Summary The NB1 glycoprotein (CD177, HNA‐2a antigen) is exclusively expressed on human neutrophils. As the clinical significance of CD177 expression is unknown, we investigated its expression in healthy individuals before and after stimulation with granulocyte colony‐stimulating factor (G‐CSF), in patients with rheumatoid arthritis, viral hepatitis, severe bacterial infections and polycythaemia vera. Expression was quantitatively determined by flow cytometry and by real time polymerase chain reaction. Only G‐CSF‐stimulated individuals and patients with severe bacterial infections and polycythaemia showed a significantly (P < 0·001) increased CD177 expression compared with healthy individuals, indicating that neutrophil CD177 expression can increase significantly in certain clinical conditions.

In pediatric and adult patients after stem cell transplantation (SCT) disseminated infections caused by human cytomegalovirus (HCMV) can cause life threatening diseases. For treatment, the three antivirals ganciclovir (GCV), foscarnet (PFA) and cidofovir (CDV) are approved and most frequently used. Resistance to all of these antiviral drugs may induce a severe problem in this patient cohort. Responsible for resistance phenomena are mutations in the HCMV phosphotransferase-gene (UL97) and the polymerase-gene (UL54). Most frequently mutations in the UL97-gene are associated with resistance to GCV. Resistance against all three drugs is associated to mutations in the UL54-gene. Monitoring of drug resistance by genotyping is mostly done by PCR-based Sanger sequencing. For phenotyping with cell culture the isolation of HCMV is a prerequisite. The development of multidrug resistance with mutation in both genes is rare, but it is often associated with a fatal outcome. The manifestation of multidrug resistance is mostly associated with combined UL97/UL54-mutations. Normally, mutations in the UL97 gene occur initially followed by UL54 mutation after therapy switch. The appearance of UL54-mutation alone without any detection of UL97-mutation is rare. Interestingly, in a number of patients the UL97 mutation could be detected in specific compartments exclusively and not in blood.

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Ganciclovir (GCV) resistance is an emerging problem for transplant recipients. A sensitive and rapid real-time PCR approach for simultaneous and semiquantitative detection of human cytomegalovirus (HCMV) UL97 mutations in codons 460/520 was established by LightCycler and confirmed by restriction fragment length polymorphism and sequencing. Results from HCMV laboratory strains were compared with results from 11 GCV-resistant clinical isolates.Mutations in the human cytomegalovirus (HCMV) phosphotransferase gene (UL97) and polymerase gene (UL54) are responsible for resistance to ganciclovir (GCV) (UL97/UL54), cidofovir (UL54), or foscarnet (UL54) (2). Over 90% of GCVresistant clinical HCMV isolates have mutations in the UL97 gene (10). Most of the GCV-resistant clinical HCMV isolates feature mutations in the UL97 gene between codons 400 and 665 (9). Some of the most frequent mutations occur in codons 460, 594, and 595 (4, 7). There is a high impact for genotypic resistance screening with respect to known UL97 mutations conferring GCV resistance (4). The ratio of the wild-type and mutant strains present in HCMV-infected allogeneic hematopoietic stem cell transplant recipients may reflect clinical outcome (5, 11). Up to now, genotypic resistance screening has been performed by PCR-based restriction fragment length polymorphism (RFLP) assay and sequencing. Combinations of RFLP assays and sequencing analysis need at least 3 to 4 days for completion. RFLP assays for the detection of mutations in codons 460 and 520 have been described previously (3, 12). The relative frequencies of the UL97 mutations M460V, M460I, and H520Q were given previously, with the mutations found in 20 out of 75 (27%) unrelated ganciclovir-resistant isolates (4).We present a new LightCycler (LC) PCR assay using specific hybridization probes with melting point analysis for simultaneous characterization of the relevant UL97 codons 460 and 520 by a dual-color format with two specific pairs of hybridization probes for both codons which are each labeled with different fluorescence dyes (LC Red dye 640 and LC Red dye 705). The binding of the hybridization probe leads to a specific melting point for the wild-type sequence. In mutant strains, however, the mismatch between the hybridization probe and the mutant target sequence results in unstable binding of the probes, decreasing the melting temperature. The evaluation for the different codons can be performed by the use of different fluorescence channels. The reaction mixture utilized a master mix containing a LightCycler Fast Start hybridization probe kit (Roche Diagnostics, Mannheim, Germany) 1ϫ, 4.5 mM MgCl 2 , 0.6 M of each primer, 0.2 M of each hybridization probe, and an additional 2.5 U/reaction Fast Start Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) for increase of the sensitivity of the amplification reaction. An enhanced concentration of Fast Start Taq DNA polymerase (5 U/l) in the reaction mixture resulted in a higher sensitivity of our assay (8). The following program was used f...