Overview

The primary objective of the microbiology component of the TBRU is to investigate microbial factors expressed during various stages of Mycobacterium tuberculosis infection and illness in order to identify surrogate markers to help follow the progress of disease or treatment. Dr. Kathleen Eisenach directs this component, based at the University of Arkansas for Medical Sciences.

Work Statement

Conduct an investigation of the microbial factors expressed during various stages of M. tuberculosis infection and illness in order to identify surrogate markers. These markers shall be used to assess the effect of interventions on human disease progression, and distinguish between infection with M. tuberculosis versus infection with or exposure to BCG and atypical mycobacteria. The types of studies shall include, but not be limited to:

• Establishing and/or maintaining appropriate animal models (e.g., aerosol infection)
  
• Identifying changes in M. tuberculosis during various stages of human clinical disease (for example: polysaccharide, protein, and/or genetic expression)
• Identifying microbial factors associated with the initiation and maintenance of the persistent stages of the infection
  
• Identifying potential vaccine targets.
  

Project Descriptions

The Work Statement asks the TBRU Microbiology Component to investigate and assess microbial factors to identify surrogate markers that can assist in the laboratory evaluation of the progress of tuberculosis disease and treatment. Our approach takes advantage of access to clinical specimens from TB patients, well described murine models of TB, recent technical advances in manipulation of RNA, and the increasing body of knowledge about M. tuberculosis genes.

Developmental Projects [1994-2000]

During the first five years [1994-2000] of the TBRU, the focus of activities of the Microbiology Component was on developmental projects. Since there were few new laboratory procedures to validate, the focus was on development of new approaches. The aim was to identify productive approaches leading to the identification of potential new surrogate markers. Validation of these approaches would follow. During this period the developmental activities of the microbiology component ran the gamut from applied to basic research, involving 10 investigators working at six international sites.

The new approaches included developing novel assays for quantifying M. tuberculosis mRNA in clinical specimens for use in the treatment interventions during pilot studies and trials. TBRU also assisted in the development of the luciferase reporter phage assay for rapid drug susceptibility testing of clinical specimens. Other efforts were focused on optimizing methods for obtaining high quality mycobacterial mRNA from infected human macrophages and animal tissues as a first step to understanding early microbial responses to treatment and to identifying genes induced in the presence of anti-TB drugs. In addition, RNA expression of selected genes in TB patients was compared using in vitro and in vivo models of TB.

To assist in the development of potential candidate vaccines, TBRU provided support for the development of the genetic tools needed to provide the means for creating non-reverting mutants of M. tuberculosis.

The objectives of the microbiology component were addressed by these investigations to improve our knowledge of how microbial markers can be used to assess the effect of therapeutic interventions on TB disease.

Current Activities

Microbial Gene Expression in Response to Anti-TB Drugs

A major focus has been to develop molecular based tests for assessing patients' response to TB therapy. These assays include quantitative RT-PCR for 16S rRNA, fbpB (85B-alpha antigen) mRNA, and hspX (alpha crystalline homologue protein) mRNA. In the context of TBRU pilot studies (patients on standard 4 drugs) and clinical trials (EBA study of Rifalazil), we demonstrated that M. tuberculosis mRNA reflects microbial viability. Recent in vitro data suggest that mRNA can be used to identify differences in bactericidal activity of drugs with different mechanisms of action. Current work involves examining changes in gene expression of broth grown M. tuberculosis exposed to various anti-TB drugs and in organisms from infected mice treated with drugs. Only preliminary data are available. It is anticipated that additional candidate surrogate markers of drug efficacy will be identified and this work will lead to an understanding of drug induced bacterial death and persistence.

Virulence Assessment Strains from the HHC Study

The TBRU Uganda HHC has generated a set of well-characterized M. tuberculosis strains that should serve as a foundation for the investigation of virulence in both in vivo and in vitro models of TB infection. TBRU investigators are in an excellent position to systematically examine a reasonable number of strains using newly developed methods for describing phenotypic and genetic characteristics. The objective of the proposed project is to assess virulence of different strains of M. tuberculosis in the mouse and human monocyte models of infection. Using an aerosol infection model, we will compare growth rates of well-characterized strains in the lungs, spleen, and liver of infected mice. Simultaneously, the granulomatous response in the infected lungs, immune responses in the infected lungs, and the clinical response to infection will be monitored. To compare the host response observed in the mouse with the human monocytes model, we plan to examine the mycobacterial growth, cell surface markers, and cytokine response following infection of human monocytes in vitro with the same strains. To further characterize the organisms' response in these models we will measure differences in microbial gene expression. Genetic diversity among strains and strain-trait correlations will be examined using a combination of genotyping methods, e.g., IS6110 RFLP analysis, mutations in katG and gryA, synonymous single nucleotide polymorphisms, and genomic deletions.

M. tuberculosis Strain Tracking

M. tuberculosis strains isolated from TB patients, both index cases and their household contacts, identified in the TBRU Uganda Household Contact Study [HHC]/Kawempe Community Health Study[KCH] are genotyped using molecular methods. All of the strains are collected, stored, and analyzed with the IS6110 restriction fragment length polymorphism [RFLP] method. Such studies provide molecular evidence for direct transmission of strains due to close contact in the household or other networks of transmission in the community. RFLP analysis is also used to document re-infection or treatment relapse/failure.

Organization and Operation

The Microbiology Component of the TBRU is led by Dr. Kathleen Eisenach. Periodically Dr. Eisenach visits each site to discuss operations and strategies. In addition to routine communications by phone and e-mail, TBRU supports weekly and monthly conference calls to promote discussion of plans and ongoing operations.

Work related to the Microbiology Component is currently focused on support for TBRU clinical trials and epidemiology studies. Efforts to coordinate and validate these activities include a well-designed study to compare laboratory performance by two technicians during routine studies at the various sites.

Laboratory Sites

Joint Clinical Research Centre (JCRC) Mycobacteriology Laboratory & Wandegeya National Laboratory-Uganda

In Kampala, the laboratory at the Joint Clinical Research Centre, is a full service TB laboratory that provides microbiologic diagnostic services [including AFB smear microscopy, culture, and drug susceptibility testing] and participates in studies to evaluate the field and laboratory performance of new diagnostics appropriate for use in Uganda. Routine and specialized lab procedures, including QC/QA, are carried out in support of TBRU studies, including the Kawempe Community Study and the Treatment Shortening Trial, and other collaborations, such as, the Burroughs Wellcome Trust study on Strategies for Management MDR-TB in Kampala, Uganda and the Centers for Disease Control Tuberculosis Trials Consortium. The laboratory also assists the TBRU Specimen Repository in processing, storage and shipment of specimens needed at international sites to complete approved TBRU studies. The on-site Scientific Director is Dr. Moses Joloba and the Supervisor is Ms. Susan Kayes.

The TBRU also utilizes the central reference laboratory (Wandegeya) of the Uganda National Tuberculosis and Leprosy Programme [NTLP] for initial microbiologic diagnostic services for all patient-related activities. Thus, TBRU works closely with Dr. Francis Adatu-Engwau, NTLP Director, and Mr. Elisha Hatanga, Supervisor of the Wandegeya NTLP Laboratory to assist the NTLP in their efforts to advance services through increased training opportunities and improved laboratory operations. An active working group (Wandegeya Laboratory Development Team) was formed in May 2001, which has conducted two microscopy workshops on site.

Universidade Federal do Espírito Santo-Núcleo Mycobacteriology Laboratory-Brazil

Dr. Moisés Palaci has the lead responsibility for TBRU microbiology studies at this site. This laboratory provides full laboratory services to support TBRU clinical trials conducted in Vitória, including AFB smear microscopy, culture, and drug susceptibility testing. Complete mycobacteriologic services are well established for the clinical studies conducted at this site. A unique computer-based program was developed by the Núcleo TB lab for tracking specimens and managing microbiologic, clinical, and epidemiologic data. This program, which was recently implemented in a number of local clinic-based labs, provides rapid notification of TB cases and potential candidates for enrollment in TBRU studies.

Tropical Disease Foundation-Makati Medical Center-Philippines

A mycobacteriology laboratory, supervised by Ms. Grace Eros, carries out the microbiological aspects of the Treatment Shortening study at the Philippines site. This laboratory has experience in evaluating new methods for drug susceptibility testing, such as the BACTEC MIGT; testing new drugs, such as fluoroquinolones; participating in a national TB prevalence survey; and identifying MDR-TB strains. The laboratory has been recently remodeled to accommodate the increase in activities and provide a safe working environment.

Staff

The Microbiology Component is led by Dr. Kathleen Eisenach, Co-Director of the TBRU. During the first five years of the TBRU, Dr. Eisenach coordinated efforts from several institutions located in Little Rock, Arkansas, Bronx, New York, St. Louis, Missouri, Ft. Collins, Colorado, New Delhi, India, Kampala, Uganda, and Vitória, Brazil. Current contract sites include Kampala, Uganda, Vitória, Brazil, and Little Rock , Arkansas for clinical support sites. Additional developmental microbiology activities have been conducted at both Stanford University and the University of Pittsburgh.