[Screening for alpha1-antitrypsin deficiency using dried blood spot: Assessment of the first 20 months]. / Dépistage du déficit en alpha1-antitrypsine sur sang capillaire recueilli sur papier-filtre : bilan des 20 premiers mois.

INTRODUCTION: Alpha1-antitrypsin deficiency is a predisposing factor for pulmonary disease and under-diagnosis is a significant problem. The results of a targeted screening in patients with respiratory symptoms possibly indicative of severe deficiency are reported here. METHODS: Data were collected from March 2016 to October 2017 on patients who had a capillary blood sample collected during a consultation with a pulmonologist and sent to the laboratory for processing to determine alpha1-antitrypsin concentration, phenotype and possibly genotype. RESULTS: In 20 months, 3728 test kits were requested by 566 pulmonologists and 718 (19 %) specimens sent: among these, 708 were analyzable and 613 were accompanied by clinical information. Of the 708 samples, 70 % had no phenotype associated with quantitative alpha1- antitrypsin deficiency, 7 % had a phenotype associated with a severe deficiency and 23 % had a phenotype associated with an intermediate deficiency. One hundred and eight patients carried at least one PI*Z allele which is considered to be a risk factor for liver disease. CONCLUSIONS: The results of this targeted screening program for alpha1- antitrypsin deficiency using a dried capillary blood sample reflect improvement in early diagnosis of this deficiency in lung disease with good adherence of the pulmonologists to this awareness campaign.

Diseases associated with calcium-sensing receptor.

The calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium metabolism by regulating parathyroid hormone secretion and urinary calcium excretion. The diseases caused by an abnormality of the CaSR are genetically determined or are more rarely acquired. The genetic diseases consist of hyper- or hypocalcemia disorders. Hypercalcaemia disorders are related to inactivating mutations of the CASR gene either heterozygous (autosomal dominant familial benign hypercalcaemia, still named hypocalciuric hypercalcaemia syndrome type 1) or homozygous (severe neonatal hyperparathyroidism). The A986S, R990G and Q1011E variants of the CASR gene are associated with higher serum calcium levels than in the general population, hypercalciuria being also associated with the R990G variant. The differential diagnosis consists in the hypocalciuric hypercalcaemia syndrome, types 2 (involving GNA11 gene) and 3 (involving AP2S1 gene); hyperparathyroidism; abnormalities of vitamin D metabolism, involving CYP24A1 and SLC34A1 genes; and reduced GFR. Hypocalcemia disorders, which are more rare, are related to heterozygous activating mutations of the CASR gene (type 1), consisting of autosomal dominant hypocalcemia disorders, sometimes with a presentation of pseudo-Bartter's syndrome. The differential diagnosis consists of the hypercalciuric hypocalcaemia syndrome type 2, involving GNA11 gene and other hypoparathyroidism aetiologies. The acquired diseases are related to the presence of anti-CaSR antibodies, which can cause hyper- or especially hypocalcemia disorders (for instance in APECED syndromes), determined by their functionality. Finally, the role of CaSR in digestive, respiratory, cardiovascular and neoplastic diseases is gradually coming to light, providing new therapeutic possibilities. Two types of CaSR modulators are known: CaSR agonists (or activators, still named calcimimetics) and calcilytic antagonists (or inhibitors of the CasR). CaSR agonists, such as cinacalcet, are indicated in secondary and primary hyperparathyroidism. Calcilytics have no efficacy in osteoporosis, but could be useful in the treatment of hypercalciuric hypocalcaemia syndromes.

[Alpha-1 antitrypsin deficiency caused by Null mutation]. / Déficit en alpha-1 antitrypsine secondaire à une mutation Null.

INTRODUCTION: Alpha-1 antitrypsin deficiency is a hereditary disease defined at the biological level by a serum alpha-1 antitrypsin level below 11µM/L. The null variants are characterized by undetectable circulating alpha-1 antitrypsin levels. Suspicion of a null variant requires the use of appropriate diagnostic techniques. CASE REPORT: We report the case of a 33-year old patient presenting with dyspnea on exertion, associated with a moderate airflow obstruction, incompletely reversible. His tobacco use was less than 3pack-years. The thoracic CT-scan showed emphysema. The serum alpha-1 antitrypsin level was collapsed. Phenotyping by isoelectrofocusing on agarose gels did not show any band. The study of the SERPINA1 gene, by PCR-sequence of the II, III, IV and V exons and the flanking intronic sequences, allowed identification of the NullQ0ourém allele in homozygous state. This mutation was found in heterozygous state in both parents of the index case and in one of his brothers. The index case showed a rapid aggravation of the airflow obstruction. CONCLUSION: In the case of a serum alpha-1 antitrypsin deficiency, the analysis of the phenotype of the protein by isoelectrofocusing must be performed as a first-line investigation. The detection of an atypical profile may suggest the presence of deficient alleles other than the PI S and PI Z alleles that can only be characterized by sequencing of the whole SERPINA1 gene. The patients carrying a null mutation have a high risk of severe chronic obstructive pulmonary disease.

Unraveling the intrafamilial correlations and heritability of tumor types in MEN1: a Groupe d'étude des Tumeurs Endocrines study.

BACKGROUND: MEN1, which is secondary to the mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Most studies demonstrated the absence of direct genotype-phenotype correlations. The existence of a higher risk of death in the Groupe d'étude des Tumeurs Endocrines-cohort associated with a mutation in the JunD interacting domain suggests heterogeneity across families in disease expressivity. This study aims to assess the existence of modifying genetic factors by estimating the intrafamilial correlations and heritability of the six main tumor types in MEN1. METHODS: The study included 797 patients from 265 kindred and studied seven phenotypic criteria: parathyroid and pancreatic neuroendocrine tumors (NETs) and pituitary, adrenal, bronchial, and thymic (thNET) tumors and the presence of metastasis. Intrafamilial correlations and heritability estimates were calculated from family tree data using specific validated statistical analysis software. RESULTS: Intrafamilial correlations were significant and decreased along parental degrees distance for pituitary, adrenal and thNETs. The heritability of these three tumor types was consistently strong and significant with 64% (s.e.m.=0.13; P<0.001) for pituitary tumor, 65% (s.e.m.=0.21; P<0.001) for adrenal tumors, and 97% (s.e.m.=0.41; P=0.006) for thNETs. CONCLUSION: The present study shows the existence of modifying genetic factors for thymus, adrenal, and pituitary MEN1 tumor types. The identification of at-risk subgroups of individuals within cohorts is the first step toward personalization of care. Next generation sequencing on this subset of tumors will help identify the molecular basis of MEN1 variable genetic expressivity.

Diagnosis of alpha-1 antitrypsin deficiency: modalities, indications and diagnosis strategy.

Alpha-1 antitrypsin (α1-AT) deficiency is an autosomal recessive genetic disorder, which predisposes affected patients to development of pulmonary emphysema or liver cirrhosis. Despite the guidelines from the American Thoracic Society and the European Respiratory Society about α1-AT deficiency screening, it remains significantly under recognized. So, it seems necessary to propose an efficient and suitable biological approach to improve diagnosis and management of α1-AT deficiency. α1-AT is a 52 kDa glycoprotein predominantly produced in the liver and its physiological serum concentration for adults ranges from 0.9 to 2.0g/L (17-39 µmol/L). It is encoded by the SERPINA1 gene, which is highly pleomorphic, and to date, more than 100 alleles have been identified. α1-AT testing would initially involve quantification of serum α1-AT concentration with possible complementary measurement of the elastase inhibitory capacity of serum. If the serum α1-AT concentration is reduced below the reference value, two strategies for laboratory testing can be used: (i) serum α1-AT phenotyping by isoelectric focusing which allows identification of the most common variant designated as the PI M variant but also of various deficient variants besides the predominant PI S and PI Z ones; (ii) genotyping by allele-specific PCR methods which allows only identification of the deficient PI S and PI Z alleles. Identification of the null alleles or of other rare deficient alleles can be performed by direct sequencing of the whole SERPINA1 gene as a reflex test.

[Hygiene and security management in medical biology laboratory]. / Hygiène et sécurité au laboratoire de biologie médicale.

Risk management in Medical Biology Laboratory (MBL) which includes hygiene and waste management, is an integrated process to the whole MBL organisation. It is composed of three stages: risks factors identification, grading and prioritization, and their evaluation in the system. From the legislation and NF EN ISO 15189 standard's requirements viewpoint, prevention and protection actions to implement are described, at premises level, but also at work station environment's one (human resources and equipments) towards biological, chemical, linked to gas, to ionizing or non ionizing radiations and fire riks, in order not to compromise patients safety, employees safety, and quality results. Then, although NF EN 15189 standard only enacts requirements in terms of prevention, curative actions after established blood or chemical exposure accident are defined.

[Recommendations for waste management]. / Recommandations pour la gestion des déchets.

Laboratory waste management must ensure the safety of patients and staff, limiting the environmental impacts and control waste disposal budget. Sorting of waste must be carried out at the source. The packaging must be adapted, allowing easy identification of specific disposal routes. With regard to wastes for human or animal health care and/or related research (DASRI), packages must comply with the regulations, standards and ADR if necessary. Storage provisions differ according to the amount of DASRI produced. Waste collection is carried out directly on the place of activity by a certified service provider. Non pre-treated DASRI is incinerated in specific approved plants for a T ° > 1,200 °C. Special provisions also exist for chemical waste and radioactive waste, the latter being regulated by ANDRA.

Anaerobic bacteria and antibiotics: What kind of unexpected resistance could I find in my laboratory tomorrow?

The purpose of this article is to set out some important considerations on the main emerging antibiotic resistance patterns among anaerobic bacteria. The first point concerns the Bacteroides fragilis group and its resistance to the combination of ß-lactam+ß-lactamase inhibitor. When there is overproduction of cephalosporinase, it results in increased resistance to the ß-lactams while maintaining susceptibility to ß-lactams/ß-lactamase inhibitor combinations. However, if another resistance mechanism is added, such as a loss of porin, resistances to ß-lactam+ß-lactamase inhibitor combinations may occur. The second point is resistance to metronidazole occurring due to nim genes. PCR detection of nim genes alone is not sufficient for predicting resistance to metronidazole; actual MIC determinations are required. Therefore, it can be assumed that other resistance mechanisms can also be involved. Although metronidazole resistance remains rare for the B. fragilis group, it has nevertheless been detected worldwide and also been observed spreading to other species. In some cases where there is only a decreased susceptibility, clinical failures may occur. The last point concerns resistance of Clostridium species to glycopeptides and lipopeptides. Low levels of resistance have been detected with these antibiotics. Van genes have been detected not only in clostridia but also in other species. In conclusion, antibiotic resistance involves different mechanisms and affects many anaerobic species and is spreading worldwide. This demonstrates the need to continue with antibiotic resistance testing and surveys in anaerobic bacteria.

Impact of infusion method on amikacin serum levels in humans.

Aminoglycosides are broad-spectrum antibiotics with peak-dependent bactericidal activity, administered by gravity infusion or for more accuracy by electronic pump infusion. The aim of this study was to assess the difference between the two systems and its pharmacokinetic impact. Twenty-four patients hospitalised for community-acquired pulmonary infections received amikacin by IV route over 1 h with a targeted peak concentration of 35 mg/L. They were randomly distributed into two groups, one receiving infusion through a pump system, the other by gravity. Amikacin serum levels were determined at the end of infusion and 24 h later. C(max) values were significantly lower with gravity than pump (40.2 +/- 12.3 vs. 50.6 +/- 17.6 mg/L, respectively; p = 0.04). Elimination half-life time, volume of distribution and clearance did not differ significantly from one group to the other. The percentage of patients who failed to achieve the targeted peak concentration was significantly higher with gravity than pump (41.7% vs. 16.7%, respectively; p < 0.001). Improving infusion flow-rate provides better control over amikacin C(max). This study underlines the fact that infusion device characteristics should be added to the physiopathological information of a patient if we are to make a better estimation of pharmacokinetic parameters.

In vitro activity against anaerobes of retapamulin, a new topical antibiotic for treatment of skin infections.

OBJECTIVES: Retapamulin is the first agent of the pleuromutilin class formulated as a topical antibacterial for treating skin infections. The aim of this study was to determine the antimicrobial activity of retapamulin by determining the minimal inhibitory concentration (MIC) values of this new drug and comparators against a wide range of anaerobic bacteria of human origin. METHODS: The in vitro activity of retapamulin and six comparators (amoxicillin, amoxicillin/clavulanic acid, ceftriaxone, imipenem, clindamycin and metronidazole) was evaluated against 232 anaerobic clinical isolates. MICs were determined by the CLSI reference agar dilution method (M11-A6). RESULTS: Ceftriaxone, clindamycin and amoxicillin/clavulanic acid resistance rates were 54%, 42% and 9.6%, respectively, within the Bacteroides fragilis group. Despite high resistance rates to various antibiotics, retapamulin inhibited 37/52 (71%) strains of the B. fragilis group and 85/87 (98%) of the other Gram-negative bacilli at a concentration of 2 mg/L or less. All the investigated strains of Clostridium perfringens were inhibited by 1 mg/L retapamulin. Three strains of C. difficile and one strain of C. clostridioforme demonstrated decreased susceptibility to retapamulin. Based on inhibitory concentrations, retapamulin was more active than clindamycin, metronidazole and ceftriaxone against Propionibacterium acnes and anaerobic Gram-positive cocci, as all isolates were inhibited by

Contribution of genetic analysis in screening for MEN1 among patients with sporadic disease and one or more typical manifestation.

Multiple Endocrine Neoplasia type 1 (MEN1) is an autosomal dominant hereditary syndrome (OMIM 131100) due to MEN1 gene mutations, predisposing to the development of hyperplasic and tumoral lesions of neuroendocrine tissues. Since the identification of the gene in 1997, more than 400 different mutations of MEN1 have been registered. Genotypic analysis of MEN1 remains fastidious and must be reserved to targeted situations. If the lesions appear in a familial assessed context, there is a strong argument to search for MEN1 mutation. This is not the case in a sporadic context. With experience acquired in our laboratory, we evaluated the frequency of MEN1 mutations in patients with sporadic presentations. Our aim was to better define criteria for MEN1 genotypic analysis. One hundred and twenty four blood samples from unrelated patients, who gave their written informed consent, were analyzed. These patients exhibited 1 to 4 manifestations of MEN1 without any familial context. After DNA extraction, the analysis was undertaken by PCR-sequencing of all the MEN1 coding exons and exon/intron boundaries or by PCR of the pre-screened fragments alone, a technique made possible by indirect screening mutation methods. Mutations were identified by comparing the sequences to the reference MEN1 sequence available from GENBANK (U93237.1). Mutations were identified in 19 patients, with variable prevalence according to clinical manifestations: 100% for patients with 4 manifestations, 45.5% for patients with 3 manifestations, 19% for patients with 2 manifestations and 2% for patients with only one manifestation. Mutations were: 11 point variations (58%), including 2 splicing sites and 8 frameshift mutations (42%) including 5 deletions, 2 insertions and 1 insertion/deletion; one mutation was identified twice. We showed a relationship between clinical presentation and MEN1 mutation identification, especially with the number of clinical manifestations but also with the type of manifestation. Pancreatic manifestations were significantly linked with probability of mutation. In a sporadic context with at least two established manifestations of MEN1, the overall probability of identifying a mutation was 26%, warranting MEN1 genotypic analysis.

[Calcium sensing receptor: physiology and pathology]. / Le récepteur sensible au calcium: physiologie et pathologie.

Calcium is a major ion in human metabolism and its level is highly controlled. This regulation is performed via the Calcium Sensing Receptor, a discovery which ten years ago led to the explanation of a number of clinical disorders. The syndromes caused by CaSR abnormalities are characterized by hypercalcemia or hypocalcemia, associated with inappropriate calciuria. An underlying genetic or auto-immune cause may be demonstrated. High blood calcium levels linked to mutations of the CaSR gene lead to familial hypocalciuric hypercalcemia and the neonatal and non neonatal forms with severe hypercalcemic. Hypocalcemia determined by mutations in the CaSR gene include autosomal dominant hypocalcemia and its sporadic form. Another clinical presentation similar to Bartter syndrome has been reported. Auto-antibodies directed against CaSRs, seen in auto-immune diseases, can lead to similar clinical presentations. Finally, CaSR polymorphisms modulate the range of blood calcium levels. With diagnosis of these diseases deleterious therapeutics can be avoided. The discovery of this receptor has led to new therapeutic prospects such as calcimimetics for hyperthyroidism.

Isolation and characterization of a porin-like protein of 45 kilodaltons from Bacteroides fragilis.

Resistance to the combination of amoxicillin and clavulanic acid in some Bacteroides fragilis strains may be associated with a lack of porin proteins. Comparison of outer membrane protein profiles from one resistant strain (B. fragilis CFPL 358) and two susceptible strains of B. fragilis (ATCC 25285 and CFPL 92125) showed that a few proteins were missing in the resistant strain, especially a 45-kDa protein. To determine whether this protein was a porin-like protein, we attempted to isolate it from the two susceptible strains by using gel filtration (Sephacryl S-200, Superose 6) and ion exchange chromatographies (DEAE Trisacryl, DEAE Sepharose Fast Flow). Elution from DEAE resins was poor compared to the 60-67-kDa region, which suggested that the 45-kDa protein exhibited stronger cationic forms. The use of sodium dodecyl sulfate during elution improved the recovery of the 45-kDa protein, showing that detergent modified its conformation and its ionic bounds with the chromatographic matrices but it was not sufficient for good purification. Superose 6 gel filtration also failed to separate this protein from the 60-67-kDa region. The only method resulting in the positive recovery of a purified 45-kDa band from both susceptible B. fragilis strains was electroelution from SDS-PAGE. The swelling assay showed that the 45-kDa protein was a porin-like protein. From this study, we concluded that the 45-kDa protein from B. fragilis was a porin-like protein which might be involved in the antibiotic resistance of a strain in which this protein was missing.