Edward B. Breitschwerdt, DVM, DACVIM
College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
Infectious Diseases Diagnosis: A Clinical Example
I will use as an example, a consultation from a practice owner in Iowa to illustrate the complexities of infectious diseases diagnosis in the practice environment. The veterinarian contacted me by e-mail because one of his "younger associates" had evaluated a dog that initially presented for acute onset of weakness and mild anemia (PCV 27%). Following "symptomatic therapy" the hematocrit normalized for several weeks (rechecked at least 4 times); however, on a subsequent recheck, the hematocrit was 12%, immune-mediated hemolytic anemia was diagnosed, and the dog was immediately referred to Iowa State University, College of Veterinary Medicine. A tick borne pathogen panel was sent to the NCSU-VBDDL (Vector Borne Diseases Diagnostic Laboratory), but the dog died before test results were available. Babesia canis antibodies (titer 1: 64) were detected by IFA testing in our laboratory and additional review of the dog's history determined that the dog had traveled to Arkansas. The client was very unhappy and the veterinarian was seeking information related to canine babesiosis in dogs in Iowa.
Implications for Veterinary Medicine
1. A diagnosis was made (perhaps), but too late to benefit the patient.
2. Serological evidence of exposure does not confirm active infection.
3. Because of Adam Birkenheuer (and others) babesiosis is a re-emerging canine infectious disease in the United States and throughout the world.
4. Babesiosis is a definitive cause of immune-mediated hemolytic anemia in dogs.
5. Based upon more recent observations, both visualization of Babesia organisms on blood smears and serological testing are insensitive diagnostic tests, as compared to PCR.
6. Arkansas is highly endemic for Babesia canis. Iowa is not endemic for B canis.
7. Dogs, owners and other animals travel farther and more frequently than at any other time in history (i.e., SARS, Monkey Pox, West Nile Virus to name a few).
8. It takes only one brown dog tick to transmit this organism. This tick is usually found in kennels (boarding), homes (visiting friends) or veterinary hospitals (heaven forbid).
9. Even the best parasitology and medicine courses may not have prepared this young graduate to deal with this patient presentation.
10. A complete medical history will always serve as the foundation of optimal medical care.
11. There is not a Center for Disease Control (CDC) or Infectious Diseases Society of America that tracks companion animal infectious diseases; therefore there is no data base to address regional, national or international prevalences.
So What Are the Solutions?
Clearly, in the context of infectious diseases, clinicians need diagnostic tests that allow for the simultaneous detection of common, uncommon and unknown infectious agents. This seemingly impossible option is becoming much more probable with the advent of molecular biology and highly sensitive and specific DNA or RNA detection systems. Although molecular diagnostics have and will continue to have limitations, their incorporation into the practice of veterinary medicine have occurred at an increasingly accelerated rate.
Immunological Imbalance and Infection
Infection is defined as the invasion and multiplication of microorganisms in body tissues. Invasion infers that infection alters the immunological balance within an individual (i.e., invasion causes an immunological battle that can range in severity from a skirmish to all out war). It is increasingly obvious that this definition lacks clinical utility in regard to organisms that can achieve long term intracellular or extracellular persistence within the host. In these instances, it appears that both the host and the organism work in concert to maintain a state of immunological balance. It is very obvious that host genetics, microbial genetics, nutrition and exposure to toxic chemicals have a major influence on disease expression.
Concurrent Infections and Disease Expression
As an example, dogs with extensive tick, flea and louse exposure can be simultaneously infected (based upon detection of DNA) with multiple organisms. Secondly, dogs are "genetically" capable of compensating to a substantial degree for simultaneous infection with bacteria, protozoa and rickettsiae. Therefore infectious disease is multifactorial and is rarely due to a singular factor and rarely precipitated by a single microorganism.
Serology and Infectious Diseases Diagnosis
Historically, serology has been the mainstay of diagnosis for difficult to isolate bacteria, protozoa and viruses. Serology still has an important role in infectious diseases diagnosis and ideally should be used in conjunction with isolation or molecular diagnostic approaches for each organism and each patient. For an acute disease process, such as Leptospirosis, Rocky Mountain spotted fever or anaplasmosis, documentation of seroconversion can be used to confirm a diagnosis. Seroconversion is defined as at least a 4-fold increase in antibody titer between acute and convalescent test samples. When a clinician plans to document seroconversion, an initial patient sample should be obtained as early in the course of illness as possible. As the antibody titer in the initial sample may be low (below the laboratory diagnostic cut off) or not detectable, this sample can be stored for 2–3 weeks in a refrigerator or freezer until the convalescent sample is obtained. When antibodies are detected in a healthy animal or in association with a chronic infection, the presence of antibodies supports prior exposure and immunological recognition of the infecting organism, but does not confirm that the animal (healthy or sick) is actively infected with the organism for which antibodies were detected. In some instances, the organism was eliminated following infection by the host immune response, which means that active infection no longer exists and treatment directed at therapeutic elimination of the organism is not warranted. In other instances, a state of immunological balance is established between the infecting organism and the host immune response (the state of premunition). Although persistently infected these animals can be outwardly and clinically healthy and may or may not have hematological or biochemical evidence of disease. If active infection in these animals is documented by molecular testing or isolation, treatment is most likely indicated, especially if the infection can be eliminated and re-infection (through repeated tick or flea exposure for example) can be prevented. Cross reactivity among various infectious agents is another limitation to serological testing. Cross reactivity among members of a genus should be an anticipated occurrence, such as the strong cross reaction between Ehrlichia canis and Ehrlichia chaffeensis or between Anaplasma phagocytophilum and Anaplasma platys. In these two examples, cross reactivity is of less clinical relevance, as in each instances both organisms with the genus respond to the same antibiotic, doxycycline. However cross reactivity between Babesia canis and Babesia gibsoni is of clinical relevance as different drugs are used to eliminate these infections.
Whenever possible, isolation, serological and molecular-based diagnostic testing should be performed in tandem. The notion that a clinician should pick a single best test or target only the most likely infectious agent in a given patient is naïve and not realistic in the clinical setting. Many infectious agents induce similar disease manifestations and it is increasingly obvious that polymicrobial infections exist more frequently than previously recognized. When confronted with a non-infectious disease process, such as cancer, thousands of dollars are spent on imaging and staging prior to the initiation of treatment. When an infectious disease is high on the list of differential diagnoses, testing dollars should be spent on determining the infectious agent or agents responsible for the disease process and thereby allow for cost effective treatment. Although molecular diagnostic tests have many inherent benefits, sensitivity is always an issue. Due to low template number in the test sample, a negative PCR result can never confirm that an animal is not infected with a specific pathogen. This is one of the most compelling arguments for combining serological and molecular testing modalities.
Novel Approaches to Culturing Bacteria
In recent years, our research group has developed a novel approach to the isolation of fastidious bacteria. In the context of clinical medicine, it is possible that too much microbiological emphasis and excessive clinical relevance has been accorded organisms that are easy to isolate (Staphylococcus and Streptococcus species, Escherichia coli) from patient samples and not enough emphasis has been placed on organisms that are highly fastidious. In the future, the use of molecular based tests that can simultaneously detect multiple organisms will clarify complex interactions that influence pathogenesis, disease expression and treatment outcomes. This is truly an exciting time for infectious disease researchers and clinicians.