Gregor Bein

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.

A longitudinal prospective study of cytomegalovirus pp65 antigenemia in renal transplant recipients

Cytomegalovirus (CMV)-encoded pp65 antigen in peripheral blood leukocytes (CMV antigenemia) was investigated in 1017 serial samples from 64 patients for 16 weeks after renal transplantation in a prospective study. In 110 samples from 24 patients, at least one antigen-positive leukocyte was identified. The median number of stained cells was 4 (range 1–1000) per 4×105 leukocytes. Twenty-one of 24 patients with serological signs of an active CMV infection were antigen-positive (sensitivity 87.5%), whereas 3 patients with antigenemia did not show serological signs of infection during the observation period (specificity 92.5%). Positive results were obtained 19 days (median) before serological response and 9 days (median) before the onset of CMV syndrome. The sensitivity in defining a CMV syndrome was 100% (n=8). In all patients who presented with CMV syndrome, antigenemia was present prior to the onset of symptoms or on the same day. In contrast, serological monitoring rendered the diagnosis of CMV infection possible at the onset of clinical symptoms in only two of eight patients. We conclude that (1) insufficient results obtained with the CMV antigenemia assay by other investigators are mainly due to technical problems that can easily be overcome by the protocol presented and (2) the detection of CMV pp65 antigen in peripheral blood leukocytes is an excellent tool for rapid and early diagnosis of CMV infection.

IgM-specific serodiagnosis of acute human cytomegalovirus infection using recombinant autologous fusion proteins

Portions of three human cytomegalovirus (HCMV) polypeptides, which were shown previously to be highly reactive with patient sera, were expressed in Escherichia coli as autologous fusion proteins. Purified recombinant polypeptides were used as antigens in enzyme linked immunosorbent assay (ELISA) and compared against assays which use natural viral antigen from cell culture for their ability to improve IgM-specific serology of acute HCMV-infection. A fusion protein (CM2) which contained two copies of the C-terminal portion of pUL44 (p52, aa 297-433) and one copy of a highly reactive fragment of the major DNA-binding protein pUL57 (aa 545-601) proved to be superior in sensitivity and specificity compared to assays which use culture derived antigen. A construct expressing one copy of the fragments from pUL44 and pUL57 in fusion with the 54 amino terminal residues of pUL32 (pp150, aa 994-1048) did not lead to an improved sensitivity compared to CM2. Adversely, this polypeptide reacted with a number of sera from asymptomatic blood donors infected latently with HCMV indicating low specificity of this antigen for the detection of acute infection. Concordant results were obtained with an antigen that combined only the C-terminal portions of pUL44 and pUL32 (CM3). ELISA experiments with sequential sera from renal transplant recipients demonstrated that detection of IgM-antibodies using CM2 as antigen correlated closely with acute infection, whereas high levels of IgM-antibodies against CM1 and CM3 persisted for a month following acute HCMV-infection. These results indicate that the application of a single autologous fusion protein like CM2 as antigen for recombinant ELISAs can improve significantly IgM-serodiagnosis of acute HCMV infection.

Semiautomated HLA-DQB1 typing by fluorescent dye photometry of amplified DNA on microtiter plates

We report a simple semiautomated HLA class II typing method that suits the demands of a 24-hour-duty transplantation service for preorgan retrieval donor typing. The procedure consists of sequence-specific amplification of HLA alleles by the polymerase chain reaction (nested PCR-SSP) followed by fluorescent dye photometry of the resulting PCR products on microtiter plates. The entire typing procedure is semiautomated and completed in less than 90 minutes after DNA isolation. The test was evaluated for the definition of the specificities DQ1-DQ9 (generic HLA-DQB1 typing). Sensitivity and specificity as judged by DNA reference typing in a different laboratory was 99.5% (n = 202 alleles).

The intracellular localization of human cytomegalovirus DNA in peripheral blood leukocytes during active infections by high-resolution fluorescence in situ hybridization

SummaryAlthough viremia is an integral part of the pathogenesis of human cytomegalovirus (HCMV) disease, the interaction between HCMV and circulating leukocytes of actively infected patients remains an area of uncertainty. It is still a matter of dispute, whether leukocytes support viral replication with subsequent production of infectious virus. In a new approach we developed and applied a sensitive fluorescence in situ hybridization assay for the precise intracellular localization of HCMV genomes in leukocytes. It was shown that in vivo HCMV genomes were exclusively localized in the cytoplasm of leukocytes, indicating that the majority of these cells are virus carriers or abortively infected. Though this method easily detects single copy genes in metaphase chromosomes, the number of HCMV DNA positive leukocytes was significantly lower than the number of HCMV pp65 antigen positive cells. In relation to the pp65 antigen positive cells, only 1–4% of these cells were DNA positive. In addition, the much lower frequency of HCMV immediate early antigen positive leukocytes in comparison to the pp65 antigen positive cells and the impossibility of detecting other viral antigens support the hypothesis that the origin of pp65 found in leukocytes results mainly from protein uptake.

HLA DNA Preorgan Retrieval Donor Typing in Renal Transplantation

For the first time, a DNA technique was used in clinical practice for preorgan donor retrieval typing in renal transplantation. The method is based on the polymerase chain reaction with nested sequence‐specific primer pairs (nested PCR‐SSP) for all serological HLA‐DR specificities of DR1‐DR18. A panel of reference‐typed individuals (n = 101) was investigated in a blind quality control study and the results revealed a sensitivity of 99.5% and a specificity of 100%. Semi‐automation of PCR‐SSP provided rapid, accurate and reliable typing results demonstrating that this DNA test is most suitable for replacing the error‐prone HLA‐DR serological technique. Twenty‐seven consecutive cadaveric organ donors have been successfully typed so far. The results were available within less than 3 h after blood sampling and in all cases prior to the retrieval of organs. In conclusion, the introduction of this accurate DNA typing in HLA matching programs may significantly improve the graft survival rate in renal transplantation.

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes.

Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level.

β2-Microglobulinuria as an early sign of cytomegalovirus infection following renal transplantation

The frequency of cytomegalovirus infection was studied in a prospective study of 106 kidney recipients. The detection of cytomegalovirus-immediate-early-antigen and cytomegalovirus-immunoglobulin (IgM) antibodies in serum was used as the reference method and showed that 23.6% (25/106) of all patients were infected. In addition, four urinary proteins (IgG and transferrin as glomerular markers and α1-microglobulin and β2-microglobulin as tubular markers) were quantitatively measured in 24-h urine samples from all of the patients using an immunoluminometric assay (ILMA). In all cytomegalovirus infection cases a pronounced but isolated increase of urinary β2-microglobulin excretion was observed. In 20 of 25 infected patients, the p2-microglo-bulinuria occurred 1–21 days (median 5.0) earlier than the appearance of the cytomegalovirus-immediate-early-antigen in blood. Thus, it can be seen that the quantitative measurement of β2-microglobulin in urine is useful for the early detection of cytomegalovirus infection following renal transplantation.