Off-label drug use in paediatric haemato-oncology patients: financial implications and proposed solutions for Belgian patients.

Treatment of children with cancer requires access to and reimbursement of effective drugs. Children with haemato-oncological diseases are often treated according to established treatment recommendations or in the framework of late-phase clinical trials. These often involve the use of drugs authorised for adults but which, however, have been used for many years in paediatrics with no perspective of authorisation in children. In Belgium, medicines are predominantly reimbursed based on their authorised indication. As a consequence, many drugs used in paediatric haemato-oncology are used off-label, despite their status of 'standard of care'. As reimbursement is often not available, alternative ways for funding need to be explored, which causes a significant administrative burden for healthcare providers and emotional distress for the parents. Solutions to organise a systematic reimbursement of standard of care off-label used drugs are described.Conclusion: A number of structural solutions are proposed, and we hope that they might guide health authorities to provide a solution to the problem caused by the lack of reimbursement of some standard of care medicines for children with cancer. What is Known: • Off-label drug use is frequently observed in paediatric haemato-oncology and compromises-in some countries-reimbursement. What is New: • An estimation of the impact of non-reimbursed drugs in Belgium is provided. • Some solutions are presented to overcome this problem in Belgium.

A cluster of Legionnaires' disease in Belgium linked to a cooling tower, August-September 2016: practical approach and challenges.

A cluster of Legionnaires' disease (LD) with 10 confirmed, three probable and four possible cases occurred in August and September 2016 in Dendermonde, Belgium. The incidence in the district was 7 cases/100 000 population, exceeding the maximum annual incidence in the previous 5 years of 1.5/100 000. Epidemiological, environmental and geographical investigations identified a cooling tower (CT) as the most likely source. The case risk around the tower decreased with increasing distance and was highest within 5 km. Legionella pneumophila serogroup 1, ST48, was identified in a human respiratory sample but could not be matched with the environmental results. Public health authorities imposed measures to control the contamination of the CT and organised follow-up sampling. We identified obstacles encountered during the cluster investigation and formulated recommendations for improved LD cluster management, including faster coordination of teams through the outbreak control team, improved communication about clinical and environmental sample analysis, more detailed documentation of potential exposures obtained through the case questionnaire and earlier use of a geographical information tool to compare potential sources and for hypothesis generation.

Incidental finding of unreported large duplication in F8 gene during prenatal analysis: Which management for genetic counselling?

Detection of incidental finding and variant of unknown significance (VUS) during prenatal diagnosis has particularly increased with the emergence of genetic tests such chromosomal microarray analysis (CMA). Many factors and clear guidelines need to be applied in the interpretation of the potential clinical consequences of unreported complex copy number variations (deletions/duplications). From a clinical case where an unreported and not completely intragenic duplication in F8 gene has been identified in a 12-week-old fetus without haemophilia A history documented in the family, we will examine and study the difficulties of interpretation and the challenges that the detection of such variant has on genetic counselling.

PCR as a test for the presence or absence of Legionella in (cooling) water.

Legionella pneumophila, a Gram-negative bacterium, is the causative agent of legionellosis. Traditionally, culture methods are normally used to detect Legionella species in different types of water (e.g. surface or tap water, circulating systems, air conditioners and their cooling devices). In this study the PCR conditions to detect Legionella were optimised based on the EnviroAmp Legionella kit (Perkin-Elmer) which is no longer commercially available. The PCR is very sensitive and specific in indicating the presence or absence (no quantification with classical PCR) of Legionella spp in general and more specifically L. pneumophila. To identify L. pneumophila. DNA sequences from the mip (macrophage infectivity potentiator) gene were amplified. The mip gene is conserved and specific for L. pneumophila although mip-like genes are also present in other Legionella spp. The PCR techniques were able to detect small amounts of Legionella in tap water samples. Cooling water, however, often contained PCR-inhibiting substances that could result in false negative PCR results for Legionella.

Detection of Naegleria spp. and Naegleria fowleri: a comparison of flagellation tests, ELISA and PCR.

To detect Naegleria spp, in particular Naegleria fowleri, the causative agent of human primary amoebic meningoencephalitis, a flagellation test (FT) is routinely used followed by a specific ELISA. A positive FT indicates the presence of Naegleria spp although some false negatives are likely to occur since parameters for enflagellation vary greatly. As negative FTs are not routinely screened any further for the presence of N. fowleri, this could result in an underestimation of the presence of this pathogen. Therefore, amoebae were further analysed using ELISA and standard PCR not only after a positive but also after a negative FT. In this study 39 cultures containing amoebae were tested with FT, ELISA and the two PCR assays with 11 positive for FT. These were submitted to ELISA and four confirmed as N. fowleri. PCR with the common primer-set on these 11 positive FTs revealed all as Naegleria spp. The specific PCR used on these cultures detected four positive for N. fowleri, corresponding totally with the ELISA results. The 28 negative flagellation tests were also submitted to ELISA and PCR. Of these, 11 were identified as Naegleria spp with common PCR and six as N. fowleri as well as with ELISA and the specific PCR. When the detection of Naegleria spp is based on intermediary processes, such as flagellation tests, false negatives are likely to occur leading to severe underestimations. This study has shown that amoebae taken from negative FTs can be identified as Naegleria spp and N. fowleri when using PCR and ELISA. The application of at least one of the specific N. fowleri tests is recommended for routine screening. The heterogeneous distribution of the false negative results between the different power plants suggested the presence of different genotypes.

A detection method for Legionella spp in (cooling) water: fluorescent in situ hybridisation (FISH) on whole bacteria.

Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.

Effects of peracetic acid and monochloramine on the inactivation of Naegleria lovaniensis.

Biocidal activities of monochloramine and peracetic acid were studied on cysts of Naegleria lovaniensis. Until recently the most commonly used biocide to disinfect cooling water systems was hypochlorite. Owing to its negative impact on the aquatic environment, ecologically less harmful alternatives have been sought. As the biocidal activity of monochloramine and peracetic acid makes them good candidates for inactivation of pathogenic Naegleria species, these biocides were tested against Naegleria lovaniensis, a relative of the pathogen Naegleria fowleri, as an alternative treatment to hypochlorite. Under laboratory conditions the biocidal activity of hypochlorite was 8- 10x stronger than that of the two investigated substances. Hypochlorite, at a concentration of 0.5 mg/L, killed 100% Naegleria lovaniensis after 1 h exposure (25 degrees C, pH 7.3- 7.4). To achieve similar results with monochloramine and peracetic acid, 3.94 mg/L or 5.33 mg/L had to be used respectively (25 degrees C, pH 8). It was known that the in situ biota of the biofilm, along with any organic material in the water column, had a negative impact on the efficiency of the biocides. There are, however, indications that the relative efficacy of monochloramine and peracetic acid was quite good under such conditions when compared with hypochlorite.

Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats.

Bone marrow (BM) cells are attractive target cells for ex vivo gene therapy of genetic diseases, including haemophilia A. However, BM-derived haematopoietic stem/progenitor cells (HSCs) transduced with factor VIII (FVIII) retroviral vectors, failed to express FVIII in vivo. To overcome the limitations of HSCs for haemophilia gene therapy, BM-derived mesenchymal cells were explored as alternative target cells. The BM mesenchymal cell population contains self-renewing mesenchymal stem/progenitor cells that give rise to different mesenchymal lineages and have been used safely in phase I gene-marking trials. Human BM mesenchymal cells were transduced in vitro with an improved retroviral vector encoding a human B-domain deleted FVIII (hFVIIIdeltaB) cDNA (MND-MFG-hFVIIIdeltaB). This vector contains multiple modifications in the cis-acting elements within the MoMLV long-terminal repeats (LTR) that prevent the binding of repressive transcription factors. These modifications were previously shown to increase and prolong gene expression in embryonic stem (ES) cells and HSCs. Transduction of BM mesenchymal cells with the MND-MFG-hFVIIIdeltaB retroviral vector resulted in high levels of functional human FVIII in vitro, ranging between 300 +/- 50 SD and 700 +/- 100 SD mU per 106 cells per 24 h. Following xenografting of the transduced human BM cells into immunodeficient NOD-SCID mice, therapeutic hFVIII levels of 12 +/- 10 ng mL-1 were detected in the plasma. Polymerase chain reaction analysis demonstrated long-term engraftment (>3 months) of the human BM mesenchymal cells. The long-term persistence of BM mesenchymal cells in the absence of myelo-ablative conditioning and the therapeutic FVIII levels in vivo underscore the potential usefulness of BM-derived mesenchymal cells for haemophilia gene therapy, as opposed to BM-derived HSCs. Despite the modifications of the MoMLV LTR, FVIII expression declined, which coincided with a decrease in FVIII mRNA transcription levels, indicating that the salutary effect of the LTR modification on transgene expression is not universally applicable to all cell types.

Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice.

The potential of using bone marrow (BM)-derived human stromal cells for ex vivo gene therapy of hemophilia A was evaluated. BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (Mo-MuLV) retroviral vector that contained the B domain-deleted human factor VIII (FVIIIdeltaB) cDNA. This FVIII-retroviral vector was pseudotyped with the gibbon ape leukemia virus envelope (GALV-env) to attain higher transduction efficiencies. Using optimized transduction methods, high in vitro FVIII expression levels of 700 to 2500 mU of FVIII/10(6) cells per 24 hr were achieved without selective enrichment of the transduced BM stromal cells. After xenografting of 1.5-3 x 106 engineered BM stromal cells into the spleen of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, human plasma FVIII levels rose to 13 +/- 4 ng/ml but declined to basal levels by 3 weeks postinjection because of promoter inactivation. About 10% of these stromal cells engrafted in the spleen and persisted for at least 4 months after transplantation in the absence of myeloablative conditioning. No human BM stromal cells could be detected in other organs. These findings indicate that retroviral vector-mediated gene therapy using engineered BM stromal cells may lead to therapeutic levels of FVIII in vivo and that long-term engraftment of human BM stromal cells was achieved in the absence of myeloablative conditioning and without neo-organs. Hence, BM stromal cells may be useful for gene therapy of hemophilia A, provided prolonged expression can be achieved by using alternative promoters.

Effects of 'contact laxatives' on intestinal and colonic epithelial cell proliferation.

Experimental studies indicate that laxatives may induce epithelial damage. In addition, some laxatives induce the release of prostaglandins. Epithelial cell damage and release of prostaglandins are two pathways by which epithelial cell proliferation could be influenced. Furthermore, fermentable laxatives like lactulose may influence large intestine cell proliferation by the trophic effect of the fermentation products such as short-chain fatty acids. For these reasons an in vivo study in rats was performed to compare the short- and long-term effect of sennosides, bisacodyl, sodium picosulfate and lactulose on epithelial cell proliferation in the ileum and large intestine. Cell proliferation was examined by the BrdUrd labelling technique after 2, 6 and 12 weeks of continuous treatment. Studies in control animals show that the Labeling Index (LI) is higher in the cecum compared with other segments of the colon, and higher in the ileum than in the colon. Treatment with sennosides, bisacodyl and sodium picosulfate does not influence the LI in the ileum and induces no statistically significant increase of the LI when the treated groups are compared with the control group. The proliferative pattern along the crypts remains unchanged with all the laxatives throughout the study. It appears therefore that 'contact' laxatives have no major influence on ileal and colonic epithelial cell proliferation and should not be regarded as tumor-promoting substances.