Parallel Validation of Three Molecular Devices for Simultaneous Detection and Identification of Influenza A and B and Respiratory Syncytial Viruses.

Rapid identification of respiratory pathogens, such as influenza virus A (FluA), influenza virus B (FluB), and respiratory syncytial virus (RSV), reduces unnecessary antimicrobial use and enhances infection control practice. We performed a comparative evaluation of three molecular methods: (i) the Aries Flu A/B & RSV, (ii) the Xpert Xpress Flu/RSV, and (iii) the Cobas Flu A/B & RSV assays. The clinical performances of the three methods were evaluated using 200 remnant nasopharyngeal swab (NPS) specimens against a combined reference standard. The limits of detection (LODs) were determined using FluA, FluB, and RSV control strains with known titers. The 95% LODs were between 1.702 and 0.0003 50% tissue culture infective dose (TCID50), with no significant differences revealed among the three assays. Perfect qualitative detection agreement was obtained in the reproducibility study. The Cobas assay failed at the first run on 13 clinical specimens, resulting in an invalid rate of 6.5%. The sensitivities and specificities for all assays were 96.0 to 100.0% and 99.3 to 100% for all three viruses. For on-demand single-specimen and batched 12-specimen workflows, the test turnaround times were 115.5 and 128.8 min for the Aries assay (12 sample capacity), 34.2 and 44.2 min for the Xpress assay (16 sample capacity), and 21.0 and 254.4 min for the Cobas assay (one instrument), respectively. In summary, the Aries, Xpress, and Cobas Liat assays demonstrated excellent sensitivities and specificities for simultaneous detection and identification of FluA, FluB, and RSV from NPS specimens in cancer patients. Test turnaround time was significantly shorter on the Xpress when instrument scalability is unlimited.

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B.

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay. The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively. This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

Clinical Evaluation of the Luminex NxTAG Respiratory Pathogen Panel.

An evaluation of the Luminex NxTAG Respiratory Pathogen Panel was performed on 404 clinical respiratory specimens. Clinical sensitivities and specificities of the assay compared to those of the reference methods were 80.0% to 100.0% and 98.9% to 100.0%, respectively. Correct genotyping information was provided for 95.5% of influenza virus A specimens. The closed-tube format of the assay simplified the workflow and minimized carryover contamination.

Macrolide-Resistant Mycoplasma pneumoniae, United States.

Macrolide-resistant Mycoplasma pneumoniae (MRMP) is highly prevalent in Asia and is now being reported from Europe. Few data on MRMP are available in the United States. Using genotypic and phenotypic methods, we detected high-level MRMP in 13.2% of 91 M. pneumoniae--positive specimens from 6 US locations.

Monitoring of cytomegalovirus viral loads by two molecular assays in whole-blood and plasma samples from hematopoietic stem cell transplant recipients.

Cytomegalovirus (CMV) viral loads in hematopoietic stem cell transplant (HSCT) recipients are typically monitored using quantitative molecular assays. The Roche Cobas AmpliPrep/Cobas TaqMan CMV test (Cobas CMV) has recently been cleared by the FDA for the monitoring of CMV viral loads in plasma samples from transplant patients. In this study, we compare and correlate the viral loads obtained by a laboratory-developed test (LC CMV) (using Roche analyte-specific reagents [ASR] on the LightCycler 2.0) on whole-blood specimens with those obtained on corresponding plasma and whole-blood specimens by the Cobas CMV assay. Testing was performed on 773 archived patient specimens. The strength of the agreement was good for the two assays performed on whole blood (κ=0.6; 95% confidence interval [CI], 0.51 to 0.7) and moderate when the tests were performed on different sample types (κ=0.54; 95% CI, 0.47 to 0.62 for the LC CMV whole blood [WB] assay versus Cobas plasma [PL], and κ=0.57; 95% CI, 0.5 to 0.65 for the Cobas WB assay versus Cobas PL), although the difference was not statistically significant. Using a combination gold standard (i.e., a true positive was a specimen that was positive by two or more methods), the sensitivity and specificity of the assays were 78.8% and 99.3% for the LC CMV assay, 85.2% and 98.1% for the Cobas CMV WB assay, and 100% and 90.5% for Cobas CMV PL assay, respectively. A comparison of the CMV viral load trends in both plasma and whole blood from a few patients with multiple positive successive samples showed similar slopes, with differences in the slope ranging from 0.01 to 0.22. However, the absolute value for individual viral load differed markedly with whole-blood viral loads, being on average 0.5- to 1.22-log higher than those in plasma. The Cobas CMV assay provides a valid option for the monitoring of viral loads in transplant patients. Due to its increased sensitivity, the detection of CMV DNA in patients with low viral loads (i.e., those below limit of quantification [LOQ]) is increased with the Cobas CMV assay in plasma specimens. Longitudinal prospective studies will be needed to examine the clinical significance of these low-level viral loads.

Clinical validation of the Lyra direct HSV 1+2/VZV assay for simultaneous detection and differentiation of three herpesviruses in cutaneous and mucocutaneous lesions.

We evaluated the Lyra Direct HSV 1+2/VZV multiplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) on 695 consecutive cutaneous and mucocutaneous lesion specimens. The intra-assay and interassay coefficient of variation values for the Lyra assay were 0.29 to 1.30% and 2.33 to 2.61%, respectively. The sensitivities, specificities, and positive and negative predictive values were 93.4 to 95.0%, 96.1 to 96.8%, 78.0 to 80.3%, and 99.0 to 99.1%, respectively, in comparison to those of viral culture. The values were further improved when a resolution analysis was performed with a laboratory-developed PCR assay.

Evaluation of Alere i Influenza A&B for rapid detection of influenza viruses A and B.

Rapid and accurate diagnosis of influenza is important for infection control, as well as for patient management. Alere i Influenza A&B is an isothermal nucleic acid amplification-based integrated system for detection and differentiation of influenza virus A and influenza virus B. The performance of the Alere i Influenza A&B was screened using frozen nasopharyngeal-swab specimens collected in viral transport medium (VTM) that were originally tested fresh with the FilmArray Respiratory Panel (RP) assay during the 2012-2013 influenza outbreak. In total, 360 VTM specimens were selected for Alere i Influenza A&B testing: 40 influenza virus A H1N1-2009 (influenza virus A-1), 40 influenza virus A H3N2 (influenza virus A-3), 37 influenza virus A "equivocal" or "no subtype detected" (influenza virus A-u), 41 influenza virus B, and 202 influenza virus-negative specimens, as initially determined by the FilmArray RP assay. The Alere assay showed sensitivities of 87.2%, 92.5%, 25.0%, and 97.4% for influenza virus A-1, influenza virus A-3, influenza virus A-u, and influenza virus B, respectively, after discordant resolution by Prodesse ProFLU+ PCR. The specificities were 100% for both influenza virus A and influenza virus B. In general, the Alere i Influenza A&B provided good sensitivity, although the assay did show poorer sensitivity with samples determined to have low influenza virus A titers by Prodesse ProFlu+ PCR (a mean real-time PCR threshold cycle [CT] value of 31.9 ± 2.0), which included the majority of the samples called influenza virus A "equivocal" or "no subtype detected" by a single BioFire FilmArray RP test. The integrated, rapid, and simple characteristics of the Alere i Influenza A&B assay make it a potential candidate for point-of-care testing, with a test turnaround time of less than 15 min.

Real-time cellular analysis coupled with a specimen enrichment accurately detects and quantifies Clostridium difficile toxins in stool.

We describe here the use of an immunomagnetic separation enrichment process coupled with a modified real-time cellular analysis (RTCA) system (RTCA version 2) for the detection of C. difficile toxin (CDT) in stool. The limit of CDT detection by RTCA version 2 was 0.12 ng/ml. Among the consecutively collected 401 diarrheal stool specimens, 53 (13.2%) were toxin-producing C. difficile strains by quantitative toxigenic culture (qTC); bacterial loads ranged from 3.00 × 10(1) to 3.69 × 10(6) CFU/ml. The RTCA version 2 method detected CDT in 51 samples, resulting in a sensitivity of 96.2%, a specificity of 99.7%, and positive and negative predictive values of 98.1% and 99.4%, respectively. The positive step time ranged from 1.43 to 35.85 h, with <24 h for 80% of the samples. The CDT concentrations in stool samples determined by RTCA version 2 correlated with toxigenic C. difficile bacterial load (R(2) = 0.554, P = 0.00002) by qTC as well as the threshold cycle (R(2) = 0.343, P = 0.014) by real-time PCR. A statistically significant correlation between the CDT concentrations and the clinical severity of CDI was observed (P = 0.015). The sensitivity of the RTCA version 2 assay for the detection of functional toxins in stool specimens was significantly improved when the immunomagnetic separation enrichment process was incorporated. More than 80% positive results can be obtained within 24 h. The stool specimen CDT concentration derived using the RTCA version 2 assay correlates with clinical severity and may be used as a marker for monitoring the status of CDI.

Comparison of the Luminex xTAG RVP Fast assay and the Idaho Technology FilmArray RP assay for detection of respiratory viruses in pediatric patients at a cancer hospital.

Respiratory viruses are increasingly recognized as serious causes of morbidity and mortality in immunocompromised patients. The rapid and sensitive detection of respiratory viruses is essential for the early diagnosis and administration of appropriate antiviral therapy, as well as for the effective implementation of infection control measures. We compared the performance of two commercial assays, xTAG RVP Fast (Luminex Diagnostics, Toronto, Canada) and FilmArray RVP (FA RVP; Idaho Technology, Salt Lake City, UT), in pediatric patients at Memorial Sloan-Kettering Cancer Center. These assays detect the following viruses: respiratory syncytial virus; influenza A and B viruses; parainfluenza viruses 1, 2, 3, and 4; human metapneumovirus; adenovirus; enterovirus-rhinovirus; coronaviruses NL63, HKU1, 229E, and OC43; and bocavirus. We tested a total of 358 respiratory specimens from 173 pediatric patients previously tested by direct fluorescence assay (DFA) and viral culture. The overall detection rate (number of positive specimens/total specimens) for viruses tested by all methods was 24% for DFA/culture, 45% for xTAG RVP Fast, and 51% for FA RVP. The agreement between the two multiplex assays was 84.5%, and the difference in detection rate was statistically significant (P < 0.0001). Overall, the FA RVP assay was more sensitive than the xTAG RVP Fast assay and had a turnaround time of approximately 1 h. The sensitivity, simplicity, and random-access platform make FA RVP an excellent choice for laboratory on-demand service with low to medium volume.

Evaluation of the Cepheid Xpert Clostridium difficile Epi assay for diagnosis of Clostridium difficile infection and typing of the NAP1 strain at a cancer hospital.

Clostridium difficile is the most common cause of health care-associated diarrhea. Accurate and rapid diagnosis is essential to improve patient outcome and prevent disease spread. We compared our two-step diagnostic algorithm, an enzyme immunoassay for glutamate dehydrogenase (GDH) followed by the cytotoxin neutralization test (CYT) with a turnaround time of 24 to 48 h, versus the Cepheid Xpert C. difficile Epi assay, a PCR-based assay with a turnaround time of <1 h. In the first phase of the study, only GDH-positive stool samples were tested by both CYT and Xpert PCR. Discordant results were resolved by toxigenic culture. In the second phase, all stool samples were tested by GDH and Xpert PCR. Only GDH-positive stools were further tested by CYT. Genotypic characterization of 45 Xpert PCR-positive stools was performed by sequencing of the tcdC gene and PCR ribotyping. In phase 1, the agreement between the GDH-CYT and the GDH-Xpert PCR was 72%. The sensitivities and specificities of GDH-CYT and GDH-Xpert PCR were 57% and 97% and 100% and 97%, respectively. In phase 2, the agreement between GDH-CYT and Xpert PCR alone was 95%. As in phase 1, sensitivity of the Xpert PCR was higher than that of the GDH-CYT. The correlation between PCR-ribotyping, sequencing, and Xpert PCR for detection of NAP1 strains was excellent (>90%). The excellent sensitivity and specificity and the rapid turnaround time of the Xpert PCR assay as well as its strain-typing capability make it an attractive option for diagnosis of C. difficile infection.

2009 H1N1 influenza infection in cancer patients and hematopoietic stem cell transplant recipients.

OBJECTIVES: Although usually mild, 2009 H1N1 Influenza has caused up to 6000 deaths in the US. To determine outcome in patients with cancer and/or hematopoietic stem cell transplant (HSCT), we reviewed our recent experience at Memorial Sloan-Kettering Cancer Center (MSKCC). METHODS: During the initial NYC outbreak (May 19-June 30, 2009), all respiratory samples at MSKCC were tested for 2009 H1N1 influenza by DFA, culture, and RT-PCR. Medical records were reviewed for all cases. RESULTS: During the 6-week period, 45(11%) of 394 tested patients were diagnosed with 2009 H1N1 Influenza. These included 29(17%) of 167 patients with hematologic conditions compared to 16(7%) of 226 with solid tumors (P < 0.01). 21(22%) of 96 tested HSCT recipients were positive. Cough (93%) and fever (91%) were common. Of 29 patients who were radiographically assessed, 8(27%) had lower airway disease. 17(37%) were hospitalized. None required mechanical ventilation. No deaths were attributed to influenza. All treated patients tolerated antiviral medication. CONCLUSIONS: 2009 H1N1 Influenza caused mild symptoms in most patients with cancer and/or HSCT. None died or required mechanical ventilation. Immunosuppression from cancer or its treatment including HSCT may not be a substantial risk for poor outcome, however further studies are needed to validate our results.

Clinical characterization of human metapneumovirus infection among patients with cancer.

BACKGROUND: Human metapneumovirus is a recently discovered RNA virus that typically causes respiratory disease in children. It has been linked to severe lower airway disease in hematopoietic stem cell and solid-organ transplant recipients. hMPV infection in a large population of patients with underlying cancer and varying degrees of immunosuppression has not been reported. We sought to characterize hMPV infection in patients with cancer. METHODS: Review of all cases of hMPV infection from two seasons (2005-6 and 2006-7) detected by DFA and/or real-time PCR at MSKCC, a tertiary cancer center in New York City. RESULTS: Among MSKCC patients with cancer, 51 (2.7%) of 1899 patients were positive for hMPV, including 3.2% with hematologic neoplasm and 1.7% with solid tumors. More children (4.5%) were positive than adults (2.2%). PCR detected twice as many cases as DFA. Cough and fever were common complaints. The longest shedding period was 80 days. 40 patients received radiographic evaluation; of these, 22 showed abnormalities including patchy (11), ground glass (5), and interstitial infiltrates (4). CONCLUSIONS: hMPV causes a nonspecific respiratory illness and was found in more than 2% of all tested persons with cancer. PCR detected substantially more cases than DFA. Unlike previous reports, we observed no fatalities due to hMPV, including 22 HSCT recipients with the infection.

Rapid expansion of cytomegalovirus-specific cytotoxic T lymphocytes by artificial antigen-presenting cells expressing a single HLA allele.

Cytomegalovirus (CMV) is a major threat in patients undergoing allogeneic bone marrow transplantation. The adoptive transfer of CMV-specific cytotoxic T lymphocytes (CTLs) expanded from the blood of CMV-seropositive donors has been shown to effectively control CMV infection. However, the requirement for safe and effective antigen-presenting cells (APCs) for each patient precludes broad applicability of this successful form of therapy. Here we analyze the ability of artificial APCs (AAPCs) to activate and expand CMV-specific CTLs from peripheral blood of seropositive HLA A2.1+ donors. We demonstrate that AAPCs expressing the CMV P495 peptide or the full-length pp65 protein stimulate P495-specific CTLs at least as effectively as autologous, peptide-pulsed, peripheral blood mononuclear cells or EBV-transformed B cells. Starting from 100 mL of blood, the AAPCs reliably yield clinically relevant CTL numbers after a single stimulation. CTLs activated on AAPCs effectively kill CMV-infected fibroblasts and have a Tc1 memory effector phenotype identical to that of CTLs generated with autologous APCs. AAPCs thus offer a rapid, controlled, convenient, and highly reproducible system for expanding CMV-specific CTLs. Furthermore, the CTL expansion obtained with AAPCs encoding full-length pp65 indicates that AAPCs may be used to present known as well as unknown CTL epitopes in the context of the AAPC's HLA.