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Febrile neutropenia and sepsis are common and life-threatening complications in hematological diseases. This study was performed retrospectively in 514 patients treated for febrile neutropenia at our institute, to investigate the clinical usefulness of a molecular tool, LightCycler® SeptiFast test (SF), to promptly recognize pathogens causing sepsis in hematological patients. We collected 1837 blood samples of 514 consecutive hematological patients. The time of processing is short. Overall, 757 microorganisms in 663 episodes were detected by molecular test and standard blood cultures (BC): 73.6% Gram-positive bacteria, 23.9% Gram-negative bacteria, and 2.5% fungal species. This large analysis demonstrated a significant episode-to episode agreement (71.9%) between the two methods, higher in negative samples (89.14%), and a specificity of 75.89%. Clinical variables that gave a statistically significant contribution to their concordance were absolute neutrophil count, ongoing antimicrobial therapy, timing of test execution, and organ localization of infection. The large analysis highlights the potential of molecular-based assays directly performed on blood samples, especially if implementing the detection of antibiotic resistance genes, which was lacking in the used study.

Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene®) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.

Background Infections and graft-vs-host disease (GvHD) still represent major, not easily predictable complications in allogeneic hematopoietic stem cell transplant (allo-HSCT). Both conditions have been correlated to altered enteric microbiome profiles during the peritransplant period. The main objective of this study was to identify possible early microbiome-based markers useful in pretransplant risk stratification. Methods Stool samples were collected from 96 consecutive patients at the beginning of the pretransplant conditioning regimen (T0) and at 10 (T1) and 30 (T2) days following transplant. When significant in univariate analysis, the identified microbiome markers were used in multivariate regression analyses, together with other significant clinical variables for allo-HSCT-related risk stratification. Four main outcomes were addressed: (1) septic complications, (2) GvHD, (3) relapse of the underlying disease, and (4) mortality. Results The presence of >5% proinflammatory Enterobacteriaceae at T0 was the only significant marker for the risk of microbiologically confirmed sepsis. Moreover, ≤10% Lachnospiraceae at T0 was the only significant factor for increased risk of overall mortality, including death from both infectious and noninfectious causes. Finally, a low bacterial alpha-diversity (Shannon index ≤ 1.3) at T1 was the only variable significantly correlating with an increased risk of GvHD within 30 days. Conclusions Microbiome markers can be useful in the very early identification of patients at risk for major transplant-related complications, offering new tools for individualized preemptive or therapeutic strategies to improve allo-HSCT outcomes.

We evaluated the Verigene Gram-negative blood culture (BC-GN) test, a microarray that detects Gram-negative bacteria and several resistance genes. A total of 102 positive blood cultures were tested, and the BC-GN test correctly identified 97.9% of the isolates within its panel. Resistance genes (CTX-M, KPC, VIM, and OXA genes) were detected in 29.8% of the isolates, with positive predictive values of 95.8% (95% confidence interval [CI], 87.7% to 98.9%) in Enterobacteriaceae and 100% (95% CI, 75.9% to 100%) in Pseudomonas aeruginosa and negative predictive values of 100% (95% CI, 93.9% to 100%) and 78.6% (95% CI, 51.0% to 93.6%), respectively.

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 We identified by multivariate analysis some associations between the relative increase of a specific phylum and clinical variables at baseline. Previous allo-HSCT and prior infections represented strong risk factors for developing colonization by Proteobacteria (exceeding 5%). Sepsis by GN-MDR bacteria was associated to increase in Enterobacteriaceae(exceeding 5%; p 0.011). Interestingly, significant microbioma changes were observed at 10 days after transplant, in patients who developed a subsequent acute GvHD, with a predominant role played by gram-positive bacteria belonging to Firmicutes. More in details, the presence of Lachnospiraceae was associated to a decreased risk of developing acute GvHD (p=0.04 and RR= 4.35), whereas dominance of Enterococcaceae (p CONCLUSIONS: Longitudinal study of microbioma profile allows early identification of patients at risk for major transplant-related complications such as sepsis by GN-MDR or acute GvHD, offering a new tool for individualized pre-emptive or therapeutical strategies to improve the outcome of allo-HSCT. Disclosures Ciceri: MolMed SpA: Consultancy.

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.

Ascaridiasis is a geohelminthiasis with a worldwide distribution, especially occurring in countries with hot-humid climates. The infection occurs most frequently in children between three and nine years of age. We report a case of ascaridiasis regarding a ten-year-old girl born in Bangladesh who arrived in Italy about one year before. During laparotomy for suspected acute appendicitis, a large number of ascaridia nematodes were seen in the small intestine.

Visceral leishmaniasis (VL) is endemic in Sicily (48 new cases in 2004, of which nine were in Agrigento). In southern Europe between 25-70 per cent of adult VL cases are related to HIV infection. The HIV cases have a high risk (1.5-9%) of developing VL either as a new infection or as the revival of a latent infection. We therefore carried out serologic screening to detect antibodies against L. infantum by IFAT in 1449 blood donors in Agrigento and the surrounding area (May-December 2005) and in 120 HIV+ in western Sicily, all of whom were asymptomatic and had no history of VL. L. DNA was assessed by nested PCR in blood samples of some seropositive donors. Of the 1449 blood donors, 11 (0.75%) were positive by IFAT and three of them were also positive in PCR. L. infantum seropositivity is most probably the expression of recent infection because the clearance of serum antibodies is rather fast (6-12 months) after VL. This is why blood donation by Leishmania seropositive donors, whether positive or negative by PCR, could constitute an infection risk especially for immunosuppressed recipients, who should receive deleukocyted blood. Moreover it could be useful to monitor HIV/Leishmania coinfection cases to avoid the risk of slatentization of L. infection when CD4+ levels are very low.

Aim: Human toxocariasis is caused by infection with the larval stage of nematode parasites of dogs and cats, Toxocara canis or Toxocara cati. These helminths are not able to complete their life cycle in undefinitive hosts and so undergo aberrant migrations in the tissues causing a wide spectrum of signs and symptoms. Eosinophilia is often severe and sometimes represents the only sign of infection, except in ocular and neurological forms. Methods: We describe the clinical features of nine children affected by toxocariasis admitted to our Infectious Diseases department from 2004 to 2006. Results: Fever and hepatomegaly were the most common clinical findings. In two cases eosinophilia was not present. Diagnosis was performed by enzyme-linked immunosorbent assay employing excretory–secretory antigens of Toxocara. canis larvae. All patients were successfully treated with oral albendazole with no side effects. Conclusion: Toxocariasis should be considered in differential diagnosis of eosinophilia and in patients with seizures of uncertain origin, isolated hepatomegaly and splenomegaly, bronchospasms or skin rash.

Actinomycosis is a bacterial infection due to Gram-positive bacteria of the Actinomyces genus. The authors describe two cases: one of them occurred in a woman with a clinical presentation of a right submandibular indurative mass. After surgery, histological examination showed a granulomatous tissue with many actinomycetes. The second case was observed in a man admitted to hospital with a diagnosis of pulmonary heteroplastic lesion. Also in this case, histological examination showed many actinomycotic colonies. Actinomycosis is an uncommon disease. Establishment of definite diagnosis requires a high index of suspicion in all cases of relapsing cervical lesions. Pulmonary actinomycosis is a rare diagnosis; respiratory physicians should be aware of this important differential when investigating patients for persistent pulmonary shadowing.

The purpose of this study was to evaluate whether the risk of transfusion-transmitted visceral leishmaniasis was present in an area of western Sicily where the incidence of the disease is higher than the regional average. From May to December 2005, 1449 blood donors from Agrigento district (Sicily, Italy) were screened for the presence of anti-Leishmania antibodies by an indirect immunofluorescent antibody test (IFAT). Blood samples from IFAT-positive donors were examined by PCR to detect Leishmania DNA. Anti-Leishmania antibodies were found in 11 (0.75%) cases, among which Leishmania DNA was detected from four (36.4%). Particular techniques to inactivate different pathogens would be considered mandatory in the case of immunosuppressed recipients.

Strongyloidiasis is caused by a small intestinal nematode with a complex life cycle. In Italy the infection is endemic in rural areas of the Po Valley. The clinical syndrome of S. stercoralis encompasses a broad spectrum of symptoms and signs and, in the immunocompromised host, larvae can migrate to different organs and tissues. Also immune response seems to play a role in the pathogenesis of the disease. We report a case of strongyloidiasis complicated by Gram-negative sepsis and nephrotic syndrome in an immigrant from South America with a normal immune response. Whereas sepsis cleared up quickly, parasitic clearance was obtained only after treatment with ivermectin and nephrotic syndrome was still present three months after the end of treatment.

Actinomycosis is a bacterial infection due to Gram positive bacteria of Actinomyces genus. Actinomyces israelii is the most common in human infection.The authors described a case occurred in a 43 aged woman with a clinical presentation of a right submandibular indurative mass.After surgery, histological examination showed a granulomatous tissue with many actinomycetes. Patient was submitted to treatment with i.v penicillin. On the 11th day antibiotic therapy was replaced with ceftriaxone and teicoplanina. Patient did not present intercurrence during this period and presented improvement of the lesion.Actinomycosis is an uncommon disease. Establishment of the definite diagnosis requires a high index of suspicion in all cases of relapsing cervical lesions. Penicillin is effective and it can be replaced by third-generation cephalosporins.

Background Typhoid fever is a systemic infection caused by the bacterium Salmonella enterica subspecies enterica serotype typhi, which is acquired by ingestion of contaminated food and water. Each year the disease affects at least 16 million persons world-wide, most of whom reside in the developing countries of Southeast Asia and Africa. In Italy the disease is uncommon with a greater number of cases in Southern regions than in Northern ones. Case presentation We report on a 57-year-old Sri-Lankan male affected by typhoid fever, the onset of which was accompanied by oropharyngeal candidiasis. This clinical sign was due to a transient cell-mediated immunity depression (CD4+ cell count was 130 cells/mm3) probably caused by Salmonella typhi infection. Human immunodeficiency virus infection was ruled out. Diagnosis of typhoid fever was made by the isolation of Salmonella typhi from two consecutive blood cultures. The patient recovered after a ten days therapy with ciprofloxacin and his CD4+ cell count improved gradually until normalization within 3 weeks. Conclusion Our patient is the first reported case of typhoid fever associated with oropharyngeal candidiasis. This finding suggests a close correlation between Salmonella typhi infection and transitory immunodepression.

Diagnosis of schistosomiasis in migrants coming from endemic areas can be difficult, especially in asymptomatic subjects. Light-intensity disease, in fact, may be missed due to the low sensitivity of the stool microscopy and serologic testing cannot distinguish between a resolved infection and an active infection in patients who have been infected and treated in the past, because specific antibodies can persist despite cure. We describe a cross-sectional study conducted on 82 migrants tested for Schistosoma mansoni on single blood (anti-schistosome antibodies, total IgE) and urine [point-of-care (POC) circulating-cathodic-antigen (CCA) test] samples. A positive POC-CCA test (active infection) resulted in two untreated patients with a positive serology while all patients (n = 66) with a past infection showed a negative POC-CCA test. POC-CCA urine test in combination with serology may be helpful in rapidly differentiate active from past S. mansoni infection in migrants coming from endemic areas.