Facing the Rising Tide of Multidrug Resistant Gram-Negative Pathogens in Allogeneic HSCT with Nanosphere's Verigene® System
Authors: Greco Raffaella , Barbanti Maria Chiara , Mancini Nicasio , Infurnari Laura , Pasciuta Renée , Forcina Alessandra , Vago Luca , Giglio Fabio , Messina Carlo , Morelli Mara , Perini Tommaso , Girlanda Stefania , Carrabba Matteo G , Lupo Stanghellini Maria Teresa , Marktel Sarah , Assanelli Andrea , Bernardi Massimo , Corti Consuelo , Peccatori Jacopo , Clementi Massimo , Ciceri Fabio 
Published: October 2019

Abstract
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.