Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay.

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

Infiltration of the lower respiratory tract by helper/inducer T lymphocytes in HTLV-1-associated adult T-cell leukemia/lymphoma.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATLL), a disorder in which peripheral blood and multiple organs are infiltrated by malignantly transformed T lymphocytes. We investigated the nature of pulmonary disease in a patient with serologic evidence of HTLV-1 infection. In this case, endobronchial biopsy specimens showed infiltration of the bronchial mucosa by pleomorphic cells exhibiting a high degree of nuclear irregularity. These cells were morphologically identical in appearance to malignant cells found in peripheral blood and infiltrating the dermis, expressed the OKT4/Leu3 phenotype and the receptor for interleukin 2, and, by analogy to gene rearrangement studies on leukemic blood cells, were monoclonal in origin. However, in situ hybridization of endobronchial biopsy specimens with full-length HTLV-1 probes failed to detect retroviral RNA or proviral DNA. These studies indicate that T lymphocytic involvement of the lower respiratory tract in HTLV-1-associated ATLL is characterized by expression of a malignant phenotype despite the inability to document actual cellular infection with this retrovirus by a molecular hybridization technique.

Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization.

Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization.

Use of in situ hybridization optimized for fluorescent detection of nuclear RNA has revealed a striking localization of specific viral RNAs within nuclei of cells latently infected with EBV. Several hundred kb of specific transcripts is sharply restricted to a small region of the nucleus, frequently in a curvilinear "track". Detection of nuclear RNA was evidenced by hybridization without denaturation, sensitivity to RNAase, inhibition by actinomycin D, and specificity of transcribed sequences. Results indicate that RNA "tracks" extend from an internal genome into the nuclear periphery, and that RNA transport may be coupled to transcription. Localized nRNA is apparent for other viral sequences, different lymphoblastoid cell lines, nuclei prepared by two different methods, and an abundant, nonviral transfected sequence. Implications for understanding nuclear organization and the investigation of gene expression are discussed.

A lymphoproliferative disorder caused by human T-lymphotropic virus type I. Demonstration of a continuum between acute and chronic adult T-cell leukemia/lymphoma.

A 35-year-old black man is described who had a human T-lymphotropic virus type I (HTLV-I) infection while living in a non-endemic region. A lymphoproliferative disorder developed that might be considered as a transition stage between acute and chronic adult T-cell leukemia/lymphoma. This suggests that HTLV-I-induced neoplasias represent a continuous disease spectrum.

HTLV-III expression in infected lymph nodes and relevance to pathogenesis of lymphadenopathy.

In situ hybridization with a 35S-labeled probe of cryosections from 14 lymph nodes of HTLV-III/LAV-seropositive individuals with lymphadenopathy showed the presence of rare cells containing HTLV-III RNA at levels consistent with virus replication. Positive cells were observed at a frequency of only 1-10 per section. In all lymph nodes studied, including those showing follicular hyperplasia or involution, most of the cells expressing viral RNA were present in the follicular areas. Only a minority of such cells were found outside the follicles. The nature of the follicular, virus-replicating cells was not evident from the processed cryosections. After prolonged autoradiography, another pattern of hybridization was observed, suggesting low levels of viral RNA diffusely distributed throughout most, but not all, follicles. This may represent extracellular virus and/or virus-antibody complexes, but was not consistent with intracellular viral RNA or phagocytosed virus particles. Comparison of hybridization levels in relation to histology suggested a decrease in both the number of HTLV-III replicating cells as well as in the diffusely distributed viral RNA with progression of follicular involution. Our observations indicate that in HTLV-III-infected subjects, lymph node follicles are an important site of virus replication and trapping, suggesting a decisive role of the virus in the characteristic reactive and cytopathogenic changes of HTLV-III-associated lymphadenopathy.

Detection of HTLV-III RNA in lungs of patients with AIDS and pulmonary involvement.

A majority of pediatric patients and rare adult patients with the acquired immunodeficiency syndrome (AIDS) develop a chronic respiratory disorder referred to as "lymphocytic interstitial pneumonitis" (LIP). Efforts to identify an infectious agent responsible for this process so far have failed. In this study, frozen sections of lungs from patients with AIDS and pulmonary disease were tested by in situ molecular hybridization for the presence of cells infected with human T-cell lymphotropic virus type III (HTLV-III) and expressing viral RNA. In the case of an infant with LIP, a relatively high frequency (0.1%) of cells in the lung were found to be positive for HTLV-III RNA. This number is the lower limit of total cells infected since the in situ hybridization technique as applied in this study depends on expression of HTLV-III genes, and previous evidence indicates that a proportion of cells infected with HTLV-III may not express viral RNA. Moreover, this degree of infection of the lung is likely limited to LIP, since in ten patients with AIDS and pulmonary diseases other than LIP, only 0% to 0.002% of cells in lung were positive for viral RNA expression. Thus, HTLV-III may play a direct causal role in the development of LIP in infected patients, implicating its involvement in yet another of the diverse clinical diseases associated with this virus.

Infectious mutants of HTLV-III with changes in the 3' region and markedly reduced cytopathic effects.

A variant of human T-lymphotropic virus type III (HTLV-III) is described that replicates but does not kill normal human T cells in vitro. This variant, designated X10-1, was derived from the genome of a cytopathic HTLV-III clone (pHXB2D) by excision of a 200-base pair segment in the 3' region of the virus, spanning the env and 3'-orf genes. Comparable variants with 55 to 109 base pairs deleted exclusively in 3'-orf produced, in contrast, virus that was extremely cytopathic. On the basis of these findings it is concluded that the 3'-orf gene is not required for cytopathogenicity or replication of HTLV-III. In addition, the results suggest that virus replication and cytotoxicity are not intrinsically coupled. Furthermore, since clone X10-1 retains the ability to trans-activate genes linked to the viral long terminal repeats, trans-activation per se is not responsible for T-cell killing by HTLV-III. These results also raise the possibility that the carboxyl terminus of the envelope gene of HTLV-III has a direct role in T-cell killing by this virus.

The trans-activator gene of HTLV-III is essential for virus replication.

Studies of the genomic structure of human T-lymphotropic virus type III (HTLV-III) and related viruses, implicated as the causal agent of acquired immune deficiency syndrome (AIDS), have identified a sixth open reading frame in addition to the five previously known within the genome (gag, pol, sor, env and 3'orf). This gene, called tat-III, lies between the sor and env genes and is able to mediate activation, in a trans configuration, of the genes linked to HTLV-III long terminal repeat (LTR) sequences. We now present evidence that the product of tat-III is an absolute requirement for virus expression. We show that derivatives of a biologically competent molecular clone of HTLV-III, in which the tat-III gene is deleted or the normal splicing abrogated, failed to produce or expressed unusually low levels of virus, respectively, when transfected into T-cell cultures. The capacity of these tat-III-defective genomes was transiently restored by co-transfection of a plasmid clone containing a functional tat-III gene or by introducing the TAT-III protein itself. As HTLV-III and related viruses are the presumed causal agents of AIDS and associated conditions, the observation that tat-III is critical for HTLV-III replication has important clinical implications, and suggests that specific inhibition of the activity of tat-III could be a novel and effective therapeutic approach to the treatment of AIDS.

Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization.

By using in situ hybridization methodology, we have directly examined primary lymph node and peripheral blood from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex for the presence of human T-lymphotropic virus type III (HTLV-III) viral RNA. Mononuclear cell preparations were hybridized with a 35S-labeled HTLV-III-specific RNA probe and exposed to autoradiographic emulsion for 2 days. HTLV-III-infected cells expressing viral RNA were detected in approximately 86% (6/7) of lymph node and 50% (7/14) of peripheral blood samples studied. However, in all patient samples examined, labeled cells were observed at very low frequency (less than 0.01% of total mononuclear cells). The HTLV-III-infected cells exhibited morphological characteristics consistent with that of lymphocytes and expressed viral RNA at relatively low abundance (20-300 copies per cell). These results demonstrate that HTLV-III expression in lymph node and peripheral blood is very low in vivo. Furthermore, the lymph node hyperplasia observed in HTLV-III-associated lymphadenopathy is not directly due to proliferation of HTLV-III-infected lymphocytes.

In situ hybridization--application to gene localization and RNA detection.

In situ hybridization offers a direct approach for localization and quantitation of nucleic acid sequences in cellular preparations. Recent improvements in technology and methodology make possible the detection of DNA and RNA of relatively low copy number. For example, development of in situ hybridization methods for detection of single copy DNA sequences on mitotic chromosomes has led to general use of this technique for gene mapping of the human genome. More recently, improvements in methodology for detection of low abundancy RNA make possible a facilitated analysis of gene expression, both from cellular genes and exogenous sequences, such as viral genomes. In situ hybridization is now a powerful method for studying nucleic acid organization and function in normal cells, as well as in malignant cells, which should contribute to better understanding of the cell transformation process.