Michele Pinon

Two Pediatric Cases of Multidrug-Resistant Tuberculosis Treated With Linezolid and Moxifloxacin

We report here 2 pediatric cases of multidrug-resistant (MDR) tuberculosis (TB) that were observed in Italy. Both families came from an Eastern European country, which is notably an area with a high prevalence of MDR TB. An increase of new cases of MDR TB in developed countries is expected over the next years because of migratory flow, and specific measures and strategies need to be taken to prevent the propagation and dissemination of MDR TB. An efficacious treatment including linezolid and moxifloxacin was administered for 13 months in 1 case. No adverse reactions were detected during close child monitoring. Linezolid and newer fluoroquinolones such as moxifloxacin have been reported to be effective for MDR-TB treatment in adults. On the contrary, there is limited available evidence regarding the effectiveness and safety of these drugs in infants and children with MDR TB. The use of second-line drugs not approved for use in children may be necessary to treat a life-threatening disease such as MDR TB, but it requires careful monitoring to quickly recognize the occurrence of dose- and duration-dependent adverse drug reactions.

Performance of interferon-γ release assay for the diagnosis of active or latent tuberculosis in children in the first 2 years of age: a multicenter study of the Italian Society of Pediatric Infectious Diseases.

Background: The diagnosis of latent or active tuberculosis in children is often challenging. Recently, interferon-&ggr; release assays have been licensed, but their diagnostic accuracy in young children remains questionable as frequent false-negative or indeterminate results have been reported. Methods: We performed a multicenter, retrospective study in children 0–24 months of age who were tested at least once with QuantiFERON-TB Gold-in-tube (QTF-IT) ± tuberculin skin test (TST), to analyze its use and performance in clinical practice. Results: Eight-hundred and twenty-three children (449 males, median age 13.5 months) were enrolled. QTF-IT sensitivity and specificity for active tuberculosis were 92.4% and 98.6%, respectively. Indeterminate tests (4.2 %) were not related to age (P = 0.838) or gender (P = 0.223); 32 children (91.4 %) with an indeterminate QTF-IT ultimately resulted uninfected. In the 616 subjects with valid paired results of QTF-IT and TST, sensitivity and specificity were comparable (91.1% vs. 85.1% and 98.1% vs. 97.9%, respectively). Diagnostic concordance between tests was higher in Bacillus Calmétte-Guerin nonvaccinated children (&kgr; = 0.802). A high rate of discordant tests was observed in latent infections. Conclusions: QTF-IT showed good sensitivity and specificity, and a low rate of indeterminate results in the first 2 years of life, supporting its use at this age. However, considering costs and the similar performance between QTF-IT and TST, it is reasonable to suggest the latter as first-line testing in young children. The complementary use of TST and interferon-&ggr; release assays may be considered in selected cases to improve the accuracy of testing.

An UPLC MS/MS method coupled with automated on-line SPE for quantification of tacrolimus in Peripheral Blood Mononuclear Cells

BACKGROUND Tacrolimus is an immunosuppressor used to treat patients undergoing liver transplantation. TDM of hematic tacrolimus by liquid chromatography became standard practice, but it does not necessarily reflect its concentration at its active site. Our aim was to validate a new method for tacrolimus quantification into target cells (peripheral blood mononuclear cells, PBMCs) and testing it on 100 real samples from 37 pediatric patients. METHODS PBMCs were collected using cell-preparation-tubes; cells number and MCV were evaluated. Tacrolimus was quantified using UPLC-MS/MS coupled with a new automated on-line SPE platform. Chromatographic run was performed on an Acquity UPLC(®) BEH C18 1.7 μm (2.1 mm × 50 mm) column for 5 min, with a gradient of water and methanol (both with 2 mM/L ammonium acetate and 1 mL/L formic acid). XBridge(®) C8 10 μm (1 mm × 10 mm) SPE cartridges were used. The internal standard was 6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline. RESULTS Full validation following FDA guidelines was performed: the method showed high sensitivity and specificity (LLOQ of 0.010 ng; LLOD of 0.005 ng). Intra- and inter-day imprecision and inaccuracy were <15%. A positive and stable matrix effect was observed, with a good recovery for tacrolimus. All drug amounts in real samples resulted within the calibration range and calibration curves were linear (r(2)=0.998). Concentrations from each patient were standardized using their evaluated MCV: intra-PBMCs concentration was meanly 12.7 times higher than the hematic one. CONCLUSION This method might be eligible and useful for a clinical routine use, giving more reliable data on drug concentration at the active site.

Pediatric Tuberculosis in Italian Children: Epidemiological and Clinical Data from the Italian Register of Pediatric Tuberculosis

Tuberculosis (TB) is one of the leading causes of death worldwide. Over the last decades, TB has also emerged in the pediatric population. Epidemiologic data of childhood TB are still limited and there is an urgent need of more data on very large cohorts. A multicenter study was conducted in 27 pediatric hospitals, pediatric wards, and public health centers in Italy using a standardized form, covering the period of time between 1 January 2010 and 31 December 2012. Children with active TB, latent TB, and those recently exposed to TB or recently adopted/immigrated from a high TB incidence country were enrolled. Overall, 4234 children were included; 554 (13.1%) children had active TB, 594 (14.0%) latent TB and 3086 (72.9%) were uninfected. Among children with active TB, 481 (86.8%) patients had pulmonary TB. The treatment of active TB cases was known for 96.4% (n = 534) of the cases. Overall, 210 (39.3%) out of these 534 children were treated with three and 216 (40.4%) with four first-line drugs. Second-line drugs where used in 87 (16.3%) children with active TB. Drug-resistant strains of Mycobacterium tuberculosis were reported in 39 (7%) children. Improving the surveillance of childhood TB is important for public health care workers and pediatricians. A non-negligible proportion of children had drug-resistant TB and was treated with second-line drugs, most of which are off-label in the pediatric age. Future efforts should concentrate on improving active surveillance, diagnostic tools, and the availability of antitubercular pediatric formulations, also in low-endemic countries.

Transient elastography for non-invasive evaluation of post-transplant liver graft fibrosis in children

As graft survival in pediatric LT is often affected by progressive fibrosis, numerous centers carry out protocol liver biopsies. Follow‐up biopsy protocols differ from center to center, but all biopsies are progressively spaced out, as time from transplant increases. Therefore, there is a need for non‐invasive techniques to evaluate graft fibrosis progression in those children who have no clinical or serological signs of liver damage. Indirect markers, such as the APRI, should be relied on with caution because their sensitivity in predicting fibrosis can be strongly influenced by the etiology of liver disease, severity of fibrosis, and patient age. A valid alternative could be TE, a non‐invasive technique already validated in adults, which estimates the stiffness of the cylindrical volume of liver tissue, 100‐fold the size of a standard needle biopsy sample. The aims of this study were to evaluate the reliability of TE in children after LT and to compare both the TE and the APRI index results with the histological scores of fibrosis on liver biopsies. A total of 36 pediatric LT recipients were studied. All patients underwent both TE and biopsy within a year (median interval ‐0.012 months) at an interval from LT of 0.36 to 19.47 years (median 3.02 years). Fibrosis was assessed on the biopsy specimens at histology and staged according to METAVIR. There was a statistically significant correlation between TE stiffness values and METAVIR scores (P = .005). The diagnostic accuracy of TE for the diagnosis of significant fibrosis (F ≥ 2) was measured as the area under the curve (AUROC = 0.865), and it demonstrated that the method had a good diagnostic performance. APRI was not so accurate in assessing graft fibrosis when compared to METAVIR (AUROC = 0.592). A liver stiffness cutoff value of 5.6 kPa at TE was identified as the best predictor for a significant graft fibrosis (METAVIR F ≥ 2) on liver biopsy, with a 75% sensitivity, a 95.8% specificity, a 90% positive predictive value, and an 88.5% negative predictive value. These data suggest that TE may represent a non‐invasive, reliable tool for the assessment of graft fibrosis in the follow‐up of LT children, alerting the clinicians to the indication for a liver biopsy, with the aim of reducing the number of protocol liver biopsies.

Hereditary pancreatitis in Paediatrics: the causative role of p.Leu104Pro mutation of cationic trypsinogen gene also in young subjects

We read with interest the letter by Nemeth et al ,1 describing a hereditary pancreatitis (HP) family of Hungarian origin carrying the heterozygous p.Leu104Pro variant of human cationic trypsinogen (PRSS1) gene. It added further data to previous publications by Schnur et al 2 and Balazs et al ,3 who proposed that a subset of PRSS1 variants caused chronic pancreatitis by inducing misfolding and endoplasmic reticulum stress, rather than increased intrapancreatic trypsin activity. However, a clinically defined relationship between the mutation and the phenotypic expression in the general population still remains to be established. Indeed, to date, p.Leu104Pro PRSS1 variant has been reported in three families: three heterozygous carriers of a German pedigree without clinically proven chronic pancreatitis, a subject of Chinese origin with …

An Unexplained Congenital Disorder of Glycosylation-II in a Child with Neurohepatic Involvement, Hypercholesterolemia and Hypoceruloplasminemia

We report on a 12-year-old adopted boy with psychomotor disability, absence seizures, and normal brain MRI. He showed increased (but initially, at 5 months, normal) serum cholesterol, increased alkaline phosphatases, transiently increased transaminases and hypoceruloplasminemia with normal serum and urinary copper. Blood levels of immunoglobulins, haptoglobin, antithrombin, and factor XI were normal. A type 2 serum transferrin isoelectrofocusing and hypoglycosylation of apoCIII pointed to a combined N- and O-glycosylation defect. Neither CDG panel analysis with 79 CDG-related genes, nor whole exome sequencing revealed the cause of this CDG. Whole genome sequencing was not performed since the biological parents of this adopted child were not available.

Osteoarticular infections in children: an Italian cohort

The efflux pump QepA confers decreased susceptibility to hydrophilic fluoroquinolones (e.g., norfloxacin, ciprofloxacin, and enrofloxacin). In this study, we characterized the third variant, named qepA3, collected from an Escherichia coli isolate in Portugal. INSRA6015 was isolated in 2005 from the urine of a 77-year-old female patient hospitalized at the Hospital Fernando Fonseca, Portugal. Susceptibility testing was performed by disk diffusion and MIC methods, (SFM and EUCAST guidelines, respectively). PCR and sequencing were used to screen and identify bla (bla TEM , bla SHV , bla OXA , bla CTX-M and plasmid-mediated ampC ) genes, as well as plasmid-mediated quinolone resistance ( qnrA , qnrB , qnrC , qnrD , qnrS , qepA and aac(6’)Ib-cr ), and the quinolone resistance-determining regions (QRDR: gyrA , gyrB , parC , and parE ) genes. PCR-mapping was used to characterize the genetic environment of the new qepA3 gene. Transfer of resistance of the QepA3 determinant, was performed through electroporation, using the E. coli TOP10 as recipient. Plasmid content was characterized by PCR-based replicon typing. Molecular characterization of INSRA6015 showed the presence of bla TEM-1, bla CMY-2 and a new variant of qepA possessing two nucleotide substitutions, leading to Phe85Leu and Val134Ile changes. This variant, named QepA3, conferred a similar phenotype to that of the QepA1 and QepA2 determinants. Sequencing of the QDRD detected substitutions Ser83Leu and Asp87Asn in the GyrA subunit and Glu84Lys in the ParC subunit, which are consistent with the high resistance to ciprofloxacin observed in the MICs. Sequence analysis of qepA3 genetic environment revealed that the gene was located inside a genetic structure identical to that of previously described for qepA1 and qepA2 . It is noteworthy that qepA3 gene, as qepA2 , was not associated with the rmtB gene encoding an aminoglycoside ribosomal methylase, contrarily to qepA1. PCR-based replicon typing indicated the presence of the IncF plasmid. We have identified and characterized a new variant of the plasmid-mediated efflux pump QepA, which is responsible for the increased levels of resistance to several clinically important quinolones, such as ciprofloxacin, and norfloxacin. This is, at our knowledge, the first description of the co-production of QepA and CMY-2. The study highlights the need of surveillance of this resistance mechanism and reinforces a more careful use of quinolones.

Off-label use of anti-infective drugs in an Italian paediatric cohort

The efflux pump QepA confers decreased susceptibility to hydrophilic fluoroquinolones (e.g., norfloxacin, ciprofloxacin, and enrofloxacin). In this study, we characterized the third variant, named qepA3, collected from an Escherichia coli isolate in Portugal. INSRA6015 was isolated in 2005 from the urine of a 77-year-old female patient hospitalized at the Hospital Fernando Fonseca, Portugal. Susceptibility testing was performed by disk diffusion and MIC methods, (SFM and EUCAST guidelines, respectively). PCR and sequencing were used to screen and identify bla (bla TEM , bla SHV , bla OXA , bla CTX-M and plasmid-mediated ampC ) genes, as well as plasmid-mediated quinolone resistance ( qnrA , qnrB , qnrC , qnrD , qnrS , qepA and aac(6’)Ib-cr ), and the quinolone resistance-determining regions (QRDR: gyrA , gyrB , parC , and parE ) genes. PCR-mapping was used to characterize the genetic environment of the new qepA3 gene. Transfer of resistance of the QepA3 determinant, was performed through electroporation, using the E. coli TOP10 as recipient. Plasmid content was characterized by PCR-based replicon typing. Molecular characterization of INSRA6015 showed the presence of bla TEM-1, bla CMY-2 and a new variant of qepA possessing two nucleotide substitutions, leading to Phe85Leu and Val134Ile changes. This variant, named QepA3, conferred a similar phenotype to that of the QepA1 and QepA2 determinants. Sequencing of the QDRD detected substitutions Ser83Leu and Asp87Asn in the GyrA subunit and Glu84Lys in the ParC subunit, which are consistent with the high resistance to ciprofloxacin observed in the MICs. Sequence analysis of qepA3 genetic environment revealed that the gene was located inside a genetic structure identical to that of previously described for qepA1 and qepA2 . It is noteworthy that qepA3 gene, as qepA2 , was not associated with the rmtB gene encoding an aminoglycoside ribosomal methylase, contrarily to qepA1. PCR-based replicon typing indicated the presence of the IncF plasmid. We have identified and characterized a new variant of the plasmid-mediated efflux pump QepA, which is responsible for the increased levels of resistance to several clinically important quinolones, such as ciprofloxacin, and norfloxacin. This is, at our knowledge, the first description of the co-production of QepA and CMY-2. The study highlights the need of surveillance of this resistance mechanism and reinforces a more careful use of quinolones.

Surveillance Study of Healthcare-Associated Infections in a Pediatric Neurosurgery Unit in Italy

Background: This prospective surveillance study was designed to estimate the incidence of healthcare-associated infections (HAIs) and to analyze the risk factors for their development in a pediatric neurosurgical unit. Methods: The study was performed in an Italian teaching hospital from October 2008 through March 2010. All children (0–18 years) undergoing neurosurgery were included and monitored daily for the development of HAIs. Results: The study included 260 patients, with a mean age of 4.3 ± 4.7 years. Thirty-six HAIs were detected in 25 patients; catheter-related infections were the most frequent. Etiological identification was available in 22 cases; Gram-negative bacteria were the most commonly isolated pathogens. The incidence density was 11.0/1,000 patient days, and the incidence rate was 13.8/100 patients. The crude mortality was 0%. The risk of developing HAIs was related to the length of hospital stay, while the higher the age of the patients, the lower the risk of developing HAIs. Conclusion: To our knowledge, this survey is the first study to evaluate the overall incidence of HAIs and to explore the risk factors implicated in their development in neurosurgical pediatric patients. The most effective strategies to prevent these infections are reduction of the length of the hospital stay and improvement in device management.