Tackling negative diagnostic test results: Focus on respiratory disease
Josepha DeLay, Davor Ojkic
Animal Health Laboratory, University of Guelph, Guelph, ON
AHL Newsletter 2022;26(4):13.
New pathogens, and new roles for known pathogens, are at the forefront of swine disease diagnostics and research. As exciting as it is to suspect a new disease syndrome, it is important to be thorough in the approach to diagnostic testing, so as not to miss more common pathogens.
Results of diagnostic testing for clinical respiratory disease can sometimes be unrewarding and frustrating. Diagnostic test results are influenced by many factors, including the specific animals and tissues tested, the time point of sampling in the disease course, the specific tests and methodologies selected for a case, and the potential involvement of new or emerging pathogens.
Bias in sampling, test selection, and result interpretation can be easily introduced at all levels of disease investigation, and veterinarians and pathologists must be vigilant in avoiding bias at all times. The economics of diagnostic testing can play a role in introducing bias as well, as we try to minimize sample and test numbers to save money. To avoid these biases and to reach a valid conclusion, the approach to disease diagnosis must be logical, well planned, and thorough.
Important points to consider in any diagnostic workup include the following:
Select acutely affected, untreated animals for sampling for diagnostic tests. This applies for both live-animal and postmortem sampling. In animals with subacute or chronic disease, the pathogen may be present at reduced levels or may be cleared, potentially leading to negative test results and erroneous conclusions regarding the cause of disease. In samples from animals recently treated with antibiotics, bacterial growth will be negatively impacted by the drug, again producing misleading test results. The number of animals tested is also important. Choosing 3 animals for sampling will optimize the chances for identifying common pathogens among affected animals (finding the ‘common disease denominator’) while being relatively economical.
Take appropriate samples from the selected animals, and treat the samples kindly. For respiratory cases, examine the entire respiratory tract, including trachea, mainstem bronchi, and both lungs. Note the character and distribution of gross lesions, and communicate these findings on the submission form. Include an estimate of the percentage of lung parenchyma or tracheal mucosa involved in the disease process. For bacterial culture and PCR tests, sample at the margin of affected and unaffected lung parenchyma. Submit a fresh sample of trachea for potential testing. For histopathology, include trachea and multiple samples from both left and right lung (e.g., 1 sample from both cranial and caudal lobes from each lung), also capturing the interface between normal and abnormal lung. Be gentle when sampling, avoiding crush injury with forceps or scissors. Place fresh samples for culture and PCR in individual whirl-pak or other sterile bags that can be sealed, and keep these samples chilled. Place samples for histopathology in a sufficiently large screw-top formalin jar to ensure there is a 1:10 ratio of tissue:formalin to allow rapid and complete fixation.
Select specific diagnostic tests that will answer the questions for a particular clinical scenario, and include histopathology whenever possible. Initial test selection (various PCRs, bacterial culture) should be based on the clinical differential diagnoses. Histologic lesions can provide valuable evidence to confirm or exclude these differentials, and can be correlated with PCR and culture results. When test results are negative, histologic features can help fine-tune additional testing. The lesions will categorize the disease process regarding pathogenesis and likely etiologies; e.g., inhaled vs blood-borne infection, bacterial pathogens vs epitheliotrophic viral injury, etc. Histologic samples also allow for direct pathogen detection using immunohistochemical (IHC) or in situ hybridization (ISH) techniques. These direct tests permit visualization of a pathogen within a lesion, and can confirm diagnoses suggested by PCR results.
A negative test result isn’t the end of the story. In the face of histologic lesions and negative test results for more common pathogens, consider known emerging pathogens or novel pathogens. Current research at other institutions has identified involvement of porcine astrovirus type 4 (PoAstV4) in tracheitis and bronchitis in young pigs, and porcine hemagglutinating encephalomyelitis virus (PHEV) in tracheitis in grow-finish pigs. Porcine parainfluenza virus has been detected in some swine pneumonia cases, although associated lesions are not well described. Porcine respiratory coronavirus is recognized but infrequently investigated. Specific PCR tests for these viruses are available at external laboratories. We don’t yet know the significance of these potential pathogens in the Ontario swine herd, but diagnosticians do consider these agents in compatible disease situations. For cases where available tests do not identify a pathogen to explain clinical disease and pathologic findings, metagenomic analysis by whole genome sequencing is an option for high-level investigation of novel pathogens.
Communication between the veterinarian and lab diagnosticians is important at every step of the diagnostic process. This includes communicating basic information, such as the clinical history and gross postmortem lesions, to more in-depth discussions when advanced diagnostic testing is warranted. AHL