METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
DESIGN AND METHODS: The activity of DPD was measured using 5-[2- (14)C]Fluorouracil (5-[2-(14)C]FUra) followed by separation of substrate and product 5-[2-(14)C]FUraH(2) with a 15 x 4.6 mm I.D., 5 microm particle size (d(p)) porous graphitic carbon (PGC) column (Hypercarb(R)) and HPLC with online detection of the radioactivity. This was standardized using the protein concentration of the cytosol (NanoOrange(R) Protein Quantitation).
RESULTS: Complete baseline separation of 5-[2-(14)C]Fluorouracil (5-[2-(14)C]FUra) and 5-[2-(14)C]Fluoro-5,6-dihydrouracil (5-[2-(14)C]FUraH(2)) was achieved using a porous graphitic carbon (PGC) column. The detection limit for 5-[2-(14)C]FUraH(2) was 0.4 pmol.
CONCLUSIONS: By using linear gradient separation (0.1% Trifluoroacetic acid [TFA] in water to 100% Methanol) protocols in concert with PGC columns (Hypercarb(R)), we have demonstrated that a PGC column has a distinct advantage over C-18 reverse phase columns in terms of column stability (pH 1-14). This method provides an improvement on the specific assay for DPD enzyme activity. It is rapid, reproducible and sensitive and can be used for routine screening for healthy and cancer patients for partial and profound DPD deficiency before treatment with 5- FUra.
METHODS: We performed a retrospective analysis of data from 759 patients with biopsy-proven NAFLD (24% with advanced fibrosis), seen at 10 centers in 9 countries in Asia, from 2006 through 2018. By using liver biopsies as the reference standard, we calculated percentages of misclassifications and indeterminate or discordant results from assessments made based on fibrosis scores (NAFLD fibrosis score [NFS] or Fibrosis-4 score) and liver stiffness measurements (LSMs), alone or in combination. The analysis was repeated using randomly selected subgroups with a different prevalence of advanced fibrosis (histologic fibrosis stage ≥F3).
RESULTS: In groups in which 3.7% and 10% of patients had advanced fibrosis, a 2-step approach (using the NFS followed by LSM only for patients with indeterminate or high NFS) and using a gray zone of 10 to 15 kPa for LSM, produced indeterminate or discordant results for 6.9% of patients and misclassified 2.7% of patients; only 25.6% of patients required LSM. In the group in which 10% of patients had advanced fibrosis, the same approach produced indeterminate or discordant results for 7.9% of patients and misclassified 6.6% of patients; only 27.4% of patients required LSM. In groups in which 24% and 50% of patients had advanced fibrosis, using LSM ≥10 kPa alone for the diagnosis of advanced fibrosis had the highest accuracy and misclassified 18.1% and 18.3% of patients, respectively. These results were similar when the Fibrosis-4 score was used in place of NFS.
CONCLUSIONS: In a retrospective analysis, we found that a 2-step approach using fibrosis scores followed by LSM most accurately detects advanced fibrosis in populations with a low prevalence of advanced fibrosis. However, LSM ≥10 kPa identifies patients with advanced fibrosis with the highest level of accuracy in populations with a high prevalence of advanced fibrosis.