Kinetics and Role of Pancreatic Stone Protein and Midregional Proadrenomedullin as Predictors of Sepsis and Bacteremia in Children with Hematological Malignancies.

Background: To investigate the kinetics and prognostic value of pancreatic stone protein (PSP) and mid-regional proadrenomedullin (MR-proADM) during episodes of febrile neutropenia (FN) in children with hematological malignancies. Material and methods: We evaluated prospectively a total of 70 FN episodes in 70 children with acute leukemias and lymphomas. CRP, PSP, and MR-proADM levels were measured at the onset of the febrile episode (day 1), day 3, and day 7. The outcome and survival of children were evaluated during the study period until day 28. The performance of each marker in identifying sepsis or severe sepsis was assessed as an area under a receiver operating characteristic (ROC) curve. ROC curves were used for each biomarker to derive cut-offs for sensitivity and specificity in distinguishing sepsis from non-sepsis. Results: During the 2-year study period, 70 febrile neutropenia episodes in 70 children with hematological malignancies were enrolled. Of 70 episodes of febrile neutropenia, in 17 (24%), a bacterial/fungal infection was documented. Criteria for sepsis were fulfilled for 31 (44%) and 7 (10%) patients were admitted to PICU. The median values of all biomarkers on day 1 differed significantly between patients with and without sepsis. PSP, MR-proADM, and CRP specificity were 0.82, 0.70, and 0.57, respectively. The sensitivity of PSP, MR-proADM, and CRP were 0.84, 0.74, and 0.88, respectively. Conclusions: PSP and MR-proADM are promising biomarkers for early diagnosis of sepsis during FN episodes in children with hematological malignancies. However, PSP has a higher sensitivity and specificity.

Association of cytokine gene polymorphisms with peripheral neuropathy susceptibility in people living with HIV in Greece.

Relatively little research has been done in recent years to understand what leads to the unceasingly high rates of HIV sensory neuropathy despite successful antiretroviral treatment. In vivo and in vitro studies demonstrate neuronal damage induced by HIV and increasingly identified ART neurotoxicity involving mitochondrial dysfunction and innate immune system activation in peripheral nerves, ultimately all pathways resulting in enhanced pro-inflammatory cytokine secretion. Furthermore, many infectious/autoimmune/malignant diseases are influenced by the production-profile of pro-inflammatory and anti-inflammatory cytokines, due to inter-individual allelic polymorphism within cytokine gene regulatory regions. Associations of cytokine gene polymorphisms are investigated with the aim of identifying potential genetic markers for susceptibility to HIV peripheral neuropathy including ART-dependent toxic neuropathy. One hundred seventy-one people living with HIV in Northern Greece, divided into two sub-groups according to the presence/absence of peripheral neuropathy, were studied over a 5-year period. Diagnosis was based on the Brief Peripheral Neuropathy Screening. Cytokine genotyping was performed by sequence-specific primer-polymerase chain reaction. Present study findings identify age as an important risk factor (p < 0.01) and support the idea that cytokine gene polymorphisms are at least involved in HIV peripheral-neuropathy pathogenesis. Specifically, carriers of IL1a-889/rs1800587 TT genotype and IL4-1098/rs2243250 GG genotype disclosed greater relative risk for developing HIV peripheral neuropathy (OR: 2.9 and 7.7 respectively), while conversely, carriers of IL2+166/rs2069763 TT genotype yielded lower probability (OR: 3.1), all however, with marginal statistical significance. The latter, if confirmed in a larger Greek population cohort, may offer in the future novel genetic markers to identify susceptibility, while it remains significant that further ethnicity-oriented studies continue to be conducted in a similar pursuit.

Molecular Advances in Sinusoidal Obstruction Syndrome/Veno-Occlusive Disease.

Sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) detected in the liver has been considered a severe complication of hematopoietic stem cell transplantation (HSCT). SOS/VOD is characterized by hepatomegaly, right upper quadrant pain, jaundice, and ascites. The severe forms of the disease may result in multi-organ dysfunction (MOD) with a high mortality rate (>80%). The development of SOS/VOD can be rapid and unpredictable. Therefore, early identification and severity assessment is crucial in facilitating prompt diagnosis and timely treatment. Effective treatment and potential prophylaxis with defibrotide highlight the need for characterizing a sub-group of patients at high risk for SOS/VOD. Moreover, antibodies that are conjugated with calicheamicin, gemtuzumab, and inotuzumab ozogamicin, have led to renewed interest in this syndrome. Evaluation and management of serious adverse events associated with gemtuzumab and inotuzumab ozogamicin are recommended. We review hepatic-, transplant- and patient-related risk factors, criteria for diagnosis and grading classification, and SOS/VOD potential biomarkers. Furthermore, we examine pathogenesis, clinical presentation, diagnostic criteria, risk factors, prophylaxis, and treatment of SOS/VOD occurring post HSCT. Moreover, we aim to provide an up-to-date summary of molecular advances in the diagnosis and management of SOS/VOD. We performed a comprehensive review of the literature and examined the recently available data, mostly using the PubMed and Medline search engines for original articles published over the last decade. In the era of precision medicine, our review provides up-to-date knowledge of genetic or sera markers for SOS/VOD with the goal of identifying a subset of high-risk patients.

Clinical Characteristics and Risk Factors for Bloodstream Infections in Children with Cancer: A Report from a Pediatric Hematology Oncology Unit.

BACKGROUND/AIM: Infections are a major cause of morbidity and mortality in children with haematologic malignancies and solid tumors as well as those undergoing hematopoietic stem cell transplantation (HSCT). The purpose of our study was to record the epidemiological characteristics and outcomes of bacteremias, focusing on pathogens, as well as risk factors and mortality rates in patients of a pediatric hematology-oncology unit from Northern Greece. MATERIALS AND METHODS: A retrospective analysis was conducted, which included all positive blood cultures from pediatric hematology oncology patients aged from 1 to 16 years old admitted to the Pediatric and Adolescent Hematology Oncology Unit of AHEPA University Hospital of Thessaloniki between January 2014 and December 2018. Data were collected from patients' printed and electronic medical records. RESULTS: 73 episodes of bacteremias were identified (41% male and 32% female with a ratio of 1.28:1; median age 6.5 years; 13.7% solid tumor, 72.6% acute lymphoblastic leukemia, 13.7% acute myeloid leukemia, and 95.8% with an indwelling permanent catheter). 49.3% of the isolates were Gram-positive bacteria and 50.7% Gram-negative, and the ratio of Gram-negative to Grampositive was 1.02. Coagulase-negative staphylococci were most frequent (39.7%), followed by E. coli (17.8%) and Klebsiella pneumoniae (17.8%). Out of all Gram-negatives, 13.5% carbapenemase producers and 8.1% ESBL-producers were found. In relation to Gram-positive, 79.3% were identified as methicillin-resistant CoNS. During the study period, 10.9% of indwelling catheters were removed, and 2.73% of episodes resulted in ICU transfer. The 3-month mortality rate was 8.2%. CONCLUSION: This study demonstrated an almost equal distribution of Gram-positive and Gramnegative bacteremias in total in this population but with an increase in the isolation of Grampositive bacteria over the last years, which is consistent with other similar studies in this patient group. Knowledge of the local epidemiology and bacterial antimicrobial resistance is important to prevent and timely treat these life-threatening infections in immunocompromised pediatric oncology patients.

Reduced expression of L-selectin in T-cells correlates with relative lymphocyte increase in patients with RRMS treated with natalizumab - functional implication towards PML risk.

Objectives: Natalizumab (NTZ), a treatment indicated for patients with highly active Relapsing - Remitting Multiple Sclerosis (RRMS), is known to induce increased relative frequency of lymphocytes. Progressive Multifocal Leukoencephalitis (PML) is a rare but serious adverse event related to NTZ. Moreover, reduced L-selectin (CD62L) expression in T-cells in cryopreserved samples of patients with RRMS under NTZ has been proposed as a biomarker of pre-PML state. We explore the association between L-selectin expression in T-cells and hematological parameters in freshly processed samples of patients with RRMS under NTZ.Methods: We studied L-selectin expression in patients with: RRMS under NTZ (n=34), fingolimod (FTY, n=14), interferon-beta (IFNß, n=22), glatiramer acetate (GA, N=17); in 9 patients with secondary progressive (SP) MS and in 6 healthy controls. Twenty-two patients under NTZ and 6 patients under FTY were followed for 18 months. One NTZ-treated patient developed PML during the study.Results: Patients under NTZ exhibited increased relative frequency of lymphocytes (40.02±1.45) compared to patients under first-line treatment (30.57±1.68, p<0.001) and to patients with SPMS (29±1.56, p=0.02), and a lower mean L-selectin expression in (69.39±1.73) compared to patients under first-line treatment (79.1±1.17, p=0.003). A negative correlation between the relative frequency of CD4+CD62L+ T-cells and the absolute lymphocyte counts (Pearson's r=0.367, p=0.033) was observed.Discussion: We hereby provide mechanistic insight in a possible pathway implicated in NTZ-related PML risk. These results further underline the need for thorough validation of L-selectin expression in T-cells as a potential pre-PML biomarker.

Evaluation of a 10color protocol as part of a 2tube screening panel for flow cytometric assessment of peripheral blood leukocytic subsets.

Peripheral blood (PB) immunophenotyping is commonly required for initial evaluation of various suspected disease entities. Several approaches have been proposed. The objective of this work is to explore the value of a 10color protocol developed in our laboratory for flow cytometric assessment of PB leukocytic subsets, as part of a 2tube screening panel. A combination of CD16/CD56/CD34/CD33/CD19/CD4/CD8/CD3/CD20/CD45 antibodies in 1 tube was applied routinely during flow cytometric analysis of PB samples for diagnostic purposes. The protocol was systematically complemented by a 2nd tube with anti-kappa, anti-lambda, CD5, CD19, and CD45 antibodies for adults and selected pediatric patients, and specifically oriented panels when necessary. 25 samples with no detectable neoplastic PB involvement and 31 samples with a hematolymphoid disorder were investigated retrospectively. The contribution of CD33 in the separation of leukocytic populations, as well as the benefits from the simultaneous assessment of CD20/CD19/CD45, CD16/CD56 and the detection of CD34+ cells were examined. The gating strategy with the use of CD33 provided additional information in certain cases. The protocol enabled recognition of differential expression of CD20 and CD45 in CD19+ cells with chronic lymphocytic leukemia phenotype, overall evaluation of NK and NK like T cells, estimation of CD16- granulocytes and CD56/CD16 expression in monocytes, as well as identification of minor cell subsets, such as CD34+ cells. The proposed 10color combination of antibodies analyzed in a standardized manner can offer significant information in the initial evaluation of PB samples, thus, guiding subsequent investigation if needed.

CD5+ B lymphoproliferative disorder with subsequent development of plasma cell leukaemia: Diagnostic and aetiologic reasoning.

BACKGROUND: Plasma cell myeloma (PCM) has been sporadically reported to occur simultaneously or subsequently to mature B lymphoproliferative disorders (LPDs), predominantly chronic lymphocytic leukaemia (CLL). METHODS: We describe the clinical and laboratory findings of a 69-year-old male patient who developed plasma cell leukaemia (PCL) 8 years after an initial diagnosis of a low stage CD5+ B LPD and 3 years after treatment for LPD. RESULTS: The transition from a clinically indolent B LPD to an aggressive PCM was documented by bone marrow (BM) biopsy, while flow cytometric (FC) immunophenotyping conferred additional information by disclosing the co-existence of both disorders in BM and the presence of abnormal monotypic PCs in peripheral blood above PCL levels. Phenotypic findings suggested a discrete clonal origin of the two disorders. CONCLUSIONS: This report of PCL development in a patient with residual CD5+ B LPD, emphasizes the need for comprehensive diagnostic evaluation of such cases and scrutiny of their aetiological relationship, including FC immunophenotyping due to its high analytical sensitivity and multiparametric capacity compared to morphology or immunohistochemistry alone. © 2017 International Clinical Cytometry Society.

A two-step approach improves the diagnosis of Clostridium difficile infection.

Clostridium difficile infection (CDI) is a major cause of health care-associated diarrhea. The aim of the present study was to evaluate a two-step approach for the diagnosis of CDI. The two-step procedure consisted of GDH-toxin A/B EIA (Enzyme immunoassay targeting enterotoxin A and Cytotoxin B), followed by PCR detecting toxigenic C. difficile. Results indicate that EIAs provide a rapid screening assay for the laboratory diagnosis of CDI but, in GDH-positive and toxins-negative samples, EIA should be always followed by PCR to distinguish toxigenic vs nontoxigenic strains. GDH-toxin A/B EIA-rapid test has high specificity but low sensitivity to detect CDI. The implementation of a two-step procedure significantly increases the diagnostic accuracy to detect CDI and provides a toxigenic type characterization of C. difficile isolates.

Utility of hematology analyzer and flow cytometry in timely and correct detection of circulating plasma cells: Report of three cases.

BACKGROUND: Circulating neoplastic plasma cells (PCs) are a common feature between classical plasma cell myeloma (PCM) and its aggressive variant, plasma cell leukemia (PCL), with their early suspicion and detection being of clinical importance for prognostic and diagnostic purposes. METHODS: Morphological and flow cytometric enumeration and characterization of peripheral blood (PB) neoplastic PCs were performed in 3 PCM patients after complete blood count (CBC) evaluation. RESULTS: Early suspicion of circulating PCs by initial CBC scatter plots and monocytosis led to morphological enumeration of PCs, with low level of concordance between morphologists. Flow cytometric PB analysis permitted accurate quantification and characterization of circulating clonal PCs. CONCLUSIONS: CBC findings can prompt early suspicion of circulating PCs in PCM patients leading to early and accurate flow cytometric analysis of clonal PCs in addition to morphological PB evaluation. The suggested combined approach proved to be most important in the PCL case with discordant morphological evaluations and marginal PC numbers according to current criteria. © 2016 International Clinical Cytometry Society.

The controversial impact of B cells subsets on immune response to pneumococcal vaccine in HIV-1 patients.

BACKGROUND: Chronic HIV infection leads to severe perturbations of the B cell populations and hypo-responsiveness to vaccines. The associations between circulating B cell subpopulations and the antibody response to pneumococcal polysaccharide vaccine in antiretroviral-naïve and treated patients were studied. METHODS: Sixty-six HIV-infected adults were grouped according to antiretroviral therapy (ART) and CD4+ cell count; 31 were ART-naïve and 35 were ART-treated, and they were matched for age, CD4 cell count, and duration of HIV infection. All subjects were immunized with the 23-valent polysaccharide vaccine against Streptococcus pneumoniae. Pre- and post-vaccination B cell subpopulations were assessed by flow cytometry. Serum IgG concentrations for vaccine serotypes were quantified by ELISA at baseline and at 4 and 48 weeks post-vaccination. RESULTS: Patients under highly active antiretroviral therapy (HAART) had significantly higher antibody levels against pneumococcal vaccine antigens, while an adequate number of patients responded to vaccination. Memory B cells were diminished over time, although treated patients maintained higher levels of all subsets studied, with the exception of activated memory and isotype-switched memory B cells. CONCLUSIONS: Low concentrations of total B cells and exhausted memory B cells was the strongest independent predictor of poor pneumococcal vaccine responsiveness, emphasizing that B cell subset disturbances are associated with a poor vaccine response among HIV-infected patients.

Effects of simvastatin on markers of inflammation, oxidative stress and endothelial cell apoptosis in patients on chronic hemodialysis.

AIM: We investigated the effects of simvastatin on markers of inflammation, oxidative stress and endothelial cell apoptosis in hyperlipidemic endstage renal disease patients on chronic hemodialysis (HD). METHODS: In 25 hyperlipidemic HD patients who received 10 mg of simvastatin for 6 months and another 25 controls, the extended lipid profile and serum hsIL-6, MCP-1, sICAM-1, sVCAM-1, and sE-selectin, plasma oxLDL, and serum sFas and sFasL levels were determined at baseline, 3 months and 6 months. In 18 patients of the simvastatin group, the expression of CD14, CD16, CD62L and CD64 on monocyfes was determined with flow cytometry. RESULT: Simvastatin treatment resulted in significant reductions in serum lipid levels at 3 months and beyond, compared to at baseline. Moreover, at 6 months, simvastatin caused a significant reduction in CRP (p < 0.001), which correlated to the decrease in total and LDL cholesterol levels, as well as a significant reduction in IL-6 (p=0.001), sICAM-1 (p < 0.001), sVCAM-1 (p < 0.001), oxLDL (p=0.001), sFas (p=0.02) and CD14 expression (p < 0.001), compared to baseline values. No significant changes in the controls were noticed during the study. CONCLUSION: In conclusion, in hyperlipidemic HD patients, simvastatin treatment resulted in a significant reduction in markers of endothelial dysfunction, inflammation, oxidative stress, endothelial cell apoptosis and peripheral blood monocyte stimulation. The reduction in CRP appears to be related to the lipid-lowering effects of simvastatin.

HLA Class II alleles and the presence of circulating Epstein-Barr virus DNA in Greek patients with nasopharyngeal carcinoma.

BACKGROUND AND PURPOSE: Nasopharyngeal carcinoma (NPC) represents a seldom malignancy in most developed countries. Nevertheless, NPC receives an endemic form in concrete racial entities. The aims of this study were to detect the presence of Epstein-Barr virus DNA (EBV-DNA) in peripheral blood of NPC patients, to molecularly define human leukocyte antigens (HLA) DRB1*, DQA1* and DQB1* allele frequencies, and, finally, to determine whether the genetic predisposition of an individual to NPC depends on the liability to EBV infection. PATIENTS AND METHODS: A total of 101 patients of Hellenic origin and nationality, with histologically proven NPC, participated in this study. EBV-DNA detection was also applied in 66 patients with EBV-related malignancies (Hodgkin's [HL] and non-Hodgkin's lymphoma [NHL]) and infectious mononucleosis (IM), as well as in 80 healthy EBV-seropositive controls. RESULTS: 81% of the NPC patients, 77.8% with HL, 72.2% with NHL, and 66.7% with IM were EBV-DNA positive, whereas the EBV genome was detected only in 15% of the healthy controls. These differences were statistically significant in all cases. Analysis of HLA class II antigens showed decreased frequency of the DRB1*07 (p = 0.003), DQA1*0103 (p = 0.002), and DQA1*0201 (p = 0.003) alleles among NPC patients. A significant association between the HLA-DR/DQ alleles and the presence of EBV-DNA in peripheral whole blood was not established. CONCLUSION: Circulating EBV-DNA and specific HLA class II alleles may predispose to or protect from NPC. However, the results of this study suggest that the genetic predisposition of an individual to NPC is independent of the liability to EBV infection.

Impact of endograft material on the inflammatory response after elective endovascular abdominal aortic aneurysm repair.

The purpose of this paper is to examine the impact of endograft material on the inflammatory response after elective endovascular abdominal aortic aneurysm repair. Consecutive patients (n = 22, all men, 53 to 82 years old) were divided into 2 groups according to the graft material used: In group A (n = 12) the endovascular device was made of polyester and in group B (n = 10) the device was made of expanded polytetrafluoroethylene (ePTFE). All patients received antiinflammatory drugs in the perioperative period. Fever, white blood cells and platelet count, serum concentrations of cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-alpha], interleukin 8 [IL-8], acute-phase proteins high-sensitivity C-reactive protein [hsCRP] and alpha1-antitrypsin [alpha1-antitrypsin]), and complement protein (C3a) were measured preoperatively and 1, 3, 6, 24, 48, and 72 hours after aneurysm exclusion. One patient in each group had a systemic inflammatory response syndrome with 2 of the systemic inflammatory response syndrome (SIRS) criteria. No other complication associated with inflammation were present in any patient. Fever was more frequent in group A patients. Increases of white blood cells and serum concentrations of IL-6, TNF-alpha, hsCRP, alpha1-antitrypsin, and C3a and decrease of platelet count were recorded in both groups, but no statistically significant difference between them was recorded. However, serum concentrations of IL-8 were significantly higher in group A patients 24 hours postoperatively (p = 0.01). No significant difference was apparent in the biological response between patients receiving a polyester or an ePTFE stent graft, except for fever and serum concentrations of IL-8.