Malte Ziemann

High prevalence of cytomegalovirus DNA in plasma samples of blood donors in connection with seroconversion

BACKGROUND: Human cytomegalovirus (CMV) is considered to latently infect blood cells. Transfusion‐transmitted infection (TT‐CMV) of immunocompromised patients occurs despite the use of CMV‐seronegative or leukoreduced units.

Increased mortality in long-term intensive care patients with active cytomegalovirus infection.

Objective:To determine the prevalence and impact on patient outcome of active human cytomegalovirus infections in patients with prolonged treatment in an intensive care unit. Design:Retrospective analysis of stored plasma samples. Setting:Anesthesiological intensive care unit of a university hospital. Patients: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. Interventions:None. Measurements and Main Results: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). Conclusions: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.

Development of a high-resolution NGS-based HLA-typing and analysis pipeline.

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.

Reliability of capillary hemoglobin screening under routine conditions

BACKGROUND: Capillary hemoglobin (Hb) measurement before admission for whole blood donation is performed in many blood donation services, in spite of several studies reporting many donors with low Hb values being missed by capillary Hb screening.

The impact of donor cytomegalovirus DNA on transfusion strategies for at-risk patients

Cytomegalovirus (CMV) DNA is frequently detected in plasma of newly seropositive donors. Selection of leukoreduced blood products from donors with remote CMV infection could avoid transfusion‐transmitted CMV infections (TT‐CMV) due to primarily infected donors. However, there are no data about the prevalence of reactivations in long‐term seropositive donors compared to the incidence of window period donations in seronegative donors. Therefore, the optimal transfusion strategy for at‐risk patients is unclear.

Prevention of Transfusion-Transmitted Cytomegalovirus Infections: Which is the Optimal Strategy?

Traditionally, leukoreduction and selection of blood products from seronegative donors have been used as alternative strategies to reduce the risk of transfusion-transmitted cytomegalovirus infections (TT-CMV) in atrisk patients. After the introduction of universal leukoreduction for red blood cell and platelet concentrates in Germany, a controversy evolved as to whether the additional selection of blood products from seronegative donors would reduce or even increase the risk of TT-CMV. This review summarizes the current knowledge about CMV infections in blood donors and the implications of this information on the effect of potential transfusion strategies. Even though there are conflicting data about the incidence of TT-CMV remaining after the introduction of leukodepletion, it has been clearly shown that both prevalence and concentration of CMV DNA in peripheral blood are highest in newly seropositive donors. Therefore, avoidance of blood products from these donors is the most important goal of any transfusion strategy. This goal can be reached by: i) selection of blood products from seronegative donors, ii) provision of CMV DNA-negative blood products, or iii) provision of blood from long-term seropositive donors. In cases of suspected TT-CMV, all implicated donors should be investigated carefully to gather further knowledge on which donors confer the lowest risk for TT-CMV.

Window period donations during primary cytomegalovirus infection and risk of transfusion-transmitted infections

Donors with short interdonation intervals (e.g., apheresis donors) have an increased risk of window period donations. The frequency of cytomegalovirus (CMV) window period donations is important information to decide whether selection of seronegative donors might be advantageous for patients at risk for transfusion‐transmitted CMV infections (TT‐CMV).

Prognostic value and cost‐effectiveness of different screening strategies for HLA antibodies prior to kidney transplantation

HLA antibody screening is conducted routinely prior to kidney transplantation, but the comparative prognostic value and cost‐effectiveness of different methods are unclear. Pre‐transplant sera of 141 patients transplanted between 1998 and 2000 were screened by ELISA and Luminex assays, and antibody specificities of reactive sera determined using bead array techniques. ELISA screening detected donor‐specific antibodies (DSA) in 19 patients, who had a higher incidence of impaired graft function (60% vs. 20%, p = 0.04) and antibody‐mediated rejection (AMR) within 90 d after transplantation (AMR, 35% vs. 5%, p = 0.02). Luminex screening detected eight additional patients with DSA, among those one with AMR. Six of eight patients with Luminex‐only‐DSA reported no prior immunizing events. Death‐censored graft survival was shorter only in patients with DSA and AMR (median, 1.7 yr instead of between 9.5 and 11.0 yr for patients without DSA or patients with DSA but no AMR, p < 0.001). Material costs per detected clinically relevant DSA were about 57% higher for Luminex screening, but this increase could be avoided by modifying the cut‐off recommended by the manufacturer. Conclusively, specification of antibodies only in sera reactive in screening tests was cost‐effective to prevent shortened graft survival. Preformed DSA were only harmful if AMR was diagnosed within 90 d after transplantation.

Evaluation of algorithms for the diagnostic assessment and the reentry of blood donors who tested reactive for antibodies against hepatitis B core antigen

BACKGROUND: Screening of blood donations for antibodies against hepatitis B core antigen (anti‐HBc) is an accepted method to prevent some transfusion‐transmitted hepatitis B virus (HBV) infections. However, anti‐HBc testing may result in donor loss due to unspecific results in the currently available anti‐HBc tests. Algorithms to distinguish true‐positive from false‐positive results and for reentry of those donors who tested false anti‐HBc positive were evaluated retrospectively.

Coxiella burnetii - Pathogenic Agent of Q (Query) Fever

1.1.1 Structure C. burnetii is a member of the family of the Coxiellaceae bacteria and replicates intracellularly in cells of different species. Phylogenetically related bacteria include Legionellaceae, Francisellaceae, Pseudomonaceae, and other Gammaproteobacteria. Coxiella are small Gram-negative, pleomorphic, coccoid bacteria with a size of 0.2–1.0 m. They occur in 3 different forms: small cells (small cell variant, SCV) which are highly infectious, large cells (large cell variant, LCV) which develop also in cell culture, as well as spore-like particles (SLP) which are infectious and very robust to environmental conditions. Dependent on the host system, Coxiella undergoes a phase variation during growth [12]. In mammalian cells, bacteria grow as LCV, and form spore-like particles and 2 different antigenic forms described as Phase I and II.