Whole Blood Bactericidal Activity during Treatment of Pulmonary Tuberculosis

Wallis, Robert S.; Vinhas, Solange Alves; Johnson, John L.; Ribeiro, Fabíola C.; Palaci, Moisés; Peres, Renata Lyrio; Tristão-Sá, Ricardo; Dietze, Reynaldo; Chiunda, Allan; Eisenach, Kathleen D.; Ellner, Jerrold J.

doi:10.1086/346053

Cited by 70 publications

(63 citation statements)

References 45 publications

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“…The isolates in the present study were passaged in vitro only twice prior to their introduction into cell culture to avoid the attenuation we had previously observed with isolate MP-28. When studied in 2000, that isolate showed nearly 1-log intracellular growth in whole blood culture (1,16); by the following year, it had become markedly attenuated (19). Similar attenuation may have occurred with CDC1551, an outbreak-associated isolate that was highly virulent when first studied in mice in 1998 (13).…”

Section: Discussionmentioning

confidence: 80%

“…Isolates were collected prospectively in a study of surrogate markers of the response to chemotherapy conducted in Vitória, Brazil (19). Briefly, subjects were 18-to 60-year-old human immunodeficiency virus type 1-seronegative patients with newly diagnosed initial episodes of sputum smear-positive pulmonary tuberculosis.…”

Section: Mycobacterial Isolatesmentioning

confidence: 99%

“…Care was taken to ensure that the recent clinical isolates were minimally passaged prior to study to avoid the selection of substrains that were adapted to in vitro replication. The reference strains, which had undergone extensive in vitro culture in broth, included HN878 (also known as 210), a group W-Beijing, outbreak-associated strain; CDC1551, a strain associated with an unusually large number of tuberculin skin test conversions; H 37 Rv, a widely studied reference isolate that is virulent in animal models; H 37 Ra, H 37 Rv's highly attenuated sibling; and MP-28, an isolate that was selected at random from a similar study conducted in Brazil in 1997 (18) and that was cultured extensively in vitro during the course of three subsequent whole-blood infection studies, in which it was identified as strain 28 (1,16,19). The objective of the present project was to examine the influence of strain variation in M. tuberculosis on the early events of human tuberculosis (TB) pathogenesis.…”

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confidence: 99%

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Survival and Replication of Clinical Mycobacterium tuberculosis Isolates in the Context of Human Innate Immunity

et al. 2005

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The initial host response to Mycobacterium tuberculosis is driven by innate immunity. For this study, we examined the ability of 18 recent clinical isolates and 5 reference strains to survive and replicate in the context of host innate immunity by using whole blood culture. Six healthy tuberculin-negative volunteers served as subjects. H 37 Ra showed the least capacity to replicate of any of the strains tested, decreasing in viability 1.3 log CFU during 72 h of whole blood culture, whereas H 37 Rv increased 0.32 log. Clinical isolates varied greatly in their ability to replicate in blood cells, ranging from ؊0.4 to ؉0.8 log (P < 0.001). Four showed significantly more growth than H 37 Rv, and one showed significantly reduced growth. Host mechanisms for restricting intracellular mycobacterial growth were more effective during the first 24 h of whole blood culture than during the 24-to 72-h period. Certain mycobacterial isolates appeared preferentially able to withstand host defenses during each of these intervals. Although there was relatively more homogeneity among subjects than among strains, one of the six subjects showed a reduced capacity to restrict intracellular mycobacterial growth due to a defect expressed during the first 24 h of culture. Our findings indicate substantial variability in the capacity of clinical tuberculosis isolates to replicate in host cells in the face of innate host immunity.The early events following inhalation by an immunocompetent, mycobacterium-naïve host of droplet nuclei containing viable Mycobacterium tuberculosis are driven by the innate immune system. The resulting influx of neutrophils, macrophages, NK cells, and other cells to the site of infection serves as a stimulus for granuloma formation and acts directly to limit the extent of mycobacterial replication at this early stage of infection. It has been suggested that the efficiency of these early innate responses may help determine whether a latent infection is established and whether that infection ultimately will progress to active disease.Through evolutionary selection, pathogenic mycobacteria have acquired means to evade specific host immune effector mechanisms, presumably including those of the innate response. The propensity of certain M. tuberculosis isolates to cause outbreaks, for example, has been linked to increased virulence in macrophages or mice in association with altered host cytokine expression profiles (2,3,6,8,12). However, even these virulent outbreak-associated isolates cause disease in only a small proportion of infected individuals. Our understanding of the interplay of biologic and genetic diversity in the host and in the mycobacterium is incomplete, in part because current models to examine the early events in mycobacterial pathogenesis have not been well suited to field or epidemiologic human studies.For the present study, we assessed the capacity of mycobacterial strains to survive phagocytosis and replicate in whole blood cultures. This model permits the involvement of neutrophils, as well as mo...

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Section: Discussionmentioning

confidence: 80%

Section: Mycobacterial Isolatesmentioning

confidence: 99%

mentioning

confidence: 99%

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Survival and Replication of Clinical Mycobacterium tuberculosis Isolates in the Context of Human Innate Immunity

et al. 2005

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“…Our findings are in agreement with those reported by Rastogi et al (13) but are in sharp contrast to the data reported by Orme and colleagues (16), in which a reduction of 3 log 10 CFU/ml over a narrow concentration range between 0 and 1 mg of isoniazid per liter was observed with intracellular M. tuberculosis Erdman in murine macrophages. The whole-blood system has advantages over a macrophage monolayer system; for example, plasma protein binding and immune components can be factored in, thereby mimicking the in vivo situation closely (18). Furthermore, exposures up to 2,560-fold higher than the concentration producing stasis can be studied in comparison to the exposures possible in the macrophage monolayer system (640-fold higher).…”

Section: Discussionmentioning

confidence: 99%

Isoniazid Pharmacokinetics-Pharmacodynamics in an Aerosol Infection Model of Tuberculosis

Jayaram

Shandil

Gaonkar

et al. 2004

Antimicrob Agents Chemother

150

119

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Limited data exist on the pharmacokinetic-pharmacodynamic (PK-PD) parameters of the bactericidal activities of the available antimycobacterial drugs. We report on the PK-PD relationships for isoniazid. Isoniazid exhibited concentration (C)-dependent killing of Mycobacterium tuberculosis H37Rv in vitro, with a maximum reduction of 4 log 10 CFU/ml. In these studies, 50% of the maximum effect was achieved at a C/MIC ratio of 0.5, and the maximum effect did not increase with exposure times of up to 21 days. Conversely, isoniazid produced less than a 0.5-log 10 CFU/ml reduction in two different intracellular infection models (J774A.1 murine macrophages and whole human blood). In a murine model of aerosol infection, isoniazid therapy for 6 days produced a reduction of 1.4 log 10 CFU/lung. Dose fractionation studies demonstrated that the 24-h area under the concentration-time curve/MIC (r 2 ‫؍‬ 0.83) correlated best with the bactericidal efficacy, followed by the maximum concentration of drug in serum/MIC (r 2 ‫؍‬ 0.73).No new drugs for the treatment of tuberculosis have been registered in the last 40 years. Discovery of new antimycobacterial agents is in part limited by the laborious and slow nature of the in vitro and in vivo studies required. Knowledge of the pharmacokinetic (PK)-pharmacodynamic (PD) properties of candidate drugs could be used to facilitate drug discovery and evaluation of the effects of combinations of agents. As a step toward such a strategy, we have recently identified the PK-PD parameter that correlated with the in vivo efficacy of rifampin in a murine aerosol model of tuberculosis (7). In the present study, we have extended this work to include isoniazid. MATERIALS AND METHODSReagents. Isoniazid (lot 36H1179) and carboxymethyl cellulose (lot 77H1077) were purchased from Sigma, St. Louis, Mo. Isoniazid stock solutions were prepared in 100% dimethyl sulfoxide (DMSO; Sigma) and diluted in normal saline prior to final use. ETDA (lot 5-4514) was purchased from Hi-Media Labs, Mumbai, India. Acetonitrile (high-pressure liquid chromatography [HPLC] grade) and trichloroacetic acid (TCA) were obtained from Spectrochem Pvt. Ltd., Mumbai, India. Cinnamaldehyde (lot 96320) was obtained from Fluka Biochemika, Bucks, Switzerland.Microbial cultures and cell lines. Mycobacterium tuberculosis H37Rv (ATCC 27294) and J774A.1 macrophages were prepared for in vitro, macrophage, and animal infection studies by previously described methods (7).Blood from healthy volunteers. Group O-positive blood from healthy volunteers collected in citrate phosphate dextrose adenine anticoagulant was obtained from a registered blood bank (TTK Blood Bank, Bangalore, India). It was stored at 4°C and was used for assays up to 1 month from the date of collection.Animals. The Institutional Animal Ethics Committee, registered with the Government of India (registration no. CPCSEA 1999/5), approved all experimental protocols with animals and the use of animals. Ethical practices recommend the use of equal numbers of animals of both sexes w...

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“…An ideal marker would measure events early during treatment and be accurate regardless of the mechanism of drug action or the regimen being tested. In addition to serial sputum culture conversion, less well studied biomarkers of response to therapy include sputum 85B protein and mRNA (2,17), sputum cytokines (15), and whole-blood bactericidal activity (18). None has been adequately validated to demonstrate long-term efficacy in phase 3 trials.…”

mentioning

confidence: 99%

Sputum Mycobacterium tuberculosis mRNA as a Marker of Bacteriologic Clearance in Response to Antituberculosis Therapy

Mahan

Palaci

et al. 2010

J Clin Microbiol

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mRNA is a marker of cell viability. Quantifying Mycobacterium tuberculosis mRNA in sputum is a promising tool for monitoring response to antituberculosis therapy and evaluating the efficacy of individual drugs. mRNA levels were measured in sputum specimens from patients with tuberculosis (TB) receiving monotherapy in an early bactericidal activity study of fluoroquinolones and in those receiving a standard rifampin-based regimen in an interleukin-2 (IL-2) trial. In the early bactericidal activity study, sputum for quantitative culture and mRNA analysis was collected for 2 days before and daily during 7 days of study drug administration. In the IL-2 trial, sputum was collected for quantitative culture, Bactec 460 liquid culture, and mRNA analysis throughout the intensive treatment phase. RNA was isolated from digested sputum and tested in quantitative reverse transcription-PCR assays for several gene targets. mRNA for the glyoxylate cycle enzyme isocitrate lyase declined at similar rates in patients receiving isoniazid, gatifloxicin, levofloxacin, and moxifloxacin monotherapy. Isocitrate lyase mRNA correlated highly with CFU in sputum prior to therapy and during 7 days of monotherapy in all treatment arms. Isocitrate lyase mRNA was detectable in sputum of culture-positive TB patients receiving a rifampin-based regimen for 1 month. At 2 months, sputum for isocitrate mRNA correlated more closely with growth in liquid culture than did growth on solid culture medium. Data suggest that isocitrate lyase mRNA is a reliable marker of M. tuberculosis viability.The development of new drugs for tuberculosis (TB) treatment has been hampered by the lack of an early surrogate marker that reflects nonrelapsing cure. Two-month sputum culture conversion on solid medium is the best-established predictor of treatment outcome (12). Early bactericidal activity (EBA), measured as the decline in sputum Mycobacterium tuberculosis colony counts (CFU) during treatment, is a commonly used tool for comparing new drugs to current drugs and dose finding, but quantitative culture is time-consuming and labor-intensive (4). An ideal marker would measure events early during treatment and be accurate regardless of the mechanism of drug action or the regimen being tested. In addition to serial sputum culture conversion, less well studied biomarkers of response to therapy include sputum 85B protein and mRNA (2, 17), sputum cytokines (15), and whole-blood bactericidal activity (18). None has been adequately validated to demonstrate long-term efficacy in phase 3 trials.In prior work we demonstrated that levels of M. tuberculosis fbpB mRNA (encoding fibronectin-binding protein, antigen 85B) and hspX (encoding alpha-crystalline homologue protein) mRNA decline rapidly in conjunction with M. tuberculosis colony counts after initiation of a rifampin-based standard drug therapy (2) and in a 14-day EBA study of rifalazil where both isoniazid and a rifamycin were given simultaneously (unpublished data). In most cases the solidmedium culture was still positive...

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Whole Blood Bactericidal Activity during Treatment of Pulmonary Tuberculosis

Cited by 70 publications

References 45 publications

Survival and Replication of Clinical Mycobacterium tuberculosis Isolates in the Context of Human Innate Immunity

Survival and Replication of Clinical Mycobacterium tuberculosis Isolates in the Context of Human Innate Immunity

Isoniazid Pharmacokinetics-Pharmacodynamics in an Aerosol Infection Model of Tuberculosis

Sputum Mycobacterium tuberculosis mRNA as a Marker of Bacteriologic Clearance in Response to Antituberculosis Therapy

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