The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis: recommendations from the Third European Conference on Infections in Leukemia.

INTRODUCTION: Timely diagnosis of invasive candidiasis (IC) remains difficult as the clinical presentation is not specific and blood cultures lack sensitivity and need a long incubation time. Thus, non-culture-based methods for diagnosing IC have been developed. Mannan antigen (Mn) and anti-mannan antibodies (A-Mn) are present in patients with IC. On behalf of the Third European Conference on Infections in Leukemia, the performance of these tests was analysed and reviewed. METHODS: The literature was searched for studies using the commercially available sandwich enzyme-linked immunosorbent assays (Platelia™, Bio-Rad Laboratories, Marnes-la-Coquette, France) for detecting Mn and A-Mn in serum. The target condition of this review was IC defined according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity, specificity and diagnostic odds ratios (DOR) were calculated for Mn, A-Mn and combined Mn/A-Mn testing. RESULTS: Overall, 14 studies that comprised 453 patients and 767 controls were reviewed. The patient populations included in the studies were mainly haematological and cancer cases in seven studies and mainly intensive care unit and surgery cases in the other seven studies. All studies but one were retrospective in design. Mn sensitivity was 58% (95% confidence interval [CI], 53-62); specificity, 93% (95% CI, 91-94) and DOR, 18 (95% CI 12-28). A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI, 79-97) and DOR, 12 (95% CI 7-21). Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity, 86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122). Significant heterogeneity of the studies was detected. The sensitivity of both Mn and A-Mn varied for different Candida species, and it was the highest for C. albicans, followed by C. glabrata and C. tropicalis. In 73% of 45 patients with candidemia, at least one of the serological tests was positive before the culture results, with mean time advantage being 6 days for Mn and 7 days for A-Mn. In 21 patients with hepatosplenic IC, 18 (86%) had Mn or A-Mn positive test results at a median of 16 days before radiological detection of liver or spleen lesions. CONCLUSIONS: Mn and A-Mn are useful for diagnosis of IC. The performance of combined Mn/A-Mn testing is superior to either Mn or A-Mn testing.

Pervasive positive selection on duplicated and nonduplicated vertebrate protein coding genes.

A stringent branch-site codon model was used to detect positive selection in vertebrate evolution. We show that the test is robust to the large evolutionary distances involved. Positive selection was detected in 77% of 884 genes studied. Most positive selection concerns a few sites on a single branch of the phylogenetic tree: Between 0.9% and 4.7% of sites are affected by positive selection depending on the branches. No functional category was overrepresented among genes under positive selection. Surprisingly, whole genome duplication had no effect on the prevalence of positive selection, whether the fish-specific genome duplication or the two rounds at the origin of vertebrates. Thus positive selection has not been limited to a few gene classes, or to specific evolutionary events such as duplication, but has been pervasive during vertebrate evolution.

Chronic beryllium disease as a re-emerging occupational disorder possibly misdiagnosed as sarcoidosis : a Swiss study project

Background: Beryllium (Be) is increasingly used worldwide for numerous industrial applications. Occupational exposure to Be may lead to Be sensitization (BeS), a CD4-mediated immune response. BeS may progress to chronic beryllium disease (CBD), a granulomatous lung disorder closely resembling sarcoidosis. The recognition of CBD requires detection of Be exposure at occupational history, and detection of BeS on blood or BAL lymphocytes. Since methods for CBD detection are not routinely available in Switzerland, we hypothesized that CBD cases are not recognized but misdiagnosis as sarcoidosis. Objective: To present an ongoing Swiss study screening patients with sarcoidosis in search of Be exposure, BeS, and CBD. Methods: Both a prospective and a retrospective cohort are being studied. In the prospective cohort, the main steps include: 1) recruitment of 100 consecutive patients with newly diagnosed pulmonary sarcoidosis at 2 centers (Lausanne, Bern). 2) screening for possible occupational Be exposure by self-administered patient questionnaire. 3) standardized detailed occupational interview and clinical visit by occupational health specialist. If step 3 is positive, then 4) blood and BAL sampling for detection of BeS by specifically developed Elispot assay and CFSE flow cytometry, with subsequent comparison to the classical Be lymphocyte proliferation test. If step 4 is positive, then 5) review of medical records and diagnostic revision from sarcoidosis to CBD. 6) appropriate measures for exposure cessation and case reporting to SUVA as occupational disease. The retrospective cohort will include 400 patients with previously diagnosed pulmonary sarcoidosis, either treated or untreated, recruited through the SIOLD Registries. Steps 2 to 5 will be peformed as above, except for a) end of study after step 2 if screening questionnaire does not reveal Be exposure, and b) step 4 done on blood sample only (BAL not needed). Current status: Self-administered screening questionnaire and tools for standardized occupational interview have been developed. BeS testing has been implemented and undergoes validation. Inclusions in the prospective phase have started at both study sites. The retrospective phase is in preparation. Conclusion: The current study status allows to conclude to technical feasibility of the project. The prospective phase if this study is funded by the SUVA. The SIOLD Registries are supported by the Swiss Pulmonary League.

Prevalence of human T cell leukemia virus-I (HTLV-I) antibody among populations living in the Amazon region of Brazil (preliminary report)

Forty-tree (31.4%) out of 137 serum samples obtained from two Indian communities living in the Amazon region were found to be positive for HTLV-I antibody, as tested by enzyme-linked immunosorbent assay (Elisa). Eighty-two sera were collected from Mekranoiti Indians, yielding 39% of positivity, whereas 11 (20.0%) or the 55 Tiriyo serum samples had antibody to HTLV-I. In addition, positive results occurred in 10 (23.2%) out of 43 sera obtained from patients living in the Belem area, who were suffering from cancer affecting different organs. Five (16.7%) out of 30 Elisa positive specimens were also shown to be positive by either Western blot analysis (WB) or indirect immunogold electron microscopy (IIG-EM).

β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3).

BACKGROUND: Invasive fungal infections (IFIs) are life-threatening complications in patients with hemato-oncological malignancies, and early diagnosis is crucial for outcome. The compound 1,3-β-D-glucan (BG), a cell wall component of most fungal species, can be detected in blood during IFI. Four commercial BG antigenemia assays are available (Fungitell, Fungitec-G, Wako, and Maruha). This meta-analysis from the Third European Conference on Infections in Leukemia (ECIL-3) assessed the performance of BG assays for the diagnosis of IFI in hemato-oncological patients. METHODS: Studies reporting the performance of BG antigenemia assays for the diagnosis of IFI (European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria) in hemato-oncological patients were identified. The analysis was focused on high-quality cohort studies with exclusion of case-control studies. Meta-analysis was performed by conventional meta-analytical pooling and bivariate analysis. RESULTS: Six cohort studies were included (1771 adult patients with 414 IFIs of which 215 were proven or probable). Similar performance was observed among the different BG assays. For the cutoff recommended by the manufacturer, the diagnostic performance of the BG assay in proven or probable IFI was better with 2 consecutive positive test results (diagnostic odds ratio for 2 consecutive vs one single positive results, 111.8 [95% confidence interval {CI}, 38.6-324.1] vs 16.3 [95% CI, 6.5-40.8], respectively; heterogeneity index for 2 consecutive vs one single positive results, 0% vs 72.6%, respectively). For 2 consecutive tests, sensitivity and specificity were 49.6% (95% CI, 34.0%-65.3%) and 98.9% (95% CI, 97.4%-99.5%), respectively. Estimated positive and negative predictive values for an IFI prevalence of 10% were 83.5% and 94.6%, respectively. CONCLUSIONS: Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.

Expression of pSTAT5 predicts FLT3 internal tandem duplications in acute myeloid leukemia.

Mutations of the Fms-like tyrosine kinase 3 (FLT3) can be detected in a significant number of acute myeloid leukemias (AML). Seventy-five cases of acute myeloid leukemia were evaluated for FLT3-internal tandem duplications (ITD) by polymerase chain reaction. Paraffin-embedded formalin-fixed trephine biopsies of these cases were evaluated for expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT1), pSTAT3, and pSTAT5. Specific expression of pSTAT5 was proven in leukemic blasts in situ by double staining with a blast-specific marker. Expression of pSTAT5 in > or =1% of blasts was highly predictive of FLT3-ITD. Neither expression of pSTAT1 nor pSTAT3 were associated with FLT3 mutations. Altogether we conclude that pSTAT5 expression can precisely be assessed by immunohistochemistry in routinely processed bone marrow trephines, STAT5 is highly likely the preferred second messenger of FLT3-mediated signaling in AML, and expression of pSTAT5 is predictive of FLT3-ITD.

Superfamily of steroid nuclear receptors: positive and negative regulators of gene expression.

The nuclear hormone receptor superfamily is characterized by an impressive functional diversity of its members despite a remarkable overall structural unity. A variety of ligands bind specifically to them and these receptors control gene networks that have profound effects on growth, development, and homeostasis. The ligand-receptor complexes recognize transcriptional enhancer DNA sequences, the hormone response elements, resulting in induction or repression of gene activity. The similarity between all these hormone response enhancer elements, as well as between the receptors themselves, indicates a conserved general strategy for the hormonal control of transcription by steroids. The activated receptors bind to responsive promoters and most likely mediate the assembly of stage- and tissue-specific transcription factor complexes that stimulate or inhibit gene expression.

Evaluation of the xenodiagnosis of chronic Chagas patients infected ten years or over in an area where transmission has been interrupted - Iguatama and Pains, west Minas Gerais State, Brazil

To evaluate the results of xenodiagnosis in chronic Chagas patients infected for ten years or over in an area where transmission has been stemmed as well as the performance of these tests applied one or more times to determine the presence of the paraiste in serum-positive patients for Trypanosoma cruzi infection, 570 xenodiagnosis were performed in 246 patients by exoposing each pacient to 40 Triatoma infestans nymphs of 3 rd/4th stage once, twice or three times, at 30 days intervals. The 570 xenodiagnosis showed overall positive results in 50.7% with a peak 78% in patients under 20 years of age, and 60.5% in those over 60. Of the l58 patients who underwent three xenodiagnosis, 51 (32.3%) had three positive tests, 48 (30.3%) had all negative results, and the remainder had alternating positve and negative findings. There was no difference in number of positive results between the 1st, 2nd and 2rd tests; however, the 1st and 2nd trials added up to 53.2% and the sum total of all three trials was 57.7%.

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

A critical role for the T cell receptor alpha-chain connecting peptide domain in positive selection of CD1-independent NKT cells.

Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.

Detection of the common acute lymphoblastic leukemia antigen in the serum of leukemia patients.

The common acute lymphoblastic leukemia antigen (CALLA) has been detected in biological fluids using a radioimmunoassay based on the inhibition of binding of 125I-labeled monoclonal anti-CALLA antibody to glutaraldehyde-fixed NALM-1 cells. With this assay, we showed first that CALLA was released in culture fluids from NALM-1 and Daudi cell lines but was absent from culture fluids from CALLA negative cell lines. Then, we found that the sera of 34 out of 42 patients (81%) with untreated common acute lymphoblastic leukemia (c-ALL) contained higher CALLA levels than any of the 42 serum samples from healthy controls. The specificity of these results was further demonstrated by testing in parallel the sera from 48 patients with CALLA negative leukemias, including 26 acute myeloid leukemia (AML), 12 T-cell acute lymphoblastic leukemia (T-ALL), and 10 acute undifferentiated leukemia (AUL). All of these sera gave negative results, except for one patient with AUL, who had a significantly elevated circulating CALLA level, and one patient with AML, who had a borderline CALLA level, 3 SD over the mean of the normal sera. Preliminary results suggest that circulating CALLA is associated with membrane fragments or vesicles, since the total CALLA antigenic activity was recovered in the pellet of the serum samples centrifuged at 100,000 g. In addition, the CALLA-positive pellets contained an enzyme considered as a membrane marker, 5'-nucleotidase. Evaluation of the clinical importance of repeated serum CALLA determinations for the monitoring of c-ALL patients deserves further investigation.

Clonal heterogeneity and chromosomal instability at disease presentation in high hyperdiploid acute lymphoblastic leukemia.

Although aneuploidy has many possible causes, it often results from underlying chromosomal instability (CIN) leading to an unstable karyotype with cell-to-cell variation and multiple subclones. To test for the presence of CIN in high hyperdiploid acute lymphoblastic leukemia (HeH ALL) at diagnosis, we investigated 20 patients (10 HeH ALL and 10 non-HeH ALL), using automated four-color interphase fluorescence in situ hybridization (I-FISH) with centromeric probes for chromosomes 4, 6, 10, and 17. In HeH ALL, the proportion of abnormal cells ranged from 36.3% to 92.4%, and a variety of aneuploid populations were identified. Compared with conventional cytogenetics, I-FISH revealed numerous additional clones, some of them very small. To investigate the nature and origin of this clonal heterogeneity, we determined average numerical CIN values for all four chromosomes together and for each chromosome and patient group. The CIN values in HeH ALL were relatively high (range, 22.2-44.7%), compared with those in non-HeH ALL (3.2-6.4%), thus accounting for the presence of numerical CIN in HeH ALL at diagnosis. We conclude that numerical CIN may be at the origin of the high level of clonal heterogeneity revealed by I-FISH in HeH ALL at presentation, which would corroborate the potential role of CIN in tumor pathogenesis.

Positive IgG Western Blot for Borrelia burgdorferi in Colombia

In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF) sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC) for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma), the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation.

Root diseases caused by fungal pathogens can be suppressed by certain rhizobacteria that effectively colonize the roots and produce extracellular antifungal compounds. To be effective, biocontrol bacteria need to be present at sufficiently high cell densities. These conditions favor the operation of positive feedback mechanisms that control the production of antifungal compounds in biocontrol strains of fluorescent pseudomonads, via both transcriptional and post-transcriptional mechanisms.

Liver kidney microsomes type 1 antibodies are associated with 80% reduction of the CYP2D6 activity in patients with chronic hepatitis C

Introduction Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease CYP2D6 activity in vitro. We investigated whether LKM-1 antibodies might reduce CYP2D6 activity also in vivo.Materials and Methods All patients with chronic hepatitis C and LKM-1 antibodies enrolled in the Swiss Hepatitis C Cohort Study (SCCS) were assessed: ten were eligible and fi tted to patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specifi c substrate using the dextromethorphan/dextrorphan (DEM/DOR) metabolic ratio to classify patients into four activity phenotypes (i.e. ultrarapid, extensive, intermediate and poor metabolizers). The concordance between phenotype based on DEM/DOR ratio and phenotype expected from genotype was examined in LKM-1 positive and negative patients. Groups were compared with respect to the DEM/DOR metabolic ratio.Results All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with CYP2D6 genotype in most LKM-negative patients, whereas only three (30%) LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was six-fold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, p = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies.Conclusion In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies in the setting of new protease inhibitor therapies