key: cord-021402-wq770ik9
authors: Relford, Roberta L.; DiMarco, Anthony
title: New Diagnostic Tools for Infectious Disease
date: 2009-05-15
journal: Consultations in Feline Internal Medicine
DOI: 10.1016/b0-72-160423-4/50009-3
sha: 
doc_id: 21402
cord_uid: wq770ik9

nan

Diseases caused by infectious agents are frequent in veterinary medicine. The signalment, history, and a thorough physical examination lead the clinician to suspect that an infectious disease may be present and lay the foundation for development of a differential diagnosis list and choice of the appropriate laboratory tests. Clinical signs associated with infectious disease can be varied and depend on the infecting agent and the organ system involved.

The first step in the laboratory evaluation is to establish a minimum database, including a complete blood count, serum biochemistry panel, and urinalysis. This minimum database, along with ancillary diagnostics such as radiographic imaging, aids in identifying specific organ involvement and can lead to a presumptive diagnosis that an infectious disease is present. Further testing often is required to identify specifically which infectious agent is involved to determine an appropriate treatment plan for the patient. This chapter discusses the laboratory tools available for infectious agent identification.

Laboratory diagnostic tools can be divided into two main categories: (1) direct identification of the infecting agent/ antigen (Table 6 -1), and (2) indirect identification by detection of antibodies directed specifically against the infecting agent/ antigen ( Table 6 -2).

The most common laboratory methodologies used to identify an infectious agent include visualization of the organism via cytology/biopsy, isolation of the agent in microbiological culture, immunodiagnostics/serology, and nucleic acid technology.

Cytology is the fastest and most inexpensive way to identify the presence of an infectious organism. Microscopic examination of body fluids, fine-needle aspirates of solid organs, and imprints or scrapings of superficial lesions are just a few examples of ways in which infectious agents can be collected and identified morphologically. One of the limitations of cytology is whether the organism occurs in sufficient numbers in circulation or in the tissues or fluids to be identified. To help identify low numbers of organisms, intracellular organisms, and viruses, special stains with affinities for certain physical characteristics of the agents can be applied to the cytology sample. Routine special stains include Gram stain, periodic acid-Schiff (PAS), and acid-fast stain.

A major advance in cytology over the years has been the development of immunocytochemistry. This methodology uses agent-specific polyclonal or monoclonal antibodies that react with unique antigens on various pathogenic organisms. The resulting antibody-antigen complexes then are detected by either fluorochromes that emit fluorescence or a chromogen that provides a color change. For example, anticoronavirus antibodies can be applied to fluid samples or granuloma aspirates from cats with suspected feline infectious peritonitis to detect the presence of coronavirus within macrophages or monocytes in the sample.

Immunohistochemistry is used classically to characterize and identify tumors for prognosis or to identify markers for therapeutic intervention, and also can be used for organism identification. Polyclonal anti-Mycobacterium bovis antibody has been shown useful as a single screening method for the detection of a number of different microorganisms in skin biopsies. 1 Cytology and biopsy samples also can be used as a source for cells, DNA, and RNA extraction. Cells and organisms can be extracted from cytology slides, formalinized tissue, and paraffin-embedded tissue and then evaluated via flow cytometry with immunomarkers or polymerase chain reaction (PCR). 2 As more antibodies are made available, the menu of organisms that can be identified will expand. These techniques are offered at many universities and reference laboratories.

Microbiological culture typically is performed to identify bacterial or fungal organisms. Challenges associated with culture include adequate sample collection, biological behavior of the organism, and interpretation of the results. The first challenge is collection of the specimen. The affected organ may not be readily accessible and the sample must be collected without contamination. At the laboratory, the great diversity of infectious microorganisms and their varied biological behaviors can make accurate identification difficult. To help overcome these challenges, many microbiology laboratories now use automated systems that can run a wider range of biological tests and compare the behavior of the sample organism on these tests with a database containing information about the reaction patterns of known pathogens. With the addition of more tests and computer algorithms to compare with a large database, the probability of correct identification of the organism is increased.

Once identified, the susceptibility of the organism to numerous antimicrobial agents can be determined. Laboratories now routinely provide the minimum inhibitory concentration (MIC), which is defined as the lowest concentration of the antimicrobial drug that inhibits the growth of the organism. These values then can be compared with the levels attainable in serum or tissue to determine the most appropriate antimicrobial agent for the treatment of that particular organism in that particular site.

Some new assays combine culture and cytology. For example the InPouch TV culture system by BioMed Diagnostics (White City, OR) for the identification of Tritrichomonas spp. uses a clear pouch containing culture media to increase low numbers of organisms rapidly in a sample. The sealed pouch then is placed directly on a microscope for reading (see Figure 15 -2). The advantages of this system include less likelihood of contamination of the sample, reduced exposure of laboratory staff to a zoonotic organism, long shelf life of the culture system, and improved sensitivity compared with wetmount preparations.

Fastidious, intracellular, and viral organisms do not lend themselves readily to growth-based technologies, and cytological detection is limited by the shedding characteristics and intracellular location of some agents. Whole organisms or their antigens can be detected immunologically in a wide range of specimens including serum, whole blood, feces, cerebrospinal fluid, body cavity effusions, synovial fluid, cell aspirates, and tissue samples. Because these assays are directed toward an antigen of the infectious organism, they usually are not speciesspecific and reagents from human assays often can be used on feline samples. However, when reagents validated for human assays are used, they should be revalidated with feline samples to ensure that interfering factors or cross-reactive antibodies are not present.

Immunodiagnostic assays include fluorescent antibody (IFA), enzyme-linked immunosorbent assay (ELISA), latex agglutination, and immunoblotting. Fluorescent antibody and immunoblot assays are performed in commercial laboratories, whereas numerous patient-side rapid ELISA and latex agglutination kits are available for antigen detection. The antigen ELISA assay comprises capturing the antigen of interest by using an antibody immobilized on a solid phase (e.g., a microtiter plate, a membrane, or microparticles) and forming a sandwich with a second antibody that is coupled to an enzyme. Formation of this sandwich occurs only if the corresponding antigen is present in the sample. After binding, the enzyme is catalyzed to give a color reaction. The most widely used ELISA for antigen determination in cats is the feline leukemia virus (FeLV) antigen assay. As with other antigen serology assays, this technique is limited based on the level of antigen load, the location of the antigen (i.e., intracellular or extracellular), and cross-reactivity with other antigens. Recent advances in ELISA assays have been directed toward improving the specificity of the antibody to the organism in question or enhancements in the detection system. Initially, nonspecific, outer surface proteins were used to develop the assay antibodies. This often resulted in cross-reactivity with multiple organisms and false-positive test results. Many tests now use purified, unique proteins to produce highly specific antibodies for the assays. These improved tests have a high diagnostic sensitivity and specificity (greater than 95 per cent) and often are discordant with less specific whole-cell IFA tests. 3, 4 Additional improvements in ELISA diagnostic assays include enhancements to the conjugate that allow shorter incubation times, superior reaction detection through the use of enzyme stabilizers, and longer conjugate stability and shelf life. Some detection systems have been changed to a non-enzyme-based conjugate for color detection using colloidal gold or colloidal carbon. 5 Latex agglutination assays use latex microspheres coated with specific antibodies directed against an infectious agent. If the organism or antigen is present, the antibody-coated particles bind to the organism and cause agglutination. Latex agglutination tests are available for Cryptococcus neoformans, Toxoplasma gondii, Histoplasma capsulatum, and Sporothrix schenckii, in addition to numerous bacterial pathogens.

One of the most rapidly expanding areas of organism identification and antigen detection has been in the area of nucleic acid-based testing. Until the introduction of nucleic acid amplification by the PCR, detection of an organism's DNA or RNA often was impossible because of the small amount of antigen present in a sample. PCR technology takes advantage of the normal function of polymerase enzymes. These enzymes are present in all living things and are responsible for copying, proofreading, and correcting errors in genetic material. Polymerase enzymes "photocopy" or amplify minute quantities of genetic material to a volume sufficient for it to be detected. To have a useful PCR assay, a nucleic acid sequence unique to a particular organism or class of organisms must be identified. This unique sequence is the material amplified and detected if the organism in question is present in the sample. During the first step, DNA is extracted carefully from the sample containing the suspected infectious agent. The extracted DNA then is heat-stressed to cause it to uncoil from its normal double-helix structure and separate into individual strands. The second step involves the addition of the two unique primers that flank each side of the sequence of interest. The primers aid in identification and amplification of the unique DNA sequence in question. When the primer finds the matching section of nucleic acid sequence, this sequence is copied to produce a small fragment of DNA (the amplicon) specific to the organism. This series of steps is repeated 20 to 50 times, with the product of each round serving as additional template for subsequent rounds of amplification. The final result is a logarithmic amplification of the original nucleic acid sequence. Detection of the multiplied nucleic acid sequence is performed during the amplification process with fluorescence emission with each amplification step (real-time PCR), after the completion of all amplification steps with the use of a secondary detection method, or from electrophoretic separation of the final DNA fragment and detection by application of a labeled complimentary nucleic acid probe.

PCR has opened a new spectrum of infectious disease tests, and numerous laboratories have started offering PCR tests for veterinary pathogens. The challenges faced today are not technological but in establishing a standardized process for accuracy, reproducibility, and quality control of such a sensitive and powerful assay. PCR testing is being offered at an increasing number of facilities. However, few laboratories have provided the important validation data that are necessary to evaluate and compare methods for diagnostic accuracy. The absence of standardization in testing protocols, primer selection, laboratory contamination control measures, quality control monitoring, and validation methods has led to increasing lab-to-lab variability and poor lot-to-lot reproducibility. Until protocols are defined and universally standardized for PCR among laboratories, the clinician should request information regarding quality control testing and controlled data supporting the primers in use at that specific laboratory.

The introduction of PCR also has opened the door for another area of debate: the issue of whether infection necessarily means "disease." The ability of PCR technology to detect very small numbers of organisms has led some to consider the level of infection required to overcome the body's immune response and cause disease, suggesting that the organism detected by PCR may not always be the infectious agent causing the animal's current illness. This is further complicated by the fact that PCR cannot distinguish between viable and nonviable organisms, reinforcing the need to establish a comprehensive clinical profile in diagnosis. In addition, diagnostic guidelines must be established to aid in determining how many organisms are needed to cause disease, or additional data must be generated to better understand the rate of clearance of infectious agents after effective treatment.

Unlike the other methods discussed, PCR testing essentially is limited to reference laboratory testing because of the complexity of the materials, equipment, and technique, and the lack of an in-clinic PCR device. However, like ELISA and other previously complex methods, new technology eventually will provide in-clinic PCR capability, accompanied by standardized methods.

Identification of the organism always has been the gold standard for diagnosing an infectious disease. However, because many organisms elude detection because of their small numbers or occult location within the body, direct identification of the organism is not always possible. Recent advances have extended the range of direct antigen detection (see discussion above on PCR). However, reliance on the detection of antibody in the serum as an indirect method for diagnosing many infectious diseases is prevalent. The methods for detecting antibody include ELISA, IFA, complement fixation (CF), hemagglutination inhibition (HI), serum virus neutralization (SVN), and Western blot analysis.

In the ELISA and IFA antibody assays, a specific antigen from the infectious agent in question is fixed to a solid surface (microtiter plate or glass slide, respectively) and the patient's serum is added. If antibodies to this organism are present in the patient's serum, they will bind to the antigen. An enzymeconjugated or fluorescent-labeled anti-species antibody then is used to detect the bound antibody. Commercial laboratories usually use antigen bound to microwell plates. Membranebound antibody is used in the small, self-contained in-clinic test kits used by most veterinary hospitals. A fluorescent microscope and trained technicians are required to perform IFA testing and these tests are available only in commercial laboratories.

The hemagglutination inhibition test is used to detect antibody against some viruses that possess a hemagglutinating antigen (HA), which is capable of agglutinating erythrocytes. The HA is placed in a microtiter well and incubated with the patient's serum. If virus-specific antibodies are present in the test serum, they will bind to HA and prevent agglutination of erythrocytes added in the last step. Unfortunately, some serum samples have interfering factors that bind to HA and produce false-positive results. In addition, the HA may have varying affinities for different species of erythrocytes and produce different results with sheep, rabbit, mouse, or guinea pig red blood cells.

Serum virus neutralization (SVN) and complement fixation (CF) assays determine whether antibody is present by evaluating some normal immunoglobulin functions. The SVN assay evaluates the ability of antibodies in a patient's serum to prevent the infection of culture cells or embryonated eggs with a known specific virus. The patient's serum first is inoculated with the virus. Then the virus-serum mixture is injected into a cell culture to detect virus infectivity. Virus-specific antibodies in the patient's serum inactivate the virus and prevent infection of the cells. The CF assay assesses the ability of antibodies to bind to a specific antigen and complement to form an antigenantibody-complement complex. This complex ties up the complement and prevents it from lysing red blood cells added as a substrate. Complement fixation assays also can be used to detect antigen.

All of the antibody tests discussed so far measure antibody to whole cell antigens or viruses. The Western blot assay separates the infectious agent into its composite proteins by gel electrophoresis. The proteins are transferred to blot paper and are incubated with the patient's serum. If antibodies to virusspecific proteins are present, they will bind to the protein bands on the blot paper and can be detected by a labeled secondary anti-species antibody. Antibody binding to a combination of bands usually is required to confirm a diagnosis.

The main limitation of using antibody detection for diagnosis is that, in most diseases, the presence of antibody against an infectious agent cannot differentiate among patients with previous exposure having lingering antibodies, patients with current active infection, or patients with antibodies generated by previous vaccination. Until the recent release of a whole virus vaccine against feline immunodeficiency virus (FIV), the presence of serum antibodies against FIV was definitive proof of infection because virus-infected cats remain persistently infected for life. Unfortunately, antibodies produced in response to this whole virus FIV vaccine are indistinguishable from antibodies generated by true FIV infection and are detected with all FIV antibody assays (IFA, ELISA, and Western blot). This severely limits the utility of FIV antibody detection in FIV-vaccinated cats.

To combat this type of interference, researchers are looking for antibodies produced in response to infection but not as a result of immunization. For some pathogens, antibodies have been identified that are directed against specific proteins that are present only on the organism when it is alive within the host. 4 New assays use these live-pathogen specific peptides instead of whole cell proteins to detect antibodies directed only against those specific peptides. An example of this type of peptide is the recently identified invariable, immunodominant region (IR 6 ) on a variable region of the lipoprotein (VlsE) of Borrelia burgdorferi. 6 The IR 6 peptide is highly antigenic and stimulates antibody production that rises rapidly during experimental infection and drops rapidly with successful treatment. Several theories have been proposed to explain this phenomenon, including the possibility that the variable region of the VlsE protein is changed rapidly in the organism and older variant molecules are degraded rapidly. This high turnover rate also would ensure that the protein would be rare in dead organisms. 7 Therefore acute infections treated immediately showed a rapid rise and drop in antibody against this antigen. It has yet to be determined whether antibody levels in chronic cases will respond similarly, and whether the IR 6 is exposed to memory cells over time, resulting in antibody levels that persist following infection and treatment. Vaccination against Borrelia spp. apparently does not induce antibodies to IR 6 , because the DNA that encodes the IR 6 sequence is not present in the laboratory strains of B. burgdorferi used for vaccine production. 8 

Rapid identification of tissue micro-organisms in skin biopsy specimens from domestic animals using polyclonal BCG antibody

Multiplexed immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor setting

Comparison of four different methods for detection of Cryptosporidium species

Comparison of results for serologic testing and a polymerase chain reaction assay to determine the prevalence of stray dogs in eastern Tennessee seropositive to Ehrlichia canis

Detection of Giardia lamblia and Cryptosporidium parvum antigens in human fecal specimens using the ColorPAC combination rapid solid-phase qualitative immunochromatographic assay

Characterization of a Borrelia burgdorferi V1sE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay

Antibody response to IR 6 , a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans

Dogs vaccinated with common Lyme disease vaccines do not respond to IR 6 , the conserved immunodominant region of the VlsE surface protein of Borrelia burgdorferi