Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.
Please cite this paper as: Couturier et al. (2010). Oseltamivir‐resistant influenza A 2009 H1N1 virus in immunocompromised patients. Influenza and Other Respiratory Viruses 4(4), 199–204. Background First‐line treatment of influenza A 2009 H1N1 relies on neuraminidase inhibitors such as oseltamivir. Resistance conferred by the H275Y neuraminidase gene mutation is concerning and likely to increase. Objectives To characterize oseltamivir resistance in a hospital‐based patient population. Patients and Methods All available respiratory specimens positive for influenza A by direct fluorescent antibody, RT‐PCR, or culture from patients at the University of Utah 5/09‐12/09 were collected. Specimens were confirmed as 2009 H1N1 by the Utah Department of Health. RT‐PCR and pyrosequencing were used to test for the H275Y mutation (CDC protocol). PyroMark Q24 AQ software (Qiagen, Valencia, CA, USA) was used to allow for quantitative H275Y mutation analysis. Medical records of patients with resistant virus were reviewed. Results We tested 191 influenza A virus‐positive samples from 187 unique patients. Fifty (27%) patients were hospitalized. Four patient specimens (2·1%) were found to carry the H275Y mutation. Three patients were hospitalized, representing 6% of inpatient samples tested. Three patients had undergone hematopoietic stem cell transplant in the past year. Two patients died. Their influenza viruses were confirmed to be oseltamivir‐resistant at an independent reference laboratory and through the Center for Disease Control and Prevention (CDC). One patient reported no history of prior oseltamivir exposure. Conclusions Widespread oseltamivir resistance among 2009 H1N1 remains a potential threat. Rapid techniques, such as pyrosequencing, which has the additional benefit of identifying mixed mutant populations of virus, may play a key role in identifying at‐risk individuals and potentially unsuspected cases. Targeted surveillance of immunocompromised patients will be critical toward improving future influenza planning and therapy.
We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.
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