Title: Assessment of whole blood bactericidal activity in the evaluation of new TB drugs

Intracellular mycobacterial subpopulations with altered metabolism and reduced replication arise in vivo as a result of pressure from host immune mechanisms. These subpopulations, which show altered susceptibility to the action of anti-TB drugs, play an important role in mycobacterial persistence and TB relapse. This chapter describes the use of whole blood cultures infected ex vivo with Mycobacterium tuberculosis to assess the effectiveness of administered anti-tuberculous treatment against non-replicating persisters. Studies in TB patients have shown that cumulative whole blood bactericidal activity is indirectly linked to outcome, and that drug activity in the ex vivo cultures is modulated by the intracellular immune milieu. Three studies in healthy volunteers have used the method for dose selection and regimen optimization. Further research in TB patients is warranted to explore the potential role of this method in accelerating the development of new TB drugs and regimens. Introduction Distinct subpopulations of Mycobacterium tuberculosis exist in patients with active tuberculosis. These organisms differ in metabolism, gene expression profiles, rates of replication, and other functional characteristics. Although these subpopulations can arise at random in small numbers in otherwise uniform bacterial populations, their numbers increase greatly in response to specific stresses created by the host immune response, often in distinct anatomic locations. These adaptations facilitate specific stages of the mycobacterial life cycle. For example, large numbers of metabolically active, replicating bacilli within caseous material in cavities are required for efficient aerosolization and transmission, whereas semi-dormant bacilli sequestered in hypoxic granulomas make possible persistence, reactivation and relapse. Current TB drugs act differentially on these subpopulations [1]. Isoniazid, for example, acts primarily on replicating, aerobic, extracellular bacilli, whereas rifampin appears equally active against both replicating and semi-dormant bacilli. Pyrazinamide requires low pH, found within granulomas and in macrophages after phagosome-lysosomal fusion, for its activity to be expressed [2]. Certain subpopulations can also emerge in response to specific stresses induced by drug treatments. Thus, the critical unit in tuberculosis is an unusually complex triad that includes host, pathogen, and therapy-related elements. The existence of sequestered mycobacterial subpopulations poses a challenge for drug developers and clinicians who wish to meaningfully assess the therapeutic potential of a new drug or regimen. Monitoring tissue sterilization and predicting relapse risk presently can be accomplished only by measuring either rates of relapse after completion of therapy or by proportions with sputum culture conversion after 2 months of treatment [3]. These endpoints have relatively large sample size requirements, and in most circumstances cannot be used to study single new drugs. Measurement of serial sputum colony counts (SSCC) during the first several weeks of therapy has been proposed as an alternative endpoint, based on a retrospective analysis of 34 subjects enrolled in separate trials of HREZ and STH [4]. These two regimens, which differ substantially in the duration of treatment required for cure (6 vs. 18 months), differed significantly in their effects on log colony counts from days 2 to 28 (-.163 vs. -.095 log/d). SSCC was measured prospectively in the OFLOTUB phase 2 study, which found moxifloxacinand gatifloxacin-containing regimens to be superior to standard therapy (-.169 and -.166 log/d, respectively, vs. -.134) [5]. The effects of moxifloxacin and gatifloxacin on 2-month conversion and relapse rates remain to be determined, as studies to date have been contradictory [5-8]. These observations indicate an urgent need for other methods that can be used to efficiently evaluate the sterilizing activity of new compounds and regimens in early clinical trials. Rationale The ex vivo whole blood model is an alternative strategy to measure drug effects on otherwise elusive but critical microbial subpopulations that arise in the context of the host immune response. The approach benefits from the simplicity of whole blood culture to study cellular immune function. Whole blood cultures consist of blood collected in a heparinized tube that is diluted with an equal volume of tissue culture medium, to which a microbe or other immune stimulus is added. Mycobcteria added to the cultures rapidly undergo essentially complete phagocytosis by neutrophils and monocytes [9,10]. Mycobacterial antigens are processed, expressed, and recognized by CD4, CD8, NK, γδ, and other lymphocytes. Cytokines, adhesion ligands, activation markers, and effector molecules are expressed, resulting in restriction of mycobacterial replication. The extent of mycobacterial growth or killing in whole blood culture therefore reflects the balance between microbial pathogenicity and host immune mechanisms. In tuberculin skin test negative healthy donors, for example, the time required for doubling of M. tuberculosis H37Rv is increased to over 34 hrs from the from the usual 18-20 in simple broth culture. Superior restriction of growth is evident in blood of TST positive subjects. The balance between host and pathogen can be shifted by removal of CD4 or CD8 T cells, addition of prednisolone, TNF antibodies, or dihydroxyvitamin D [9-12]. Growth inhibition in whole blood culture has improved in clinical trials in which patients were vaccinated with BCG or given oral vitamin D supplementation, or in AIDS patients following initiation of antiretroviral therapy [13-15]. Of greatest relevance to this review is the finding that whole blood cultures of patients with newly diagnosed active TB are capable of full restriction of growth of virulent clinical isolates, including the patient’s own strain [16,17]. This presumably reflects the influence of antigen-driven in vivo expansion and activation of M. tuberculosis-reactive T cells. The resulting bacteriostasis can therefore serve to model the killing of non-replicating M. tuberculosis by a single drug or by drug combinations in vitro as it occurs in granulomas in vivo. This differs fundamentally from simple macrophage cultures, which are generally very permissive of intracellular mycobacterial replication. Drug levels in whole blood culture reflect those in the blood at the time of phlebotomy. By collecting blood samples at various intervals before and after dosing, the full magnitude of therapeutic effects can be determined as drug concentrations rise and fall due to absorption, distribution, metabolism and elimination. The parameter of greatest interest therefore is the area under the curve over 24 hours of dosing. Technical aspects Fresh blood with viable cells is required. Blood can be collected in heparin-containing Vacutainers (BD) or similar pre-filled tubes. Sodium heparin is preferred, though lithium may be acceptable. Other anticoagulants such as ACD adversely affect cell function and should not be used. A total volume of approximately 1 ml blood per time point is sufficient. The specific times and number of time points depends on the kinetics of the drugs to be studied, but generally at least 4 and preferably 5 are preferred. Sampling at the same time points for PK sampling is desirable, as it facilitates additional analyses of the PK/PD relationship. Practical considerations favor reserving samples for transport as a batch to the laboratory for processing by mid-afternoon. For drugs administered once daily in the morning, this can be facilitated by collecting specimens for later time points the prior evening. While awaiting transport, tubes are maintained at room temperature with slow constant rotation. Variability was increased in baseline (pretreatment) samples in one study in which samples were stored on a table top without mixing. Sixteen hours is the maximum recommended duration of such temporary storage. Cultures are performed in sealed 2 ml tubes that are rotated slowly during incubation. The slow mixing enhances immune restriction of mycobacterial growth. At the conclusion of the period of incubation (usually 72 hrs), cells are pelleted. The supernatant is removed, and may be reserved for analysis of induced cytokines. Sterile distilled water is added to lyse red blood cells. The cells are pelleted again, and the liquid phase again removed. The remaining pellet resuspended in a small volume of 7H9 broth and added to a Mycobacterial Growth Indicator Tube (MGIT, Becton Dickinson). MGIT time to positivity (TTP) is recorded. In early studies, Bactec culture bottles were used instead. Mycobacterial growth in Bactec is measured daily as production of CO2 from radiolabeled palmitic acid. A subsequent study has shown a high correlation between time to positivity in Bactec and MGIT. The disadvantages of the Bactec system are its requirements that CO2 production by each culture must be measured daily, and that each completed culture must be disposed in a manner appropriate for low-level radioisotopes. The MGIT 960 system has largely replaced Bactec in clinical mycobacteriology laboratories. MGIT tubes contain Middlebrook 7H9 liquid media plus an oxygen-quenched fluorochrome (tris 4, 7diphenyl-1, 10-phenonthroline ruthenium chloride pentahydrate) embedded in silicone at the bottom of the tube. Bacterial growth within the tube results in depletion of free oxygen, which in turn results in fluorescence within the MGIT tube when visualized under UV light. The intensity of fluorescence is directly proportional to the extent of oxygen depletion. MGIT tubes placed in a MGIT 960 instrument are maintained at 37C and monitored every 60 minutes for increasing fluorescence. The instrument declares a culture positive if either it reaches a predetermined level of fluorescence or if it shows a rapid rate of increase in