Preformed Donor-Specific HLA Antibodies in Living and Deceased Donor Transplantation: A Multicenter Study.

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.

Correlation between genotypic (V3 population sequencing) and phenotypic (Trofile ES) methods of characterizing co-receptor usage of HIV-1 from 200 treatment-naïve HIV patients screened for Study A4001078.

Assessment of HIV-1 co-receptor usage is essential to identify patients who are likely to respond to maraviroc (MVC)-containing regimens. Co-receptor usage of plasma virus from all treatment-naïve patients screened for a MVC clinical trial was assessed using phenotypic and genotypic methodologies to evaluate concordance between testing methods and to assess the quantity of CXCR4-using (non-R5) virus in samples giving discordant results. Co-receptor usage was prospectively measured using the enhanced sensitivity Trofile assay (Trofile ES) to screen patients for enrollment in Study A4001078. Population and deep sequencing methodologies were utilized retrospectively to analyze all screening samples, with co-receptor usage determined using the geno2pheno algorithm. Concordance between methods was explored using descriptive statistics. The quantity of non-R5 virus in all samples was measured using deep sequencing. Trofile ES and matched genotype results were obtained for 199screening samples. Concordance of Trofile ES with population genotyping (5.75% false-positive rate [FPR]) and deep sequencing (3.5% FPR; 2% non-R5 threshold) was 91.7% and 89.6%, respectively. Population genotype data were available for all samples with non-reportable Trofile ES results; the distribution of co-receptor usage in this set was consistent with that in the overall population: 75% (12/16) R5 and 25% (4/16) non-R5. The majority of samples contained non-R5 plasma HIV-1 RNA estimated at either <1 log(10) (62.0%) or ⩾4 log(10) (30.5%) copies/mL; the absolute amount of detectable non-R5 virus remained stable between screening and baseline visits. Samples originally classified as non-R5 by Trofile ES but R5 by population sequencing had a relatively low absolute amount of non-R5 virus (mean 2.1%, median 0.1%). The determination of co-receptor usage using either Trofile ES or genotyping methodologies showed similar frequencies of R5 and non-R5 virus in this treatment-naïve study population. For both concordant and discordant samples, population sequencing appropriately identified R5 samples with low levels of non-R5-using virus.

Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis.

BACKGROUND: Inferring viral tropism from genotype is a fast and inexpensive alternative to phenotypic testing. While being highly predictive when performed on clonal samples, sensitivity of predicting CXCR4-using (X4) variants drops substantially in clinical isolates. This is mainly attributed to minor variants not detected by standard bulk-sequencing. Massively parallel sequencing (MPS) detects single clones thereby being much more sensitive. Using this technology we wanted to improve genotypic prediction of coreceptor usage. METHODS: Plasma samples from 55 antiretroviral-treated patients tested for coreceptor usage with the Monogram Trofile Assay were sequenced with standard population-based approaches. Fourteen of these samples were selected for further analysis with MPS. Tropism was predicted from each sequence with geno2pheno[coreceptor]. RESULTS: Prediction based on bulk-sequencing yielded 59.1% sensitivity and 90.9% specificity compared to the trofile assay. With MPS, 7600 reads were generated on average per isolate. Minorities of sequences with high confidence in CXCR4-usage were found in all samples, irrespective of phenotype. When using the default false-positive-rate of geno2pheno[coreceptor] (10%), and defining a minority cutoff of 5%, the results were concordant in all but one isolate. CONCLUSIONS: The combination of MPS and coreceptor usage prediction results in a fast and accurate alternative to phenotypic assays. The detection of X4-viruses in all isolates suggests that coreceptor usage as well as fitness of minorities is important for therapy outcome. The high sensitivity of this technology in combination with a quantitative description of the viral population may allow implementing meaningful cutoffs for predicting response to CCR5-antagonists in the presence of X4-minorities.

Short communication: selection of thymidine analogue resistance mutational patterns in children infected from a common HIV type 1 subtype G source.

In HIV-1, thymidine analogue mutations (TAMs) cluster in one of two groups (215Y, 41L, 210W, or 215F, 219E/Q), representing two independent mutational patterns (T215Y and T215F cluster, respectively). The mechanisms by which these pathways are selected are not fully understood. To investigate possible factors driving the selection of the TAMs, we analyzed the TAM patterns with regard to the respective treatment, viral load, and HLA in 18 children all infected from a common source of HIV-1 clade G virus and initially treated with zidovudine. The HIV reverse transcriptase sequences of 14/18 children carried at least one TAM. At first sampling date, the T215Y-linked pattern was observed in five cases and the T215F cluster was seen in nine. During the follow-up period, three patients changed their patterns. Children treated with identical NRTI combinations at the first sampling date developed different pathways. Under AZT/d4T therapies, an association was found between the HLA B*13 (in combination with HLA DRB1*0701) and the mutation T215Y. The mutation T215Y reverted in three out of four patients who discontinued AZT/d4T treatment. We speculate that in the context of these subtype G viruses, the development of the T215Y mutation may be strongly disfavored whereas the presence of HLA B*13 may counteract this effect and permit its development.

Improved efficiency in detecting cellular immunity towards M. tuberculosis in patients receiving immunosuppressive drug therapy.

BACKGROUND: Reactivation of a latent Mycobacterium tuberculosis infection in immunocompromised individuals is associated with significant morbidity and mortality. The limited sensitivity of the established tuberculin skin-test in identifying latently infected patients on immunosuppressive drug therapy represents a major obstacle to better tuberculosis control after transplantation. METHODS: In this study, a quantitative flow-cytometric whole-blood assay and the skin-test were comparatively evaluated towards both diagnostic power and practicability in 117 long-term renal transplant recipients (age 53.1+/-14.8 years; 7.0+/-5.0 years after transplantation) in a low-prevalence region. RESULTS: Among the aforementioned renal transplant recipients, a high proportion (52.14%) had purified protein-derivative (PPD)-specific T-cell-immunity in vitro. Despite immunosuppression, prevalence as well as median frequencies of PPD-specific T-cells (0.22%; >0.05-4.71%) were as high as previously reported for immunocompetent individuals and haemodialysis patients. In contrast to in vitro testing, skin testing was less practicable in an ambulatory setting. Moreover, skin-test reactivity was significantly reduced as only 50.0% of patients with PPD-reactivity in vitro were skin-test positive. T-cell reactivity towards early secretory antigenic target-6 (ESAT-6), a protein specific for M. tuberculosis but absent from the bacillus Calmette-Guerin BCG-vaccine strain, was found in 52.9% of all individuals with PPD-reactivity in vitro. CONCLUSIONS: In conclusion, the whole-blood assay reveals a high prevalence of latent tuberculosis infection in renal transplant recipients. It may represent a valuable alternative to skin testing as the test result is not adversely affected by immunosuppression. Moreover, reactivity towards ESAT-6 allows the distinction of a latent infection from BCG-induced reactivity. The assay is well-suited for use in screening programmes and may facilitate the management of tuberculosis infection in immunocompromised individuals.

Effectivity of Alternative Adjuvants in Comparison to Freund's Complete Adjuvant.

In order to replace the use of mammals for the production of hyperimmune sera and to replace Freund"s complete adjuvant (FCA) with its undesirable side effects, the production and immunological features of IgY were investigated systematically. An important advantage of IgY is that large amounts can easily be extracted from the yolk of immunized laying hens. With special regard to animal welfare, several biocompatible adjuvants (ABM system, Gerbu adjuvant, TiterMax) were tested with FCA for their usage and effectiveness in the immunization of chickens. Under appropriate immunization schedules which strictly avoid intramuscular administration, IgY antibodies with very high titers and a strong binding capacity (avidity) comparable to rabbit hyperimmune sera can be obtained with minimal adjuvant side effects.

Effectivity of Different Methods for the Extraction and Purification of IgY.

Extraction of IgY from the egg yolk of immunized laying hens differs from the conventional way of antiserum preparation following blood sampling or exsanguination also with regard to the preparation technique. Six methods of IgY preparation were studied comparatively. As a result, the ammonium sulfate precipitation proved to be the most environmentally compatible and economical one. The advantage of this method becomes particularly obvious when the method is followed by affinity chromatography.