Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection.

To facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote regions in which the disease is endemic, a simple cheap sampling method is required for miltefosine quantification. The aims of this study were to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spot (DBS) samples and to validate its use with Ethiopian patients with visceral leishmaniasis (VL). Since hematocrit (Ht) levels are typically severely decreased in VL patients, returning to normal during treatment, the method was evaluated over a range of clinically relevant Ht values. Miltefosine was extracted from DBS samples using a simple method of pretreatment with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10 to 2,000 ng/ml, and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at the lower limit of quantification), respectively. The method was accurate and precise for blood spot volumes between 10 and 30 µl and for Ht levels of 20 to 35%, although a linear effect of Ht levels on miltefosine quantification was observed in the bioanalytical validation. DBS samples were stable for at least 162 days at 37°C. Clinical validation of the method using paired DBS and plasma samples from 16 VL patients showed a median observed DBS/plasma miltefosine concentration ratio of 0.99, with good correlation (Pearson'sr= 0.946). Correcting for patient-specific Ht levels did not further improve the concordance between the sampling methods. This successfully validated method to quantify miltefosine in DBS samples was demonstrated to be a valid and practical alternative to venous blood sampling that can be applied in future miltefosine pharmacokinetic studies with leishmaniasis patients, without Ht correction.

Fetal phenotypes in otopalatodigital spectrum disorders.

Otopalatodigital spectrum disorders (OPDSD) include OPD syndromes types 1 and type 2 (OPD1, OPD2), Melnick-Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). These conditions are clinically characterized by variable skeletal dysplasia associated in males, with extra-skeletal features including brain malformations, cleft palate, cardiac anomalies, omphalocele and obstructive uropathy. Mutations in the FLNA gene have been reported in most FMD and OPD2 cases and in all instances of typical OPD1 and MNS. Here, we report a series of 10 fetuses and a neonatally deceased newborn displaying a multiple congenital anomalies syndrome suggestive of OPDSD and in whom we performed FLNA analysis. We found a global mutation rate of 44%. This series allows expanding the clinical and FLNA mutational spectrum in OPDSD. However, we emphasize difficulties to correctly discriminate OPDSD based on clinical criteria in fetuses due to the major overlap between these conditions. Molecular analyses may help pathologists to refine clinical diagnosis according to the type and the location of FLNA mutations. Discriminating the type of OPDSD is of importance in order to improve the genetic counseling to provide to families.

Genetic counselling difficulties and ethical implications of incidental findings from array-CGH: a 7-year national survey.

Microarray-based comparative genomic hybridization (aCGH) is commonly used in diagnosing patients with intellectual disability (ID) with or without congenital malformation. Because aCGH interrogates with the whole genome, there is a risk of being confronted with incidental findings (IF). In order to anticipate the ethical issues of IF with the generalization of new genome-wide analysis technologies, we questioned French clinicians and cytogeneticists about the situations they have faced regarding IF from aCGH. Sixty-five IF were reported. Forty corresponded to autosomal dominant diseases with incomplete penetrance, 7 to autosomal dominant diseases with complete penetrance, 14 to X-linked diseases, and 4 were heterozygotes for autosomal recessive diseases with a high prevalence of heterozygotes in the population. Therapeutic/preventive measures or genetic counselling could be argued for all cases except four. These four IF were intentionally not returned to the patients. Clinicians reported difficulties in returning the results in 29% of the cases, mainly when the question of IF had not been anticipated. Indeed, at the time of the investigation, only 48% of the clinicians used consents mentioning the risk of IF. With the emergence of new technologies, there is a need to report such national experiences; they show the importance of pre-test information on IF.

A series of 38 novel germline and somatic mutations of NIPBL in Cornelia de Lange syndrome.

Cornelia de Lange syndrome is a multisystemic developmental disorder mainly related to de novo heterozygous NIPBL mutation. Recently, NIPBL somatic mosaicism has been highlighted through buccal cell DNA study in some patients with a negative molecular analysis on leukocyte DNA. Here, we present a series of 38 patients with a Cornelia de Lange syndrome related to a heterozygous NIPBL mutation identified by Sanger sequencing. The diagnosis was based on the following criteria: (i) intrauterine growth retardation and postnatal short stature, (ii) feeding difficulties and/or gastro-oesophageal reflux, (iii) microcephaly, (iv) intellectual disability, and (v) characteristic facial features. We identified 37 novel NIPBL mutations including 34 in leukocytes and 3 in buccal cells only. All mutations shown to have arisen de novo when parent blood samples were available. The present series confirms the difficulty in predicting the phenotype according to the NIPBL mutation. Until now, somatic mosaicism has been observed for 20 cases which do not seem to be consistently associated with a milder phenotype. Besides, several reports support a postzygotic event for those cases. Considering these elements, we recommend a first-line buccal cell DNA analysis in order to improve gene testing sensitivity in Cornelia de Lange syndrome and genetic counselling.

Compliance with infection control practices in sputum microscopy centres: a study from Kerala, India.

BACKGROUND: One of the strategies of the Revised National Tuberculosis Control Programme in India to achieve tuberculosis control is by increasing case detection through a nationwide network of designated microscopy centres (DMC). Practice of standard precautions for infection control in these DMCs is very important to prevent transmission of infection not only to the laboratory personnel, but also to the general population. However, in India this has not been evaluated by an external agency. METHOD: A cross-sectional study was carried out to assess knowledge, facilities and compliance regarding infection control practices (ICP) in all 38 DMCs in Kannur district, Kerala, India, in 2015. Using observations and interviews, the investigators collected data in a structured format. RESULTS: Overall knowledge about infection control was found to be satisfactory among 29% of laboratory technicians. Overall facilities for infection control were satisfactory in 61% of the DMCs, while adherence to ICP was satisfactory in 45% of the DMCs. Knowledge regarding ICP was better in government DMCs, whereas facilities for ICP and adherence to biomedical waste management guidelines were better in private DMCs. CONCLUSION: Given the higher risk of infection among laboratory technicians, there is an urgent need to address the shortcomings in infection control practices.

Contexte : Une des stratégies du Programme national révisé de lutte contre la tuberculose (RNTCP) en Inde consiste à lutter contre la tuberculose en augmentant la détection des cas à travers un réseau national de Centres de Diagnostic par Microscopie (DMC). La mise en oeuvre des précautions standard de lutte contre les infections dans ces DMC est très importante pour prévenir la transmission des infections, non seulement vis-à-vis du personnel de laboratoire, mais également vis-à-vis de la population générale. Cependant, en Inde, ceci n'a pas été évalué par une agence externe.Méthode : Une étude transversale a été réalisée pour évaluer les connaissances, la structure et l'adhésion aux pratiques de lutte contre l'infection (ICP) dans les 38 DMC du district de Kannur, état du Kerala, Inde en 2015. Grâce à des observations et à des entretiens, les investigateurs ont recueilli des données dans un format structuré.Résultats : Les connaissances d'ensemble relatives à la lutte contre l'infection ont été jugées satisfaisantes pour 29% des techniciens de laboratoire. Le niveau d'ensemble des centres en matière de lutte contre l'infection a été satisfaisant dans 61% des DMC. L'adhésion aux pratiques de lutte contre l'infection a été satisfaisante dans 45% des DMC. Les connaissances relatives aux pratiques d'ICP ont été meilleures dans les DMC du gouvernement tandis que les installations et l'adhésion aux directives de gestion des déchets biomédicaux ont été meilleures dans les DMC privés.Conclusion : Connaissant le risque très élevé d'infection parmi les techniciens de laboratoire, il y a un besoin urgent de corriger les déficiences des pratiques de lutte contre l'infection.

Marco de referencia: Una de las estrategias del Programa Nacional Revisado contra la Tuberculosis de la India consiste en controlar la enfermedad al aumentar la detección de casos por conducto de una red nacional de centros de microscopia diagnóstica (DMC). La práctica de las precauciones corrientes de control de las infecciones en estos centros es muy importante con el fin de evitar la transmisión no solo al personal de laboratorio, sino también a la población general. Sin embargo, ningún organismo externo ha evaluado estas prácticas en el país.Método: Se llevó a cabo un estudio transversal con el fin de evaluar los conocimientos, la adecuación de los establecimientos y el cumplimiento de las prácticas en materia de control de las infecciones (ICP) en 38 DMC del distrito de Kannur en el estado de Kerala en la India en 2015. Los investigadores recogieron datos mediante observaciones y entrevistas estructuradas.Resultados: En general, el 29% del personal auxiliar de laboratorio poseía un grado satisfactorio de conocimientos sobre el control de las infecciones; el 61% de los DMC contaba con dispositivos adecuados de ICP y en el 45% se observó el cumplimiento de las normas. El conocimiento de las ICP fue mayor en los DMC del sector público, pero los dispositivos de los establecimientos y el cumplimiento de las directrices de eliminación de los desechos biológicos fueron de mejor calidad en los DMC del sector privado.Conclusión: Teniendo en cuenta el mayor riesgo de exposición a las infecciones que presenta el personal auxiliar de laboratorio, existe una necesidad urgente de abordar las deficiencias observadas en las prácticas de control de las infecciones.

Twenty-five novel mutations including duplications in the ATP7A gene.

Twenty-five novel mutations including duplications in the ATP7A gene. Menkes disease (MD) and occipital horn syndrome (OHS) are allelic X-linked recessive copper deficiency disorders resulting from ATP7A gene mutations. MD is a severe condition leading to progressive neurological degeneration and death in early childhood, whereas OHS has a milder phenotype with mainly connective tissue abnormalities. Until now, molecular analyses have revealed only deletions and point mutations in both diseases. This study reports new molecular data in a series of 40 patients referred for either MD or OHS. We describe 23 point mutations (9 missense mutations, 7 splice site variants, 4 nonsense mutations, and 3 small insertions or deletions) and 7 intragenic deletions. Of these, 18 point mutations and 3 deletions are novel. Furthermore, our finding of four whole exon duplications enlarges the mutation spectrum in the ATP7A gene. ATP7A alterations were found in 85% of cases. Of these alterations, two thirds were point mutations and the remaining one third consisted of large rearrangements. We found that 66.6% of point mutations resulted in impaired ATP7A transcript splicing, a phenomenon more frequent than expected. This finding enabled us to confirm the pathogenic role of ATP7A mutations, particularly in missense and splice site variants.

Mutations of the UPF3B gene, which encodes a protein widely expressed in neurons, are associated with nonspecific mental retardation with or without autism.

Mutations in the UPF3B gene, which encodes a protein involved in nonsense-mediated mRNA decay, have recently been described in four families with specific (Lujan-Fryns and FG syndromes), nonspecific X-linked mental retardation (XLMR) and autism. To further elucidate the contribution of UPF3B to mental retardation (MR), we screened its coding sequence in 397 families collected by the EuroMRX consortium. We identified one nonsense mutation, c.1081C>T/p.Arg361(*), in a family with nonspecific MR (MRX62) and two amino-acid substitutions in two other, unrelated families with MR and/or autism (c.1136G>A/p.Arg379His and c.1103G>A/p.Arg368Gln). Functional studies using lymphoblastoid cell lines from affected patients revealed that c.1081C>T mutation resulted in UPF3B mRNA degradation and consequent absence of the UPF3B protein. We also studied the subcellular localization of the wild-type and mutated UPF3B proteins in mouse primary hippocampal neurons. We did not detect any obvious difference in the localization between the wild-type UPF3B and the proteins carrying the two missense changes identified. However, we show that UPF3B is widely expressed in neurons and also presents in dendritic spines, which are essential structures for proper neurotransmission and thus learning and memory processes. Our results demonstrate that in addition to Lujan-Fryns and FG syndromes, UPF3B protein truncation mutations can cause also nonspecific XLMR. We also identify comorbidity of MR and autism in another family with UPF3B mutation. The neuronal localization pattern of the UPF3B protein and its function in mRNA surveillance suggests a potential function in the regulation of the expression and degradation of various mRNAs present at the synapse.

[Ophthalmologic signs in mucopolysaccharidoses: two case reports]. / Un point d'appel ophtalmologique à l'origine du diagnostic de deux cas de mucopolysaccharidose.

BACKGROUND: The mucopolysaccharidoses (MPS) form a group of heterogeneous hereditary lysosomal storage diseases, distinguished by facial dysmorphy in gargoyle-like facies. The enzymatic deficiency involves the degradation of glycosaminoglycans, whose accumulation manifests in severe general and ophthalmologic problems. CASES REPORT: We report the cases of two 18-month-old girls consulting for corneal clouding and photophobia. The diagnosis was made based on the facial dysmorphy, then biologically corroborated: Scheie's syndrome (MPS type I-S) and Hurler's syndrome (MPS type I-H). The corneal clouding was isolated or associated with bilateral disc swelling. Enzyme replacement therapy was instituted in both cases while waiting for bone marrow transplantation, with a better prognosis in the first case because of the type of MPS and the less severe neurological involvement. DISCUSSION: The accumulation of glycosaminoglycans in ocular tissues can involve stromal opacities, glaucoma, retinopathy, and optic nerve swelling. Whereas the ophthalmological involvement is often secondary, it can lead the ophthalmologist to the diagnosis of MPS. The early diagnosis of MPS, before the onset of neurological signs, is vital, since treatment can stop disease progression. CONCLUSION: Better knowledge of the clinical signs of MPS on the part of the ophthalmologists could improve the prognosis of these patients.

Prenatal diagnosis of trisomy 21 by i(21q): a rare case of fetoplacental chromosomal discrepancy.

OBJECTIVE: A study was conducted to explain the mechanism of an unusual discrepancy between short- and long-term culture examination methods of chorionic villus sampling (CVS). METHOD: In a 29-year-old Caucasian woman, transabdominal CVS was carried out at 12 weeks of gestation. Non-mosaic karyotype 46,XX,i(21q) was found on long-term CVS culture but number and morphology of chromosomes were normal on short-term culture, amniocyte culture, hygroma colli fluid and fetal fibroblast. RESULTS: Chromosomal aberration probably appeared after the trophoblast cell line differentiation, four days after fertilization, by means of a 21 centromere misdivision and formation of a i(21q) with secondary positive selection of the 46,XX,i(21q) cell line and loss of the 46,XX in the fetus. CONCLUSION: The restricted number of cases with this type of discrepancy limits the possibility of drawing generalised conclusions. In case of discrepancy, we recommend confirmation by amniocentesis or by fetal blood combined with sonographic examination to provide a more definitive diagnosis.

Differential effect of high doses versus low doses of interleukin-12 on the adoptive transfer of human specific cytotoxic T lymphocyte in autologous lung tumors engrafted into severe combined immunodeficiency disease-nonobese diabetic mice: relation with interleukin-10 induction.

BACKGROUND: Interleukin (IL)-12 can enhance the development of effective immune responses against tumors as well as against certain infectious agents. It is therefore a potential candidate for therapeutic use in cancer therapy and in design of vaccines against several infectious diseases. METHODS: The authors have established a specific cytotoxic T-cell line (TIL-Heu) from lymphocytes infiltrating a human large cell carcinoma of the lung (LCC). In the current report, the authors have investigated the in vivo effect of recombinant human IL-12 (rhIL-12) on the adoptive transfer of TIL-Heu cells in autologous tumor (Heu-n) engrafted into severe combined immunodeficiency disease-nonobese diabetic (SCID-NOD) mice. RESULTS: Initial in vitro experiments indicated that rhIL-12 increased the cytotoxic potential of TIL-Heu cells in a dose-dependent manner. Heu-n tumors transplanted into SCID-NOD mice were injected with TIL-Heu cells, resulting in a significant tumor growth inhibition. When low doses of rhIL-12 were injected intratumorally after TIL-Heu transfer, a clear increase in tumor growth suppression was observed. Surprisingly, higher doses of rhIL-12 had no effect on cytotoxic T lymphocyte (CTL)-induced prevention of tumor growth. Further in vitro experiments revealed an inhibition of tumor cell lysis after incubation with supernatant of TIL-Heu cells stimulated with high doses of rhIL-12, strongly suggesting that an immunosuppressive factor secreted by the high dose IL-12-stimulated CTL may be responsible for the tumor escape observed in vivo. CONCLUSIONS: The authors' data indicate that IL-10 may play a critical role in the lack of effect of high dose IL-12, by mediating tumor cell resistance to CTL killing. Therefore, understanding the cross-talk between immunoregulatory and immunosuppressive cytokines ultimately may provide new approaches to improve cytokine-mediated cancer immunotherapy.

[Hemolytic anemia after kidney transplantation]. / Anémie hémolytique après transplantation rénale.

Several cases of haemolysis after bone marrow or organ transplantation have been reported. An allospecific anti-erythrocyte antibody has been described in each case. We report a similar case after renal transplantation. Berger's disease led to end-stage renal failure in an 18-year-old girl. During haemodialysis carried out for 6 years, the patient had received 9 transfusions of phenotyped, filtered, packed red blood cells. The recipient was grouped as O Rh positive, CcDee, Kell negative, HLA-A24 A11 B35 B-DR1 DR2. The donor was her father, grouped as O Rh negative, ccddee, Kell negative, HLA-A24 A- B35 B- DR2 DR-. Screening for erythrocyte antibodies was negative in the recipient as were screening for lymphocytotoxic antibodies and compatibility testing. However anti-D antibody was present in the donor who had received a transfusion of Rh positive blood several years before renal donation. The recipient was given 2 units of O Rh positive, phenotyped and filtered red blood cell concentrates during transplantation. Immunosuppressive therapy associated azathioprine and prednisone. The onset of graft function was immediate. Haemoglobin fell to 52 g/l on day 14 post transplantation (103 g/l on day 6). Anti-D antibody was identified in the serum. The direct antiglobulin test was positive with anti-IgG, antiglobulin and the eluate contained an antibody exhibiting also anti-D specificity. The anti-D of the patient and the donor had the same Gm allotyping. The outcome was favourable though the antibody was persistent for several months. This case of haemolysis is consistent with the fact that immunocompetent B lymphocytes transferred with the grafted organ are still able to produce clinically significant antibodies.