Evaluation of the selection of cerebrospinal fluid testing in suspected meningitis and encephalitis.

Diagnostic stewardship interventions can decrease unnecessary antimicrobial therapy and microbiology laboratory resources and costs. This retrospective cross-sectional study evaluated factors associated with inappropriate initial cerebrospinal fluid (CSF) testing in patients with suspected community-acquired meningitis or encephalitis. In 250 patients, 202 (80.8%) and 48 (19.2%) were suspected meningitis and encephalitis, respectively. 207 (82.8%) patients had inappropriate and 43 (17.2%) appropriate testing. Any inappropriate CSF test was greatest in the immunocompromised (IC) group (n = 54, 91.5%), followed by non-IC (n = 109, 80.1%) and HIV (n = 44, 80%). Ordering performed on the general ward was associated with inappropriate CSF test orders (adjOR 2.81, 95% CI [1.08-7.34]). Laboratory fee costs associated with excessive testing was close to $300,000 per year. A stepwise algorithm defining empiric and add on tests according to CSF parameters and patient characteristics could improve CSF test ordering in patients with suspected meningitis or encephalitis.

Decreased expression of circulating microRNA-126 in patients with type 2 diabetic nephropathy: A potential blood-based biomarker.

Circulating microRNAs (miRNAs) have been proposed as promising biomarkers for multiple diseases. miR-126 is reported to be associated with type 2 diabetes mellitus (T2D), diabetic nephropathy (DN) and end stage renal disease. The aim of this study was to investigate the expression of circulating miR-126 and to assess its potential as a blood-based biomarker for DN in T2D patients. In 52 patients with T2D without history of DN (with noromoalbuminuria), 50 patients with T2D and DN (29 with microalbuminuria and 21 with macroalbuminuria), and 50 non-diabetic healthy controls, the expression of circulating miR-126 in peripheral whole blood was evaluated by quantitative polymerase chain reaction. The expression levels of circulating miR-126 were significantly decreased in T2D patients and further decreased in DN patients compared with those in the controls. Multivariate logistic regression analysis confirmed the independent association of lower miR-126 levels with T2D [adjusted odds ratio (OR), 0.797; 95% confidence interval (CI), 0.613-0.960] and DN (adjusted OR, 0.513; 95% CI, 0.371-0.708). miR-126 levels were associated with the degree of albuminuria and showed significantly low expression in DN patients with microalbuminuria (adjusted OR, 0.781; 95% CI; 0.698-0.952) and further lower expression in DN patients with macroalbuminuria (adjusted OR, 0.433; 95% CI, 0.299-0.701), respectively compared with T2D patients with normoalbuminuria. miR-126 levels negatively correlated with albuminuria positively with glomerular filtration rate (P<0.05), and in addition, negatively correlated with fasting glucose, glycated hemoglobin, triglyceride and LDL (P<0.05). Stepwise multiple regression analysis identified albuminuria as a significant predictor of miR-126 (P<0.001). miR-126 in peripheral blood yielded area under the receiver operating characteristic curves of 0.854 (95% CI, 0.779-0.929) and 0.959 (95% CI, 0.916-1.000) in the differentiation of DN patients from T2D patients and DN patients from non-diabetic controls respectively. These data suggest that decreased expression of circulating miR-126 is associated with the development of DN in T2D patients, and may be a promising blood-based biomarker for DN risk estimation.

Development and Validation of a Simple Diagnostic Method to Detect Gain and Loss of Function Defects in Fibroblast Growth Factor-23.

BACKGROUND: Fibroblast growth factor-23 (FGF23) is a bone-derived hormone that regulates the homeostasis of phosphate and vitamin D. Three substitutions in the hormone are reported to cause autosomal dominant hypophosphatemic rickets and seven substitutions to cause autosomal recessive hyperphosphatemic familial tumoral calcinosis (HFTC). Both disorders are rare in the general population and occur most often in the Eastern Mediterranean region and Africa. None of the mutations could be identified using standard restriction fragment length polymorphism. The only technique currently available to confirm the clinical diagnosis is DNA sequencing. METHODS: Using a tri-primer ARMS-PCR, in vitro site-directed mutagenesis and DNA sequencing, we developed, verified and validated a rapid and reliable diagnostic test for the ten mutations in FGF23. RESULTS: We generated a test for all ten mutations and confirmed each test by DNA sequencing. We increased the specificity of the test by introducing a mismatch at position -2 in the 3'-terminus of the reverse primer of the normal and the mutant sequences. Finally, using DNA sequencing, we validated the technique for FGF23/S129F substitution by testing samples from 80 individuals from two unrelated Arab families harboring HFTC. CONCLUSIONS: This inexpensive and specific method could be adopted where DNA sequencing is not available or affordable.

FGF23-S129F mutant bypasses ER/Golgi to the circulation of hyperphosphatemic familial tumoral calcinosis patients.

FGF23 is essential for the homeostasis of phosphate, and vitamin D. Loss-of-function mutations in this hormone cause hyperphosphatemic familial tumoral calcinosis (HFTC). Earlier reports suggested that intact FGF23 from loss of function mutants such as FGF23/S129F (iFGF23/S129F) is retained intracellularly while the carboxy-terminal fragment is secreted. We sought to investigate the fate of iFGF23/S129F mutant hormone in vivo and in vitro. Five patients clinically diagnosed with HFTC and confirmed by DNA sequencing to carry the c.386 C>T; p.S129F mutation in the homozygous state were studied. Healthy and heterozygous individuals were used as controls in the study. Using ELISA assays, we showed that iFGF23/S129F was 2-5 folds higher in patients' plasma, compared to heterozygous or healthy controls. Importantly, the mutant hormone could not be detected in the patients' sera. However, using proteinase inhibition profiling, we found that a serum metalloproteinase degraded the iFGF23/S129F explaining our failure to detect it in sera. The serum metalloproteinase degrades the WT and the mutant at different rates. Also, confocal microscopy imaging using wild-type (WT) FGF23 or FGF23/S129F mutant in transiently transfected HEK293 and HeLa cells showed weak staining of the Golgi complex with some vesicular staining resembling the ER. Additionally, FGF23 variants (FGF23/WT, FGF23/S129F, FGF23/S71G, and FGF23/R176Q) from stably transfected HEK293 cells secreted high levels into a serum-free medium that can be detected by ELISA and Western blot. Our results suggest that iFGF23/S129F mutant bypasses the ER/Golgi quality control system to the circulation of HFTC patients by an unknown pathway. Finally, we hypothesize that either the mutant hormone is unable to bind α-Klotho-FGFR1c, or it binds the dyad receptor with low affinity and, therefore, incapable of initiating maximal intracellular signaling. Our findings raise the potential use of the WT hormone in therapies of some HFTC patients.

Haptoglobin polymorphism in relation to antioxidative enzymes activity in type 2 diabetes mellitus.

BACKGROUND: Increased oxidative stress and impaired antioxidative capacity are common findings in diabetics. This study reports on the status of antioxidative enzymes in relation to haptoglobin (Hp) polymorphism in type 2 diabetes. METHODS: The study comprised 165 type 2 diabetic patients and 94 controls. Erythrocytic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), and plasmatic ceruloplasmin ferroxidase (Cp) were measured by spectrophotometry and Hp phenotypes were determined by gel electrophoresis. RESULTS: Irrespective of Hp phenotype, while the activities of Cp ferroxidase and GPx were significantly higher in patients than in controls, those of SOD were significantly lower. No significant differences observed for CAT. However, significant Hp-phenotype dependent differences were observed between patients and controls regarding the activity of these enzymes. While ferroxidase activity in Hp2-2 patients was significantly higher than that in Hp1-1 or Hp2-1 patients, that of SOD and GPx were significantly lower. When patients were analyzed as a single group, Spearman's univariate analysis has demonstrated that HbA1c positively correlates with ferroxidase activity and negatively correlates with levels of GPx and SOD. However, when patients were treated as separate Hp-dependent groups, similar but stronger correlations between these variable were noted only in the case of Hp2-2 patients. CONCLUSIONS: These findings suggest that Hp polymorphism has some bearing on the activity of antioxidative enzymes in type 2 diabetes and that Hp2-2 diabetics are under increased oxidative stress as compared with those expressing Hp1-1 or Hp2-1.

The Arabic allele: a single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron 11. Our results were confirmed by RFLP on 2% agarose gel. In silico analysis predicted a new cryptic splice site downstream of the original position generating a 10-base deletion from the beginning of exon 12; (c.1706-1715del.ATCTCCTCAG). cDNA sequencing of exon 12 confirmed the computational analysis. The deletion was visualized on 4% agarose gel. The deletion generates a frameshift and a premature termination codon (c.1991-1993; p.(Asp569Valfs*93). RT-PCR revealed that LDLR mRNA is 9.3%±6.5 and 17.9%±8.0 for FH homozygote and heterozygote individuals respectively, compared to a healthy family control. We predict a class II LDLR mutation that leads to a truncated receptor missing exons 14-18. We called this mutation "the Arabic allele". We expect a significant contribution of this mutation to the prevalence of FH among Arabs. Also, we propose that the severe down regulation of LDLR mRNA expression is due to nonsense-mediated-decay.