Ranka Serventi-Seiwerth

LightCycler SeptiFast assay as a tool for the rapid diagnosis of sepsis in patients during antimicrobial therapy.

Rapid aetiological diagnosis and early administration of adequate antimicrobial therapy soon after a patient’s arrival at the hospital are crucial for the successful management of sepsis and septic shock (Dombrovskiy et al., 2007; Raghavan & Marik, 2006). Routine aetiological diagnosis of sepsis relies on standard culture methods that take at least 24 h or more to give the initial information. Therefore, quick identification of pathogens causing sepsis within the clinically relevant time frame should be based on state-of-the-art molecular methods that target bacterial and fungal DNA (Abdul-Redha et al., 2007; Breitkopf et al., 2005; Dombrovskiy et al., 2007). In this letter, we describe the clinical utility of the first standardized, CE-certified, multiplex real-time PCR assay for the molecular diagnosis of sepsis that has been approved for in vitro diagnostic use (LightCycler SeptiFast assay; Roche Diagnostics). The aim of our preliminary study was to determine the additional diagnostic value of the LightCycler SeptiFast assay, which enables detection of DNA from 25 human pathogens (Gram-positive and Gram-negative bacteria as well as fungi) in the blood of patients with suspected sepsis even after empirical antimicrobial therapy has been started. The study enrolled 36 patients (a total of 39 samples) with a clinical diagnosis of sepsis that were treated at the University Hospital for Infectious Diseases, Zagreb, and Zagreb University Clinical Center in Croatia. Seventeen patients were hospitalized in the intensive care unit (ICU), nine patients outside the ICU and ten patients in the Department of Haematology following bone marrow or peripheral blood stem cell transplantation (BSCT). All patients enrolled in the study were already receiving empirical antimicrobial therapy at the time of testing. Blood cultures were taken from all patients as well. Thirteen out of 39 (33 %) samples tested positive with the SeptiFast assay for bacterial or fungal DNA (Table 1). Gramnegative bacterial DNA was detected in 11 of 13 samples (Klebsiella pneumoniae/oxytoca n54; Escherichia coli n53; Pseudomonas aeruginosa n54). Gram-positive bacterial DNA (Enterococcus faecium n51; Streptococcus pneumoniae n51) was detected in two patients with polymicrobial sepsis (in combination with K. pneumoniae/ oxytoca in both patients). Aspergillus fumigatus DNA was detected in two patients with fever and pulmonary infiltrates. Additional SeptiFast-positive results (blood culture-negative) were obtained in nine of 36 patients. Four of 39 samples (10.3 %) were negative by the SeptiFast assay but positive by culture and those results were interpreted as false-negatives. Four of 39 samples were positive by both the SeptiFast assay and culture. Twenty-two samples tested negative by both techniques. We observed one discordant result between the SeptiFast assay and culture (Enterobacter cloacae was detected by culture and K. pneumoniae/oxytoca by SeptiFast assay). Incorrect interpretation of Enterobacter aerogenes/cloacae as K. pneumoniae/oxytoca due to the similarities of internal transcribed spacer regions of the target micro-organisms was also described by the manufacturer of the assay (4.2 % error rate in the evaluation study). The SeptiFast assay does not detect Actinobacillus sp. DNA, but this microorganism was detected in one patient by culture only. In the group of 10 haematological patients, SeptiFast results were positive for six of the 10 patients (60 %), whereas blood cultures were positive in only two out of 10 patients (20 %). The declared analytical sensitivity of the SeptiFast assay ranges between 30 and 100 c.f.u. (depending on the micro-organism). The SeptiFast assay provides a rapid identification of the causative microorganism within 6 h (3 h of technician hands-on time) whereas blood cultures usually require 2 days for Gram-staining results and a total of 3 days for the species to be identified. However, the use of the SeptiFast assay is limited to well-established molecular laboratories, requires excellent technical skills and is very expensive compared to culture. Additionally, the SeptiFast assay cannot provide information regarding the antibiotic susceptibility of micro-organisms. We conclude that the SeptiFast assay is a clinically valuable add-on to conventional culture methods for rapid aetiological diagnosis of sepsis in patients where empirical antimicrobial therapy has already been started and pretreatment blood cultures were negative. This assay showed particular sensitivity in haematological patients following BSCT,

Biological features and outcome of biphenotypic acute leukemia: a case series

BACKGROUNDnBiphenotypic acute leukemia (BAL) is a distinct entity that is immunophenotypically defined by the European Group for the Immunological Classification of Leukemia (EGIL) scoring system and accounts for less than 5% of all acute leukemia cases. Since it is a rare and heterogeneous form of acute leukemia with an allegedly poor outcome, there is no consensus on the best treatment approach in these patients. Our objective was to analyze the biological features and outcome of patients diagnosed with BAL in our institution.nnnPATIENTS AND METHODSnUsing the EGIL system, we identified 21 cases (3.9%) of BAL from 535 newly diagnosed acute leukemia patients in an 11-year period.nnnRESULTSnThere were ten cases of myeloid+B-lymphoid leukemia, eight cases of myeloid+T-lymphoid, one case of B+T-lymphoid and two cases of trilineage (myeloid+B+T-lymphoid leukemia). The complete remission (CR) rate with high-dose chemotherapy was 72% and overall survival at 5 years was 21%. Patients that received acute lymphoblastic leukemia-oriented chemotherapy had a higher CR rate compared with those who received acute myeloid leukemia-oriented chemotherapy (100% vs. 60%, P = .007). The white blood cell count at diagnosis was found to have statistically significant impact on survival.nnnCONCLUSIONnDespite the progress in the treatment of acute leukemia, the prognosis of BAL remains poor and treatment protocols devised explicitly for this entity should be investigated in prospective collaborative studies.

Comparison of Branded and Generic Imatinib Plasma Concentrations in Patients With Chronic Myelogenous Leukemia: Unicentric Study

INTRODUCTIONnFor over a decade, imatinib has been the first-line treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML). Doubts on the bioequivalence and bioavailability of emerging generic compounds have been expressed. Adequate imatinib plasma concentration ([IPC]xa0≥1000 μmol/L) is associated with a better chance of optimal treatment response in patients with CML. In this study, we compared the achieved IPCs between the branded compound and its 2 generic forms.nnnPATIENTS AND METHODSnIPCs were compared in 24 consecutive patients with CML in the first chronic phase who changed from branded to generic imatinib. The median age was 49 years (range, 22-76 years). Fifteen of them were male. Six patients were switched to Neopax, 13 to Imakrebin, and 5 patients received both generics consecutively. All compounds were used in an equivalent dose of 400 mg orally once daily for at least 1 month before plasma concentrations were measured. High-performance liquid chromatography was used to determine imatinib plasma concentration from a specimen collected 21 to 24 hours after the last dose.nnnRESULTSnThe median IPC achieved with branded imatinib was 1454 μmol/L (range, 485-2707 μmol/L) with 18 patients (75%) having IPCxa0≥ 1000 μmol/L. For Neopax and Imakrebin, median IPCs were 1717 μmol/L (range, 1249-3630 μmol/L) and 1458 μmol/L (range, 707-880 μmol/L), respectively, with 11 of 11 (100%) and 16 of 18 (89%) patients having IPCxa0≥ 1000 μmol/L. No significant difference in measured IPCs between all 3 compounds was found (P > .257).nnnCONCLUSIONnWhen taken at equivalent doses, imatinib generics are bioequivalent and comparable in clinical efficacy and have the potential for substantial savings in the treatment cost for CML.

The influence of tumor necrosis factor microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation

Aim To investigate the influence of tumor necrosis factor (TNF) microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation. Methods We analyzed TNFa, TNFb, and TNFd microsatellites among 100 patients who underwent allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen (HLA)-identical sibling donor at the Internal Clinic of the University Hospital Center Zagreb in the period 2001-2009. The analysis was performed using polymerase chain reaction amplification and electrophoresis on a polyacrylamide gel in an automated sequencer. Results There was no significant difference in patient survival with respect to the allele length at a given microsatellite. However, a significantly lower survival rate was noticed among patients who were positive for TNFa8 allele (Pu2009<u20090.001) and a significantly higher survival rate among those who were positive for TNFa10 allele (Pu2009=u20090.0220). Conclusion These results for the first time suggest an influence of TNFa microsatellite on patient survival following HSCT and indicate a need for further studies of this microsatellite.