Analysis of hepatitis B virus drug-resistant mutant haplotypes by ultra-deep pyrosequencing

Direct sequencing and reverse hybridization are currently the main methods for detecting drug-resistance mutations of hepatitis B virus (HBV). However, these methods do not enable haplotype analysis so they cannot be used to determine whether the mutations are co-located on the same viral genome. This limits the accurate identification of viral mutants that are resistant to drugs with a high genetic barrier. In our current study, ultra-deep pyrosequencing (UDPS) was used to detect HBV drug-resistance mutations in 25 entecavir-treated and five treatment-naive patients. Of the 25 entecavir-treated patients, 18 had experienced virological breakthrough and two exhibited reduced susceptibility to entecavir. The results obtained by UDPS were compared with those of direct sequencing, and the haplotypes of the drug-resistant HBV mutants were analysed. The average number of reads per patient covering the region in which drug-resistance mutations are located was 1735 (range 451-4526). UDPS detected additional drug-resistance mutations not detected by direct sequencing in 19 patients (mutation frequency range 1.1-23.8%). Entecavir-resistance mutations were found to be co-located on the same viral genome in all 20 patients displaying virological breakthrough or reduced susceptibility to entecavir. In conclusion, UDPS was not only sensitive and accurate in identifying drug-resistance mutations of HBV but also enabled haplotype analysis of the mutants. This method may offer significant advantages in explaining and predicting the responses of patients with HBV to antiviral therapy.     

Erratum: Correction of Nomenclature of Mutations

[This corrects the article on p. 1111 in vol. 26, PMID: 21860566.].     

A Case of Korean Ginseng-Induced Anaphylaxis Confirmed by Open Oral Challenge and Basophil Activation Test

Two case reports discussing Korean ginseng-induced allergic reactions have been published; both were inhalation-induced respiratory allergies in occupational settings. In this report we discuss the first case of anaphylaxis that developed after an oral intake of ginseng, confirmed by an open oral challenge, a skin prick test (SPT), and a basophil activation test (BAT). A 44-year-old man experienced rhinorrhea and nasal stiffness, followed by respiratory difficulty with wheeze and abdominal pain 10 minutes after oral intake of fresh ginseng. He had suffered from episodes of allergic rhinitis during the spring season for several years. Upon presentation, a physical examination, chest radiograph, and routine laboratory tests were unremarkable. Total serum IgE level was 41 IU/mL. The SPT results showed strong positive responses to alder, birch pollens, and ginseng extracts (1:500 w/v). The methacholine bronchial challenge test revealed a positive result at PC20 of 5.83 mg/mL. The open oral challenge was performed using 50 g of fresh ginseng and showed immediate onset of facial flushing, cough, respiratory difficulty with wheeze, and abdominal pain combined with a significant decrease in FEV1 levels (54% from the baseline). Serum-specific IgE and IgG4 antibodies were not detectable by enzyme-linked immunosorbent assay. BAT showed a remarkable increase in the expression of CD203c and CD63 with the addition of ginseng extract in a dose-dependent manner, while no changes were noted in the controls. In conclusion, oral intake of Korean ginseng could induce anaphylaxis, which is mediated by non-IgE-dependent direct activation of basophil/mast cells.     

Initial work-up for cervical lymphadenopathy: back to basics

The aim of this study was to evaluate the value of FNA, Tb-PCR, and CBC as an initial work-up protocol for cervical lymphadenopathy to reiterate the importance of CBC in terms of predicting the clinical course. In this consecutive case series, 158 patients with cervical lymphadenopathy were enrolled. All patients underwent FNA and CBC, with or without Tb-PCR. The validity of combined FNA?±?Tb-PCR and CBC in the diagnosis of diseases requiring definitive treatment was evaluated. Final diagnoses were self-limiting disease in 110 (69.6%), malignancy in 19 (12.0%), and tuberculosis in 26 (16.5%). Sensitivity of FNA?±?Tb-PCR was 66.7% and of added CBC profile was 97.9%. Patients with neutropenia or lymphocytosis were found to have a higher chance of spontaneous recovery than patients with a normal WBC profile. FNA and Tb-PCR were found to be important in patients aged more than 20. The results indicate that FNA, Tb-PCR, and CBC are basic and essential in initial work-up for cervical lymphadenopathy. In particular, CBC was found to aid in detecting critical diseases and predicting the likelihood of open biopsy in patients with negative FNA and Tb-PCR. Patient age was also found to be an important determinant of the work-up protocol.