Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

[Castleman disease: retrospective single-center study of therapeutic results in 10 patients]. / Castlemanova choroba: retrospektivní studie lécebných výsledku u 10 pacientu z jednoho centra.

BACKGROUND: Castleman disease is a non-clonal lymphoproliferative disorder with 2 clinical (unicentric, multicentric) and 4 histomorphological (hyaline vascular, plasma cell, mixed, plasmablastic) forms which combine creating a pleomorphic picture of this rare entity. In our work, the largest documented cohort in the Czech Republic was analyzed focusing on diagnostics and particularly on therapy. PATIENTS AND METHODS: The retrospective study (1998-2013) included 10 patients, 6 males, 4 females. Patients with unicentric form (3) underwent surgical sanitation. Patients with multicentric form (7) were followed-up only (2) or extirpation of the largest mass was carried out (1) or a systemic therapy was administered (4) which comprised the following regimens: R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), CTD/CAD/CVD (cyclophosphamide, thalidomide/adriamycin/bortezomib, dexamethasone), further including monotherapies with tocilizumab, thalidomide and lenalidomide and in one case (associated POEMS syndrome, i.e. polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) autologous stem cell transplantation after melphalan conditioning was performed. During treatment response monitoring, all patients underwent PET/CT examination (fluorodeoxyglucose positron emission tomography/computed tomography). RESULTS: The remission rate was 50% (3 unicentric forms with remission lasting 51, 8 and 9 months, resp.; 2 multicentric forms with remission lasting 3 months during thalidomide therapy and 12 months after lenalidomide therapy), stable disease was observed in 40% of cases (multicentric forms, 2 without any treatment followed-up for 171 and 24 months, resp.; 1 after systemic therapy followed-up for 23 months; 1 after two extirpations with stable lymphadenopathy for 15 years, where the first operation was 27 years ago). In one patient (10%), the associated POEMS syndrome progressed rapidly with fatal consequences (4 months follow-up). CONCLUSION: Unlike unicentric forms completely curable by excision, multicentric forms are often treatment-refractory. Concerning high cost-effectiveness, good tolerability and documented efficacy also in rituximab-resistant cases, we prefer immunomodulatory drugs (particularly thalidomide) for managing multicentric Castleman disease in our center.

Long-term results of allogeneic hematopoietic stem cell transplantation after reduced-intensity conditioning with busulfan, fludarabine, and antithymocyte globulin.

UNLABELLED: Reduced-intensity conditioning (RIC) is widely used for allogeneic stem cell transplantation (SCT). Here we present our long-term experience with RIC regimen consisting of fludarabine (30 mg/m2/day on days -10 to -5), busulfan (4mg/kg/day on days -6 and -5) and antithymocyte globulin (ATG Fresenius, 10 mg/kg/day on days -4 to -1) (Flu-Bu-ATG) in a cohort of 71 patients with various hematological malignancies including chronic myeloid leukemia (24 patients), acute myeloid leukemia (19 patients), lymphoma (20 patients), multiple myeloma (3 patients), myelodysplastic syndrome (3 patients), and myelofibrosis (2 patients). The median age was 50 years. The overall response rate was 87%, including 83% CR and 4% PR. The incidence of acute and chronic GVHD was 35% and 52% and the cumulative incidence of non-relapse mortality at 1 year and 4 years was 8% and 14%. With the median follow-up of 55.0 months, the 2- and 4-year event-free survival (EFS) was 49.0% and 40.3%, and the overall survival (OS) was 73.2% and 62.6%, respectively. Gender, age at SCT, type of donor, disease status at SCT, previous autologous transplantation, and complete chimerism by day +100 did not significantly influence EFS and OS. In a multivariate analysis, no presence of chronic GVHD (p=0.029, HR: 2.5),and diagnosis other than CML (p=0.018, HR: 4.6), and CD34+ dose < 5x106/kg (p=0.010, HR: 2.8) were significant predictors of poor OS. Flu-Bu-ATG protocol is a RIC regimen that combines effective disease control with low non-relapse mortality and acceptable toxicity profile. KEYWORDS: reduced-intensity conditioning, fludarabine, busulfan, antithymocyte globulin.

Mucositis does not lead to false-positivity of the Platelia Aspergillus enzyme-linked immunosorbent assay.

We are reporting a study evaluating the crossover of antigens reacting in Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) from faeces to vessels during mucositis as a possible cause of false-positivity of this test. In our series of 102 episodes of different grades of mucositis, we found strong reactivity of faeces in the PA ELISA test irrespective of the grade of mucositis, the percentage of oral food intake or the presence of total parenteral nutrition. However, none of the patients included in the study were positive in the serum (when the criterion of two samples with cut-off index of positivity [IP] > 0.5 was used).

[Detection of 1,3-beta-D glucan for diagnosis of invasive fungal infections in hematooncological patients: usefulness for screening of invasive mycosis and for confirmation of galactomannan positive results]. / Detekce 1,3-beta-D glukanu v diagnostice invazivních mykotických infekcí u nemocných s hematologickou malignitou: prínos metody ve skríningu invazivních mykóz a v konfirmaci galaktomanan pozitivních vzorku.

BACKGROUND AND AIMS: 1,3-beta-D glucan (BG) -- the antigen of fungal cell wall can be detected by a commercially available test for early detection of invasive fungal infections (IFI). The main advantage of this test is its broad coverage of fungal species. The aim of our study was to evaluate usefulness of BG detection for screening of IFI and for confirmation of galactomannan (GM) positive blood samples. Combination of the results of both tests could lead to correct and early diagnosis of invasive aspergillosis (IA). PATIENTS AND METHODS: Between January 2005 and July 2007 blood samples were collected in patients from intermediate to high risk of IFI. Moreover, between February and October 2007 all patients that had consecutive positive results of GM had their positive symplex tested also for BG. RESULTS: In BG screening study, 1154 of blood samples from 104 treatment cycles were tested for BG. The incidence of IFI was 17.3 % (n = 18) and probable or proven IFI was detected in 9 cases (8.6%). The highest sensitivity, specificity, PPV and NPV (88.9 %, 40.7 %, 13.6 % and 97.2 %) were obtained when as criteria for positivity cut off 80 pg/ml and one positive result were used. When consecutive positivity of the test was applied as criterium, cut off 60 pg/ml was found more useful (sensitivity 66.7 %, specificity 47.7 %, PPV 11.8 % and NPV 93.2 %). Low PPV, caused by frequent false positive results, was identified as main limitation of this assay. 65 treatment cycles were positive if 1 sample above 80 pg/ml was used as a cut of for positivity. If consecutive positivity with cut off 60 pg/ml was used, 58 treatment cycles were positive. But in 51 (78.4 %) and 45 (77.5 %) cases, respectively, the positivity was not associated with IFI (false positivity). We did not find any correlation between positive BG assay result and frequency of empirical antifungal treatment, mucositis, yeast colonization, administration of selected antibiotics or infusion solutions or bacteriaemia. In our confirmation study, 40 GM positive episodes in 39 patients were identified. In 31 (78 %) GM positivity was false and was not associated with clinical signs and symptoms of IA. Sensitivity of GM detection in IA was 100 % but PPV only 18 %. Confirmation of consecutive GM positive samples (using cut off index positivity 0,5) by consecutive positivity of BG (with cut off 60 pg/ml) was found very useful for diagnosis of IA -- most of GM false positive results were eliminated and PPV increased to 88 %. CONCLUSIONS: Our analysis focused on routine use of BG test for panfungal screening of IFI in patients with hematological malignancy and confirmed limited usefulness of this test in such setting. Low sensitivity together with low PPV are major limits of this test. On the other hand, BG testing seems to be a promising tool for confirmation of consecutive GM positive result in serum in patients with IA. Positivity of both tests could increase their PPV of tests and eliminate false positive results.

[The use of galactomannan detection in diagnosing invasive aspergillosis in hemato-oncological patients]. / Vyuzití detekce galaktomananu pro diagnostiku invazivní aspergilózy u hematoonkologických nemocných.

UNLABELLED: PREMISES AND OBJECTIVES: Timely diagnosis is of critical importance for the prognosis of invasive aspergilosis (IA) patients. Over recent years, IA detection of galactomannan using the ELISA method has assumed growing importance. The objective of the study was to analyse the usability of the method in current clinical practice of a hemato-oncological ward. PATIENTS AND METHODS: From May 2003 to October 2006, blood samples were taken from patients at IA risk to detect galactomannan (GM) in serum using the ELISA method. The patients who underwent the tests were classified by the probability of IA presence on the basis of the results of conventional diagnostic methods and section findings. RESULTS: A total of 11,360 serum samples from 911 adult patients were tested for GM presence. IA (probable/proven) was diagnosed in 42 (4.6%) of them. The rates of sensitivity, specificity, positive and negative predictive value of galactomannan detection for IA diagnosis in our ward were, respectively, 95.2%, 90.0%, 31.5% and 99.7%. The principal causes of the limited positive predictive value of the test were the high percentage of false-positive test results (mainly caused by concomitant administration of some penicillin antibiotics or Plasma-Lyte infusion solution), as well as the fact that a large percentage of patients we examined fell within the group of patients with hematological malignity with a very low prevalence of IA. CONCLUSION: GM detection in serum is associated with high sensitivity and excellent negative predictive value in IA diagnosis in hemato-oncological patients. Knowledge and elimination of possible causes of false-positive results as well as focusing the screening on patients at greatest risk of infection are necessary for an even better exploitation of the test.

[Invasive aspergillosis in hematooncological patients: advantages and disadvantages of various diagnostic methods, treatment options and financial costs of therapy]. / Invazivní aspergilové infekce u hematoonkologických nemocných: význam jednotlivých diagnostických metod, lécebných postupu a nástin financní nárocnosti lécby.

BACKGROUND: Invasive aspergillosis (IA) is a leading invasive fungal infection in hematooncological patients. The aim of this study was to analyse the incidence, diagnostic procedures and treatment of IA in hematooncological department in large hospital in the Czech Republic. PATIENTS AND METHODS: A retrospective analysis of medical and laboratory records from patients hospitalised in our department with proven/probable IA between January 2000 and December 2006 was performed. RESULTS: 52 cases of IA in 51 patients were identified (17.3% proven IA/82.7% probable IA). Number of IA cases notably increased during study period (1 case of IA in 2000 vs 21 cases of IA in 2006) and majority of them was of nosocomial origin (61.5%). Pulmonary aspergillosis was diagnosed in 46 cases (88.5%). Patients treated for acute leukemia or undergoing allogeneic stem cell transplantation represent the group at the highest risk of IA (in total 52% of cases). Fever and signs of pulmonary involvement were the most common clinical signs of infection (presented in 92.3% and 69.2 cases respectively). Conventional diagnostic methods including autopsy were able to diagnose only 15 cases of IA (28.8%). In all other cases (71.2%) the diagnosis was done by detection of galactomannan (GM) in serum. Introduction of GM monitoring enabled erlier initiation of antifungal treatment by 4 days. Initial therapy of IA led to the treatment response (partial and complete) in 18 (34.6%) of infections--the highest percentage of response has been seen in voriconazole monotherapy group (42%) and when combination of voriconazole and caspofungin has been used (83%). Salvage therapy was initiated due to the failure of initial treatment in 21 (40.3%) of cases. Patients were treated mostly with combination ofvoriconazole and caspofungin and/or monotherapy with voriconazole has been used with treatment response 55% and 50% respectively. Introduction of new antifungal drugs together with increased number of patients with IA led to the marked increase of total costs spent on treatment of IA per year--from 11,5 thousands CZK in 2000 to 6,2 millions CZK in 2006. CONCLUSIONS: IA is the most frequent cause of infection-related mortality in patients with haematological malignancies. Routine use of non-culture base methods in diagnosis of IA together with treatment using new, effective antifungals can improve prognosis of patients with this life threatening infection.

[Early diagnosis of invasive fungal infections in hematooncological patients by serological methods]. / Casná diagnostika invazivních mykotických infekcí u hematoonkologických nemocných pomocí sérologických metod.

Invasive fungal infections have become one of the major causes of morbidity and mortality in hematooncological patients over the past 2 decades. Even there is an increasing incidence of invasive fungal infections caused by rare filamentous fungi, the majority of infections are caused by Candida sp. and especially Aspergillus sp. Early diagnosis and prompt initiation of antifungal treatment are leading factors influencing prognosis of patients with invasive fungal infection. Important advances in the field of early diagnosis of invasive fungal infections have been realized over the last years. Beside of new radiological methods the major progress has been done in serological methods. In this paperwe review the most important of these serological methods and their position in routine clinical practice.

Clinical outcomes and direct hospital costs of reduced-intensity allogeneic transplantation in chronic myeloid leukemia.

A reduced-intensity conditioning allogeneic stem cell transplantation was given to 19 patients (aged 15-59 years) in the first chronic phase and one patient in the accelerated phase with chronic myeloid leukemia (CML) after a regimen consisting of fludarabine (Flu), busulfan (Bu) and ATG Fresenius. The median follow-up was 27 months. Until day +100, no transplant-related mortality was recorded. The incidence of acute and chronic graft-versus-host disease (GvHD) was 55 and 75%, respectively. Two patients (10%) died from GvHD. Fourteen (70%) patients achieved molecular remission. Additional post-transplant intervention (donor lymphocyte infusion, imatinib) was necessary, however, in 10 patients (50% of the patients; non-achievement of stable molecular remission or later relapses). The total direct cost of the transplantation treatment for all of the patients came to 1,572,880 euro. If the patients had been treated with imatinib and followed-up with the same time period as they were following a transplantation, the direct cost of the imatinib treatment would have been 2,005,117 euro. The transplantation treatment appears to be less expensive after approximately 2 years of follow-up. Flu+Bu+ATG is a low-toxicity regimen for patients with CML. However, a close follow-up is necessary and about 50% of the patients require further therapeutic intervention.

Multicolor FISH mapping of the dioecious model plant, Silene latifolia.

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.

Sex chromatin and nucleolar analyses in Rumex acetosa L.

Rumex acetosa (sorrel) is a dioecious plant with a XX/XY1Y2 sex chromosome system. Both the Y chromosomes are nearly entirely heterochromatic and it has been hypothesised that they can persist as chromocenters in male interphase nuclei. Using specific antibodies against 5-methylcytosine and histone H4 acetylated at terminal lysine 5, global levels of DNA methylation and histone acetylation were studied on the sex chromosomes and autosomes of both sexes. The heterochromatic Y chromosomes did not display a higher methylation level compared to the autosomes. The only prominent hypermethylation signals were found at two nucleolar organising regions located on the autosome pair V, as confirmed by in situ hybridisation with 25S rDNA probe and staining. Immuno-analysis of DNA methylation on female and male interphase nuclei neither revealed any sex-specific differences. Two active (silver-positive) nucleoli and two likely inactive nucleolar organising regions (displaying prominent methylation signals) were found in both sexes. In a fraction of nuclei isolated from leaf cells, two peripheral bodies strongly positive for 4',6-diamidino-2-phenylindole were observed only in males, never in females. These heterochromatin regions were depleted in histone H4 acetylation at terminal lysine 5 and corresponded, according to in situ hybridisation with a Y-chromosome-specific repetitive probe, to the two Y chromosomes. We conclude that the peripheral condensed bodies observed exclusively in male nuclei represent the constitutive heterochromatin of the Y chromosomes which is characterised by a substantial histone H4 underacetylation.