The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in transplantation medicine and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. High-quality NGS-based methods are in proprietary use and not publicly available. Here, we introduce the first highly automated open-kit/open-source HLA-typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm allows for highly confident allele-calling to three-field resolution (cDNA nucleotide variants). The method was validated on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, our method provides the flexibility to add further enrichment target regions.

Background and objectives: The prognostic value of preformed donor-specific HLA antibodies (DSA), which are only detectable by sensitive methods, remains controversial for kidney transplantation. Design, setting, participants, & measurements: The outcome of 4233 consecutive kidney transplants performed between 2012 and 2015 in 18 German transplant centers was evaluated. Most centers used a stepwise pretransplant antibody screening with bead array tests and differentiation of positive samples by single antigen assays. Using these screening results, DSA against HLA-A, -B, -C, -DRB1 and -DQB1 were determined. Data on clinical outcome and possible covariates were collected retrospectively. Results: Pretransplant DSA were associated with lower overall graft survival, with a hazard ratio of 2.53 for living donation (95% confidence interval [95% CI], 1.49 to 4.29; P<0.001) and 1.59 for deceased donation (95% CI, 1.21 to 2.11; P=0.001). ABO-incompatible transplantation was associated with worse graft survival (hazard ratio, 2.09; 95% CI, 1.33 to 3.27; P=0.001) independent from DSA. There was no difference between DSA against class 1, class 2, or both. Stratification into DSA <3000 medium fluorescence intensity (MFI) and DSA ≥3000 MFI resulted in overlapping survival curves. Therefore, separate analyses were performed for 3-month and long-term graft survival. Although DSA <3000 MFI tended to be associated with both lower 3-month and long-term transplant survival in deceased donation, DSA ≥3000 MFI were only associated with worse long-term transplant survival in deceased donation. In living donation, only strong DSA were associated with reduced graft survival in the first 3 months, but both weak and strong DSA were associated with reduced long-term graft survival. A higher incidence of antibody-mediated rejection within 6 months was only associated with DSA ≥3000 MFI. Conclusions: Preformed DSA were associated with an increased risk for graft loss in kidney transplantation, which was greater in living than in deceased donation. Even weak DSA <3000 MFI were associated with worse graft survival. This association was stronger in living than deceased donation.

To determine the prevalence and impact on patient outcome of active human cytomegalovirus infections in patients with prolonged treatment in an intensive care unit. Retrospective analysis of stored plasma samples. Anesthesiological intensive care unit of a university hospital. All 138 patients treated for at least 14 days (of a total of 4940 patients admitted during the study period). Immunocompromised patients and patients with inconclusive results for cytomegalovirus DNA were excluded. None. Stored plasma samples of patients with prolonged intensive care unit stay were tested for cytomegalovirus DNA. Sixty-four of 255 evaluable samples from 99 immunocompetent patients tested cytomegalovirus DNA-positive with a mean DNA concentration of 8,600 genome equivalents per milliliter. Active cytomegalovirus infection was diagnosed by reproducibly positive results in 35 patients (35%). Only one case had been diagnosed clinically. Patients with and without active cytomegalovirus infection were not significantly different in parameters, such as age, sex, admission category, source of admission, or comorbidities. Even review of specific surgical procedures or the use of a heart-lung–machine showed no significant differences between the groups. The mortality rate in patients with cytomegalovirus infection was significantly increased (28.6% vs. 10.9%, p = 0.048), and surviving patients had a longer intensive care unit stay (32.6 vs. 22.1 days, p <0.001). Active cytomegalovirus infection is a frequent but seldom diagnosed finding in surgical patients with prolonged intensive care unit stay, which is associated with increased mortality and prolonged intensive care unit stay of surviving patients.