Development and Validation of a Novel LC-MS/MS Method for a TDM-Guided Personalization of HSCT Conditioning with High-Dose Busulfan in Children.

Personalization of busulfan (Bu) exposure via therapeutic drug monitoring (TDM) is recommended for patients treated with high-dose conditioning regimens. Several laboratories' developed methods are available in the literature with a lack of standardization. The aim of this study is to develop a new standardized LC-MS/MS method and validate it according to the international ICH M10 (EMA) guidelines. Our method is based on rapid protein precipitation from 50 µL plasma followed by separation on a reversed-phase C-18 UHPLC column after the addition of deuterated internal standard and has been tested on real samples from pediatric patients treated with myeloablative conditioning regimens, including Bu, before autologous or allogeneic hematopoietic stem cell transplantation (HSCT). The validated LC-MS/MS method is linear over wide concentration ranges (125-2000 ng/mL), accurate, and reproducible in the absence of matrix effects, allowing for the specific and rapid quantification of Bu and allowing next-dose recommendations to be made in a timely fashion to answer clinicians' needs. Given the lack of data on the stability of Bu in real clinical samples, stability was assessed both on quality controls and on real samples to set up a robust protocol in real-life conditions. This novel LC-MS/MS method is suitable for application to the TDM-guided personalization of conditioning treatments with high-dose busulfan in pediatric patients undergoing HSCT.

Azole resistance in Aspergillus isolates by different types of patients and correlation with environment - An Italian prospective multicentre study (ARiA study).

BACKGROUND: A wide range of frequency of azole-resistance in A fumigatus in different patient populations worldwide was observed threatening to reduce therapeutic options. OBJECTIVES: Estimate the prevalence of azole-resistance, investigate the molecular mechanisms of resistance, compare the genotypes of resistant clinical isolates with those from the surrounding environment. METHODS: Aspergillus isolates were collected by seven Italian hospital microbiology laboratories. Strains were isolated from different clinical samples from unselected patients. The azole-resistance was evaluated using screening test and microdilution EUCAST method. The molecular mechanism of resistance was performed sequencing the cyp51A gene. Resistant isolates were genotyped by microsatellite analysis and their profiles compared with those of azole-resistant isolates from previous Italian studies. RESULTS: 425 Aspergillus isolates from 367 patients were analysed. The azole-resistance rates were 4.9% and 6.6% considering all Aspergillus spp. isolates and the A fumigatus sensu stricto, respectively. All resistant isolates except one were from a single hospital. Two rare azole-resistant species were identified: A thermomutatus and A lentulus. The predominant resistance mechanism was TR34 /L98H. No correlation between the clinical resistant strains and environmental isolates from patients' home/work/ward was observed. The analysis of the molecular correlation between the resistant clinical strains collected in the present study and those of environmental and clinical origin collected in previous Italian studies reveals a progressive diversification of azole-resistant genotypes starting from a founder azole-resistant genotype. CONCLUSIONS: This study confirms the trend of azole-resistance rate in Italy, showing a geographical difference. Data reinforce the importance of surveillance programmes to monitor the local epidemiological situation.

Resistance to Antibiotics of Uropathogen Bacteria Isolated From Urine and Blood in Pediatric Cancer Patients: A Single Center, 12-year Study.

Resistant pathogens have become a major healthcare problem in children with cancer, causing different kinds of infections such as the bloodstream ones, most common, and most frequently described and the urinary tract ones, of which less data are available. We analyzed and compared the proportions, and the trends of resistance in pathogens isolated from blood and urines in children with cancer followed in IRCCS Istituto Giannina Gaslini, Genova, Italy, from January 2007 to December 2018. Overall, 345 strains detected in urines and 282 in bloodstream infections were analyzed. Enterobacteriales were the most frequently isolated pathogens. During the study period in urines, there was a significant increase of resistance to ceftazidime, ciprofloxacin, piperacillin/tazobactam, and trimethoprim-sulfamethoxazole, but pathogens from blood were significantly more frequently resistant to amikacin, piperacillin/tazobactam, and combination therapy piperacillin/tazobactam+amikacin, even if with a decreasing trend during the study period. These data confirm the importance of surveillance of isolated microorganism and antibiotic resistance in cancer children.

Intravenous isavuconazole can be administered 5 days-a-week. A possibility suggested by a real-life observation.

In this report it is shown that intravenous formulation of isavuconazole could be administered 5/7 days a week in patients who can not swallow capsules, once the steady state has been stably reached and maintained, thanks to its very long half-life. In this case TDM should be highly recommended.

Diagnosis of invasive fungal disease in children: a narrative review.

Introduction: Invasive fungal diseases (IFD) represent important causes of morbidity and mortality in pediatrics. Early diagnosis and treatment of IFD is associated with better outcome and this entails the need to use fast and highly sensitive and specific methods that can support clinicians in the management of IFD.Areas covered: A narrative review was performed on conventional diagnostic methods such as culture, microscopy and histopathology are still gold standard but are burdened by a lack of sensitivity and specificity; on the other hand, imaging and noninvasive antigen-based such as beta-D-glucan, galactomannan and molecular biomarkers are the most convenient nonculture methods for diagnosis and monitoring effects of therapy. Aim of the present review is to summarize what is available in these fields at end of the second decade of the third millennium and look for future perspectives.Expert opinion: Promising and useful diagnostic methods have been applied in infectious disease diagnosis in clinical practice or in designing platforms. Unfortunately, most of them are not standardized or validated in pediatric population. However, clinicians should be aware of all innovative diagnostic tools to use in combination with conventional diagnostic methods for a better management of pathology and patient.

Magnusiomyces clavatus infection in a child after allogeneic hematotopoetic stem cell transplantation: Diagnostic and therapeutic implications.

Magnusiomyces clavatus is an ascomycetous fungus causing invasive disease in immuno-compromised patients. Neutropenia, contaminated venous catheters, previous antifungal treatment are risk factors for this infection. We report a case of Magnusiomyces clavatus fungemia with pulmonary, renal and skin localizations in a 6-year-old boy with prolonged neutropenia because of three allogeneic hematopoietic stem cell transplantations. The infection was controlled by aggressive and strictly monitored combination therapy with voriconazole and liposomial-ampthotericine-B along with durable recover from neutropenia.

Prolonged antibiotic administration for surgical site infection in pediatric laryngotracheal surgery.

OBJECTIVES/HYPOTHESIS: Incidence of surgical site infection (SSI) after laryngotracheal (LT) surgery is relatively high, especially in children. STUDY DESIGN: Retrospective and prospective cohort study. METHODS: Clinical records of children who underwent open LT surgery at the Istituto Giannina Gaslini Children's Hospital from January 2008 to August 2017 were reviewed for development of SSI. Standard antibiotic prophylaxis was administered until February 2015. In March 2015, an antibiotic treatment tailored on pathogens isolated from surveillance cultures and prolonged until extubation was introduced. Incidence and risk factors for SSI before and after the new protocol implementation were analyzed by means of univariate and multivariable analyses. RESULTS: A total of 57 procedures were analyzed. SSI incidence was 36% in patients receiving standard prophylaxis and 4% in those treated with the new strategy (P = .004), with an absolute benefit increase of 32% (95% confidence interval: 11%-52%), in absence of any difference in clinical conditions between the two groups. CONCLUSIONS: The new management protocol had a highly favorable impact on the development of an SSI. LEVEL OF EVIDENCE: 2b Laryngoscope, 129:2634-2639, 2019.

Granulated sugar for adjuvant treatment of surgical wound infection due to multi-drug-resistant pathogens in a child with sarcoma: a case report and literature review.

The use of sugar for treating wounds which are difficult to heal and positive to resistant pathogens has already been documented. The authors describe the successful treatment by direct instillation of granular sugar in the antibiotic-resistant infected surgical site wound of a child with sarcoma. Sugar instillation in the extended spectrum beta-lactamase (ESBL)-producing Enterobacter cloacae positive wound, in addition to systemic treatment with meropenem and levofloxacin, allowed culture negativization in six days and complete wound healing in 30 days. These results make the use of sugar an attractive option for wounds which are difficult to treat, even in an immunocompromised child.

Clostridium difficile causing pediatric infections: New findings from a hospital-based study in Italy.

Recent studies support a change of Clostridium difficile infections (CDIs) epidemiology in pediatric patients. Since limited information is available about C. difficile in this population, we investigated the epidemiology of CDI in a large pediatric hospital that acts as reference centre in Italy and analyzed C. difficile isolates to identify the prevalent PCR-ribotypes (RTs), the binary toxin (CDT)-positive strains and the antibiotic susceptibility patterns. The CDI incidence was 6.6 cases/1000 admissions and the majority (92%) of CDI were healthcare-associated (47% occurred in the Hematology-Oncology and in the Gastroenterology units). Most of symptomatic children <3 years with a positive culture for C. difficile were negative for other gastrointestinal pathogens, supporting C. difficile as cause of disease in these patients, including those showing recurrences. Strains RT020 (16%) and RT014 (14%) were identified as the main cause of infection, while RT356/607 and RT018, predominant in Italian adult patients, were absent (RT356/607) or rarely found (RT018) among children. CDT-positive strains represented the 20% of the total number of isolates analyzed. In particular, two emerging types, RT033 and RT442, were recognized as Toxin A-/Toxin B-/CDT+. Resistance to antibiotics characterized almost 50% of the toxigenic isolates analyzed in this study and, in particular, 20% of them were multidrug resistant (MDR). The emergence and circulation of strains with peculiar toxins profiles and/or MDR strongly highlight the necessity of a rapid CDI diagnosis, a careful monitoring of C. difficile in pediatric patients and a more strict control of antibiotics usage in the Italian pediatric hospitals.

Performance of 1,3-ß-D-glucan for diagnosing invasive fungal diseases in children.

Plasma 1,3-ß-D-glucan (BDG) is indicated as a tool for early diagnosis of invasive fungal diseases (IFD). However, data on its diagnostic value are scarce in children. Therefore, definition of BDG test performance in paediatrics is needed. BDG was evaluated in children admitted to "Istituto Giannina Gaslini," Genoa, Italy, who developed clinical conditions at risk for IFD. Results were analysed for sensitivity, specificity, predictive values, likelihood ratios, accuracy, informedness and probability of missing one case by a negative test. A total of 1577 BDG determinations were performed on 255 patients (49% males, median age 5.4 years). Overall 46 IFD were diagnosed, 72% proven/probable. The test performance was evaluated for 80 pg/mL, 120 pg/mL, 200 pg/mL, 350 pg/mL, 400 pg/mL cut offs. Sensitivity was always <0.80 and specificity > 0.90 only for cut offs ≥200 pg/mL. Negative predictive value was ≥0.90 for all the cut offs evaluated, while positive predictive value resulted barely 0.50 (8% IFD prevalence). Accuracy was never >0.90, and informedness was at best 0.50. The risk of missing one IFD by a negative result was < 10%. Analyses in haemato-oncological or newborn patients did not show major differences. Detection of serum BDG does not appear a valuable adjunctive diagnostic tool for IFD in paediatrics.

Epidemiology of invasive fungal diseases in children with solid tumours undergoing autologous haematopoietic stem cell transplantation: a 10-year experience in a tertiary Italian centre.

The objective of the study was to determine the incidence of invasive fungal disease (IFD) in children undergoing autologous haematopoietic stem cell transplantation (auHSCT) for solid tumours (ST). Retrospective study on auHSCT was performed in children with ST (January 2006-December 2015). Data on the number of patient-days at risk (pdr) during the first 30 and 90 days after auHSCT and cases of proven/probable IFDs were collected. Infection rate (IR, episodes/1000 pdr) and proportions and cumulative risk (CR) of IFD were evaluated. In 186 patients, 270 auHSCT were performed, for a total of 8327 pdr during the first 30 days and 24 366 up to day 90. Median age was 5 years (interquartile range 2;8), 63% were male. At day 30, seven procedures were complicated by IFD, with an IR of 0.84 (95% CI 0.66-1.02) and aCR of 2.6% (95% CI 1.4-5.4) at 18 days after HSCT. Within day 90, two further IFDs were detected with an IR of 0.37 (95% CI -0.49 to 1.23) and a CR of 3.3% (95% CI 1.7-6.3) at day 69. Children undergoing auHSCT for ST have a low incidence of IFDs in the first 90 days after the procedure.

Candida infections in paediatrics: Results from a prospective single-centre study in a tertiary care children's hospital.

To describe the epidemiology of invasive Candida infection in a tertiary care paediatric hospital. Prospective single-centre survey on all Candida strains isolated from normally sterile fluids and urines in the period 2005-2015 . A total of 299 ICI were documented in 262 patients. Urinary tract infection represented the most frequent diagnosis (62%), followed by fungaemia (34%) and peritonitis (4%). Fungaemia was most frequent in children with cancer (59%) or in low birth weight neonates (61%), while urinary tract infections were more frequent in patients with urinary tract malformation. C.albicans was the most frequently isolated species (60%) compared with C. non-albicans, but differences were present according to the site of isolation and underlying conditions. Overall 90-day mortality was 7%, 13% in fungaemias, 8% in peritonitis and 2% in urinary tract infections. The rates of invasive Candida infection increased during the study period. Invasive Candida infection is diagnosed with increasing frequency in children. Site of isolation and aetiology are frequently related with the presence of underlying, favouring conditions. Mortality was not negligible, especially in the presence of more invasive infections and specific underlying conditions.

Primary Hydatid Cyst of the Brain in a Child: A Case Report.

BACKGROUND: Primary intracranial hydatid cyst is a rare location of human echinococcosis whose spontaneous, traumatic or even iatrogenic rupture, as in case of misdiagnosis, may cause anaphylactic reactions and dissemination. CASE REPORT: We discuss the management of a 9-year-old boy who was admitted to our Emergency Department with an intracranial hypertension syndrome. Head CT scan and brain MRI showed a huge intra-axial right temporo-parieto-occipital cyst with a marginal calcification, associated with left ventricular uncompensated hydrocephalus. DTI showed displacement of the ipsilateral corticospinal tract, whereas MR spectroscopy showed absence of normal brain metabolites and presence of succinate and lactate within the cyst. A diagnosis of hydatid cyst was then presumed on the basis of the neuroradiological findings. Empiric chemotherapy with albendazole was instituted and surgical en bloc removal of the cyst was obtained, allowing the patient to recover without complications. Diagnosis of brain echinococcosis was confirmed by laboratory tests. CONCLUSIONS: HE is still an endemic manifestation in some rural areas of the world, and it should be included in the differential diagnosis of children living in or coming from an endemic country who present with an intracerebral cyst. Early diagnosis and complete surgical removal of the intact cyst are the main factors that determine a favourable outcome.

Primary intestinal mold infection in children with solid tumors: a case report in an adolescent with Ewing sarcoma, and literature review.

We report a case of primary intestinal infection due to filamentous fungi in an adolescent with Ewing sarcoma. The clinical picture was that of peritonitis secondary to intestinal perforation and the diagnosis was established only on histopathological bases. This condition is very rare, and only one case of primary intestinal mold infection in children with solid tumors has been reported in the literature, although more records can be found describing similar conditions in other cancer patient populations (i.e. adults with solid tumors or children with hematological malignancies or patients receiving hemopoietic stem cell transplant). Clinicians must be aware of this possibility since only an aggressive medical and surgical approach can improve patients' prognosis.

Characterization of Staphylococcus aureus small colony variant strains isolated from Italian patients attending a regional cystic fibrosis care centre.

Small colony variant (SCV) Staphylococcus aureus are a subpopulation of auxotroph, slow-growing strains causing persisting and relapsing infections in cystic fibrosis (CF) patients. Twenty-eight SCV and 29 normal S. aureus strains were isolated from 42 out of 222 Italian CF patients. The isolates were characterized for: susceptibility to antibiotics, methicillin-resistance (MR), Panton Valentine leukocidin, auxotrophy, hypermutability and biofilm formation. Clonal identity of SCV and normal strains was determined by pulsed-field gel electrophoresis. We found that 27 out of 28 SCVs were thymidine-dependent. Furthermore, in contrast to normal phenotype, SCVs were characterized by antibiotic resistance. We also found that 39.3% SCV vs 20.7% normal strains were strong mutators. Moreover, SCVs showed a higher capability to form biofilm compared to normal strains (100% vs 59%). Importantly, we found evidence of clonal spread of SCV strain among CF patients. Using molecular typing, we found that five patients shared the same type A and five out of seven MR-SCV belonged to the same clone (Clone C). The particular virulence and spreading ability of MR-SCV observed highlights the importance of accurate identification and susceptibility testing of these strains. It is important to adopt the optimal approach to treat patients and to prevent cross-infection in CF centres.

Antibiotic susceptibility of Gram-negatives isolated from bacteremia in children with cancer. Implications for empirical therapy of febrile neutropenia.

BACKGROUND: Monotherapy is recommended as the first choice for initial empirical therapy of febrile neutropenia, but local epidemiological and antibiotic susceptibility data are now considered pivotal to design a correct management strategy. AIM: To evaluate the proportion of Gram-negative rods isolated in bloodstream infections in children with cancer resistant to antibiotics recommended for this indication. MATERIALS & METHODS: The in vitro susceptibility to ceftazidime, piperacillin-tazobactam, meropenem and amikacin of Gram-negatives isolated in bacteremic episodes in children with cancer followed at the Istituto "Giannina Gaslini", Genoa, Italy in the period of 2001-2013 was retrospectively analyzed using the definitions recommended by EUCAST in 2014. Data were analyzed for any single drug and to the combination of amikacin with each ß-lactam. The combination was considered effective in absence of concomitant resistance to both drugs, and not evaluated by means of in vitro analysis of antibiotic combinations (e.g., checkerboard). RESULTS: A total of 263 strains were evaluated: 27% were resistant to piperacillin-tazobactam, 23% to ceftazidime, 12% to meropenem and 13% to amikacin. Concomitant resistance to ß-lactam and amikacin was detected in 6% of strains for piperacillin-tazobactam, 5% for ceftazidime and 5% for meropenem. During the study period there was a nonsignificant increase in the proportions of strains resistant to ß-lactams indicated for monotherapy, and also increase in the resistance to combined therapies. CONCLUSION: in an era of increasing resistance to antibiotics guideline-recommended monotherapy could be not appropriate for initial empirical therapy of febrile neutropenia. Strict local survey on etiology and antibiotic susceptibility is mandatory for a correct management of this complication in cancer patients.

Role of acute graft-versus-host disease in the risk of bacteremia and invasive fungal disease after allogeneic hemopoietic stem cell transplantation in children. Results from a single-center observational study.

Data on epidemiology of severe infectious complications, ie, bacteremia or invasive fungal disease (IFD), in children with acute graft-versus-host disease (aGVHD) after allogeneic hemopoietic stem cell transplantation (HSCT) are scarce. In a retrospective, single-center study, we analyzed the risk (hazard ratio [HR]) and the rate (episodes/1000 patients days at risk) of bacteremias and IFD in children receiving allogeneic HSCT, according to the type of donor (matched related [MRD] or alternative [AD]) and presence and grade of aGVHD. From 2000 to 2009, 198 children receiving 217 allogeneic HSCT developed 134 severe infectious episodes (103 bacteremias and 31 IFD). The type of donor (AD versus MRD) was the most important risk factor for the severe infections (P = .0052). In separate multivariable analysis for bacteremia and IFD, children receiving an AD HSCT had increased HR and rate of bacteremia compared with those receiving a MRD transplantation (P = .0171 and P = .0001, respectively), whereas the HR and the rate of IFD were significantly influenced by the grade of aGVHD (P = .0002 and P < .0001, respectively). Finally, infectious episodes occurred late after HSCT, especially in presence of severe aGVHD, and bacteremias were 3 to 6 times more frequent than IFD. These data may be important to design management strategies of infections in pediatric allogeneic HSCT.