ABSTRACT
A new method for the detection by flow cytometry of anti-double-stranded DNA antibodies and of circulating immune complexes (IC) containing endogenous DNA (IC-eDNA) is described. From each serum sample, two samples were taken, one was used to detect IC-eDNA. The other to detect anti-DNA antibodies was incubated with calf thymus DNA. ICs were isolated by polyethylene glycol precipitation or by cryoprecipitation, after which immunoglobulins were labeled with FITC-conjugated anti-human globulin. Serum samples from 63 systemic lupus erythematosus (SLE) patients, 32 incomplete lupus, and 87 control patients were tested. Detection of anti-dsDNA antibodies by flow cytometry had a diagnostic sensitivity and specificity almost comparable to routine tests, the fluorescent enzyme immunoassay EliA™-dsDNA test, and the ultrasensitive Crithidia luciliae indirect immunofluorescence test. In 21 (33%) out of 63 SLE serum samples, IC-eDNA was detected. In these samples, free anti-dsDNA antibodies were hardly detectable or undetectable by flow cytometry or by routine tests. When anti-DNA antibodies are neutralized by endogenous DNA and can no longer be detected by routine tests, the serologic diagnosis and the follow-up of relapses in patients with SLE is compromised. To overcome this obstacle, we propose an accessible solution: the detection of circulating IC-eDNA by flow cytometry.
Subject(s)
Antibodies, Antinuclear/blood , Antibodies, Antinuclear/immunology , Antigen-Antibody Complex/blood , Antigen-Antibody Complex/immunology , DNA/immunology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/immunology , Biomarkers , Flow Cytometry , Humans , Lupus Erythematosus, Systemic/diagnosis , Sensitivity and SpecificitySubject(s)
Anti-Bacterial Agents/therapeutic use , Clostridioides difficile/isolation & purification , Enterobacter cloacae/isolation & purification , Pneumonia/diagnosis , Pneumonia/drug therapy , Acute Kidney Injury/diagnosis , Acute Kidney Injury/therapy , Adult , Bacterial Proteins/isolation & purification , Bacterial Toxins/isolation & purification , Clostridioides difficile/drug effects , Enterobacter cloacae/drug effects , Female , Humans , Imipenem/therapeutic use , Kidney Failure, Chronic/diagnosis , Kidney Failure, Chronic/therapy , Kidney Transplantation/adverse effects , Metronidazole/therapeutic use , Penicillanic Acid/analogs & derivatives , Penicillanic Acid/therapeutic use , Piperacillin/therapeutic use , Piperacillin, Tazobactam Drug Combination , Pneumonia/microbiology , Respiratory Insufficiency/therapy , Vancomycin/therapeutic use , beta-Lactamases/metabolismSubject(s)
Colitis/microbiology , Helicobacter Infections/microbiology , Helicobacter , Acute Disease , Adult , Colitis/immunology , Female , Helicobacter/classification , Helicobacter/genetics , Helicobacter/immunology , Helicobacter Infections/diagnosis , Helicobacter Infections/immunology , Humans , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Tomography, X-Ray ComputedSubject(s)
Bacteremia/microbiology , Bacteriological Techniques/methods , Cephalosporins/pharmacology , Chromogenic Compounds/metabolism , Enterobacteriaceae/drug effects , Reagent Kits, Diagnostic , beta-Lactam Resistance , Blood/microbiology , Culture Media/chemistry , Enterobacteriaceae/isolation & purification , Humans , Prospective Studies , Time FactorsABSTRACT
BACKGROUND: Cefoxitin has demonstrated in vitro resistance to hydrolysis by extended-spectrum beta-lactamases. METHODS: We evaluated the microbiological and clinical efficacy of cefoxitin in 33 patients treated for an infection related to extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E). Clinical and microbiological outcomes were assessed from the initiation of cefoxitin therapy to the latest information available in the patient's medical file. RESULTS: The 33 patients were mainly males (n = 26), aged 70 years (median, minimum-maximum: 23-93) and main sites of infection were urinary (n = 23) and catheter-related bloodstream infections (n = 4). Escherichia coli and Klebsiella pneumoniae were isolated in 19 and 14 subjects, respectively. The clinical outcome was favorable in 30 of 33 patients in the first 48 h after the start of cefoxitin, and in 20 (of 24 evaluable) at the end of follow-up. Six microbiological failures were documented and resistance to cefoxitin emerged in two strains of K. pneumoniae. CONCLUSIONS: Cefoxitin could be considered as a carbapenem-sparing antibiotic for some ESBL-E infections, preferentially those related to E. coli.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Cefoxitin/administration & dosage , Escherichia coli Infections/drug therapy , Escherichia coli/drug effects , Klebsiella Infections/drug therapy , Klebsiella pneumoniae/drug effects , Adult , Aged , Aged, 80 and over , Carbapenems/pharmacology , Escherichia coli/enzymology , Escherichia coli/physiology , Escherichia coli Infections/microbiology , Female , Humans , Klebsiella Infections/microbiology , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/physiology , Male , Microbial Sensitivity Tests , Middle Aged , Treatment Outcome , Young Adult , beta-Lactamases/metabolismSubject(s)
Acinetobacter baumannii/isolation & purification , Bacterial Proteins/metabolism , Enterobacteriaceae/isolation & purification , Pseudomonas/isolation & purification , beta-Lactamases/metabolism , Acinetobacter Infections/metabolism , Acinetobacter Infections/microbiology , Acinetobacter baumannii/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Child , Enterobacteriaceae/metabolism , Enterobacteriaceae Infections/metabolism , Enterobacteriaceae Infections/microbiology , Female , France , Hospitals , Humans , Male , Middle Aged , Pseudomonas/metabolism , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Young AdultABSTRACT
Carnitine palmitoyltransferase 2 (CPT2) deficiency, the most common inherited disease of the mitochondrial long-chain fatty acid (LCFA) oxidation, may result in distinct clinical phenotypes, namely a mild adult muscular form and a severe hepatocardiomuscular disease with an onset in the neonatal period or in infancy. In order to understand the mechanisms underlying the difference in severity between these phenotypes, we analyzed a cohort of 20 CPT2-deficient patients being affected either with the infantile (seven patients) or the adult onset form of the disease (13 patients). Using a combination of direct sequencing and denaturing gradient gel electrophoresis, 13 CPT2 mutations were identified, including five novel ones, namely: 371G>A (R124Q), 437A>C (N146T), 481C>T (R161W), 983A>G (D328G), and 1823G>C (D608H). After updating the spectrum of CPT2 mutations (n=39) and genotypes (n=38) as well as their consequences on CPT2 activity and LCFA oxidation, it appears that both the type and location of CPT2 mutations and one or several additional genetic factors to be identified would modulate the LCFA flux and therefore the severity of the disease.
Subject(s)
Carnitine O-Palmitoyltransferase/deficiency , Carnitine O-Palmitoyltransferase/genetics , Adult , Amino Acid Sequence , DNA/chemistry , DNA/genetics , DNA Mutational Analysis , Fatty Acids/metabolism , Genotype , Humans , Infant , Mitochondrial Myopathies/enzymology , Mitochondrial Myopathies/genetics , Mitochondrial Myopathies/pathology , Mutation , Oxidation-Reduction , Sequence Homology, Amino AcidABSTRACT
Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a rare autosomal recessive disorder of mitochondrial fatty acid oxidation. CPT1 controls the import of long-chain fatty acids into the mitochondria, where they are oxidized. Two CPT1 isoforms, the so-called "liver" and "muscle" CPT1s encoded by the CPT1Aand CPT1Bgenes, respectively, have been identified so far. While the cDNA sequences of both isoforms are known, only CPT1Bgene organization has yet been described. We took advantage of the working draft data to characterize the organization of the human CPT1A gene. We have shown the existence of 20 exons, spanning 60 kb of DNA. Two alternate promoters and numerous transcription factor-binding sites were identified within the 5' upstream region of the gene. In the 3' untranslated region, the major polyA signal was suggested to lie about 2 kb downstream of the stop codon. These data enabled us to characterize six novel mutations in four CPT1A-deficient patients; namely Q100X (exon 4), A414 V (exon 11), Y498C (exon 13), 1876-1G>A (intron 15), a 113-bp intronic insertion in the mature CPT1A mRNA (exon 13-14 junction), and a large 8-kb deletion encompassing intron 14 to exon 17. Thus, identification of the CPT1A gene organization contributes to improve the molecular screening in patients and provides tools for the study of the human CPT1A gene expression.