Anthony R. Sambol

Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans

Generation of Swine Flu As the newly emerged influenza virus starts its journey to infect the worlds human population, the genetic secrets of the 2009 outbreak of swine influenza A(H1N1) are being revealed. In extensive phylogenetic analyses, Garten et al. (p. 197, published online 22 May) confirm that of the eight elements of the virus, the basic components encoded by the hemagglutinin, nucleoprotein, and nonstructural genes originated in birds and transferred to pigs in 1918. Subsequently, these formed a triple reassortant with the RNA polymerase PB1 that transferred from birds in 1968 to humans and then to pigs in 1998, coupled with RNA polymerases PA and PB2 that transferred from birds to pigs in 1998. The neuraminidase and matrix protein genes that complete the virus came from birds and entered pigs in 1979. The analysis offers insights into drug susceptibility and virulence, as well as raising the possibility of hitherto unknown factors determining host specificity. A significant question is, what is the potential for the H1 component of the current seasonal flu vaccine to act as a booster? Apart from the need for ongoing sequencing to monitor for the emergence of new reassortants, future pig populations need to be closely monitored for emerging influenza viruses. Evolutionary analysis suggests a triple reassortant avian-to-pig origin for the 2009 influenza A(H1N1) outbreak. Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).

Validation of the Cepheid Xpert Flu A real time RT-PCR detection panel for emergency use authorization.

BACKGROUND In April 2009, the United States Secretary of the Department of Health and Human Services declared a public health emergency concerning the 2009 influenza H1N1 outbreak. This declaration allowed the FDA to issue Emergency Use Authorization (EUA) of approved in vitro diagnostics to detect the 2009 influenza H1N1 in clinical specimens. OBJECTIVES This report outlines the validation testing of the Cepheid Xpert Flu A Panel for the qualitative detection of 2009 H1N1 viral RNA. STUDY DESIGN This study was a multi-site, dual-method clinical evaluation comparing the results of testing between the Xpert Panel assay to the FDA-cleared Luminex Molecular Diagnostics xTAG Respiratory Viral Panel (Luminex RVP) assay and the EUA-granted Focus Diagnostics Influenza A/H1N1 (2009) Real Time RT-PCR (Focus H1N1) assay. RESULTS When compared to Luminex RVP (n=300) for influenza A detection, the Xpert Panel had a sensitivity of 91.2% (95% CI: 85.1-95.4), specificity of 99.4% (95% CI: 96.7-100), positive predictive value (PPV) of 99.2% (95% CI: 95.6-100), and a negative predictive value (NPV) of 93.1% (95% CI: 88.3-96.4). When compared to the Focus H1N1 (n=258) for detection of H1N1, the Xpert Panel had a sensitivity of 92.1% (95% CI: 82.4-97.4), specificity of 100% (95% CI: 98.5-100), PPV of 100% (95% CI: 95.0-100), and a NPV of 97.5% (95% CI: 94.3-99.2). CONCLUSIONS The results show the Cepheid Xpert Flu A Panel to be comparable to both the Luminex RVP and the Focus H1N1 assays. The Cepheid Xpert Panel was granted an EUA on 24 Dec 2009.

Use of rapid influenza diagnostic tests under field conditions as a screening tool during an outbreak of the 2009 novel influenza virus: Practical considerations

BACKGROUND Rapid influenza diagnostic tests (RIDTs) are used in various settings as a first-line screen of patient specimens. During the initial outbreak of the 2009 novel influenza A/H1N1 virus, the Nebraska Public Health Laboratory (NPHL) adopted a testing algorithm, attempting to maximize the usefulness of RIDTs. However, it became apparent that a high percentage of the positive specimens received from off-site facilities were negative for influenza viruses by the confirmatory test, the Luminex xTAG Respiratory Viral Panel (RVP) molecular assay. OBJECTIVES To explore the cause of discrepancies between RIDTs results obtained from on-site facility testing versus confirmatory testing performed at NPHL. STUDY DESIGN Specimens (n=336) tested with RIDTs at off-site facilities and screened for high-probability of containing H1N1 were sent to the NPHL for confirmatory testing by RVP. RESULTS Of 336 specimens analyzed, 104 were negative for influenza A or B by both RIDT and RVP; 127 were positive by both tests; 102 were positive by RIDT only; and 3 were positive by RVP only. Using the RVP assay as the gold standard, overall RIDT characteristics in this screened population were: sensitivity=97.7% (95%CI: 92.5, 99.3); specificity=48.1% (95%CI: 40.4, 55.8); positive predictive value=54.3% (95%CI: 47.0, 61.4); and negative predicative value=97.1% (95%CI: 90.6, 99.1). CONCLUSIONS The results show that the confirmation of RIDT-positive results varied widely by testing site. Possible explanations for the discrepancies in performance characteristics include testing a narrowly defined sample population, test facility characteristics, facility work load, and seasonal timing.

Comparison of FilmArray and Quantitative Real-Time Reverse Transcriptase PCR for Detection of Zaire Ebolavirus from Contrived and Clinical Specimens

ABSTRACT Rapid, reliable, and easy-to-use diagnostic assays for detection of Zaire ebolavirus (ZEBOV) are urgently needed. The goal of this study was to examine the agreement among emergency use authorization (EUA) tests for the detection of ZEBOV nucleic acids, including the BioFire FilmArray BioThreat (BT) panel, the FilmArray BT-E panel, and the NP2 and VP40 quantitative real-time reverse transcriptase (qRT) PCR assays from the Centers for Disease Control and Prevention (CDC). Specimens used in this study included whole blood spiked with inactivated ZEBOV at known titers and whole-blood, plasma, and urine clinical specimens collected from persons diagnosed with Ebola virus disease (EVD). The agreement for FilmArray and qRT-PCR results using contrived whole-blood specimens was 100% (6/6 specimens) for each ZEBOV dilution from 4 × 107 to 4 × 102 50% tissue culture infective dose (TCID50)/ml, as well as the no-virus negative-control sample. The limit of detection for FilmArray and qRT-PCR assays with inactivated ZEBOV, based on duplicate positive results, was determined to be 4 × 102 TCID50/ml. Rates of agreement between FilmArray and qRT-PCR results for clinical specimens from patients with EVD were 85% (23/27 specimens) for whole-blood specimens, 90% (18/20 specimens) for whole-blood specimens tested by FilmArray testing and matched plasma specimens tested by qRT-PCR testing, and 85% (11/13 specimens) for urine specimens. Among 60 specimens, eight discordant results were noted, with ZEBOV nucleic acids being detected only by FilmArray testing in four specimens and only by qRT-PCR testing in the remaining four specimens. These findings demonstrate that the rapid and easy-to-use FilmArray panels are effective tests for evaluating patients with EVD.

Safety considerations in the laboratory testing of specimens suspected or known to contain Ebola virus.

Reference to the Ebola virus causes concern among all individuals, whether from the public or within the medical community. Realization that patients with Ebola virus disease (EVD) have now been recognized in the United States in response to the major outbreak occurring in West Africa has heightened this fear. Recently, the World Health Organization declared the Ebola epidemic to be a Public Health Emergency of International Concern to provide containment of this major international health threat. In response to this threat to public health, the United States has stepped up efforts to provide care for infected patients, which include bringing individuals with EVD into the United States for treatment. These activities, along with the increased possibility of having more individuals recognized with EVD in the United States, have caused hospitals to evaluate how to contain and care for patients suspecting of having EVD. As a part of this response, laboratorians have been asked to be prepared to test specimens from persons under investigation (PUIs) for EVD or patients known to have EVD. Recently, the Centers for Disease Control and Prevention (CDC) provided an interim guideline followed by a supplemental document for how US laboratories could safely manage specimens from …

An Integrated Approach to Laboratory Testing for Patients with Ebola Virus Disease

Beginning in 2003, the Nebraska Medical Center in Omaha developed a laboratory capability plan in conjunction with the creation of a biocontainment unit (BCU) for treatment of patients harboring emerging infectious organisms. The laboratory response planning involved experts at the Nebraska Public Health

Geographic factors contributing to a high seroprevalence of West Nile virus-specific antibodies in humans following an epidemic.

ABSTRACT Sera of 624 blood donors were evaluated to determine seroprevalence of West Nile virus (WNV) antibodies following the 2003 WNV epidemic in Nebraska. Geographic factors contributing to differences in WNV seropositivity were evaluated. The overall prevalence of WNV in Nebraska was higher than reported previously in other U.S. locations (9.5% WNV immunoglobulin G seroprevalence rate), with the highest prevalence identified in the western part of the state (19.7%), followed by the central (13.8%) and the eastern (4.2%) parts. Regions of the state with the highest WNV-positive mosquito rates correlated with the highest human WNV seroprevalence rates. The results showed that both the western and central parts of the state, where mosquito positivity rates were highest, had significantly higher seroprevalence rates than the eastern region. Additional studies are needed to determine whether the high prevalence rates in Nebraska will be reflected in other states and what impact environmental and geographical factors may have on future outbreaks of WNV infection.

Seroprevalence of West Nile virus infection in solid organ transplant recipients

A.G. Freifeld, J. Meza, B. Schweitzer, L. Shafer, A.C. Kalil. A.R. Sambol. Seroprevalence of West Nile virus infection in solid organ transplant recipients.
Transpl Infect Dis 2010: 12: 120–126. All rights reserved

Performance of a Commercial Immunoglobulin M Antibody Capture Assay Using Analyte-Specific Reagents To Screen for Interfering Factors during a West Nile Virus Epidemic Season in Nebraska

ABSTRACT In 2003, the Nebraska Public Health Laboratory tested more than 10,371 serum and 516 cerebral spinal fluid specimens. Results showed that without performing the interfering factors screen for specimens in the low positive index value range of >1.1 to ≤3.5, a false positivity rate of 6.5% would have been realized.

Evaluation of a new West Nile Virus lateral-flow rapid IgM assay

This study evaluated the performance of a new Food and Drug Administration-approved lateral-flow diagnostic screening test for qualitative detection of West Nile Virus (WNV) immunoglobulin M (IgM) in serum or plasma. Five public health laboratories across the United States performed retrospective testing on blinded serum samples from patients with physician reported diagnoses of WNV infection. The results of the RapidWN WNV IgM assay were compared with two commercially available WNV IgM enzyme-linked immunosorbent assays (EIA) and two public health-developed WNV-IgM tests. After discrepancies were resolved, the RapidWN WNV IgM EIA demonstrated a 98.8% sensitivity (range: 96.0-100%), a 95.3% specificity (range: 90.9-100%), a positive predictive value of 96.3% (range: 94.7-100%), and a negative predictive value of 98.4% (range: 95.5-100%), as compared to the predicate assays. The study results suggest that the RapidWN WNV IgM EIA is an effective, qualitative screening test that produces results comparable to that of predicate assays and can be employed rapidly to detect WNV IgM in patients suspected of having WNV infection.