Expression of HLA-DRA and CD74 mRNA in whole blood during the course of complicated and uncomplicated Staphylococcus aureus bacteremia.

To improve management of Staphylococcus aureus bacteremia (SAB), better understanding of host-pathogen interactions is needed. In vitro studies have shown that S. aureus bacteria induce dose-dependent immunosuppression that is evidenced by reduced expression of major histocompatibility complex (MHC) class II on antigen presenting cells. Thus, the aim of this study was to determine whether expression of the MHC class II-related genes HLA-DRA and CD74 is more greatly reduced in complicated SAB, with its probable higher loads of S. aureus, than in uncomplicated SAB. Adult patients with SAB were prospectively included and blood samples taken on the day of confirmation of SAB (Day 1) and on Days 2, 3, 5 and 7. HLA-DRA and CD74 mRNA expression was determined by quantitative reverse transcription PCR. Sepsis was defined according to the Sepsis-3 classification and SAB was categorized as complicated in patients with deep-seated infection and/or hematogenous seeding. Twenty patients with SAB were enrolled and samples obtained on all assessment days. HLA-DRA and CD74 expression did not differ significantly between patients with SAB and sepsis (n = 13) and those without sepsis (n = 7) on any assessment day. However, patients with complicated SAB (n = 14) had significantly weaker HLA-DRA expression on all five assessment days than patients with uncomplicated SAB (n = 6). Additionally, they tended to have weaker CD74 expressions. Neutrophil, monocyte and leukocyte counts did not differ significantly between complicated and uncomplicated SAB. In conclusion, patients with complicated SAB show weaker HLA-DRA expression than those with uncomplicated SAB during the first week of bacteremia.

Quantitative Real-Time Polymerase Chain Reaction Measurement of HLA-DRA Gene Expression in Whole Blood Is Highly Reproducible and Shows Changes That Reflect Dynamic Shifts in Monocyte Surface HLA-DR Expression during the Course of Sepsis.

INTRODUCTION: A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). AIMS: The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA-mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis. METHODS AND FINDINGS: Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1-28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1-2, HLA-DRA 0.40 (0.28-0.59) p<0.001, CIITA 0.48 (0.32-0.72) p = 0.005, mHLA-DR 0.63 (0.45-1.00) p = 0.04, day 7 HLA-DRA 0.59 (0.46-0.77) p<0.001, CIITA 0.56 (0.41-0.76) p<0.001, mHLA-DR 0.81 (0.66-1.00) p = 0.28. CONCLUSION: We conclude that qRT-PCR measurement of HLA-DRA expression is robust, and that this method appears to be preferable to FCM in identifying patients with severe sepsis that may benefit from immunostimulation.