A real-time PCR assay using the LightCycler (LC) instrument for the specific identification of Mycobacterium tuberculosis complex (MTB) was employed to detect organisms in 135 acid-fast bacillus (AFB) smear-positive respiratory specimens and in 232 BacT/ALERT MP (MP) culture bottles of respiratory specimens. The LC PCR assay was directed at the amplification of the internal transcribed spacer region of the Mycobacterium genome with real-time detection using fluorescence resonance energy transfer probes specific for MTB. The results from the respiratory specimens were compared to those from the Amplicor M. tuberculosis PCR test. Specimens from MP culture bottles were analyzed by Accuprobe and conventional identification methods. MTB was cultured from 105 (77.7%) respiratory AFB smear-positive specimens; 103 of these samples were positive by LC PCR and Amplicor PCR. Two samples negative in the LC assay contained rare numbers of organisms; both were positive in the Amplicor assay. Two separate samples negative by Amplicor PCR contained low and moderate numbers of AFB, respectively, and both of these were positive in the LC assay. There were 30 AFB smear-positive respiratory specimens that grew mycobacteria other than tuberculosis (MOTT), and all tested negative in both assays. Of the 231 MP culture bottles, 114 cultures were positive for MTB and all were positive by the LC assay. The remaining 117 culture bottles were negative in the LC assay and grew various MOTT. This real-time MTB assay is sensitive and specific; a result was available within 1 h of having a DNA sample available for testing.Mycobacterium tuberculosis remains a serious public health issue due to its high risk of person-to-person transmission, morbidity, and mortality (15,20). Currently, approximately 8 million new infections and 3 million deaths are attributed to tuberculosis (TB) each year (L. B. Reichman, Letter, Chest 112:855, 1997). Progressive increases in TB infections are expected, and a worldwide annual incidence of 12 million cases by 2005 is predicted by the World Health Organization (20). The resurgence of TB in industrialized countries since the mid-1980s, primarily due to the increased incidence of immunocompromised patients with AIDS, and the emergence of multidrug-resistant strains of M. tuberculosis has accented the need for rapid diagnosis of this disease (15,20). Rapid detection of active TB infection is critical for effective patient management and implementation of infection control measures.Conventional detection of mycobacteria is based on a number of protocols, including microscopic examination of smears stained with the Ziehl-Neelsen stain or auramine fluorescent dye and selective culture techniques (8,17,26,39). The key aspect of TB control is rapid diagnosis, which for many years has been based on the staining of smears for the presence of acid-fast bacilli (AFB). The AFB smear test lacks specificity, so there is a need for a laboratory test for specific detection of the M. tuberculosis complex (MTB) that can be performe...
RNase P complexes have been proposed as a novel RNA-based gene interference strategy to inhibit gene expression in human malignancies and infectious diseases. This approach is based on the sequencespecific design of an external guide sequence (EGS) RNA molecule that can specifically hybridize to almost any complementary target mRNA and facilitate its cleavage by the RNase P enzyme component. We designed a truncated RNase P-associated EGS molecule to specifically recognize the U5 region of HIV-1 mRNA and mediate cleavage of hybridized mRNA by the RNase P enzyme. Genes encoding for this U5-EGS (560) molecule, as well as a U5 EGS (560D) antisense control, were cloned into retroviral plasmids and transferred into a CD4 ؉ T cell line. Transfected cells were exposed to increasing concentrations of HIV-1 clinical isolates from clades A, B, C, and F. Heterogeneous cultures of CD4 ؉ T cells expressing the U5 EGS (560) molecule were observed to maintain CD4 levels, were devoid of cytopathology, and did not produce HIV p24 gag antigen through 30 days after exposure to all HIV-1 clades at a multiplicity of infection of 0.01. Identical cells expressing the U5 EGS (560D) antisense control molecule underwent a loss of CD4 expression, produced elevated levels of HIV-1, and formed large syncytia similar to untreated cells. When the viral inoculum was increased at the time of exposure (multiplicity of infection ؍ 0.05), the inhibitory effect of the U5 EGS (560) molecule was overwhelmed, but viral-mediated cytopathology and particle production were delayed compared with control cell populations. Viral replication and cytopathology associated with infection of multiple HIV-1 clades can be effectively inhibited in CD4 ؉ cells expressing the RNase P-associated U5 EGS (560) molecule.
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