Elitza Theel, Ph.D.
Professor of Laboratory Medicine and Pathology
Division of Clinical Microbiology
Mayo Clinic, Rochester, Minnesota
Hello everyone and thank you for joining us for today’s Hot Topic. My name is Dr. Elli Theel. I direct the Infectious Diseases Serology Laboratory as well as co-direct the Vector-borne Diseases Service Line here at Mayo Clinic in Rochester, Minnesota.
Today, I’ll be sharing with you our recommended approach to serologic testing for assessment of infection with Coccidioides.
And before diving in, these are my disclosures. I serve as a consultant for the companies listed here.
So, as a reminder, there are two species of Coccidioides, C. immitis and C. posadasii, that are most frequently associated with human infections, and these are dimorphic fungi, meaning that they exist in the environment as filamentous molds, and inhalation of released arthroconidia can lead to infection in some hosts. Once in the patients, the arthroconidia develop into spherules which are full of endospores, as you can see on the image to the upper right, and each of those endospores can individually develop into new spherules, continuing the infectious process.
Coccidioides is endemic throughout the southwest United States, although its range has expanded and continues to expand into the upper Northwest and into the mountain states. Coccidioides posadasii is also widely found throughout Central America and into South America, as you can see on the map.
Clinically though, the majority of infections, or roughly 60%, are largely asymptomatic; however certain populations are at higher risk of manifesting disease, including those individuals that are immunocompromised, and in the United States, approximately 150,000 cases are documented annually, with patients primarily presenting with pulmonary symptoms.
When it comes to the diagnosis of coccidioidomycosis, it often takes a multipronged approach, including the use of both direct and indirect detection methods.
Direct detection methods include culture and molecular testing, both of which are available through Mayo Clinic Laboratories. While these diagnostic strategies offer definitive evidence of infection, there are a number of limitations to be cognizant of, including the longer turnaround time associated with culture, primarily due to the time it takes for the organism to grow, and also when it comes to molecular testing, sensitivity can be variable, depending on the specimen type and extent of disease. Also, both of these methods often require invasive specimen collection procedures, which may be contraindicated in some patients.
As a result, indirect detection methods, which primarily includes detection of antibodies against Coccidioidesis, is also frequently ordered in suspected cases or for pre-immunosuppression screening, particularly in regions where Coccidioides is prevalent.
We offer a number of serologic tests for Coccidioides, including both the classic reference methods of complement fixation and immunodiffusion as a single, standalone panel. But we also offer a reflexive algorithm, which starts with a highly sensitive screening EIA, followed by complement fixation (CF) and ID confirmation of EIA reactive samples. And for multiple reasons that I’ll list on the following slides, we actually strongly encourage and recommend that clinicians order this reflexive algorithm first for suspected cases or for screening purposes to assess for Coccidioides infections, rather than first ordering the CF/ID only panel.
So, in order to understand the benefit of starting with an EIA screen for Coccidioides serologic testing, I think it’s first really important to understand the nature and processes that go into complement fixation and immunodiffusion testing methods. Again, these two methods were first developed in the 1940s and 1950s, and they remain the reference standard methods for fungal serology because when used together the CF assay provides increased sensitivity generally speaking, whereas the ID assay provides increased specificity.
However, there are a number of challenges and limitations associated with these assays, starting first with the high sample volume that’s required to perform both of these tests. Whether we are testing serum or spinal fluid by these two assays, the minimum required volume is 1.2 mL, which we frequently do not get, and so that requires the lab to contact the client site and request more, which ultimately leads to delayed testing.
These assays also take a long time to perform. For example, from start to finish, due to incubation times, it takes anywhere from 48-72 hours to result the Coccidioides IgG and IgM immunodiffusion plates. These assays are also very challenging to perform and to maintain, with a multitude of individual reagent quality control steps and potentially subjective result interpretation, leading our lab to have a set group of technologists trained to perform this testing in order to maintain standardization and consistency.
From a client and patient perspective, I think it’s really crucial for users to know that the vast majority of CF/ID test results for Coccidioides are negative. Our positivity rate for these two assays combined ranges anywhere from 6%-8% per month. So, regardless of whether patient results are positive or negative, the time to results is 48-72 hours, which begs the question of whether we can improve on this, which I’ll show you next, we can.
And finally, due to the number of different reagents, the time it takes to perform the testing, and technologist effort, the cost of testing to the patient is significant and similar to the turnaround time issue, it can be reduced for the majority of patients if the screen with reflex algorithmic approach is undertaken to test for Coccidioides infections.
So, the algorithm approach I’ve been referring to begins with testing all samples using an automated Coccidioides enzyme immunoassay. This EIA uses a proprietary mixture of recombinant and native antigens from Coccidioides, and we detect both IgM and IgG-class antibodies, without differentiation between the two immunoglobulin classes. While the assay is FDA, cleared, we have modified it to optimize positive percent agreement to 100% relative to complement fixation and immunodiffusion; however, this is at a cost of negative percent agreement, which decreases to roughly 80%. As a result, all EIA reactive samples are automatically reflexed to the CF/ID panel for confirmation.
So, what are the benefits of this approach? Well, given that our EIA reactivity rate varies from 15%-20% monthly, one of the most significant patient benefits is that for the vast majority of patients, so over 80%, the turnaround time for negative results will be within 24 hours of sample receipt, as compared to the current turnaround time of 72 hours from sample receipt if the complement fixation and immunodiffusion panel is ordered first. Additionally, the cost will be lower, as testing will be for a single EIA versus multiple, technically challenging and complex assays.
And then finally, from a laboratory perspective, screening out all the negative samples will alleviate some of the technologist burnout on this high-volume test and allow them to focus on interpretation of positive sample results. Also, given the challenges with reagents and plastics backorders that many of us have struggled with over the past few years, testing only EIA reactive samples by CF/ID will help to alleviate some of these supply chain issues we have been experiencing.
So, in conclusion, our recommendations for initial Coccidioides testing or screening, we strongly encourage and recommend that clients transition to ordering the Coccidioides EIA with reflex to CF/ID test code, or COXIS. And as always, we recommend that this be done alongside fungal and molecular testing when possible.
In contrast, for previously diagnosed patients, if there is a need to assess serologic changes over time, only in those cases would we recommend ordering the CF/ID-only panel, using the test code SCOC.
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