Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants.

Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.

Identification of invasive fungal diseases in immunocompromised patients by combining an Aspergillus specific PCR with a multifungal DNA-microarray from primary clinical samples.

The increasing incidence of invasive fungal diseases (IFD), most of all invasive aspergillosis (IA) in immunocompromised patients emphasises the need to improve the diagnostic tools for detection of fungal pathogens. We investigated the diagnostic performance of a multifungal DNA-microarray detecting 15 different fungi [Aspergillus, Candida, Fusarium, Mucor, Rhizopus, Scedosporium and Trichosporon species (spp.)] in addition to an Aspergillus specific polymerase chain reaction (PCR) assay. Biopsies, bronchoalveolar lavage and peripheral blood samples of 133 immunocompromised patients (pts) were investigated by a multifungal DNA-microarray as well as a nested Aspergillus specific PCR assay. Patients had proven (n = 18), probable (n = 29), possible (n = 48) and no IFD (n = 38) and were mostly under antifungal therapy at the time of sampling. The results were compared to culture, histopathology, imaging and serology, respectively. For the non-Aspergillus IFD the microarray analysis yielded in all samples a sensitivity of 64% and a specificity of 80%. Best results for the detection of all IFD were achieved by combining DNA-microarray and Aspergillus specific PCR in biopsy samples (sensitivity 79%; specificity 71%). The molecular assays in combination identify genomic DNA of fungal pathogens and may improve identification of causative pathogens of IFD and help overcoming the diagnostic uncertainty of culture and/or histopathology findings, even during antifungal therapy.

Emergence of azole-resistant invasive aspergillosis in HSCT recipients in Germany.

OBJECTIVES: Aspergillus fumigatus is the most common agent of invasive aspergillosis (IA). In recent years, resistance to triazoles, the mainstay of IA therapy, has emerged in different countries worldwide. IA caused by azole-resistant A. fumigatus (ARAF) shows an exceedingly high mortality. In this study, IA due to ARAF isolates in HSCT recipients in Germany was investigated. METHODS: The epidemiology of azole resistance in IA was analysed in two German haematology departments. Between 2012 and 2013, 762 patients received HSCT in Essen (n = 388) and Cologne (n = 374). Susceptibility testing of A. fumigatus isolates was performed by Etest, followed by EUCAST broth microdilution testing if elevated MICs were recorded. In all ARAF isolates the cyp51A gene was sequenced and the genotype was determined by microsatellite typing using nine short tandem repeats. RESULTS: In total, A. fumigatus was recovered from 27 HSCT recipients. Eight patients had azole-resistant IA after HSCT, and seven of the cases were fatal (88%). All except one patient received antifungal prophylaxis (in five cases triazoles). TR34/L98H was the most common mutation (n = 5), followed by TR46/Y121F/T289A (n = 2). In one resistant isolate no cyp51A mutation was detected. Genotyping revealed genetic diversity within the German ARAF isolates and no clustering with resistant isolates from the Netherlands, India and France. CONCLUSIONS: This report highlights the emergence of azole-resistant IA with TR34/L98H and TR46/Y121F/T289A mutations in HSCT patients in Germany and underscores the need for systematic antifungal susceptibility testing of A. fumigatus.

Assessment of Aspergillus-specific PCR as a screening method for invasive aspergillosis in paediatric cancer patients and allogeneic haematopoietic stem cell recipients with suspected infections.

Invasive aspergillosis (IA) remains difficult to diagnose in immunocompromised patients, because diagnostic EORTC/MSG criteria are often not met. As biomarkers might elucidate the pathogen, we analysed the performance of an Aspergillus PCR assay in blood for diagnosis of IA in immunocompromised paediatric patients with suspected infections. Ninety-five haemato-oncological paediatric patients were included over a period of 3 years, the underlying diseases consisting of acute leukaemia, solid tumours, non-malignant immunocompromising disorders and haematopoietic stem cell transplantation recipients. We retrospectively analysed 253 consecutive episodes of suspected infections. Thirty-eight patients had possible IA, none of the patients fulfilled EORTC/MSG criteria of probable/proven IA. PCR positivity was observed in 97/967 analyses. Sensitivity, specificity, positive and negative predictive value of the PCR per episode were 34%, 78%, 31% and 81% using possible IA as endpoint. Taken together, an undirected blood screening by Aspergillus-specific PCR is of little diagnostic value in a heterogenous paediatric patient cohort. Harnessing PCR for diagnosis of IA should thus be focused on blood analyses of more homogenous high-risk patients and/or analyses of bronchoalveolar lavage, tissue or cerebrospinal fluid specimens.

Aspergillus PCR-based investigation of fresh tissue and effusion samples in patients with suspected invasive Aspergillosis enhances diagnostic capabilities.

Although it is a severe complication in immunocompromised patients, diagnosing invasive fungal disease (IFD), especially invasive aspergillosis (IA), remains difficult. In certain clinical scenarios, examining tissue samples for identification of the infectious organism becomes important. As culture-based methods rarely yield results, the performance of an Aspergillus-specific nested PCR in fresh tissue or pleural effusion samples was evaluated. Fresh tissue (n = 59) and effusion (n = 47) specimens from 79 immunocompromised patients were subjected to an Aspergillus-specific PCR assay. Twenty-six patients had proven (n = 20) or probable (n = 6) IFD, according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria, while the remaining patients were classified as having either possible IFD (n = 30) or no IFD (n = 23). IA was identified as the underlying IFD in 21/26 proven/probable cases. PCR positivity was observed for 18/21 proven/probable and 6 possible IA cases; cases classified as no IA did not show positive signals. Patients with proven IFD (n = 5) with cultures positive for non-Aspergillus molds also had negative Aspergillus PCR results. Aspergillus PCR performance analysis yielded sensitivity and specificity values of 86% (95% confidence interval [CI], 65% to 95%) and 100% (95% CI, 86% to 100%), respectively, thus leading to a diagnostic odds ratio of >200. In this analysis, good diagnostic performance of the PCR assay for detection of IA was observed for tissue samples, while effusion samples showed lower sensitivity rates. PCR testing represents a complementary tool; a positive PCR result strengthens the likelihood of IA, whereas IA seems unlikely in cases with negative results but findings could indicate non-Aspergillus IFD. Thus, PCR testing of these specimens enhances the diagnostic capabilities.

Azole-resistant invasive aspergillosis in a patient with acute myeloid leukaemia in Germany.

We report the first culture-proven case of invasive aspergillosis (IA) caused by azole-resistant Aspergillus fumigatus in a patient with acute myeloid leukaemia in Germany. IA presented as breakthrough infection under posaconazole prophylaxis. Analysis of the resistance mechanism revealed the TR/L98H mutation in the cyp51A gene, which indicates an environmental origin of the strain. This case underscores the need for monitoring azole resistance in Aspergillus spp. and for routine susceptibility testing of moulds.

[Intraocular hemorrhage in a goat with thrombocytopenia and metastasis of an adenocarcinoma in the iris]. / Intraokuläre Blutung bei einer Ziege mit Thrombozytopenie und Karzinommetastase in der Iris.

This case report describes the findings in a 10-year-old goat with metastasis of an adenocarcinoma in the iris. Two weeks before admission, the owner had noticed blepharospasm of the left eye. Clinical examination by the referring veterinarian revealed unilateral intraocular hemorrhage. The goat was referred to our clinic for further work-up. The rectal temperature was 40 degrees C. The most important haematological result was severe thrombocytopenia. There was mild corneal oedema of the left eye. Approximately 75 per cent of the anterior chamber was filled with non-coagulated blood. The fluid in the anterior chamber dorsal to the blood was cloudy, and the corpora nigra could not be seen clearly. All other internal parts of the eye could not be seen. Ultrasonography of the left eye confirmed cloudiness of the anterior chamber and revealed moderate thickening of the iris. The right prescapular lymph node was markedly enlarged. Cytological examination of a fine needle aspirate of the lymph node showed a mixed population of neoplastic cells. Based on immunohistochemical evaluation of the cells metastasis of a carcinoma was diagnosed. The goat was subjected to euthanasia, and a postmortem examination was carried out. The anterior chamber of the left eye contained blood, and the iris was thicker than normal and adhered to the posterior surface of the cornea. There were neoplastic alterations in the iris, the oesophagus, the lung lobes, the liver, the kidney and in the prescapular, retropharyngeal, mediastinal and hepatic lymph nodes. Histologically, a diagnosis of carcinoma was confirmed, but the origin of the tumour could not be determined.

Validity of S-100 B in patients after brain radiation.

BACKGROUND: S-100B is a calcium binding acute phase protein and a potential biomarker for brain injury. In prior studies elevated plasma S-100B levels were detected in stroke and severe head trauma. The aim of this study was to evaluate whether S-100 B is elevated during cerebral radiotherapy and whether that is associated with adverse outcomes. MATERIAL AND METHODS: In this prospective pilot study, 45 patients (25 males, 20 females, median age 58 (17-81)) underwent cerebral radiation therapy because of a primary or metastaic cerebral malignancy. 39 patients were included in the evaluation. 6 patients died during the study period. S-100 plasma concentrations were measured with an electrochemiluminescence immunoassay on admission and weekly during radiation therapy for the duration of 6 weeks. In 10 healthy young volunteers (5 males, 5 females, median age 32 (28-36)) S-100 B plasma levels were measured weekly for 6 weeks as a negative control. Furthermore, in an active control 10 patients (4 males, 6 females, median age 68 (64-76)) with stroke (7 = major stroke, 3 = lacunar infarct) S- 100 B plasma levels were measured for 7 consecutive days after the event. RESULTS: During radiotherapy S-100 B plasma concentrations increased from median baseline values of 0.030 microg/l to 0.044 microg/l. For the time of radiation therapy most patients showed a mild increase, but absolute plasma values were still within the normal range. In the control group of healthy volunteers S-100 B remained unchanged. In stroke patients S-100 B increased to maximum values of 1.7 microg/l three days after the event. In the 3 patients with lacunar infarcts no increase of S-100 B levels could be detected. CONCLUSION: Brain irradiation leads to a mild increase of S-100 B plasma levels. However, the absolute rise was far weaker compared to that seen in major brain injuries.

Intravenous perfluorocarbon emulsion increases nitrogen washout after venous gas emboli in rabbits.

Intravenous perfluorocarbon emulsion (IV-PFC) has been shown to provide hemodynamic protection from gas embolism (Venous-VGE or arterial-AGE). The objective of this study was to investigate the mechanism of PFC protection from controlled VGE by quantifying the effects of IV-PFC emulsion on pulmonary elimination of nitrogen (N2). All rabbits received an intravenous pretreatment of PFC emulsion (Oxygent, 2.7 g/kg) or saline, then either a continuous room air infusion (0.25 ml/kg for 10 minutes) or a bolus of air (0.8 ml/kg within 10 seconds) through the femoral vein. Expiratory N2 peaked higher with PFC infusion immediately after air injection. The recovery to baseline of end tidal N2 was faster for PFC-treated animals (40 +/- 4.7 vs. 58 +/- 6.5 minutes). In PFC-treated animals, expired CO2, O2, arterial pressure and central venous pressure returned to baseline faster than the saline group. This study demonstrated that PFC increased pulmonary N2 washout. Correspondingly, PFC treatment better preserved the animals' hemodynamics after VGE injury. The use of IV-PFC promises to be a breakthrough non-recompression therapy for gas embolism in the treatment of Decompression Sickness (DCS) and in surgery.

Detection of Aspergillus DNA in cerebrospinal fluid from patients with cerebral aspergillosis by a nested PCR assay.

Invasive aspergillosis (IA), a complication with high mortality rates, especially in disseminated IA with cerebral involvement, is difficult to diagnose. Biopsy of cerebral lesions is often not feasible, and culture of Aspergillus spp. from cerebrospinal fluid (CSF) is frequently negative. New molecular methods have emerged for diagnosing IA. So far, there are only few reports of Aspergillus DNA detection in CSF. After modifying the DNA extraction protocol, we detected Aspergillus DNA in CSF samples by a previously described nested PCR assay. In six patients with hematologic malignancy and cerebral aspergillosis, CSF samples were investigated for Aspergillus DNA. IA was classified according to the EORTC/MSG 2002 criteria. Two patients each had proven, probable, and possible IA. Thirty-five CSF samples were investigated for Aspergillus DNA by nested PCR. Samples with positive results in the nested PCR assay were quantified by LightCycler PCR assay. Fourteen CSF samples showed positive results in the nested PCR assay. Of these, six samples gave positive results in real-time PCR. The range of CFU per ml was 2,154 to 63,100,000. The highest number of CFU per ml was found in a CSF sample of a patient with acute lymphocytic leukemia and probable cerebral aspergillosis. Detection of Aspergillus DNA in CSF samples is thus possible and has the potential to improve diagnosis of cerebral aspergillosis. Further prospective studies with larger numbers of patients must be performed to evaluate the clinical significance of Aspergillus PCR with CSF samples.

Molecular markers in stored kidneys using perfluorocarbon-based preservation solution: preliminary results.

BACKGROUND: Delayed graft function (DGF) is a problem in kidney transplantation and cold ischemia has been identified as a risk factor. Perfluorocarbons (PFC) have an enhanced ability to dissolve and release oxygen. We evaluated histologically and a number of molecular changes induced by ischemia in stored kidneys with University of Wisconsin (UW) and PFC-based preservation solutions (PFC-UW). MATERIALS AND METHODS: ACI rats were used as kidney donors. UW (control group) or PFC-UW (study group) preservation solutions were used for kidney perfusion. All kidneys were stored at 4 degrees C for 12, 24, and 36 hours. After this time, intragraft histologic evaluation as well as mRNA HO-1 and iNOS levels were also analyzed. RESULTS: In the kidneys stored at 24 hours, mRNA HO-1 levels were elevated in the study group when compared with the control and mRNA iNOS was decreased. CONCLUSION: We observed overexpression of HO-1 and underexpression of iNOS in the kidney tissue stored with PFC-UW solution at 24 hours. These preliminary data suggest that increasing oxygen delivery by PFC added to the perfusion solution triggers cytoprotective mechanism in kidney transplantation.

Unilateral evisceration of an eye following cornea and lens perforation in a sulfur-crested cockatoo (Cacatua galerita).

A 24-year old male sulfur-crested Cockatoo (Cacatua galerita) was presented with a subacute perforation of the cornea without involvement of the lens. The bird was treated conservatively and the eye remained quiescent up to a second traumatic corneal perforation associated with a lens capsule rupture 15 months later. Due to the second perforating trauma of an already blind eye involving the lens, evisceration of the eye was performed. Two months after surgery the cosmetic result was excellent. Treatment options for perforating ocular traumas in captive birds are discussed in detail.

Comparison of bioimpedance versus thermodilution cardiac output during cardiac surgery: evaluation of a second-generation bioimpedance device.

OBJECTIVE: To compare a second-generation thoracic electrical bioimpedance (TEB) hemodynamic monitoring system with the clinically used pulmonary artery catheter thermodilution (TD-PAC) system. DESIGN: Blinded, simultaneous measurements at specified key time points during surgery. SETTING: University teaching hospital cardiac surgical operating rooms. PARTICIPANTS: Forty-seven patients undergoing primary elective coronary artery bypass surgery. INTERVENTIONS: Timed cardiac output measurements by thermodilution and continuous monitoring of bioimpedance were performed. MEASUREMENTS AND MAIN RESULTS: Cardiac index (TEB and TD-PAC) and other hemodynamic parameters were measured at 4 time points: (1) after anesthesia induction, (2) with the mediastinum open, (3) immediately after cardiopulmonary bypass, and (4) at the end of the case. Pearson's correlation and Bland-Altman analysis were carried out. Cardiac index by TEB and TD-PAC had an overall correlation of r = 0.71 (p < 0.0001). The Bland-Altman statistics showed a mean difference of -0.28 L/min/m2 and precision of 0.67 L/min/m2. The best correlation was at time 1, and the lowest correlation was at time 4. Mediastinal opening and cardiopulmonary bypass had little or no effect on the correlation between technologies. CONCLUSION: TEB reporting of cardiac index during coronary artery surgery generally agreed with TD-PAC cardiac index except at the end of the case (time 4).

Blood transfusion: the silent epidemic.

Blood transfusion has been widely studied and the risk/benefit ratio remains unclear. Focus historically has been upon viral transmission, particularly hepatitis and HIV. Today, with advanced screening for these viruses, the risk for such transmission has become vanishingly small. Immunosuppression, with consequent postoperative bacterial infection and ABO incompatibility are now risks that physicians should consider as associated with allogeneic blood transfusion. Other inflammatory events, such as transfusion associated acute lung injury, also occur. The benefits of transfusion have never been well studied and there is scant literature on that area. Therefore, in an evidence-based medical practice the physician should regard transfusion with a skewed risk/benefit ratio. The following article examines that risk/benefit ratio in the post-AIDS era.

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.

Systemic infections with Aspergillus species in patients with hematological malignancies: current serological and molecular diagnostic approaches.

Within the recent years, novel molecular and serological methods have been developed to improve the diagnosis of invasive aspergillosis in patients with malignant hematological diseases being at highest risk for this life-threatening infection. Early diagnosis and treatment are essential for adequate therapeutic management, which, however, often remains difficult since most of the diagnostic tools used clinically at present either lack specificity or sensitivity, or both at the worst. The clinical value, advantages and remaining problems of recently developed serological assays and molecular approaches to detect this emerging pathogen are reviewed.

Institutional variability in red blood cell conservation practices for coronary artery bypass graft surgery. Institutions of the MultiCenter Study of Perioperative Ischemia Research Group.

OBJECTIVE: To assess whether substantial institutional variability exists in red blood cell conservation practices associated with coronary artery bypass graft (CABG) surgery. DESIGN: Prospective, randomized patient enrollment and data collection. SETTING: Twenty-four U.S. academic institutions participating in the Multicenter Study of Perioperative Ischemia. PARTICIPANTS: A well-defined subset of primary CABG surgery patients (n = 713) expected to be at low risk for bleeding and exposure to allogeneic transfusion. INTERVENTIONS: None (observational study). MEASUREMENTS AND MAIN RESULTS: Frequency of use of red blood cell conservation techniques was determined among institutions. Correlation was determined between use of each technique and transfusion of allogeneic red blood cells and between use of each technique and median institutional blood loss. Significant variability (p < 0.01) was detected in institutional transfusion practice with respect to the use of predonated autologous whole blood, normovolemic hemodilution, red cell salvage, and reinfusion of shed mediastinal blood. The frequency of institutional use of these techniques was not associated with allogeneic transfusion (r2 < 0.15) or blood loss (r2 < 0.10) in the low-risk population of patients examined. CONCLUSIONS: Institutions vary significantly in perioperative blood conservation practices for CABG surgery. Further study to determine the appropriate use of these techniques is warranted.