Comparison of BACTEC™ blood culture media for the detection of fungemia.

The aim of the present study was to investigate whether addition of the BACTEC™ Mycosis bottle to the standard BACTEC™ aerobic and anaerobic bottles contributed to a higher detection rate and a faster time to detection (TTD) of fungi. This was a retrospective cohort study of all patients with a positive blood culture with Candida species delivered to the Department of Clinical Microbiology, Herlev and Gentofte Hospital, Denmark in the 8-year period 2006 through 2014. The patients had at least one BACTEC™ aerobic and one Mycosis bottle sampled at the same time and at least one of the bottles yielded growth of fungi. Among 184 patients included, 173 were examined using BACTEC™ aerobic, anaerobic and Mycosis bottles. The anaerobic vial generally had the lowest detection rate and the longest TTD. The detection rate of BACTEC™ aerobic plus anaerobic with the BACTEC™ Mycosis bottle was significantly higher than the detection rate of BACTEC™ aerobic plus anaerobic without BACTEC™ Mycosis bottle for all species after 1-5 days, and specially for Candida glabrata at 2, 3, 4 and 5 days. TTD for C. glabrata was significantly shorter for BACTEC™ Mycosis than TTD for BACTEC™ aerobic or anaerobic bottles after ½ to 4 days. When combining "first or only" detection, the BACTEC™ Mycosis bottle had a significantly higher detection as compared to the aerobic bottle. Addition of the BACTEC™ Mycosis bottle to the standard BACTEC™ aerobic and anaerobic bottles significantly contributed to a higher detection rate and a faster TTD of fungemia.

Semi-national surveillance of fungaemia in Denmark 2004-2006: increasing incidence of fungaemia and numbers of isolates with reduced azole susceptibility.

A semi-national laboratory-based surveillance programme for fungaemia was initiated in 2003 that now covers c. 3.5 million inhabitants (64%) of the Danish population. In total, 1089 episodes of fungaemia were recorded during 2004-2006, corresponding to an annual incidence of 10.4/100 000 inhabitants. The annual number of episodes increased by 17% during the study period. Candida spp. accounted for 98% of the fungal pathogens. Although Candida albicans remained predominant, the proportion of C. albicans decreased from 66.1% in 2004 to 53.8% in 2006 (p <0.01), and varied considerably among participating departments, e.g., from 51.1% at a university hospital in Copenhagen to 67.6% in North Jutland County. Candida glabrata ranked second, and increased in proportion from 16.7% to 22.7% (p 0.04). Candida krusei was isolated rarely (4.1%), but the proportion doubled during the study period from 3.2% to 6.4% (p 0.06). MIC distributions of amphotericin B and caspofungin were in close agreement with the patterns predicted by species identification; however, decreased susceptibility to voriconazole, defined as an MIC of >1 mg/L, was detected in one (2.5%) C. glabrata isolate in 2004 and in 12 (14.0%) isolates in 2006 (p 0.03). Overall, the proportion of isolates with decreased susceptibility to fluconazole exceeded 30% in 2006. The incidence of fungaemia in Denmark was three-fold higher than that reported from other Nordic countries and is increasing. Decreased susceptibility to fluconazole is frequent, and a new trend towards C. glabrata isolates with elevated voriconazole MICs was observed.

Comparison of Etest and a tablet diffusion test with the NCCLS broth microdilution method for fluconazole and amphotericin B susceptibility testing of Candida isolates.

Three methods were compared for the susceptibility testing of yeast isolates to fluconazole and amphotericin B: two fagar diffusion methods (Etest and a tablet diffusion test) and the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. Given as MIC(50)s (range), fluconazole endpoints were: for the 24 h broth microdilution test, 0.25 mg/L (0.06-32 mg/L); for the Etest, 0.38 mg/L (0.064-24 mg/L); and for the NCCLS broth microdilution test, 2 mg/L (0.06->or=64 mg/L). With breakpoints of <3 mg/L for susceptible and >16 mg/L for resistant, the Etest and the 24 h microdilution test classified the isolates in agreement with the classification obtained by the NCCLS method. Results obtained by Etest were in closer NCCLS method than those obtained with the tablet test. Amphotericin B endpoints were lower for the 24 h microdilution and Etests than MICs obtained by the NCCLS broth microdilution method. Reproducibility was high for all tests; however, disadvantages of both diffusion tests were microcolonies in the inhibition zone and dependence on stringent standardization of inoculum.

Parvovirus B19 infection infrequently involved in children and adults with myelodysplastic syndrome.

Parvovirus B19 infection has occasionally been reported to mimic myelodysplastic syndrome (MDS) or to cause worsening of anemia in MDS. We examined the presence of parvovirus DNA in a series of children (n=19) and adults (n=39) with a diagnosis of MDS. The series of adults included only refractory anemia (RA) and RA with ring sideroblasts (RARS). Investigation for parvovirus B19 DNA in bone marrow cells was performed employing the nested form of the polymerase chain reaction (PCR). Only a 51-year-old male with RA tested positive for parvovirus DNA. Serial examinations demonstrated the disappearance of parvovirus DNA from the bone marrow. We conclude that parvovirus infection may only rarely mimic MDS or be a superimposed infection in childhood MDS or in RA and RARS in adults.

Myelopoiesis in myelodysplasia evaluated by multiparameter flow cytometry.

Evaluating the proliferative activity of the immature erythro- and myelopoiesis as well as the mature myelopoiesis in 21 MDS patients and 14 healthy controls by simultaneously staining bone marrow cells for surface phenotype and DNA content, we found the percentages of proliferating S-phase cells in the early stage of MDS were higher. With disease progression evaluated by the FAB classification this parameter decreased significantly for both the immature myelo- and erythropoiesis. Evaluation of the proliferative activity of the mature myelopoiesis defined by the CD66 antigen revealed no difference between the normal controls and the MDS patients. Using another assay simultaneously labelling bone marrow cells for three leucocyte differentiation antigens during treatment with GM-CSF and low-dose AraC the cells clearly differentiated in one case. In another patient the disease seemed to progress as evaluated by cells only expressing immature antigens. The above mentioned immunophenotypic changes persisted at least one month after termination of treatment. In conclusion, the evaluation of proliferation and differentiation of leucocyte subsets using multiparameter flowcytometric assays in myelodysplastic patients from different FAB groups before as well as during treatment with haemopoietic growth factors may prove valuable in the future.

[Erythropoiesis in myelodysplastic syndrome detected by multiparameter flow cytometry]. / Erytropoiesen ved myelodysplastiske syndromer belyst ved multiparameter-flowcytometri.

By staining human bone marrow cells with a monoclonal antibody reacting with surface antigens on erythroid precursor cells (AS-E1) and with propidium iodide reacting with nuclear DNA, we have evaluated the proliferative activity of erythropoiesis in patients with myelodysplastic syndromes using flow cytometric analysis. Comparing 36 patients (13 RA/RAS, 13 RAEB, 10 RAEB-t) with seven normal controls, significant differences in both the percentage of erythroid precursor cells and the fraction of these cells in the S or S-G2M-phase of the DNA cell cycle between the four groups were found. Since neither the percentage of erythroid precursor cells nor their fraction in S or S-G2M phase alone was found to characterize their proliferative activity, we calculated the proliferative fractions of the erythroid cells, i.e. the number of the erythroid precursor cells in S or S-G2M related to all bone marrow cells in S or S-G2M phase. Applying these parameters, we found significantly increased proliferative fractions of erythroid precursor cells in the RA/RAS patients compared to the normal controls (p-0.03 and 0.002 respectively), as well as a highly significant decrease with disease progression.

The proliferative activity of myelopoiesis in myelodysplasia evaluated by multiparameter flow cytometry.

Double staining of bone marrow cells for CD13 and CD33 leucocyte differentiatian antigens and for DNA content has allowed us to evaluate the proliferative capacity of myelopoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. By analysing 39 patients (15 RA/RAS, 14 RAEB and 10 RAEB-t) and eight normal controls, we found significant differences in both the percentage of cells positive for these immature myeloid antigens between the FAB groups as well as in the fractions of CD13 and CD33 positive cells in S or S-G2M phase of the cell cycle. Moreover, a clear decrease in the immature myeloid cell proliferative activity upon progression within the FAB groups was evident. Finally, we found a significant negative association between the percentage of myeloblasts in the bone marrow and the proliferative activity of the immature myeloid cells, indicating that the block in differentiation in MDS patients might be coupled to a simultaneous block in proliferation, especially in advanced stages. These data suggest that the use of double parameter assays in the longitudinal follow-up of MDS patients might yield new information about the biology of MDS.

Differences in relative DNA content between human peripheral blood and bone marrow subpopulations--consequences for DNA index calculations.

By sequentially staining for leukocyte differentiation antigens with monoclonal antibodies and for stainable DNA content with propidium iodide, we have evaluated the DNA ratios of normal peripheral blood and bone marrow subpopulations. In 19 peripheral blood samples the relative DNA content of the monocytes was higher than that of other subpopulations, with highly significant differences between the DNA ratios of the CD14+ monocytes, on the one hand, unlabeled controls, CD3+ T-lymphocytes, and CD20+ B-lymphocytes, on the other. In bone marrows from 16 healthy volunteers, the relative DNA content of the myelomonocytic subpopulations was higher than that of the T-lymphocytes with significant differences between the CD3+ lymphocytes, on the one hand, and the CD14+ monocytes, the CD13+ and CD33+ immature myeloid, the NAT9+ mature myeloid, and the AS-E1+ nucleated erythroid subpopulation, on the other. Since a mixture of peripheral blood mononuclear cells or T lymphocytes from healthy volunteers is often used as an external standard for DNA ploidy determinations, these data suggest that such a procedure could result in an overestimation of the DNA indices of most of the bone marrow subpopulations. Instead, we suggest the use of the DNA ratio of the CD14+ subpopulation from peripheral blood as standards for ploidy determinations for the myeloid bone marrow subpopulations.

A quantitative evaluation of erythropoiesis in myelodysplastic syndromes using multiparameter flow cytometry.

By staining human bone marrow cells with a monoclonal antibody reacting with erythroid precursor cells (AS-E1) and propidium iodide, we have evaluated the proliferative capacity of erythropoiesis in patients with myelodysplastic syndromes (MDS) using flow cytometry. Comparing 36 patients (13 RA/RAS, 13 RAEB, 10 RAEB-t) with 7 normal controls, significant differences in both the percentage of AS-E1+ cells and the fraction of AS-E1+ cells in the S or S-G2M-phase between the four groups were found. Since neither the percentage of AS-E1+ cells nor their fraction in S or S-G2M alone was found to characterize their proliferative activity, we introduced the proliferative fractions of the erythroid cell, i.e. the number of the AS-E1+ cells in S or S-G2M related to all bone marrow cells in S or S-G2M. Applying these parameters, we found significantly increased proliferative AS-E1 fractions in the RA/RAS group compared to the normal controls (p = 0.03 and 0.002) respectively, as well as a highly significant decrease with disease progression.

Optimization of a method for simultaneous analysis of cell surface phenotype and cell cycle in human bone marrow and peripheral blood leukocytes by flow cytometry: the value of both internal and external standards.

Three different methods for the simultaneous analysis of surface phenotype and DNA quantification were compared. One method, involving the fixation of cells in 70% ethanol, was convincingly superior, both with regard to the CV of the G0G1 peak and the intensity of the DNA labelling. Furthermore, the correlation between the surface antigen densities before and after fixation were high. Experiments evaluating the intraday and the interday variation of the DNA ratio (the mean channel of the G0G1 peak of the sample divided by the mean channel of the G0G1 peak of chicken erythrocytes), documented the former to be small, with S.D. values varying from 0.0 to 0.016, while the latter were considerably higher with S.D. values varying from 0.077 to 0.123. Since the intraday variation of the DNA ratio was consistently low and the interday variation strongly correlated to the position of the red fluorescence test beads, it was possible to minimize the interday variation of the DNA ratio, by calculating the DNA index as the ratio between the DNA ratio of the sample and that of an external control (buffy coat leukocytes). Analyzing normal bone marrow and calculating the DNA index (DI) on the basis of these ratios, the confidence limits of the DI were decreased by more than half the values obtained when DI calculation was based solely on an internal standard, thereby making subsequent ploidy determinations of patient samples more precise. We conclude that this setup of internal and external standards allows accurate determinations of DNA aneuploidy even in an assay where whole cells labelled for surface antigen and DNA content are analyzed.

[Granulocyte-macrophage colony-stimulating factor. Treatment of patients with highly malignant lymphomas and acute lymphoblastic leukemias after autologous bone marrow transplantation]. / Granulocyt-makrofag-kolonistimulerende faktor. Behandling af patienter med højmaligne lymfomer og akut lymfoblastleukoemi efter autolog knoglemarvstransplantation.

We have evaluated the effect of intravenous infusions of granulocyte-macrophage colony-stimulating factor (GM-CSF) in ten patients undergoing autologous bone marrow transplantation after total body irradiation (11 Gy) and cyclophosphamide (120 mg/kg) conditioning for malignant lymphomas or acute lymphoblastic leukaemias. Mild side effects related to GM-CSF were seen in six patients, of whom one chose to discontinue treatment. Compared to a historic control group consisting of six patients treated with an identical conditioning regimen, a tendency was seen towards a more rapid neutrophil regeneration and a decreased need for erythrocyte transfusions. Moreover, a statistically significant improvement in thrombocyte related parameters was demonstrated. In contrast, there were no significant differences in severe toxicity, days on i.v. antibiotics, septic episodes or duration of hospitalization. In conclusion, it seems that GM-CSF treatment is of value in patients undergoing AKMT.

Hybrid acute leukemia: therapeutical implications of immunological phenotyping.

We phenotyped blood and bone marrow cells from a patient with acute Ph1+ acute leukemia longitudinally during the four months he received intensive chemotherapy. At presentation this case of biphenotypic acute leukemia had two immunologically different types of blast cells, one expressed CD10 (CALLA), CD13 (MY7) and CD33 (MY9) but lacked CD20 (B1), the other type expressed no CD10 or CD33. The phenotype, during AML induction therapy, changed to a more CD10+, CD20+ ALL one. ALL therapy based on these findings induced improvement in bone marrow function but the patient died of septicemia at day 134. The use of concomitant immunophenotyping (IP) and cell cycle analysis had shown proliferation advantage of the more lymphoid malignant cells. These results suggest that it is possible to induce lineage-associated changes in the phenotype of hybrid malignant cells and that these leukemias might be treated best according to longitudinal immunophenotyping of the blast cells.

Trisomy 13: a preferentially male chromosome aberration interfering specifically with myeloid proliferation and differentiation? Report of a case and review of the literature.

A case of trisomy 13 is presented: a 73-year-old man with acute nonlymphocytic leukemia (ANLL), FAB borderline M1/M2, and peripheral leukocyte and platelet counts that were difficult to control with chemotherapy. A literature review shows that 35 cases of trisomy 13 are known at present. They are characterized by male predominance (76%), preferentially myeloid disorders (ANLL, myelodysplastic syndromes, chronic myeloid leukemia), leucocytosis, and relatively high platelet counts and hemoglobins. It is suggested that trisomy 13 is a specific nosologic entity with male predominance and characterized by interference with proliferation and differentiation in the myeloid differentiation series.

Clinical and prognostic implications of chromosome 5q deletions: 96 high resolution studied patients.

Interstitial deletions of the long arm of chromosome 5 (5q-) is the most common structural rearrangement in acute nonlymphocytic leukemia and myelodysplastic syndromes. Owing to the variability of both breakpoints a large number of different deletions occur. However, as banding problems have hitherto precluded the precise localization of the breakpoints, it is still uncertain whether or not different deletions are associated with clinical differences. In this paper relationships between cytogenetic and clinical data were analyzed in 96 5q- cases investigated with high resolution techniques, which allow more precise breakpoint localizations. Twenty-nine cases have not been published before; the others were extracted from the literature. The analyses show: (1) With higher age at diagnosis the proximal breakpoint approaches the centromere. (2) The female patients are older, develop additional chromosome aberrations less often, and tend to live longer than the male patients. (3) del(5)(qI3q33), which is more frequent than any other deletion, tends to be associated with female sex and older age, and shows lower frequencies of additional chromosome abnormalities, and longer survival than the other deletions. It seems to constitute a more benign subgroup than the other deletions. (4) 5q31 is missing in 91 of 93 informative cases and is the most common deleted segment. (5) These results suggest that the deletion of 5q31 may be central in the pathogenesis of 5q- related disorders and that the deletion of other bands has important consequences for prognosis.

[Extramedullary manifestations among adult patients with acute lymphoblastic leukemia (ALL)]. / Ekstramedullaere manifestationer hos voksne patienter med akut lymfoblastleukaemi (ALL).

In 56 adult acute lymphoblastic leukemia (ALL) patients, 71% presented with extramedullary leukemic infiltration in lymphoid tissues (EML- spleen, liver, lymphnodes or thymus). EML was seen most often in patients with T-ALL (0.05 greater than p greater than 0.02) and in patients with high white blood counts (0.1 greater than p greater than 0.05). Surprisingly, these more often achieved complete remissions (0.1 greater than p greater than 0.05), of which both the duration and disease-free survival were longer. Thus, EML at diagnosis seems to be a favourable prognostic factor. At diagnosis, 23% of the patients had extramedullary leukemic infiltration in non-lymphoid tissue (EMIL-CNS, testis, skin, pleural cavities or gingiva). While more of these patients were of T-cell origin (61%, p less than 0.01), they were less likely to achieve CR, both the duration of remissions (p less than 0.01) as well as the disease free survival were shorter. Not unexpectedly, during the course of disease, the incidence of relapse localised at EMIL (the majority presenting in "sanctuary sites") increased, while that in the bone marrow decreased. Interestingly, in patients in CR presenting with EMIL, the first sign of relapse was unilateral peripheral facial paralysis in 60%. Finally, it should be stressed that the course of disease in patients presenting with simultaneous EML- and EMIL involvement was like that seen for EMIL patients. We conclude that while involvement of leukemia in EMIL represents a bad prognostic sign, the affection of leukemia in EML does not seem to confer a poorer prognosis, for adult ALL patients.

D-[3H]glucosamine labelling of epidermal and dermal glycosaminoglycans in cultured human skin.

An in vitro model is presented for the study of glycosaminoglycans in human skin. The synthesis of six glycosaminoglycan species in both dermis and epidermis was measured by D-[3H]glucosamine labelling. Punched biopsies (epidermis + entire dermis) of 3 mm in diameter were cultured at 37 degrees C in 5% carbon dioxide-95% air. When the label was added 18 h after explantation, the incorporation started immediately, and for all glycosaminoglycans the time-dependent incorporation was linear for 16 h. The experimental variation was minimized by expressing the measurements in epidermis "per explant" and in dermis "per mg of wet explant". A ratio to dermal hydroxyproline did not improve the precision. Most of the variation arose "before" isolation and separation of the glycosaminoglycans. The labelled products were macromolecules and were converted to small molecules by chondroitinase ABC + heparinase. The total incorporation in dermis was 2 1/2 times higher than in epidermis. Hyaluronic acid was the predominant synthesized product in dermis, and hyaluronic acid and heparan sulphate were the predominant products in epidermis. The proportions (%) in dermis/epidermis were as follows: hyaluronic acid, 61/44; heparan sulphate, 18/31; dermatan sulphate, 5/8; chondroitin 4/6-sulphate, 10/7 and heparin-like glycosaminoglycan, 1/2. The same species were also demonstrated as native constituents of uncultured human skin. Hyaluronic acid and dermatan sulphate predominated in dermis, whereas no single species predominated in epidermis. Their concentrations in uronic acid equivalents per mg of wet skin (pmol/mg of epidermis + dermis) were as follows in dermis/epidermis: hyaluronic acid, 243/0.48; heparan sulphate, 22/0.44; dermatan sulphate, 170/0.56; chondroitin 4/6-sulphate, 72/0.50; and heparin-like glycosaminoglycan, 5/0.22. Thus, only 0.4% of the in vivo synthesized glycosaminoglycan was present in epidermis.