Accurate quantification of mycobacterial load is important to evaluate disease severity and to monitor the course of treatment in tuberculosis (TB). We evaluated the quantitative capability of the AdvanSure TB/NTM real-time PCR kit (LG Life Science, Korea) to determine the cycle threshold (Ct) for mycobacterial burden. We retrospectively analyzed data from 108 patients whose respiratory specimens (sputums and bronchoalveolar lavage fluids) were positive for Mycobacterium tuberculosis complex (85 culture-positive and 23 culture-negative specimens). We compared Ct values with grades of acid-fast bacilli (AFB) staining, semi-quantitative colony count on solid medium, and time to positivity (TTP) in liquid and solid media. We also investigated the cutoff Ct value for predicting stain-positive status. Ct value showed significant reverse correlation with AFB staining grade (r(s)=-0.635, P<0.01). Ct value significantly decreased as the semi-quantitative counts on the solid medium increased (P<0.001), and the mean Ct value of each of the groups 1+, 2+, 3+, and 4+ were 29.0, 30.0, 27.1, and 25.5, respectively. A weak correlation between Ct value and TTP in liquid and solid media was observed (r(s)=0.468 and 0.365, respectively). A cutoff Ct value of <33.2 best predicted stain positivity, with a sensitivity of 95.0% and a specificity of 32.0%. Our findings suggest the potential use of AdvanSure TB/NTM real-time PCR kit for quantitatively determining bacterial burden, albeit with some enhancements.
Area Under Curve ; Bronchoalveolar Lavage Fluid/microbiology ; DNA, Bacterial/*analysis ; Humans ; Mycobacterium tuberculosis/genetics/*isolation & purification ; Phenotype ; ROC Curve ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction ; Retrospective Studies ; Sensitivity and Specificity ; Sputum/microbiology ; Tuberculosis/diagnosis/microbiology

BACKGROUND: We evaluated a sensitive and quantitative method utilizing fragment analysis of the fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), simultaneously measuring mutant allele burden and length, and verified the analytical performance. METHODS: The number and allelic burden of FLT3-ITD mutations was determined by fragment analysis. Serial mixtures of mutant and wild-type plasmid DNA were used to calculate the limit of detection of fragment analysis, conventional PCR, and Sanger sequencing. Specificity was evaluated using DNA samples derived from 50 normal donors. Results of fragment analysis were compared to those of conventional PCR, using 481 AML specimens. RESULTS: Defined mixtures were consistently and accurately identified by fragment analysis at a 5% relative concentration of mutant to wild-type, and at 10% and 20% ratios by conventional PCR and direct sequencing, respectively. No false positivity was identified. Among 481 AML specimens, 40.1% (193/481) had FLT3-ITD mutations. The mutant allele burden (1.7-94.1%; median, 28.2%) and repeated length of the mutation (14-153 bp; median, 49 bp) were variable. The concordance rate between fragment analysis and conventional PCR was 97.7% (470/481). Fragment analysis was more sensitive than conventional PCR and detected 11 additional cases: seven had mutations below 10%, three cases represented conventional PCR failure, and one case showed false negativity because of short ITD length (14 bp). CONCLUSIONS: The new fragment analysis method proved to be sensitive and reliable for the detection and monitoring of FLT3-ITD in patients with AML. This could be used to simultaneously assess ITD mutant allele burden and length.
Alleles ; DNA ; Humans ; Leukemia, Myeloid, Acute ; Limit of Detection ; Methods ; Plasmids ; Polymerase Chain Reaction* ; Sensitivity and Specificity ; Tissue Donors ; Vascular Endothelial Growth Factor Receptor-1

BACKGROUND: The purpose of this retrospective and prospective study is to evaluate the efficiency of ultrasound (US) guidance as a method of decreasing the malposition rate of central venous catheterization (CVC) in the emergency department (ED). METHODS: We retrospectively enrolled 379 patients who underwent landmark-guided CVC (Group A) and prospectively enrolled 411 patients who underwent US-guided CVC (Group B) in the ED of a tertiary hospital. Malposition of the CVC tip is identified when the tip is not located in the superior vena cava (SVC). In Group B, we performed US-guided intravascular guide-wire repositioning and then confirmed the location of the CVC tip with chest radiography when the guide-wire was visible in any three other vessels rather than in the approached vessel. In the case of a guide-wire inserted into the right subclavian vein (SCV), the left SCV and both internal jugular veins (IJV) were referred to as the three other vessels. The two subject groups were compared in terms of the malposition rate using Fisher's exact test (significance = p < 0.05). RESULTS: There were 38 malposition cases out of a total of 790 CVCs. The malposition rates of Groups A and B were 5.5% (21) and 4.1% (17), respectively, and no statistically significant difference in malposition rate between the two groups was found. In Group B, the malposition rate was decreased from 4.1% (17) to 1.2% (5) after the guide-wire was repositioned with US guidance, which led to a statistically significant difference in malposition rate (p < 0.01). CONCLUSIONS: The authors concluded that repositioning the guide-wire with US guidance increased correct placement of central venous catheters toward the SVC.
Catheterization, Central Venous* ; Central Venous Catheters* ; Emergencies ; Humans ; Jugular Veins ; Prospective Studies ; Radiography ; Retrospective Studies ; Subclavian Vein ; Tertiary Care Centers ; Thorax ; Ultrasonography ; Vena Cava, Superior