Hisae Fusegawa

Screening School Children for Albuminuria, Proteinuria and Occult Blood with Dipsticks

Abstract Beginning in 1974, the Japanese Ministry of Health Welfare directed the screening of schoolchildren for proteinuria. We studied their procedure and methods in 6197 school children and also evaluated a new urine dipstick that measures albumin concentrations down to about 10 mg/l and creatinine down to about 300 mg/l. We used specimens from adult in- and outpatients to test the accuracy of the dipsticks. Based on the quantitative results, we set as cutoffs < 150 mg/l for protein and < 30 mg/l for albumin as the concentrations representing “low risk.” The quantitative values were assumed to be correct, and the dipstick results were judged accordingly, i.e., a dipstick protein of ≥ “150” mg/l or an albumin of | “30” mg/l indicated increased risk of developing or having a genitourinary disorder. The sensitivity/specificity of the protein dipstick was 95.1%/95.5%, and the same for the albumin dipstick was 83.8%/93.8%. The cut-off for the albumin dipsticks probably should be set somewhat lower to reduce the number of false negatives and increase the sensitivity of the dipstick. When we compared the quantitative albumin to the protein dipsticks with the above cut-offs, we found the sensitivity/specificity to be 79.3%/94.4%, i.e., much like the albumin dipstick results. The many reports on the association of albuminuria and risk of renal disease recommend that screening should be done for albumin rather than protein. Based on the data from the school children, we estimate that a dipstick albumin of “30” mg/l is borderline increased risk, and that a protein dipstick of “150” mg/l is the same. If we call the dipstick “10” mg/l albumin, “30” mg/l albumin and the “150” mg/l protein results “low risk,” then we estimate the prevalence of albuminuria in the school children to be about 2.1% and proteinuria to be about 4.3%. Children with these values should have a quantitative test for albumin and protein. We also tested a dipstick for creatinine and found increasing values with increasing age in both genders; the older boys had significantly higher creatinine values than the older girls and younger boys. For the albumin/creatinine ratio, we found 6028 children with a ratio of < 30 mg/g indicating low risk and 159 children with a ratio of ≥30 mg/g indicating increased risk. The ratio may be more useful owing to the likely reduction of the number of false negatives and false positives. Note: 1 mg/l albumin = 8.85 μmol/l .

Monitoring of inhibitors of enzymatic amplification in polymerase chain reaction and evaluation of efficacy of RNA extraction for the detection of hepatitis C virus using the internal control

Abstract Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, coamplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.

Detection of mycobacterial DNA with polymerase chain reaction in eye discharge and gastric juices in a case of scleritis

We report a case of mycobacterial scleritis in which prompt diagnosis was made by the detection of mycobacterial DNA with polymerase chain reaction (PCR) in eye discharge and gastric juices, when conventional tests were negative. A 77-year-old woman who had a past history of pulmonary tuberculosis visited the outpatient clinic of Tokai University Hospital complaining of pain in her right eye. She was diagnosed as having scleritis and uveitis. There were no indications of active tuberculosis. We examined the gastric juices, sputum, and eye discharge by microscopy, culture, and PCR for detection of mycobacterium. The results of microscopy and culture were negative, but with PCR we detected atypical mycobacterium in eye discharge and gastric juices. After oral treatment with antituberculosis agents, the patients eye symptoms disappeared. Detecting mycobacterial DNA with PCR could be useful for early diagnosis of mycobacterial scleritis, so that treatment with antituberculosis agents could be started.