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MHC class I-restricted CD8+ T cells play an important role in controlling HIV and SIV replication. In SIV-infected Indian rhesus macaques (Macaca mulatta), comprehensive CD8+ T cell epitope identification has only been undertaken for two alleles, Mamu-A*01 and Mamu-B*17. As a result, these two molecules account for virtually all known MHC class I-restricted SIV-derived CD8+ T cell epitopes. SIV pathogenesis research and vaccine testing have intensified the demand for epitopes restricted by additional MHC class I alleles due to the shortage of Mamu-A*01+ animals. Mamu-A*02 is a high frequency allele present in over 20% of macaques. In this study, we characterized the peptide binding of Mamu-A*02 using a panel of single amino acid substitution analogues and a library of 497 unrelated peptides. Of 230 SIVmac239 peptides that fit the Mamu-A*02 peptide-binding motif, 75 peptides bound Mamu-A*02 with IC50 values of ≤500 nM. We assessed the antigenicity of these 75 peptides using an IFN-γ ELISPOT assay with freshly isolated PBMC from eight Mamu-A*02+ SIV-infected macaques and identified 17 new epitopes for Mamu-A*02. The synthesis of five Mamu-A*02 tetramers demonstrated the discrepancy between tetramer binding and IFN-γ secretion by SIV-specific CD8+ T cells during chronic SIV infection. Bulk sequencing determined that 2 of the 17 epitopes accumulated amino acid replacements in SIV-infected macaques by the chronic phase of infection, suggestive of CD8+ T cell escape in vivo. This work enhances the use of the SIV-infected macaque model for HIV and increases our understanding of the breadth of CD8+ T cell responses in SIV infection.

(2) and is associated with prolonged hospitalization, with higher health care costs (3). The clinical microbiology laboratory plays a vital role in the treatment of patients with bloodstream infections. While current methods for the detection and characterization of bloodstream pathogens can take days, the risk of death from sepsis increases by 6 to 10% per hour from the onset of shock to the start of effective antimicrobial treatment (4).Over 50% of organisms identified in positive blood cultures are Gram-positive bacteria (5). Gram-positive organisms also are common contaminants of blood cultures. Therefore, rapid differentiation of pathogens from contaminants would be clinically useful.The Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (IUO) (Nanosphere, Northbrook, IL) is a random-access, automated test that performs nucleic acid extraction directly from positive blood culture media, hybridization onto a microarray, and analysis in 2.5 h. Targets in the assay include Staphylococcus spp., Streptococcus spp., Micrococcus spp., Listeria spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus anginosus group, Enterococcus faecalis, and Enterococcus faecium. In addition, if S. aureus or S. epidermidis is detected, then the presence or absence of the mecA gene is reported. Similarly, if E. faecium or E. faecalis is detected, then the presence or absence of the vanA and vanB genes is reported. The BC-GP test was approved by the U.S. Food and Drug Administration (FDA) on 27 June 2012 for the reporting of all IUO targets except Micrococcus spp. (6).We evaluated the performance of the Verigene BC-GP IUO assay in comparison with routine laboratory methods used in the clinical laboratory. A recent evaluation of the BC-GP assay (7) tested blood cultures from a blood culture system (VersaTREK; Trek Diagnostic Systems, Cleveland, OH) that is used by Ͻ10% of U.S. laboratories, while our study utilized the BacT/Alert instrument (bioMérieux, Durham, NC), which is found in ϳ50% of laboratories (N. Safwat, personal communication). Another novel aspect of our study is the assessment of the time from Gram stain result reporting to identification and susceptibility result reporting for current laboratory methods, in comparison to the time for BC-GP results. This research was conducted at the Cleveland Clinic, after approval by the institutional internal review board.(This study was presented in part at ID Week, San Diego, CA, 20 October 2012.) MATERIALS AND METHODSBlood culture samples. The BC-GP IUO test was evaluated with blood cultures submitted for testing at the Cleveland Clinic microbiology laboratory in February to August 2012. Positive aerobic blood culture samples (in BacT/Alert FA bottles [bioMérieux, Durham, NC]) that contained Gram-positive cocci from unique patients were included in the study. Specimens were excluded if multiple morphologies were observed ...

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