Expression, transport properties, and chromosomal location of organic anion transporter subtype 3.

The rat and mouse organic anion-transporting polypeptides (oatp) subtype 3 (oatp3) were cloned to further define components of the intestinal bile acid transport system. In transfected COS cells, oatp3 mediated Na(+)-independent, DIDS-inhibited taurocholate uptake (Michaelis-Menten constant approximately 30 microM). The oatp3-mediated uptake rates and affinities were highest for glycine-conjugated dihydroxy bile acids. In stably transfected, polarized Madin-Darby canine kidney (MDCK) cells, oatp3 mediated only apical uptake of taurocholate. RT-PCR analysis revealed that rat oatp3, but not oatp1 or oatp2, was expressed in small intestine. By RNase protection assay, oatp3 mRNA was readily detected down the length of the small intestine as well as in brain, lung, and retina. An antibody directed to the carboxy terminus localized oatp3 to the apical brush-border membrane of rat jejunal enterocytes. The mouse oatp3 gene was localized to a region of mouse chromosome 6. This region is syntenic with human chromosome 12p12, where the human OATP-A gene was mapped, suggesting that rodent oatp3 is orthologous to the human OATP-A. These transport and expression properties suggest that rat oatp3 mediates the anion exchange-driven absorption of bile acids previously described for the proximal small intestine.

[A case of AIDS complicated with liver tuberculosis].

We experienced a double infection of tuberculosis and amebiasis of the liver. A 28 year old male with AIDS was admitted to our hospital because of severe diarrhea and liver abscess by Entamoeba histolytica. In spite of improvement of the diarrhea and liver abscess by the therapy against E. historicica, serum levels of gamma-GTP and ALP remained high and hepatosplenomegaly gradually increased. A liver biopsy was performed. Pathology showed a granulomatous lesion with Langhans' giant cells. From this specimen, IS6110 gene, a specific DNA for Mycobacterium tuberculosis was detected by PCR method. After anti-tuberculosis treatment was given for 6 months the increased serum gamma-GTP, ALP decreased and hepatosplenomegaly diminished.

Screening school children for albuminuria, proteinuria and occult blood with dipsticks.

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 > or = "150" mg/l or an albumin of I "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 > or = 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.

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.

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, co-amplification 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.

Brief survey of common intestinal parasites in the Tokyo Metropolitan Area.

Since 1955, when sanitary conditions were poor, the incidence of intestinal parasitism has steadily decreased. Similarly, the number of requests for fecal examinations by physicians has also decreased. However, in our hospital, the incidence of parasites detected in fecal material has been increasing since 1994, regardless of the decreasing number of stool exams performed. Possible reasons for this situation can be summarized as follows: First, an effective drug for treating Trichuris trichiura and Enterobius vermicularis infections has reduced the incidence of these two helminths. Second, an apparent increase in the incidence of infections with the tapeworms Diphyllobothrium latum and Diplogonoporus grandis may just be a reflection of patients gathering at a few facilities for treatment. Third, the number of individuals infected with a single Ascaris is significantly increasing. Fourth, parasites related with travel abroad (Schistosoma haematobium and Opisthorchis viverrini) are appearing due to the increase in travel to and from foreign countries. Of the above, we think particular attention should be paid to the increase in A. lumbricoides infections.

[Monitoring of inhibitors of the polymerase chain reaction for the detection of hepatitis C virus using the positive internal control].

In the direct detection of pathogens by polymerase chain reaction (PCR) from clinical samples, false negative results due to the presence of inhibitor are problematic. In order to monitor such an inhibitor, we evaluated the detection of the positive internal control in PCR assay for HCV-RNA in serum samples positive for anti-HCV antibodies, of which 41 samples were positive for HCV-RNA by competitive RT-PCR assay. The positive internal control was coamplified with HCV-RNA and hybridized to the specific probe on magnetic beads and then hybrids were detected by colorimetric measurement using automatic PCR machine (COBAS AMPLICOR). Detection of the positive internal control was negatively correlated with viral copy number in sera assayed by competitive RT-PCR. Five of 52 samples (9.6%) with high HCV-RNA copy number (10(7) or 10(8) copies/ml) showed negative results for the internal control. The negative results for the internal control turned out to be positive when the sera were diluted and re-assayed, suggesting competitively inhibitory effects of high viral copy number on amplification of the internal control. Addition of heparin in the serum sample showed an inhibitory effect with a dose dependent manner on the detection of both HCV-RNA and the internal control, with a more effect on the lower copy number of HCV. On the other hand, addition of hemoglobin in the sample with concentration of up to 450mg/dl had no inhibitory effect on the detection of either HCV-RNA or the internal control. Coamplification and detection of the positive internal control was demonstrated to be useful to estimate effects of inhibitors, which may be present in clinical samples, in the detection of HCV-RNA by PCR.

[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 patient's 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.

[Clinical diagnostic value of the PCR assay for detection of Mycobacterium tuberculosis].

To evaluate the clinical diagnostic value of the PCR assay for detection of M. tuberculosis, a total of 758 samples from 387 patients were assayed by the polymerase chain reaction (PCR) using the IS6110 gene as a target, and the results of the PCR assay were compared with those obtained by conventional culture. The patient's profiles such as clinical situation that required prompt differential diagnosis by the PCR assay, underlying disease and final diagnosis were analyzed. The PCR assay lead to the rapid diagnosis of tuberculosis which was confirmed by culture in all 56 cases including elderly and immunocompromised patients. One hundred and seventy-six of the 231 (76.2%) cases had abnormal shadows in the chest X-ray film that required the PCR assay for diagnosis of tuberculosis and differential diagnosis of pleural effusion, lung cancer or bacterial pneumonia. Negative PCR results were also used when active pulmonary tuberculosis had to be excluded. Forty-nine out of 95 cases (51.6%) with a final diagnosis of tuberculosis were in PCR-negative. The basis of the diagnosis in spite of the PCR negative results included positive culture results (8 cases) and histological findings (9 cases) in other specimens that were not subjected to PCR, radiological findings (24 cases), biochemical characteristics of pleural effusion (6 cases) or cerebrospinal fluid (1 case), and bronchoscopical findings (1 case). In 56 smear-negative patients with final diagnosis of pulmonary tuberculosis, the larger the number of specimens received for the PCR assay from each patient, the higher the frequency of positive cases; with 1, 2 and 3 specimens received for the PCR assay, 13.3% (4/30), 45.5% (5/11) and 60.0% (3/5) of cases were PCR positive, respectively. For the prompt diagnosis of tuberculosis by detection of tuberculous DNA using PCR, it is important to examine appropriate samples from the affected part of the patient and also to repeat the tests especially when the smear is negative.

[Direct detection of human cytomegalovirus in urine specimens from bone marrow transplant patients by polymerase chain reaction].

Human cytomegalovirus (HCMV) infection is a major causative life-threatening agent that results in opportunistic infections in bone marrow transplant patients. Since antiviral therapy is available for severe HCMV infections, methods to rapidly identify infected patients are needed so that therapy can be promptly instituted. In this study, we used direct method to detect HCMV in urine specimens by nested polymerase chain reaction (nested-PCR) to monitor urinary excretion of HCMV in patients undergoing bone marrow transplantation. The viral DNA was amplified directly from preheated urine without further treatment prior to amplification. Five microliter of urine proved to give the most efficient amplification. The detection limit of the PCR assay for detection of HCMV was 10 copies/microl. The positive rate of the PCR assay and the tissue culture method for detection of HCMV were 15 of 61 (24.6%) and 4 of 61 (6.6%), respectively. Sensitivity and specificity of the PCR assay were 100% and 81% respectively. Based on these data, urinary excretion of HCMV in 21 patients undergoing bone marrow transplantation were monitored weekly by the PCR assay. As a result, in addition to a higher sensitivity, the PCR assay allowed to identify HCMV infection 3.1 (1 approximately 5) weeks earlier than culture method or antibody elevation. Repeated monitoring of virus excretion by this rapid and simple method was useful to promptly detect HCMV infection, allowing proper institution of antiviral drug therapy in patients undergoing bone marrow transplantation.

[Rapid detection and identification of mycobacteria by the PCR assay based on the co-amplification of the gene IS6110 and groEL].

In definite diagnosis of mycobacterial infection, prompt and adequate differential diagnosis leads to an appropriate treatment. We developed and evaluated a PCR assay based on co-amplification of the insertion sequence IS6110 and groEL gene that are species-specific for Mycobacterium tuberculosis complex and genus-specific, respectively. The detection limit of the assay system for cultured M. tuberculosis was 2 cells/ml, as compared with 200 cells/ml by culture onto Ogawa's medium. To assess the value of the assay in routine laboratory works, the results obtained by PCR were compared with those by standard microbiological methods for 758 specimens collected for the examinations of mycobacterial infection. The PCR system for detection of mycobacteria gave overall positive rate of 27.6% (209/758), as compared to 6.1% (46/758) by smear and 7.7% (58/758) by culture onto Ogawa's medium. Sensitivity and specificity were 97.8% and 97.3%, respectively, for the IS6110 and groEL gene for detection of M. tuberculosis complex; 91.7% and 80.3%, respectively, for only the groEL gene for detection of atypical mycobacteria. The PCR assay based on co-amplification of the IS6110 and groEL gene would be useful for diagnosis of mycobacterial infections, allowing not only more sensitive detection of mycobacteria but also rapid discrimination between M. tuberculosis complex and atypical mycobacteria. This assay would help to eliminate time-consuming confirmation, and to avoid both unnecessary treatment and hospitalization of the patient.

[Detection of Mycobacterium tuberculosis by amplification of mycobacterial DNA from pleural fluid and gastric juice: the comparison of IS6110 gene with Amplicor mycobacterium].

The polymerase chain reaction (PCR) using oligonucleotides based on the repetitive sequence (IS6110) of Mycobacterium tuberculosis and Amplicor Mycobacterium, which combines a PCR with the hybridization assay, were evaluated for detection of M. tuberculosis in pleural fluid and gastric juice. The detection limits of these two assay systems for cultured M. tuberculosis were less than 2 cells/ml, as compared with 200 cells/ml by culture. A total of 42 pleural fluid and 94 gastric juice specimens were examined. A total of 5 pleural fluid and 5 gastric juice were culture positive for M. tuberculosis. Only the PCR gave positive results in 2 pleural fluid of which laboratory findings are characteristic of tuberculous pleuritis, and in one gastric juice of the patient who was diagnosed as having pulmonary tuberculosis. Sensitivity, specificity and positive predictive value for IS6110 gene were 75%, 94% and 71.1%, respectively, in pleural fluid. Two of three positive specimens for IS6110 gene from pleural fluid were negative for Amplicor Mycobacterium. These specimens resulted in positive with Amplicor Mycobacterium when solvent extracted DNA was used. Both the PCR systems had the same sensitivity (80%), specificity (98.8%) and positive predictive value (83.3%) in gastric juice. The PCR systems would be useful for rapid detection of M. tuberculosis without long term culture and for detection of non-cultured one from pleural fluid as well as gastric juice. However, when the presence of an inhibitor of PCR is suspected in a specimen, DNA should be purified from the specimen before amplification.

[Gene level diagnostics of infectious diseases].

Recent developments in medical technology have caused a great change in infectious diseases, as characterized by epidemics of antibiotics-resistant bacteria, opportunistic infection in compromised hosts, and blood-borne viral infections such as hepatitis virus and human immunodeficiency virus. In the diagnosis of such new aspects of infectious diseases, conventional immunological, culture, and microscopical techniques are not always practical. By contrast, evaluation of infectious agents using molecular biological technology frequently offers the rapid, most accurate and sensitive method of diagnosis. Amplification methods are particularly attractive for the detection of small numbers of microorganisms, as in latent conditions, or for the fastest identification of the pathogen without laborious isolation. For introduction of the tests into routine procedures, their standardization as well as simplicity and low cost are required. Gene level diagnostics should be applied appropriately to management of infectious diseases along with the conventional techniques, while further roles of the tests must be determined, on the basis of the molecular elucidation of infectious diseases.

[Detection of mycobacterial DNA by in vitro enzymatic amplification in lung nodular lesions in a patient with ovarian immature teratoma].

We experienced a 13-year-old girl with ovarian immature teratoma, who presented multiple pulmonary nodular lesions. Thoracotomy was performed to rule out the metastatic tumor. Histopathological study of the resected tumor suggested a possible diagnosis of tuberculosis. However, neither the acid-fast staining nor the mycobacterial culture of the specimens confirmed the diagnosis. Polymerase chain reaction (PCR) and hybridization technique successfully detected specific DNA sequence for M. tuberculosis from the specimens. The lesions regressed after anti-mycobacterial therapy. Detecting mycobacterial DNA by PCR, which led to a proper diagnosis and treatment of this patient, is a rapid and sensitive method in diagnosis of tuberculosis. This method is recommended to distinguish tuberculosis from neoplastic lesions of the lung, where surgical intervention is necessary.

[Importance of primer selection in 5'NC region for the detection of hepatitis C virus RNA by polymerase chain reaction].

We compared two primer sets (A: 167bp, B: 269bp) derived from highly conserved domains within the 5' noncoding region (5'NC) of the hepatitis C virus (HCV) genome for their ability to detect HCV-RNA in a nested cDNA polymerase chain reaction assay (nested-PCR) in sera from 31 patients suspected of having HCV infection. Seventeen (54%) of 31 patients were positive for HCV-RNA with both primer sets. Using primer set A, 14(93%) of 15 samples with positive and 3(19%) of 16 samples with negative anti-HCV antibody test gave positive results for HCV-RNA. With primer set B, 15(100%) of 15 antibody positive samples and 2(13%) of 16 negative samples were positive for HCV-RNA. One antibody negative sample from a patient with alcoholic liver cirrhosis was positive for HCV-RNA only with primer set A. Another sample with positive antibody test, from a patient with chronic renal failure, was positive for HCV-RNA only with primer set B. A combination of more than one set of primers directed to the highly conserved 5'NC region, as well as proper selection of the exact nucleotide sequences, are important in improving the detection rate of HCV-RNA by PCR in serum of infected patients.

[Detection of methicillin-resistant Staphylococcus aureus by in vitro enzymatic amplification of mecA and femA genes].

In the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid detection of MRSA is extremely important. The mecA gene codes the new drug resistant polypeptides called penicillin-binding protein 2a (PBP2a) or 2'(PBP2'), which mediates the clinically relevant resistance to all beta-lactam antibiotics. This gene could be beneficial in the detection of MRSA using the polymerase chain reaction (PCR). However, the identical mecA gene has been found in both coagulase-positive and coagulase-negative Staphylococcus with the appropriate methicillin-resistant phenotype. The second gene related to the expression of methicillin-resistance has been called femA. In this study, we amplified both mecA and femA genes by PCR in 97 strains and 32 clinical specimens. The mecA gene was positive in all 63(100%) MRSA strains and 2(6%) of the 34 methicillin-sensitive Staphylococcus aureus (MSSA) strains. Two strains with the methicillin-sensitive phenotype and the mecA gene resulted in methicillin-resistance when cultured on an agar plate containing 4.5% NaCl. The mecA gene was also present in all 10(100%) coagulase-negative Staphylococcus strains with the methicillin-resistant phenotype. The femA gene was positive in all 97(100%) MRSA and MSSA strains. On the other hand, the femA gene was absent from coagulase -negative Staphylococcus strains with the methicillin-resistant phenotype. Although the mechanism by which the product of femA gene influences the expression of methicillin-resistance is unknown, the gene appears to be restricted only in coagulase-positive Staphylococcus, regardless of methicillin-resistance. In conclusion, in vitro enzymatic amplification of both mecA and femA genes would lead to rapid and definite diagnosis of the MRSA infection.

[Rapid diagnosis of tuberculous lymphadenitis by amplification of mycobacterial DNA].

In patients with cervical lymph node swelling, prompt differential diagnosis of tuberculosis leads to an appropriate treatment. A method based on DNA amplification and hybridization for the rapid detection of Mycobacterium tuberculosis was used for demonstrating its specific DNA in cervical lymph nodes biopsied from seven patients. The DNA specific for Mycobacterium tuberculosis was detected in 5 patients. Four of them were histologically diagnosed as tuberculous lymphadenitis. In another case with necrotizing lesions but without granulomatous reactions, culture method subsequently revealed a positive result. In other 2 cases, the diagnosis of tuberculosis was promptly excluded by both this method and conventional methods (histologic diagnosis/direct microscopy). The rapid detection of Mycobacterium tuberculosis by amplification of mycobacterial DNA is helpful particularly in differential diagnosis of cervical lymphadenopathy.

[Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in blood diseases].

In patients with malignant blood disease, infection is the most serious complication. The prompt differential diagnosis of tuberculosis leads to an appropriate treatment. A method based on DNA amplification and hybridization for the rapid detection of Mycobacterium tuberculosis was used to test 10 clinical specimens (sputum, gastric aspirate and pleural effusion) from blood disease patients in whom tuberculosis was suspected. Mycobacterium tuberculosis DNA was detected in 3 specimens, including one which was negative on standard microbiological criteria (microscopy and/or culture). The other 7 specimens with fever or abnormal shadow on a chest X-ray were negative by both our method and the Standard microbiological criteria. Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in cases of blood disease in clinically useful.