No abstract

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.Brucella, one of the world's major zoonotic pathogens, is responsible for enormous economic losses, as well as considerable human disease in areas of endemicity (7). The detection of Brucella DNA by real-time PCR (RT-PCR) in serum samples simplifies the technique and shortens the turnaround time compared with that for conventional PCR techniques. While much attention has been directed toward minimizing falsepositive reactions resulting from specimen contamination or amplicon carryover, relatively little attention has been given to the causes of false-negative PCR results.Our group has recently developed a LightCycler-based RT-PCR assay for serum samples for the diagnosis of human brucellosis; this test is more sensitive than blood cultures and more specific than the serologic tests commonly used (8, 10). We chose boiling as the DNA preparation method for the diagnosis of brucellosis because the technique is simple, is reproducible, can be performed rapidly, and is effective with other clinical samples, such as urine and cerebrospinal fluid (4, 9), and because no sophisticated equipment is necessary. The most important reason, however, is because the number of circulating bacterial cells in serum samples from patients with brucellosis is probably very small, and moreover, the nucleic acids from the pathogen are likely released into the circulation as breakdown products during bacteremia (11). Although AlSoud and colleagues (1, 2) did not recommend the use of this method, De Medici et al. (6) selected boiling as their preferred extraction method for the detection of Salm...

C3 glomerulonephritis is a clinicopathologic entity defined by the presence of isolated or dominant deposits of C3 on immunofluorescence. To explore the effect of immunosuppression on C3 glomerulonephritis, we studied a series of 60 patients in whom a complete registry of treatments was available over a median follow-up of 47 months. Twenty patients had not received immunosuppressive treatments. In the remaining 40 patients, 22 had been treated with corticosteroids plus mycophenolate mofetil while 18 were treated with other immunosuppressive regimens (corticosteroids alone or corticosteroids plus cyclophosphamide). The number of patients developing end-stage renal disease was significantly lower among treated compared with untreated patients (3 vs. 7 patients, respectively). No patient in the corticosteroids plus mycophenolate mofetil group doubled serum creatinine nor developed end-stage renal disease, as compared with 7 (significant) and 3 (not significant), respectively, in patients treated with other immunosuppressive regimens. Renal survival (100, 80, and 72% at 5 years) and the number of patients achieving clinical remission (86, 50, and 25%) were significantly higher in patients treated with corticosteroids plus mycophenolate mofetil as compared with patients treated with other immunosuppressive regimens and untreated patients, respectively. Thus, immunosuppressive treatments, particularly corticosteroids plus mycophenolate mofetil, can be beneficial in C3 glomerulonephritis.

Hyponatremia is an alteration in patients with advanced liver disease. Although survival is significantly reduced in patients with spontaneous development of hyponatremia, a reduced sodium concentration cannot be considered as a independent predictor of the risk for death.