ALBERT

Description: Infections are the major cause of treatment-related morbidity and mortality in hematological patients, mostly after allogeneic hematopoietic stem cell transplantation (HSCT). Although the proportion of infection-related deaths has decreased in the past two decades, the alarming emergence of multidrug resistant (MDR) gram-negative pathogens, increasingly reported from various cancer treatment centers, represents a significant challenge. Dramatic infection-related mortality (64.4%) have been observed in allogeneic HSCT, due to carbapenem-resistant Klebsiella pneumoniae (CRKp). Timely microbiological diagnosis, active surveillance and early therapeutic strategies represent critical aspects for the management of MDR infections after allogeneic HSCT. In this study, we investigated whether a new molecular tool could be clinically relevant in the management of high-risk hematological patients, particularly in allogeneic HSCT. At San Raffaele Hematology and BMT Unit, all blood cultures positive for gram-negative bacteria in the last year, were prospectively collected and in parallel tested with Verigene® Gram-negative blood culture (BC-GN) test (Nanosphere, Northbrook, IL, USA), a microarray-based system allowing a rapid identification of genus, species, and genetic resistance determinants for a broad panel of gram-negative bacteria directly from positive blood cultures. Initially, we evaluated the reliability of the BC-GN test on 50 consecutive patients undergoing chemotherapy (n=16), autologous (n=4) or allogeneic (n=30) HSCT. The concordance with the standard blood culture was 100% considering the bacteria detectable by the system. Resistance genes (CTX-M or carbapenemases, as KPC and VIM) were detected in 15% of the isolates, and 100% were associated to the same phenotypic antibiotic resistance. We observed only 8% of phenotypic resistances not detected by the test, belonging to other kinds of resistance mechanisms not related to the genes included in the panel. Overall, Verigene BC-GN assay correctly identified 40/50 of all the gram-negative pathogens (3 Klebsiella pneumonia, 25 Escherichia coli, 5 Pseudomonas aeruginosa, 4 Acinetobacter spp, 1 Enterobacter spp, 2 Citrobacter spp), yielding a general sensitivity of 80%, which increased to 100% if only the genera and species included in the panel were considered. Then, we examined the potential clinical impact of this molecular approach in allogeneic HSCT recipients (n=30), either in an inpatient (n=24) or outpatient (n=6) management. As far as transplanted patients' care is concerned, the BC-GN test strongly influenced clinical practice. In the majority of cases we were able to early start targeted antibiotic therapy (78%), sparing or interrupting non-specific antimicrobial therapy (56%), thus reducing useless and/or potentially more toxic antibiotics and their potential impact in favouring antimicrobial resistance. Moreover, in 5/8 cases the 'triple therapy', recommended at fever onset for all CRKp-colonized patients, was spared thanks to BC-GN test results, showing the absence of resistance markers. In 95% of patients immunosuppressive prophylaxis was not reduced, thanks to a rapid control of sepsis, avoiding the risk of undesired GVHD. Early contact isolation was possible in 32% of patients, preventing the spread of infections from a patient to the others and allowing infection control. Infection-related mortality was reported only in one patient. An outpatient management was continued in 3/6 haemodynamically stable patients, avoiding unnecessary hospitalization. While conventional microbiological methods required 2-4 days, BC-GN test provided detailed results within 2 hours from blood culture positivity. The mean gain in time comparing BC-GN test to fast blood cultures was reported to be about 20 hours. In our experience, this new microarray test has provided highly accurate identification results and earlier potentially important information on antibiotic susceptibility, both confirming and excluding the presence of an MDR phenotype. The contribution to a rapid diagnosis and an earlier targeted antimicrobial therapy of Verigene BC-GN test is of high clinical relevance for the infections by MDR bacteria, a significant public health challenge worldwide, particularly in allogeneic HSCT recipients. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Allogeneic HSCT (allo-HSCT) is the only cure for several patients with hematological malignancies. Recent advances in therapies have significantly reduced transplant-related mortality (TRM); nevertheless infections and graft-versus-host disease (GvHD) still represent major complications. Recent studies indicate that patients undergo dramatic alterations of intestinal microbiota during allo-HSCT, potentially affecting the outcome. An alarming emergence of gram-negative multi-drug-resistant (GN-MDR) pathogens has been increasingly reported from various cancer centers, and the primary site of colonization is mainly the gut. Moreover, GvHD was associated with major shifts in the composition of intestinal microbiota, able to modulate the severity of intestinal inflammation. METHODS: Between October 2014 and March 2016, we have conducted a prospective observational study to examine the intestinal microbiota by NGS techniques in 100 consecutive adult patients, who received allo-HSCT for high-risk hematological malignancies (63.5% acute leukemia). Stem cell donors were family haploidentical (n=45), HLA identical sibling (n=15), unrelated volunteer (n=35), cord blood (n=5). Stem cell source was mainly T-cell replete PBSCs. Fecal specimens have been collected before conditioning (T0), during aplasia (T10) and after engraftment (T30). The fecal microbiome have been analyzed using the 454 GS Junior System, and QIIME software. We defined a threshold of normality for the main phyla on the basis of the control. RESULTS: We observed an important difference in relative percentages of phyla populating the gut between our pts and control. Our patients showed a progressive reduction of the intestinal microbial diversity (alpha diversity) during the transplant process, with a specific decrease of anaerobic species belonging to both Bacteroidetes and Firmicutes phyla, and a relative increase of facultative anaerobic gram-positive families such as Enterococcaceae and Staphylococcaceae. A great variation was observed, particularly between T0 and T30, where we found a significant decrease in alpha diversity (p 〈 0.001). A significantly different distribution in the baseline microbiome was reported in patients who will experience different clinical outcomes. Patients (n=23) who developed a microbiologically-confirmed sepsis show an increase of Proteobacteria phylum (cut-off 5%; p