The CDC reports that over 40 million Americans suffer from chronic disease, the majority of whom report multiple diagnoses of chronic disorders. The causes of many of these chronic diseases are not well understood and doctors struggle to effectively diagnose and treat these disorders. In recent years, attention has turned towards the possibility that many chronic disorders, especially those involving autoimmune response, may be caused by highly fastidious, immune-evasive pathogens. Bacteria of the genus Bartonella (a member of the Rickettsiales order) have been linked in the medical case series literature to a wide spectrum of diseases in human and veterinary medicine, including polyarthritis, cutaneous vasculitis, endocarditis, myocarditis, epistaxis, peliosis hepatis, granulomatous inflammatory disease, and, most recently, neurological dysfunction and arthritis. Traditional methods for detecting bacterial infections in patient samples have proven to be largely ineffective for the accurate diagnosis of Bartonellosis. Researchers at the North Carolina State University College of Veterinary Medicine have developed a highly sensitive Bartonella testing platform which combines the pre- enrichment culture of blood in a novel liquid media followed by a Bartonella PCR assay using genus level primers and sequence id of positive results. This innovative Bartonella testing platform was developed to facilitate the detection of Bartonella infections in immuno-competent patients where this highly fastidious bacteria infects at such low levels that sampling error at the point of testing introduces extremely high false negative rates for direct PCR and that the host immune system has little opportunity to develop antibodies against this bacteria. The cornerstone of this molecular diagnostic platform is a patented pre-enrichment medium called Bartonella Alpha Proteobacteria Growth Medium (BAPGM). Galaxy Diagnostics holds an exclusive license on BAPGM. Preliminary data support the usefulness of the BAPGM microbiological approach in human medicine. This novel Bartonella testing platform has resulted in the detection of single and polymicrobial Bartonella infections in blood samples obtained from both chronically-ill and healthy humans and animals. Perhaps more importantly research using the BAPGM-PCR testing platform have revealed important clinical associations between Bartonella infection and serious chronic disease. Our expectation is that the development of this testing platform will lead to medical breakthroughs in the diagnosis and treatment of chronic disease and will provide extensive savings in healthcare expenditures through early detection and prevention of debilitating chronic conditions. Of particular importance is research regarding modes of transmission and the identification as a cause or contributing factor in specific chronic disorders, like arthritis, neurological disease, organ failure and other hard- to-diagnose chronic conditions. As Bartonella spp. are transmitted by a large number of arthropod vectors, including fleas, lice, sand flies, ticks, biting flies and potentially others, determining the appropriate bacteria to screen for may be complicated by regional variations in the transmission of various Bartonella species to the blood donor public. Incidence of different Bartonella species by regions due to the existence of chronic carriers and/or vectors should be addressed in the future in order to establish the actual occurrence of this genus (and each of its species) in the US. The overall goal of Phase I will be to develop a BAPGM Axenic Culture Bottle formulation to be used for immediate inoculation or for transport of microbiological specimens. Specimens will be then transported to Galaxy Diagnostics for Bartonella testing using our current culture plus molecular testing protocol;
Aim 1 develop and validate a new formulation in the standard BAPGM platform culture medium to fit the requirements of the BAPGM Axenic Culture Bottle design proposed;
Aim 2 will test the feasibility that reasonable storage conditions on the new formulations can be achieved. This will be accomplished by examining growth sensitivity testing as performed previously after storage at room temperature, 4-8oC and -20oC. Phase II will focus on the product development and regulatory milestones required to develop test kits including the BAPGM Axenic Culture Bottle which will allow worldwide sale and distribution. Phase Ii research would also address the potential changes in the BAPGM Axenic Culture Bottle head-space gas composition required to maximize bacteria recovery and growth and that might lead to an optimization of the time required for the enrichment culture step in the testing platform.
As a result of the use of the BAPGM pre-enrichment culture approach during the last four years, our NCSU research group has contributed greatly to an expanded understanding of the medical implications of Bartonella infection in immunocompetent people experiencing neurological and neuro-cognitive dysfunction and in individuals with frequent animal and arthropod contact [1-2]. Medical research using this novel diagnostic technology has also contributed to the isolation and identification of an expanding list of subspecies [3-10]. Importantly, preliminary data from our NCSU lab has revealed a strong association between Bartonellosis and several forms of arthritis. The development of the BAPGM media for use in a Bartonella testing platform represents a great advance for the early detection of Bartonella infection in people. Accurate molecular detection and isolation of a microbial pathogen provides clinicians with a rational basis for accurate diagnosis of an infectious disease and also provides vital information for the selection of an effective antimicrobial regimen. Failure to isolate and characterize bacteria in patient samples would generally result in failure to treat the patient with antibiotics, which could have very detrimental consequences. Already, patients with diagnostic elevations in anti-neutrophil cytoplasmic antibodies (ANCA) have been immunosuppressed, only to develop Bartonella-induced endocarditis as a consequence of misdiagnosis [11-12]. In addition, bacterial isolates are vital to determine virulence factors, to characterize the immunological response of the infected host, and to determine the mechanisms by which the pathogen induces cellular injury or potentially evades immune recognition by the host. Phase II will focus on additional product design research to determine the optimum head-space gas composition in the sealed BAPGM Axenic Culture Bottle design (i.e. 5% CO2 atmosphere head-space inoculation by sterile transient needle venting after inoculation vs no venting) in order to maximize bacteria culture growth as well as test kit design milestones and the research required to meet regulatory approval requirements for commercialization.