Seven hundred fifty-eight processed sputum sediments received for the diagnosis of tuberculosis or other mycobacterial infections were tested by utilizing a rRNA target amplification assay and traditional culture techniques. The results from the rRNA target amplification assay (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test), available in 5 h, were compared with the results from standard culture techniques held for 6 weeks. A total of 119 specimens (16%) were culture positive for Mycobacterium tuberculosis. Overall sensitivity, specificity, positive predictive value, and negative predictive value were 82, 99, 97, and 96%, respectively, for the Gen-Probe assay; 88, 100, 100, and 97%, respectively, for culture; and 53, 99.8, 99.6, and 91%, respectively, for fluorochrome stain. The Gen-Probe assay employs the isothermal enzymatic amplification ofM. tuberculosis complex rRNA followed by detection of the amplicon with an acridinium ester-labeled DNA probe. This assay has the potential of reducing the time for diagnosis of tuberculosis to 1 day.

The chemical compositions of the cell walls obtained from 10 strains (serotypes 1a, 3a, 4a, 4b, 4c, 4d, 4e, 4e, 4f, 6, and 7) of Listeria monocytogenes were analyzed. These cell walls were shown to be mainly composed of peptidoglycan and ribitol teichoic acids. Considerable variations in the composition of neutral sugars were observed among these cell walls. Chemical and NMR analyses indicated that the teichoic acids from L. monocytogenes serotypes 4a and 4d are composed of the following repeating units: Formula: See Text.

The cell-wall skeletons of Listeria monocytogenes strain EGD and Propionibacterium acnes strain C7, which have the ability to induce macrophage activation, were analyzed, and the structures of the peptidoglycans were investigated. The analytical data indicate that both peptidoglycans have glucosamine residues with free amino groups, which are responsible for the resistance to lysozyme. Possible structures of these peptidoglycans were deduced from the composition and the results of determination of N- and C-terminal amino acids, together with the characterization of fragments obtained by enzymatic treatment and partial acid hydrolysis of both peptidoglycans. The results suggested that the peptidoglycan of L. monocytogenes contains a cross-linkage region of peptide chains with meso-diaminopimelic acid and D-alanine, which belongs to the A1 gamma type (Schleifer, K.H. & Kandler, O. (1972) Bacteriol. Rev. 36, 407-477), whereas the peptidoglycan of P. acnes contains a cross-linkage region of peptide chains with L,L-diaminopimelic acid and D-alanine, in which two glycine residues combine with amino and carboxyl groups of two L,L-diaminopimelic acid residues. The latter type should be classified as a new type. These cell-wall skeletons and peptidoglycans were shown to have immunoadjuvant activity on the induction of delayed-type hypersensitivity and suppressive activity on the growth of 3-methylcholanthrene-induced fibrosarcoma in BALB/c mice, and the peptidoglycans were shown to be an immunological-active principle of these cell-wall skeletons.

We have developed a sensitive and quantitative assay using transcription-mediated amplification and hybridization protection assay for the detection of hepatitis B virus (HBV) DNA in serum. The transcription-mediated amplification was carried out in a single tube. The hybridization protection assay was carried out in a microtiter plate with two probes with different specific activities to obtain a broad detection range. As a result, the assay had a detection range of 5 × 103 to 5 × 108 genome equivalents (GE)/ml and good quantitative accuracy on a logarithmic scale. A moderately sized manual assay run can be completed within 5 h. Measurements of the amounts of HBV DNA in clinical samples by the assay showed the amounts under various disease conditions to be widely distributed (more than 5 logs, from approximately 5 × 103 to 5 × 108 GE/ml). It was also shown that the amount of HBV DNA in one chronic hepatitis patient varied widely, with a range of more than 5 logs during long-term monitoring. Our assay has the potential to be used to monitor and determine the prognosis of HBV patients and carriers, especially during interferon treatment.

Background: Detection of telomere repeats by Southern hybridization of genomic DNA is time consuming, and the reading of a mean terminal restriction fragment (TRF) length from a smear pattern of an autoradiogram can be inaccurate. We developed a hybridization protection assay (HPA) for telomere repeats. Methods: We heated 5 μL of DNA solution or 10 μL of cell or tissue lysate at 95 °C for 5 min, mixed it with 100 μL of hybridization solution containing 3 × 106 relative light units of acridinium ester-labeled probe, and incubated the mixture for 20 min at 60 °C. We then added 300 μL of selection buffer and incubated the mixture for 10 min at 60 °C to differentially hydrolyze unhybridized probe. Chemiluminescence was measured for 2 s per tube. Results: The amount of telomere repeats was assayed by HPA within linearity from 10 to 3000 ng of purified genomic DNA or from 1000 to 100 000 cell equivalents of lysate. To normalize the amount of DNA in lysate, the amount of Alu sequence was measured by HPA. A ratio of telomere to Alu (TA ratio) = 0.01 corresponded to ∼2 kbp of mean TRF length determined by Southern blotting in cultured fibroblast and colorectal tissue samples. The TA ratio decreased from 0.06 to 0.02 with increasing division age from 30 to 90 population doubling levels of cultured human fetal fibroblasts. The assay required ∼45 min from collection of cell or tissue samples. Conclusions: The amount of telomere repeats was quantitatively measured by HPA in 10 ng of sheared genomic DNA or in the lysate of 1000 cells. This method is simple, rapid, quantitative, sensitive, and applicable to the measurement of telomere repeats in clinical samples such as needle biopsy specimen or as few as 1000 cells in body fluid or washings.