Emerging resistant bacteria strains in bloodstream infections of acute leukaemia patients: results of a prospective study by the Rete Ematologica Lombarda (Rel).

Multiresistant bacterial infections are a potentially life-threatening condition in acute leukaemia (AL) patients. We aimed to better define the very recent epidemiology and outcome of bloodstream infections (BSIs) in a real-life setting. We prospectively collected all consecutive febrile/infectious episodes occurring in AL patients admitted to 9 haematology units. In 293 AL patients, 433 BSIs were diagnosed. Gram-positive (GP) bacteria were isolated in 44.8 % BSI and Gram-negative (GN) in 38.3 %, while polymicrobial aetiology- or fungi-related events were identified in 15.7 and 1.1 % of the cases, respectively. GP was observed more frequently in patients not in complete remission (p = 0.04), while GN during consolidation cycles (p = 0.003). Extended spectrum ß-lactamase-producing strains accounted for 23.2 % of enterobacteria. They were associated with previous antibiotic exposure, including fluoroquinolones prophylaxis (p = 0.01). Carbapenem-resistant (CR) strains occurred in 9 % of enterobacteria. Among Pseudomonas aeruginosa strains, 21.6 % were multiresistant. Overall 30-day mortality was 8.5 %. CR GN and multiresistant P. aeruginosa BSIs were independent predictors of death (p = 0.002), as well as relapsed/resistant AL (18.3 %; p = 0.0002) and the presence of pulmonary infiltrates (26.6 %; p < 0.001). Although GP still predominate over GN BSI, the percentage of antibiotic resistant GN strains is considerable in AL patients and it is associated with poor prognosis.

Development of simple equations for effective screening of spurious hemolysis in whole-blood specimens.

INTRODUCTION: We aimed to identify simple but reliable indices for effective screening of spurious hemolysis in whole-blood specimens. METHODS: Thirteen inpatient whole-blood samples were divided in two aliquots. The former was left untreated, whereas the latter was mechanically hemolyzed by forced aspiration with an insulin syringe. All aliquots were tested on Siemens Advia 2120 and Sysmex XE-2100. The hemolysis index (HI) was also assessed in centrifuged plasma. RESULTS: The mechanical hemolysis generated a 4-40% decrease in red blood cells (RBCs). A statistically significant decrease was observed for hematocrit (Ht) and mean corpuscular volume (MCV), whereas mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelet count were increased. The values of hemoglobin (Hb) and white blood cells remained substantially unchanged. Two specific equations ([Ht/Hb] × âˆšMCV and [Ht/Hb] × 100) were developed. Both equations displayed an area under the curve of ≥0.99 for identifying spurious hemolysis, much greater than that of both RBC ghosts and immature platelet fraction. A highly significant correlation was also observed between results of these equations and percentage reduction in RBCs or HI increase. CONCLUSION: Provided that these results will be confirmed in further studies, these equations may provide a reliable means for screening spurious hemolysis in whole-blood samples.

Early molecular diagnosis of aspergillosis in a patient with acute myeloid leukaemia.

Diagnosis of invasive fungal infection remains challenging. Here we report a case of early diagnosis of invasive aspergillosis in a neutropenic patient affected by acute myeloid leukaemia, achieved through the detection of Aspergillus fumigatus species-specific ribonucleic acid sequences by a sensitive multiplex real-time polymerase chain reaction-based molecular assay. Thanks to the early diagnosis, targeted therapy was promptly established and the severe fungal infection controlled, allowing the patient to subsequently receive allogeneic hematopoietic stem cell transplantation from a haploidentical donor, her only curative option. Also in this instance, targeted secondary antifungal prophylaxis with voriconazole avoided any other fungal infection afterwards. This report suggests how the implementation of molecular assays in combination with routine diagnostic procedures, can improve microbiological diagnosis in sepsis, particularly in case of fungal infection, difficult to detect with standard microbiological culture methods.

Influence of in vitro hemolysis on nucleated red blood cells and reticulocyte counts.

INTRODUCTION: Nucleated red blood cells (NRBCs) and reticulocytes are early and important measures of red blood cells' (RBCs) turnover, but little is known on how spurious hemolysis may affect the reliability of these parameters. MATERIALS AND METHODS: Ten EDTA-anticoagulated samples were divided into three aliquots. The first was immediately tested, where-as the others (defined A and B) were mechanically hemolyzed by aspiration 5 and 10 times through a small-gauge needle. RBC, NRBC, and reticulocyte counts were performed on Sysmex XE-2100. RESULTS: An increasing amount of hemolysis was produced in hemolyzed aliquots A and B. The RBC and reticulocyte counts progressively decreased from the nonhemolyzed sample to hemolyzed aliquots 'A' and 'B'. The NRBC count increased in 3 of the 10 samples and decreased in the remaining seven. CONCLUSIONS: Hemolysis of venous blood samples may seriously jeopardize NRBC and reticulocyte counts.

Evaluation of the TiMo12Zr6Fe2 alloy for orthopaedic implants: in vitro biocompatibility study by using primary human fibroblasts and osteoblasts.

To reveal the biocompatibility of TiMo12Zr6Fe2 (TMZF), a new titanium alloy used since 1998 for orthopaedic prosthesis, we compared the behavior of primary human fibroblasts and osteoblasts grown on TMZF discs or on plastic tissue culture dishes, a widely used material specifically treated by the manufacturer to enhance cell growth. Proliferation, differentiation. RNA and collagen type I expression level of human cells were carried out. The analysis were performed over a period of 96 h. Fibroblasts behaved at the same way on the two different supports after 48 h, their number increased after 96 h when cells were grown on the alloy. Osteoblasts adhered and proliferated on the alloy discs as well as on plastic. RNA expression level was not affected. Interestingly, cell number at each time point was higher for fibroblasts than for osteoblasts. The RNA expression level was higher for the osteoblasts. Both cell types cultured on the alloy revealed an increase in the amount of type I collagen and a similar electrophoretic pattern was found for collagen produced by fibroblasts and osteoblasts grown on either supports. These results indicate good biocompatibility of the TMZF alloy, which allowed adhesion and proliferation of both the examined cell types and suggest that TMZF is a promising material for orthopaedic implants.

Neer capsular shift surgery in the treatment of recurrent antero-inferior shoulder dislocations.

A total of 47 patients affected with recurrent multidirectional shoulder dislocation with a prevalent anteroinferior component were submitted to Neer inferior capsular shift surgery. Bankart lesion, present in 24 patients, was repaired prior to performing capsuloligamentous surgery. The patients included in the study presented with generalized ligamentous hyperlaxity in 20 cases, while hyperlaxity was localized in the pathological shoulder in the remaining cases, presumably related to the high number of dislocations. The choice of performing Neer inferior capsular shift surgery was initially based on the data and the modern conceptions that emerged in the literature. Successively, the good results obtained encouraged us to follow the path taken, and we are currently encouraged to advise this surgical procedure for the category of patients mentioned above.

Circulating CA 549 and other associated antigens in breast cancer patients.

The new marker CA 549 was determined in the serum of 258 breast cancer patients, classified according to TNM (148 at diagnosis and 110 at relapse), using a RIA method (cut-off: 10 U/ml). CEA, CA 15-3 and MCA were also evaluated. At diagnosis, CA 549 was more sensitive than the other markers, and cut-off values of 11 and 12 U/ml did not significantly reduce sensitivity. No significant correlation existed between the markers, except for CA 15-3 and CA 549 (r = 0.65). A new quantitative approach to the four markers was effected in the relapsed patients: an X value was calculated for each marker by dividing serum concentration by its cut-off. In these patients, grouped according to the area involved, marker sensitivities were similar except in locoregional relapse, where CA 549 and MCA were the most sensitive. From the data obtained, the more defined cut-off and the good specificity, it is suggested that CA 549 be routinely determined in the follow-up of the disease.

Tumour associated antigens CA 15.3 and CA 125 in ovarian cancer.

CA 125 and CA 15.3 antigens were determined by enzyme immunoassay in 78 patients with ovarian cancer for a total of 540 determinations. The antigens were also investigated in sera from 100 women with other gynaecological diseases, 82 lung cancer patients and in 39 pleural fluids of varying origin. CA 15.3 reference values were evaluated in 91 healthy women (cut-off: 25 U/ml). CA 15.3 sensitivity at diagnosis (60%) and for detecting relapse (44%) was lower than that of CA 125 (90% and 64.7%, respectively). However, CA 15.3 does not increase with aspecific mesothelial cell reaction and thus it is more specific than CA 125. Combined use of the markers during follow-up improves early detection of relapse (at least one of the two was positive in 79% of cases). Therefore both CA 15.3 and CA 125 should be routinely determined for the detection and monitoring of ovarian cancer.

Long-term effects of parenteral dichloromethylene bisphosphonate (CL2MBP) on bone disease of myeloma patients treated with chemotherapy.

Data on the long-term treatment of myeloma bone disease with bisphosphonates are scanty. In a prospective pilot trial we evaluated the effect of long-term parenteral administration of dichloromethylene bisphosphonate (Clodronate), in addition to standard chemotherapy, in 30 patients with active myeloma bone disease. Patients were treated with a mean of 4 courses (range 2-8) of Clodronate: 300 mg/day i.v. for seven days followed by 100 mg/day i.m. for 10 days, administered at a mean interval of 4 months (range 3-6). The median follow-up was 24 months (range 8-36). Clodronate reduced bone pain rapidly and significantly, and reduced the mean values of the biochemical indices of bone resorption to within normal limits; these effects were maintained throughout the follow-up. In three hypercalcemic episodes serum calcium became normal after 2-5 days of treatment with Clodronate. No toxic or side effects were noticed. The occurrence of skeletal morbidity in patients treated with Clodronate was compared with that observed in the control group of myeloma patients (p less than 0.001) in severe bone pain as well as in the incidence of new osteolytic lesions and pathological fractures (p less than 0.001). Supportive Clodronate therapy contributes significantly in controlling the progression of myeloma bone disease.

Efficiency of composite laboratory tests in the diagnosis of liver malignancies.

The utility of the markers CEA, beta-HCG, CA-50, alpha-fetoprotein (APF), ferritin, alkaline phosphatase (AP), its isoenzyme liver-1 (APL1), gamma-glutamyltransferase (gGT), its fast migrating isoenzyme (gGT1) and 5'nucleotidase (5'N) in differentiating liver malignancies and benign involvement was evaluated in the sera of 85 patients with hepatocellular carcinoma (HCC), 157 with chronic liver disease (CLD) and 91 with liver metastases (LM) derived from different tumors. The mean concentrations of all the parameters except CEA and GGT1 were significantly different in HCC and CLD, but a broad overlap existed in the two groups, so different cut-offs were considered to assess the positive and negative predictive values and test efficiency (Eff). The best results were observed considering AFP greater than 100 IU/m (Eff0.86), ferritin greater than 800 ng/ml (Eff0.69), CA-50 greater than 100 U/ml (Eff 0.63), beta-HCG greater than 10 mU/ml (Eff 0.61), AP greater than 300 IU/ml (Eff 0.66), the presence of APL1 (Eff 0.78), 5'N greater than 25 mU/ml (Eff 0.70), gGT greater than 100 mIU/ml (Eff 0.63). Among HCC patients 17% did not secrete AFP; in 26% the protein was less than 100 IU/ml and in 36% less than 400 IU/ml. Apart from AFP the most effective marker was APL1. At the above cut-offs more than three parameters were simultaneously positive in 71% of HCC and 9.9% of CLD. CEA, CA50, AFP were the only parameters that distinguished the HCC from the LM group; in the latter, APL1 was also a very sensitive marker (87%) for neoplastic involvement of the liver.

Detection of malignant pleural effusions by tumor marker evaluation.

Cytologic examination and determination of tumor markers (PHI, LDH, alpha-1-glycoprotein, alpha-2-HS-glycoprotein, beta 2-microglobulin, ferritin [corrected], sialic acid, IgE, fetoprotein, CEA, beta HCG and beta 1-SP-glycoprotein) were carried out in pleural fluid samples obtained from 70 patients with suspected neoplasia. Tumor markers were also determined in sera. The protein content of all pleural effusions was greater than or equal to 3 g/dl. Patients were grouped according to diagnosis as follows: (a) 42 with neoplastic diseases (7 mesotheliomas and 19 lung, 4 ovarian, 3 breast and 8 miscellaneous cancers), (b) 22 with benign inflammations and (c) 6 with congestive effusions. Of the parameters examined, only CEA and beta-HCG [corrected] gave information that the effusion was probably malignant. Using 6 ng/ml as cut-off for CEA and 10 mIU/ml for beta HCG, the sensitivity was 57.1% and 45.2%, respectively, specificity was 92.8% for both parameters and test efficiency 0.75 and 0.69, respectively. When CEA and beta HCG were considered together sensitivity increased to 73.8% and efficiency to 0.78. CEA and/or beta HCG were positive in the pleural effusions of 19 of the 20 malignant pleural effusions, all with a negative cytologic examination, which subsequently became positive in 8. Because of their high specificity, these two parameters are a useful tool and can be routinely measured to evaluate pleural effusions of dubious origin, even if CEA and beta HCG cannot, on [corrected] their own, define the primary malignancy.

Beta-2-microglobulin as a tumor marker in solid malignancies.

Beta-2-microglobulin (beta 2-MG) and CEA were measured in the sera of 186 cancer patients divided into two groups: at diagnosis (D) and at follow-up (F). Four groups of patients at diagnosis (D-I, D-II, D-III and D-IV according to TNM classification) and two at follow-up (in remission, F-RS, and in relapse, F-RP) were considered. All patients had normal serum creatinine and urea concentrations. beta 2-MG values in D-I were significantly (p less than 0.01) lower than those for D-II and D-III, while in D-IV they overlapped those of group D-I. No significant difference was observed between F-RS and F-RP patients. Patients with serum CEA concentration greater than 100 ng/ml revealed beta 2-MG values close to those of groups D-I and D-IV. In 10% of patients in stage IV or with CEA greater than 100 ng/ml beta 2-MG was lower than the mean value of the healthy population. From data beta 2-MG is probably produced by an aspecific reaction to the tumor and the decrease in advanced stages could express a decreased immunologic response. On the other hand, high serum beta 2-MG in the initial stages of the neoplasia may reflect an elevated cell turnover, while low beta 2-MG during the final stages may be due to a weak expression of the protein by highly undifferentiated cells.

Variations in alpha-2-HS glycoprotein level in neoplastic disease with and without involvement of bone.

Variations in the serum level of alpha-HS-glycoprotein may be observed in all pathological conditions which induce changes in bone turn-over, as well as in inflammatory and neoplastic diseases. This study includes 162 patients divided into four groups according to the TNM classification (tumour, lymph node metastasis). The first consisted of patients with neoplasms at TNM stages 1 and 2 with no bone metastases; the second of similar patients at TNM stages 3 and 4. The third group were patients with primary or secondary neoplasms of bone, and the fourth were patients with viral or bacterial diseases. The levels of alpha-2-HS-glycoprotein serum were determined for all the groups and these were compared with AAG (alpha-1-glycoprotein) and CEA (carcinoembryonic antigen). There were no significant differences in the levels of alpha-2-HS-glycoprotein for the first group as compared with normal controls, while in the other groups the differences were significant. The levels of alpha-2-HS-glycoprotein were diminished when the levels of CEA and AAG were both high, but increased when only one of these other parameters was high.

Selective bone marrow involvement of lymphoplasmacytic cells secreting monoclonal IgA rheumatoid factor in a patient with Sjögren's syndrome and serum hyperviscosity.

The clinical features and results of serological studies of a patient with Sjögren's syndrome, IgA kappa monoclonal gammopathy, and hyperviscosity syndrome are reported. The novel aspect of this case is the selective localisation to the bone marrow of lymphoplasmacytoid cells secreting IgA kappa morphologically identical to the cells infiltrating the salivary glands. The serum of the patient contained large amounts of immunoglobulin-anti-immunoglobulin immune complexes. By gel filtration chromatography it was shown that the immune complexes formed a peak of molecular weight 680 kilodaltons. The immune complexes were dissociable under acidic conditions. The immunoglobulin with rheumatoid activity was characterised as monoclonal IgA kappa protein. Treatment with plasmapheresis combined with immunosuppressive treatment with cyclophosphamide reduced the serum viscosity with concomitant clinical improvement.

Effects of a new aminodiphosphonate (aminohydroxybutylidene diphosphonate) in patients with osteolytic lesions from metastases and myelomatosis. Comparison with dichloromethylene diphosphonate.

We have compared in an open trial the clinical and biochemical effects of a new aminodiphosphonate, aminohydroxybutylidene diphosphonate, with those of dichloromethylene diphosphonate, which has been proved effective. The patients presented extensive and symptomatic bone involvement from multiple myeloma, breast cancer, and other metastatic tumors. The treatment consisted of aminohydroxybutylidene diphosphonate, 2.5 mg/d intravenously for five days, or dichloromethylene diphosphonate, 300 mg/d intravenously for seven days, followed by 100 mg/d intramuscularly for ten days. Twelve patients treated with aminohydroxybutylidene diphosphonate and 16 patients treated with dichloromethylene diphosphonate were assessable and were followed up for one to six months. Therapy with aminohydroxybutylidene diphosphonate showed a quicker action in reducing bone pain and reduced significantly more the serum calcium level than did therapy with dichloromethylene diphosphonate. Aminohydroxybutylidene diphosphonate therapy also affected urinary calcium levels and hydroxyproline excretion more markedly than did dichloromethylene diphosphonate, although the differences are not statistically significant. However, the biochemical indexes rebounded more quickly in patients treated with aminohydroxybutylidene diphosphonate, indicating that the loading amount (only 12.5 mg) used in this preliminary study is insufficient to sustain a prolonged effect. The effectiveness and lack of side effects render aminohydroxybutylidene diphosphonate an attractive treatment for malignant bone resorption.

A sulfhydryl-rich IgM protein with multiple serological specificities.

A monoclonal IgM lambda protein from a patient (E.T.) suffering from a lymphocytic lymphoma agglutinated Salmonella typhi bacteria and uncoated acryl particles. The antigenic determinant on Salmonella typhi bacteria was found to be 0-12 (alpha-D-Galp-(1-2)-alpha-D-Manp) while the structure on acryl particles recognized by IgM ET has not been defined. Both binding sites for bacteria and acryl particle determinants are localized on the same IgM molecule. The uncommon affinity of this IgM protein for some divalent heavy metal ions led to the finding of an unusually high content of sulfhydryl groups in the Fab portion of the molecule.