Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Accelerated Loss of Islet β Cells in Sucrose-Fed Goto-Kakizaki Rats, a Genetic Model of Non-Insulin-Dependent Diabetes Mellitus

The Goto-Kakizaki (GK) rat is a spontaneously diabetic animal model of non-insulin-dependent diabetes mellitus, which is characterized by progressive loss of β cells in the pancreatic islets with fibrosis. In the present study, we examined the effects of sucrose feeding on the islet pathology in this model. Six-week-old GK rats were fed with 30% sucrose for 6 weeks to induce severe hyperglycemia, and their condition was compared with that of nontreated rats. Age-matched normal Wistar rats were also given sucrose for the same periods and used for comparison. The sucrose-treated GK rats showed elevated blood glucose levels on oral glucose tolerance tests at 60 minutes and 120 minutes, representing 123% and 127% of values in untreated GK rats, respectively. At the end of the study, the mean β-cell volume density in GK rats was 50% less than that in untreated Wistar rats. Sucrose feeding further reduced the volume densities of β cells to only 50% of the levels of age-matched GK rats. Apoptotic cells were found in islet β cells only in GK rats fed sucrose (mean 0.067%). There appeared to be more islets that immunohistochemically stained strongly positive with 8-hydroxy-deoxyguanosine as a marker of oxidative damage of DNA in GK rats fed sucrose compared with those not given sucrose. GK rats not fed sucrose showed significantly lower proliferative activity of β cells measured by 5-bromo-2′-deoxyuridine uptake and intensified expression of Bcl-2 immunoreactivities at 6 weeks of age compared with those in age-matched Wistar rats. These two indices were reduced in both GK and Wistar rats with increasing age and were not affected by sucrose feeding in either group. The present study thus indicated that sucrose feeding promoted the apoptosis of β cells in GK rats through increased oxidative stress without altering their proliferative activity.     

Histocytologic diagnosis of pancreatic cancer by percutaneous aspiration biopsy under ultrasonic guidance

Percutaneous aspiration biopsy of the pancreas using a heparinized 22-gauge fine needle was performed under ultrasonic guidance in five patients with benign pancreatic diseases and in 18 patients with pancreatic cancer. Using a heparinized needle and syringe, it was possible to make good smears containing abundant tumor cells and to obtain small tissue specimens. Using egg albumin as binding material, a new cell-block technic was developed to conveniently obtain histologic specimens. In this way, a correct diagnosis was made cytologically in all 23 patients suspected of having a pancreatic malignancy. Histologic specimens were obtained in 22 (95.6%) our of 23 patients. A correct diagnosis was established histologically in all patients from whom histologic materials were obtained. This procedure thus has proved a very reliable method for diagnosing pancreatic cancer.     

Morphologic demonstration of tubular obstruction in acute renal failure.

We used the qualitative Hanssen technique in albino rats to seek morphologic demonstration of tubular obstruction in two types of acute renal failure: one induced by folic acid and another by methemoglobin. Immediately after the intravenous injection of folic acid, 250 mg/kg body weight, the animals became almost anuric. Two to three hours after the injection, sodium ferrocyanide remained within the proximal convoluted tubules. After the intravenous injection of methemoglobin, 0.5 to 1.0 g/kg body weight, the animals became oliguric but not anuric. Sodium ferrocyanide injected with methemoglobin was seen mainly in distal tubules and collecting ducts 2 to 3 hours after the injection. The degree of tubular dilatation was more marked in the former model than in the latter, in agreement with the degree of oliguria. These morphologic findings were taken to indicate that the above two types of acute renal failure were caused by tubular obstruction rather than by intrarenal vasoconstriction and subsequent cessation of glomerular filtration. (Am J Pathol 87:323-330, 1977).     

Characterization of the UL4 gene product of herpes simplex virus type 2

Summary.  We have identified the herpes simplex virus type 2 (HSV-2) UL4 gene product using a rabbit polyclonal antiserum raised against a recombinant 6xHis-UL4 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 27-kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor, suggesting that the UL4 gene was expressed as a γ₂ gene. Indirect immunofluorescence studies localized the UL4 protein within the nucleus as discrete punctate forms at late times postinfection. However, when expressed in the absence of other viral proteins, the UL4 protein was limited to the cytoplasm, indicating that an interaction with one or more other virus-induced proteins was responsible for the nuclear localization during infection. Subnuclear fractionation studies showed that the protein was released from the nuclear structure of infected cells by high salt treatment. Moreover, the UL4 protein was detected in purified virions and light particles. Received December 24, 1997 Accepted February 4, 1998     

Intravenous ulinastatin therapy for Stevens-Johnson syndrome and toxic epidermal necrolysis in pediatric patients. Three case reports

BACKGROUND: More effective therapy is needed for the treatment of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The clinical efficacy of intravenous ulinastatin therapy was investigated in 3 Japanese pediatric patients with SJS or TEN. METHODS: Ulinastatin was given to 1 pediatric SJS patient and 2 pediatric TEN patients within 7 days (patient 1; SJS), 6 days (patient 2; TEN), or 4 days (patient 3; TEN) after the onset of the skin rash. Ulinastatin was administered intravenously at a dose of 7,500 U/kg/day (maximum dose: 300,000 U/day). No corticosteroids were given. After the skin lesions resolved, the ulinastatin dose was reduced to between 2,500 and 5,000 U/kg/day as maintenance therapy and then the drug was withdrawn. RESULTS: Erythema, fatigue, and fever improved within 12-36 h of starting the ulinastatin infusion, and the skin lesions resolved completely after 4-7 days of ulinastatin therapy. None of the patients had cutaneous or ocular sequelae. No patient developed secondary infection or relapse and ulinastatin therapy caused no side effects. CONCLUSION: Ulinastatin dramatically reduced the febrile period with no adverse effects and was very safe in this study. Ulinastatin appears to be a useful and effective therapy for controlling SJS and TEN without sequelae.     

Syndrome of the sea-blue histiocyte--the first case report in Japan and review of the literature--.

A case of the syndrome of sea-blue histiocyte is presented in a 53-year-old Japanese woman, which is the first recorded case in Japan. The patient had hepatosplenomegaly, bleeding manifestations, mild thrombocytopenia, fatty metamorphosis and cirrhosis of the liver, as well as abnormal serum lipid profiles. Her parents were consanguineous and her maternal grandmother with hepatomegaly died of hepatic failure. Histologically, peculiar histiocytes containing numerous, intracytoplasmic sea-blue stained granules on May-Giemsa stain were demonstrated in biopsy materials of the bone marrow, lymph node and liver. The sea-blue granules in these histiocytes proved to have histochemical staining characteristics of lipogenic ceroid-like pigment. Ultrastructurally, these granules showed membrane-bound, pleomorphic inclusions of heterogeneous nature, including electron-dense amorphous or variegatedly osmiophilic, frequently laminated materials. Enzyme cytochemically, localization of acid phosphatase activity was demonstrated in and around the intracytoplasmic inclusions. With regard to the pathogenesis of the sea-blue histiocytes in this case, it may be suggested that the existence of the abnormality in lipid metabolism plays an important role in intralysosomal ceroidogenesis in these histiocytes.     

Detection of Neisseria meningitidis and Yersinia pestis with a novel silicon-based sensor.

A light-addressable potentiometric (silicon) sensor was used in an immunofiltration procedure for the detection of pathogenic bacteria. Yersinia pestis was detected by filtering the cells onto nitrocellulose membranes and then filtering anti-Y. pestis mouse monoclonal antibody and anti-mouse immunoglobulin G-horseradish peroxidase conjugate. For Neisseria meningitidis detection, mouse monoclonal antibody to the major outer membrane protein of this bacterium was coupled directly to horseradish peroxidase. N. meningitidis cell suspensions were filtered onto polycarbonate membranes, and the enzyme conjugate was allowed to react with the filtered bacteria. The presence of both enzyme conjugates was determined potentiometrically with the silicon sensor. The sensitivity of this technique relative to that of an enzyme-linked immunosorbent assay for N. meningitidis was determined. Fewer than 1,000 bacterial cells could be detected with the silicon sensor in a 20-min assay, whereas a 2.5-h enzyme-linked immunosorbent assay with the same antigen and antibody preparations was significantly less sensitive.     

Argyrophil cell carcinoma (apudoma) of the esophagus

Summary In a series of 79 cases of primary esophageal carcinoma resected at The Center for Adult Diseases, Osaka, there were six tumors with specific histopathologic features valid for the diagnosis of argyrophil cell carcinoma. Of the 6 tumors, 3 were studied electron microscopically and assay for ACTH content was performed on 4 tumors.Clinically, the ages of the 6 patients ranged from 56 to 71 years; two were women and four men. Four of the 6 patients died with widespread tumor recurrences within 9 months of operation.Microscopically, the 6 tumors were composed largely or almost entirely of small, spindle-shaped cells resembling those of oat cell carcinoma of the lung, and were characterized by the arrangement of tumor cells in solid sheets or anastomosing cords, the presence of argyrophil tumor cells, and the deposits of amyloid. Electron microscopically, the three tumors contained neurosecretory-type granules. Using bioassay or radioimmunoassay ACTH activity in the tumor tissues was detected in 3 out of the 4 tumors determined.From the light and electron microscopic characteristics and the assay evidence, it seems reasonable to conclude that the 6 tumors are endocrine polypeptide producing tumors (apudomas) that arise from argyrophil cells normally found among the basal cells of the esophageal mucosa, and that they represent a distinct histopathologic entity clearly distinguishable from other types of esophageal carcinomas.Supported in part by a Grant-in Aid for Cancer Research from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, JapanThe authors are grateful to Prof. H. Imura and Dr. Y. Hirate, Department of Internal Medicine, Kobe University School of Medicine for their interest and performing the assays for ACTH on the tumor tissues.