The lengthy chemotherapy of tuberculosis reflects the ability of a small subpopulation of Mycobacterium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these persisting bacteria is not known. Lung lesions in guinea pigs infected with M. tuberculosis have striking similarities, such as necrosis, mineralization, and hypoxia, to natural infections in humans. Guinea pigs develop necrotic primary lesions after aerosol infection that differ in their morphology compared to secondary lesions resulting from hematogenous dissemination. In infected guinea pigs conventional therapy for tuberculosis during 6 weeks reduced the bacterial load by 1.7 logs in the lungs and, although this completely reversed lung inflammation associated with secondary lesions, the primary granulomas remained largely unaffected. Treatment of animals with the experimental drug R207910 (TMC207) for 6 weeks was highly effective with almost complete eradication of the bacteria throughout both the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli remaining after R207910 treatment were to be found extracellular, in a microenvironment of residual primary lesion necrosis with incomplete dystrophic calcification. This zone of the primary granuloma is hypoxic and is morphologically similar to what has been described for human lung lesions. These results show that this acellular rim may, therefore, be a primary location of persisting bacilli withstanding drug treatment. Tuberculosis (TB) is treatable by drugs, and the World HealthOrganization has promoted the use of "directly observed therapy" to administer effective regimens to infected patients. However, despite this, no new drug classes have been introduced in the last two to three decades and new, effective compounds are badly needed. A central problem, however, even when compliance problems are dealt with, is the sheer length of time needed for current drug regimens to ensure clearance of the infection without relapse (44). As a result, conventional drug regimens are usually of 6 to 9 months in length.The length of treatment is believed to represent the need to eradicate a small population of bacteria that persist within the lung and extrapulmonary tissues. In addition, these "persisters" likely are the source of reactivation TB that can then occur at a later date, including as a result of human immunodeficiency virus infection. The different mechanisms underlying mycobacterial persistence are not known but appear to represent some form of refractory state rather than true drug resistance (7,26,31). In vitro a subpopulation of 5 to 10% of Mycobacterium tuberculosis appears far less responsive to killing by drugs. In vivo ca. 99% of bacteria in mice are killed within 2 weeks of drug treatment, but then it requires at least 3 more months of treatment to clear the remaining 1% (19-21, 28, 40). This apparent drug tolerance of M. tuberculosis can be readily demonstrated and modeled in mice.Treatment of persisting bacteria in h...

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Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV‐1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor‐α stimulated and unstimulated ACH‐2 cells demonstrated the ability to detect <5% HIV RNA positive cells with a sensitivity of <30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV‐seropositive patients on no, single, combination, or triple drug therapy and 8 HIV‐seronegative patients were examined. The majority of HIV‐positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV‐infected cell populations in the peripheral blood of HIV‐positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease. Cytometry 31:265–274, 1998. © 1998 Wiley‐Liss, Inc.