) spores/ml of feces, a value which represented a significant improvement over that achieved by staining (> or =1.0 x 10(6) spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis, E. cuniculi, and E. hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

Fatal sepsis in a patient with rheumatoid arthritis treated with etanercept.

Patients with long-standing, severe, erosive rheumatoid arthritis who have extra-articular manifestations and have undergone joint replacement surgery are at increased risk for serious infection and premature mortality. New therapies, including cytokine antagonists, hold great promise for improving the course of rheumatoid arthritis. However, they have powerful anti-inflammatory effects that may mask symptoms of serious infection. We report a case of fatal pneumococcal sepsis occurring in a 37-year-old woman with rheumatoid arthritis treated with the tumor necrosis factor antagonist etanercept and suggest management strategies for early detection and management of this complication.

Laboratory diagnosis of viral hepatitis.

Both serologic and molecular assays are useful in the diagnosis of viral hepatitis. They may detect early infections before other signs of disease appear, differentiate acute from chronic infections, and detect persistence of viremia or verify development of immunity. Molecular assays may also be used to monitor responses to antiviral therapy, and in the future, be a primary method to screen blood and organ donors (NAT). EIA serologies are used to diagnose acute HAV infections or establish immune status. Similar immunoassays are used to detect HBV infections, verify persistence of antigenemia and degree of infectivity, and indicate immunity (including the response to vaccination). HBV molecular assays can shorten the diagnostic window period, verify persistence of viremia, including monitoring response to antiviral therapy, and be useful in NAT screening of donors. Molecular assays play a major role in HCV diagnosis where serologic tests can document past or present infection but cannot differentiate one from the other. A variety of molecular tests can be used as sensitive (and early) detectors of viremia (and serve as confirmatory tests for positive serologies and as donor NAT methods), document its persistence as an indicator of chronic infection, and monitor responses to antiviral therapy. Both qualitative and quantitative molecular assays are available, and their efficient use requires familiarity with the sensitivity and dynamic ranges of each method.

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis.

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.