Takafumi Fukui

Use of Real-Time PCR on Blood Samples for Diagnosis of Invasive Aspergillosis

We developed a new quantitative system for diagnosis of invasive pulmonary aspergillosis (IPA) using real-time automated polymerase chain reaction (PCR). Intra-assay and interassay precision rates for in vitro examination were 2.53% and 2.20%, respectively, and the linearity of this assay was obtained when there were >20 copies/well. We examined 323 samples taken from 122 patients with hematological malignancies, including 33 patients with IPA and 89 control patients. Blood samples were subjected to PCR antigen detection methods, using enzyme-linked immunosorbent assay (ELISA) and determination of plasma (1-->3)-beta-D-glucan (BDG) concentration. The sensitivities of PCR, ELISA, and BDG measurement for diagnosis of IPA were 79%, 58%, and 67%, respectively; the specificities were 92%, 97%, and 84%. Positive findings on PCR preceded those of computed tomography by -0.3+/-6.6 days, those of BDG measurement by 6.5+/-4.9 days, and those of ELISA by 2.8+/-4.1 days. Real-time PCR was sensitive for IPA diagnosis, and quantitation was accurate.

Detection of Three Genetic Polymorphisms in the 5' ''-Flanking Region and Intron 1 of Human CYP1A2 in the Japanese Population

Interindividual variability of the activity of CYP1A2 may be expected to affect cancer susceptibility, since the enzyme is capable of activating several carcinogens. In the present study, we found three new polymorphisms in the 5′‐flanking region (CYP1A2/B) and intron 1 (CYP1A2/C and CYP1A2/D) of CYP1A2 in Japanese by using polymerase chain reaction‐single strand conformation polymorphism. We developed methods to detect these polymorphisms by polymerase chain reaction‐restriction fragment length polymorphism and performed a population study (159 subjects) to estimate the frequencies of the alleles. The frequencies of the CYP1A2/A (adenine), CYP1A2/B (thymine‐deleted), CYP1A2/C (guanine) and CYP1A2/D (adenine) variants were 21.1, 42.0, 8.2 and 61.3%, respectively. The results of family study supported the idea that these CYP1A2 genotypes are inherited with an autosomal codominant transmission.

Quantitative analysis of hTERT mRNA expression in colorectal cancer

OBJECTIVES:Telomerase is highly activated in a variety of malignant neoplasms including colon cancer. Among the major components of telomerase, human telomerase reverse transcriptase (hTERT) is thought to regulate telomerase activity. To assess the importance of telomerase for the diagnosis of colorectal cancer, we measured the expression of hTERT mRNA and telomerase activity in a large series of 140 colorectal cancers, 140 adjacent normal tissues, and 20 adenomas.METHODS:The expression level of hTERT was measured quantitatively by competitive reverse transcriptase–polymerase chain reaction (RT-PCR), and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay in the same samples.RESULTS:The median expression level of hTERT mRNA in carcinomas was significantly higher than that in either adenomas or normal tissues. The median level of hTERT in adenomas was significantly higher than that in normal tissues. Telomerase activities in carcinomas were significantly higher than those in either adenomas or normal tissues. Telomerase activities in adenomas were also significantly higher than those in normal tissues. Furthermore, the relative expression levels of hTERT mRNA in adenomas and carcinomas were significantly correlated with the relative telomerase activities; the Spearman rank correlation was 0.53 (p = 0.021) and 0.18 (p = 0.031), respectively.CONCLUSIONS:Our data suggest that determination of hTERT mRNA by competitive RT-PCR is superior in quantitative accuracy and sensitivity and would support the importance of telomerase activity for the diagnosis of colorectal cancer.

A detection method for point mutation in the precore region of human hepatitis B virus (HBV)-DNA using mutation-site-specific assay

In the natural progression of acute active hepatitis and chronic active hepatitis in human hepatitis B virus (HBV)-infected patients, inactive hepatitis develops by seroconversion, which can be explained by the disappearance of HBe antigen. However, it has been found that in some patients with hepatitis, alanine aminotransferase levels undergo fluctuation even though their serum is negative for HBe antigen. In these patients, HBV-DNA has been detected in the serum and the HBV-DNA so detected has been considered a cause of worsening liver function. Most HBV-DNA detected in these cases has a point mutation from G to A at the 83rd base in the precore region. As a result of this point mutation, HBV is unable to produce HBe antigen. We have devised a sensitive polymerase chain reaction (PCR) method, a mutation-site-specific assay, for the detection of point mutations at the 83rd base in the precore region using a specific mutation-trapped oligonucleotide primer for the mutant HBV genome.

Diagnosis and Monitoring of Human Cytomegalovirus Diseases in Patients with Human Immunodeficiency Virus Infection by Use of a Real-Time PCR Assay

We used a real-time PCR assay to measure human cytomegalovirus (HCMV) DNA load in whole blood and plasma of 70 patients who were infected with human immunodeficiency virus type 1. Break points of 3.0 x 10(3) copies/mL in whole blood and 1.0 x 10(3) copies/mL in plasma were well-correlated with the existence of definite HCMV disease (sensitivity, 93% and 86%; specificity, 89% and 85%; positive predictive value, 70% and 63%; and negative predictive value, 98% and 95%, respectively). In patients with < 50 cells/microL of CD4(+) T lymphocytes, positive predictive values increased to 78% and 71%, respectively. The viral loads of all patients who received anti-HCMV therapy declined to < or =2.0 x 10(2) copies/mL in parallel with the improvement of clinical symptoms. These findings show that the HCMV DNA load quantified with our method is a useful tool for diagnosis of HCMV diseases and for monitoring the disease activity in patients infected with HIV-1.

Technical Evaluation: Identification of Pathogenic Mutations in PKD1 and PKD2 in Patients with Autosomal Dominant Polycystic Kidney Disease by Next-Generation Sequencing and Use of a Comprehensive New Classification System.

Genetic testing of PKD1 and PKD2 is expected to play an increasingly important role in determining allelic influences in autosomal dominant polycystic kidney disease (ADPKD) in the near future. However, to date, genetic testing is not commonly employed because it is expensive, complicated because of genetic heterogeneity, and does not easily identify pathogenic variants. In this study, we developed a genetic testing system based on next-generation sequencing (NGS), long-range polymerase chain reaction, and a new software package. The new software package integrated seven databases and provided access to five cloud-based computing systems. The database integrated 241 polymorphic nonpathogenic variants detected in 140 healthy Japanese volunteers aged >35 years, who were confirmed by ultrasonography as having no cysts in either kidney. Using this system, we identified 60 novel and 30 known pathogenic mutations in 101 Japanese patients with ADPKD, with an overall detection rate of 89.1% (90/101) [95% confidence interval (CI), 83.0%–95.2%]. The sensitivity of the system increased to 93.1% (94/101) (95% CI, 88.1%–98.0%) when combined with multiplex ligation-dependent probe amplification analysis, making it sufficient for use in a clinical setting. In 82 (87.2%) of the patients, pathogenic mutations were detected in PKD1 (95% CI, 79.0%–92.5%), whereas in 12 (12.8%) patients pathogenic mutations were detected in PKD2 (95% CI, 7.5%–21.0%); this is consistent with previously reported findings. In addition, we were able to reconfirm our pathogenic mutation identification results using Sanger sequencing. In conclusion, we developed a high-sensitivity NGS-based system and successfully employed it to identify pathogenic mutations in PKD1 and PKD2 in Japanese patients with ADPKD.