ABSTRACT
A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 10² and 10³ leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine.
Subject(s)
Bacteriological Techniques/methods , Freeze Drying , Leptospira/genetics , Leptospira/isolation & purification , Leptospirosis/diagnosis , Molecular Diagnostic Techniques/methods , Polymerase Chain Reaction/methods , Animals , Blood/microbiology , Cricetinae , Disease Models, Animal , Humans , Lung/microbiology , Mesocricetus , Reagent Kits, Diagnostic , Sensitivity and Specificity , Urine/microbiologyABSTRACT
A simple and reliable tool for the early diagnosis of leptospirosis is urgently needed. We report the development of a lyophilized reagent-based polymerase chain reaction (PCR) assay targeting lipL32 gene, which is present only in pathogenic leptospires. To determine the effectiveness of the newly developed assay in the early diagnosis of leptospirosis, the sensitivity and specificity was evaluated. In simulated clinical samples, the assay was able to detect 102 and 103 leptospires/ml in spiked urine and blood samples, respectively. In experimentally infected animals, leptospiral DNA could be detected in blood and lung samples as early as Day 1 post infection. This assay was also shown to be stable and remained sensitive for up to five months at ambient temperature. Hence, this lyophilized reagent-based PCR assay with high specificity, sensitivity and stability would provide a simple, rapid and reliable method in diagnosing acute leptospirosis, especially in the field of veterinary medicine
ABSTRACT
Agarose gel electrophoresis is the standard method that is used to separate, identify, and purify DNA fragments. However, this method is time-consuming and capable of separating limited range of fragments. A new technique of gel preparation was developed to improve the DNA fragment analysis via electrophoresis.
Subject(s)
DNA, Bacterial/analysis , Electrophoresis, Agar Gel , DNA Fragmentation , Sepharose/chemistryABSTRACT
The Sunnybrook Gallstone Study was a randomized, double-blind, controlled trial of chenodeoxycholic acid treatment over 2 years in 160 patients with radiolucent gallstones. Sixty-four patients received 750 mg daily, 53 received 375 mg daily and 43 received placebo. Total dissolution of gallstones occurred in 10.9% of patients on 750 mg daily, 13.2% of those on 375 mg daily and in no patient on placebo. The drug was tolerated well. Diarrhea severe enough to cause withdrawal from the study occurred in two patients. No patient developed clinically significant hepatotoxicity. Serum cholesterol rose 10% or more above baseline after 2 years in 33% of patients treated with chenodeoxycholic acid and in 30% of those on placebo. Cholecystectomy was performed in 10.9% of patients on 750 mg daily, 17% on 375 mg daily and 13.6% on placebo. Chenodeoxycholic acid given at these doses dissolved radiolucent gallstones safely but the efficacy was limited.
