A Synonymous Change in the Influenza A Virus Neuraminidase Gene Interferes with PCR-Based Subtyping and Oseltamivir Resistance Mutation Detection

Stanford University Medical Center, Stanford, CA.
Journal of clinical microbiology (Impact Factor: 3.99). 06/2011; 49(8):3101-2. DOI: 10.1128/JCM.00642-11
Source: PubMed


Rapid detection of the emergence of oseltamivir-resistant influenza A is critically important for both public health surveillance and the selection of appropriate antiviral therapy.…

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    • "The dengue virus is an RNA virus that replicates via an error prone RNA polymerase with high mutation rates [11]. This phenomenon has been observed with another RNA virus, influenza, where a mutation prevented RT-PCR subtyping [12] of isolates. Similar clusters of untypable dengue RNA positive samples have not been observed in prior CYD dengue studies, including those conducted in Asia [10,13–14] "
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    ABSTRACT: The CYD tetravalent dengue vaccine candidate is being evaluated for protective efficacy against symptomatic dengue in Phase 3 efficacy trials. The laboratory test algorithm to confirm dengue cases was evaluated prior to Phase 3 trials. During a Phase 2 trial in Latin America a dengue epidemic occurred in the study countries. A total of 72 suspected dengue cases were reported and assessed: virological confirmation comprised qRT-PCR methods and a commercial ELISA kit for NS1 protein (Bio-Rad). The qRT-PCR included a screening assay targeting a conserved dengue region of the 3′-UTR (dengue screen assay) followed by 4 individual serotype assays targeting the conserved dengue NS5 genomic region (WT dengue qRT-PCR assays). The NS1 and WT dengue qRT-PCR were endpoint assays for protocol virological confirmation (PVC). Of the 72 suspected cases, 14 were PVC. However, a unique pattern of dengue qRT-PCR results were observed in 5 suspected cases from Honduras: the dengue screen qRT-PCR assay was positive but WT dengue qRT-PCR and NS1 Ag ELISA were negative. To investigate these observations, additional molecular methods were applied: a SYBR® Green-based RT-PCR assay, sequencing assays directed at the genome regions covered by the WT dengue qRT-PCR, and a modified commercial dengue RT-PCR test (Simplexa™ Dengue, Focus Diagnostics). The exploratory data confirmed these additional cases as dengue and indicated the serotype 2 WT dengue qRT-PCR assay was unable to detect a circulating Latin American strain (DENV-2/NI/BID-V608/2006) due to a sequence variation in the isolate. The Simplexa Dengue RT-PCR test was able to detect and serotype dengue. Based on these findings an updated molecular test algorithm for the virological confirmation of dengue cases was developed and implemented in the Phase 3 efficacy trials.
    Trials in Vaccinology 12/2014; 3(1):127–133. DOI:10.1016/j.trivac.2014.07.002
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    • "Amplification of RNase P RNA confirmed adequate extraction and the absence of reaction inhibitors. Influenza A positive extracted nucleic acids were subtyped for 2009 H1N1 using a lab-developed rRT-PCR assay (Trevino et al., 2011). Extracts negative for 2009 H1N1 by the lab-developed test were subtyped using Prodesse ProFAST+ reagents (Gen-Probe Prodesse, Waukesha, WI). "
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    ABSTRACT: Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B.
    Journal of virological methods 07/2012; 186(1-2):137-140. DOI:10.1016/j.jviromet.2012.07.023 · 1.78 Impact Factor
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    ABSTRACT: A new influenza B variant was discovered in Singapore in April 2011 during diagnostic testing of a 3-year-old boy with respiratory symptoms. Influenza B virus was isolated from culture and confirmed by standard immunofluorescence testing, but was not detected by the routine, in-house influenza screening reverse-transcription polymerase chain reaction assay that targets the nucleoprotein (NP) gene. Subsequent sequencing investigations demonstrated that several other published assays targeting NP could also fail to detect this novel variant.
    Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 08/2011; 16(33). · 5.72 Impact Factor
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