A case of recurrent Corynebacterium jeikeium endocarditis: Unanswered questions for the treatment of chronic endovascular infections.

A 53-year-old woman with end-stage renal impairment on hemodialysis was evaluated for recurrent episodes of Corynebacterium jeikeium bacteremia and endocarditis. Her recurrences occurred despite surgical debridement and extended courses of culture-directed antimicrobial therapy. The clinical course was complicated by the requirement of various endovascular prostheses for vascular access. To our knowledge this degree of relapse with guideline medical and surgical therapy is rare and challenges current practice and understanding of this group of infection.

HLA class I sequence-based typing using DNA recovered from frozen plasma.

We describe a rapid, reliable and cost-effective method for intermediate-to-high-resolution sequence-based HLA class I typing using frozen plasma as a source of genomic DNA. The plasma samples investigated had a median age of 8.5 years. Total nucleic acids were isolated from matched frozen PBMC (~2.5 million) and plasma (500 µl) samples from a panel of 25 individuals using commercial silica-based kits. Extractions yielded median [IQR] nucleic acid concentrations of 85.7 [47.0-130.0]ng/µl and 2.2 [1.7-2.6]ng/µl from PBMC and plasma, respectively. Following extraction, ~1000 base pair regions spanning exons 2 and 3 of HLA-A, -B and -C were amplified independently via nested PCR using universal, locus-specific primers and sequenced directly. Chromatogram analysis was performed using commercial DNA sequence analysis software and allele interpretation was performed using a free web-based tool. HLA-A, -B and -C amplification rates were 100% and chromatograms were of uniformly high quality with clearly distinguishable mixed bases regardless of DNA source. Concordance between PBMC and plasma-derived HLA types was 100% at the allele and protein levels. At the nucleotide level, a single partially discordant base (resulting from a failure to call both peaks in a mixed base) was observed out of >46,975 bases sequenced (>99.9% concordance). This protocol has previously been used to perform HLA class I typing from a variety of genomic DNA sources including PBMC, whole blood, granulocyte pellets and serum, from specimens up to 30 years old. This method provides comparable specificity to conventional sequence-based approaches and could be applied in situations where cell samples are unavailable or DNA quantities are limiting.