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1 This is the written version of our Hot Topic video presentation available at: MayoMedicalLaboratories.com/hot-topics Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice. Today our topic is serologic testing for the diagnosis and monitoring of some of the most common fungal infections in the United States. 1

2 Our speaker for this program is Dr. Elli Theel, Director of the Infectious Disease Serology Laboratory at Mayo Clinic, Rochester, Minnesota. Dr. Theel, thank you for presenting today. Thank you. 2

3 I have nothing to disclose. 3

4 Serologic testing plays an important role in the diagnosis of some of the common fungal infectious agents in the United States, including Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides and Cryptococcus species. However, the decision of which fungal serologic test to order should be based on the patient s clinical presentation, their exposure history and risk factors for infection, all of which may differ between these fungal organisms. Serologic evaluation for all 4 of these agents simultaneously may lead to falsely positive results in low-risk patients. Therefore, in an effort to minimize inappropriate testing, as of March 2014 Mayo Medical Laboratories will no longer offer the Fungal Antibody Survey on serum and CSF specimens. Instead, fungal serologic testing will be available for each agent individually and as a panel for Histoplasma and Blastomyces due to the overlapping endemicity and similar clinical presentation of these 2 fungi. The focus of this hot topic will be to review the available serologic assays for these fungal agents and to discuss result interpretation. Identification and detection of these fungi by direct staining, culture, and molecular methods has been described in previous Hot Topics. 4

5 We will begin with Blastomyces dermatitidis, which is a dimorphic fungus that grows as a yeast at body temperature and as a filamentous mold at 25 degrees Celsius. Blastomyces is endemic throughout the Ohio and Mississippi river valleys in the US and encroaches into southern parts of Canada as well. The incidence of infection among these regions is low and the primary route of infection is through inhalation of fungal microconidia. While roughly half of those exposed will remain asymptomatic, patients may present with a wide range of clinical manifestations, including pulmonary flulike symptoms, disseminated disease, and more severe complications like acute respiratory distress syndrome. 5

6 Detection of antibodies to Blastomyces can be useful as an aid for the diagnosis of blastomycosis, alongside other laboratory methods and clinical evaluation. Mayo Medical Laboratories currently offers an FDA-cleared, qualitative enzyme immunoassay or EIA for detection of total antibodies to yeast phase Blastomyces antigens from serum and has also validated this assay for testing of cerebrospinal fluid. Compared to patients with culture-positive Blastomyces, this EIA has a sensitivity of approximately 85% in serum and therefore a negative EIA result cannot be used to definitively exclude the diagnosis of blastomycosis. If acute or pulmonary infection is suspected, a new specimen collected 2 to 3 weeks later should be submitted and tested. The specificity of the Blastomyces EIA is high and therefore samples positive by the EIA do not require further confirmatory testing. Finally, a distinct advantage of this assay, compared to more traditional techniques including immunodiffusion which we will discuss next, is its rapid turnaround time. 6

7 Mayo Medical Laboratories also offers the classic immunodiffusion assay for detection of precipitating antibodies to the Blastomyces A antigen, and similar to the Blastomyces EIA, results from the immunodiffusion assay should be used as an aid for the diagnosis of blastomcyosis. Briefly, immunodiffusion assays are performed by adding patient samples to wells in an agar plate which surround a central well that contains a purified fungal antigen, as shown in the image on this slide. During the incubation process, antibodies, if present in the patient sample, and antigen will diffuse out of their respective wells and an antigen-antibody complex will form between the wells and become visible between the wells, as indicated. The presence of a precipitation band is indicative of acute or active infection. Compared to the Blastomyces EIA, the performance characteristics of the ID assay are similar, likewise showing decreased sensitivity in patients with localized infection, and therefore, a single negative immunodiffusion result should not be used to exclude diagnosis. However, the immunodiffusion assay has a prolonged turnaround time compared to the EIA, and therefore we recommend ordering the Blastomyces EIA for routine evaluation of patients with suspected blastomycosis. 7

8 Histoplasma capsulatum, similar to Blastomyces, is a dimorphic fungus which is also endemic along the Ohio and Mississippi River valleys, with infection occurring through inhalation of aerosolized Histoplasma microconidia. Clinically, the vast majority of primary infections are unrecognized or result in mild influenzalike symptoms for which medical attention is not sought out. In a small proportion of individuals however and particularly those who are at the extremes of age or who are immunosuppressed, exposure can lead to acute pulmonary disease characterized by a dry cough, fever, and fatigue and in some cases may progress to disseminated infection with extrapulmonary foci. 8

9 Detection of antibodies to Histoplasma provides additional laboratory evidence of disease when used alongside other laboratory findings and clinical presentation. All specimens submitted for anti-histoplasma antibody detection, are first screened by an EIA. Patients who are nonreactive by this assay may be considered as negative for histoplasmosis, unless their exposure was recent and early acute infection is suspected. In such cases, collection and submission of a new specimen in 2 to 3 weeks is recommended. Alternatively, all samples which are reactive or equivocal by the EIA are automatically reflexed for confirmatory testing by the Histoplasma immunodiffusion and complement fixation assays. It is important to remember that clinical decisions regarding histoplasmosis should not be based on reactive results from the EIA alone. The Histoplasma immunodiffusion assay utilizes 2 different antigens, the H and the M proteins, and we can therefore detect up to 2 different precipitating bands in the agar plate. Antibodies to the M antigen, appear shortly following exposure and can remain detectable by immunodiffusion for up to 3 years following disease resolution. Therefore, presence of the M band alone cannot be used to discriminate between acute or remote infection. On the other hand, the presence of an H band either alone or in combination with the M band is indicative of active or recent histoplasmosis. Importantly, a negative immunodiffusion result should not be used to exclude histoplasmosis as the specimen may have been collected early following exposure and prior to the development of detectable precipitating antibodies. 9

10 Alongside immunodiffusion, we also perform Histoplasma complement fixation testing. Briefly, complement fixation assays are based on the ability of antibodyantigen complexes, formed between antibodies present in the patient sample and added fungal antigens, to fix and inactivate exogenously added complement. Sensitized RBCs are added to this mix and since the complement pathway is inactivated, the red blood cells will remain intact and settle to the bottom of the well as a compact pellet, indicative of a positive reaction and the presence of specific antibodies. 10

11 In the absence of patient antibodies however, the added complement will remain active and lead to lysis of the sensitized red blood cells. Therefore, the presence of lysis is indicative of a negative reaction and the absence of specific antibodies. 11

12 The Histoplasma complement fixation assay is performed using 2 different Histoplasma antigens, one purified from the yeast phase and the second purified from the mycelial phase of growth. Serial dilution of patient samples, as shown in the image below, allows us to determine an endpoint titer which can be used to guide diagnosis. For either antigen, serially increasing titers or an endpoint titer greater than or equal to 1:32 is associated with the presence of active histoplasmosis. A titer of 1:8 or 1:16 is considered positive, but only presumptive evidence of infection and additional clinical correlation is necessary. Titers less than 1:8 are not considered to be significant and therefore are not reported. Importantly, low-level titers may be detected in individuals who reside in endemic areas and are otherwise healthy. Finally, declining complement fixation titers over months to years have been associated with disease resolution, but should not be used to monitor response to therapy. The complement fixation assay has a higher sensitivity for histoplasmosis as compared to immunodiffusion assays, particularly during early disease, however their specificity is lower and for this reason complement fixation and immunodiffusion are performed concurrently. Also these assays are fairly labor intensive with long incubation times which leads to an average turnaround time of 48 to 72 hours. 12

13 The final dimorphic fungus we will discuss today is Coccidioides which grows as a filamentous mold in the environment, but forms spherules and endospores in the host. Coccidioides is endemic throughout the southwestern United States and infection occurs through inhalation of aerosolized arthroconidia. In the United States, the incidence of medically significant Coccidioides infections is estimated to be approximately 150,000 individuals annually. Though many more people are exposed to the fungus, in up to 60% of them infection will be inapparent or sufficiently mild to not prompt clinical evaluation. Individuals who succumb to disease typically present with pulmonary symptoms, fever, and fatigue. 13

14 As with the other fungal agents, serologic testing for anti-coccidioides antibodies should be used as an aid to diagnose coccidioidomycosis. Similar to the testing process for anti-histoplasma antibodies, specimens submitted for detection of antibodies to Coccidioides are first screened by an enzyme immunoassay. Patients who are nonreactive by the EIA may be considered as negative for coccidioidomycoses, unless patient exposure was recent and early infection is suspected. In such cases, collection and submission of a new specimen in 2-3 weeks is recommended. Alternately, all samples which are reactive by the EIA are automatically reflexed for confirmatory testing by the Coccidioides immunodiffusion and complement fixation assays. Again, it is important to remember that clinical decisions regarding coccidioidomycosis should not be based on a reactive EIA alone. The Coccidioides immunodiffusion assay detects IgM- and IgG-class antibodies separately, using 2 different purified antigens. Following infection, IgM antibodies develop to the TP antigen and are detectable within 3 weeks of symptom onset in over 80% of infected individuals. IgM-class antibodies will subsequently decline over the next 6 to 7 months following disease resolution. IgG-class antibodies to Coccidioidies develop to the CF antigen, are detectable 3 to 5 weeks following infection and remain detectable following the disappearance of IgM-class antibodies. 14

15 The complement fixation assay for Coccidioides uses antigens derived from a culture filtrate of the organism and similar to the other fungi, endpoint titers can be indicative of disease progression. Specifically, titers of 1:2 to 1:8 are indicative of either prior exposure or acute, focal infection. Discrimination between these 2 clinical entities requires correlation with clinical presentation and other laboratory findings. Additionally, submission of a convalescent sample is recommended as a 4- fold or greater rise in titers between acute and convalescent sera would be indicative of active disease. Traditionally, complement fixation titers greater than or equal to 1:16 have been associated with more severe disease manifestations. Finally however, single time point negative immunodiffusion or complement fixation results should not be used to exclude infection as specimens may have been collected prior to the development of detectable antibodies. 15

16 Cryptococcus neoformans and Cryptococcus gattii are encapsulated yeast which are transmitted following environmental aerosolization and inhalation. Epidemiologically, Cryptococcus neoformans can be isolated throughout the United States, whereas Cryptococcus gattii is primarily isolated from states along the pacific coast and more recently has been reported to be endemic to the southeastern United States. Both of these agents are primarily found in the lungs and have a tropism for the central nervous system. Interestingly though, while Cryptococcus neoformans is considered primarily an opportunistic pathogen among immunocompromised hosts, Cryptococcus gattii is commonly associated with infection in otherwise healthy individuals and has a higher predilection for the central nervous system. While the incidence of cryptococcosis as a whole is low, immunocompromised patients are at increased risk of infection and severe disease. 16

17 Detection of the Cryptococcus polysaccharide antigen can be used as definitive laboratory based evidence of infection. Prior to March of 2014, Mayo Medical Laboratories used a latex agglutination assay to detect this antigen in both serum and CSF specimens. As of then however, MML has transitioned to using a new lateral flow immunoassay or LFA to detect the cryptococcal antigen. 17

18 The LFA is a dip-stick based method and FDA-cleared for use in both serum and CSF specimens. This assay offers increased sensitivity for detection of both Cryptococcus neoformans and Cryptococcus gattii compared to the previous latex agglutination assay and will be used as both a screen and to determine an endpoint titer. It is important to understand however, that endpoint titers between the LFA and latex agglutination assays do not correlate and are not interchangeable. We and others have found that endpoint titers acquired by the LFA are often much higher than those acquired on the same sample by the latex agglutination assay. Therefore, for patients who are being monitored for disease regression by the Cryptococcus antigen test, the same method, either LFA or LA must be used on serial samples. In order to help providers transition to the new LFA method, MML will report both the LFA and latex titers for a 3-month period following implementation of the LFA. The latex titer will be performed and reported at no charge to the patient. 18

19 The following 3 slides are a summary of the available and preferred fungal serologic tests performed at Mayo Medical Laboratories and are included for your reference. 19

20 Finally, to conclude, the College of American Pathologists, recently recommended that all CSF specimens submitted for initial diagnostic testing and which are positive by the Cryptococcus antigen assay, must also be submitted for routine fungal culture. CSF specimens submitted for repeat or serial cryptococcal antigen testing do not need to be cultured. To comply with this requirement, MML will offer 3 different test codes for Cryptococcus antigen testing from CSF. First, the LFACX test should be ordered if the specimen is submitted for initial diagnostic purposes and the referring laboratory does not perform routine fungal culture on CSF. If the CSF is positive for cryptococcal antigen in this case, the sample will be reflexed to fungal culture and for determination of an endpoint titer. Alternatively, if the specimen is submitted for initial diagnostic purposes and the referral laboratory performs fungal culture on site, test code CLFA should be requested. In this case, Cryptococcus antigen-positive CSF specimens will be reflexed to titer only. Finally, for CSF specimens submitted to monitor antigen levels, the CLFAT test code should be ordered which will determine an endpoint titer only. 20

21 Thank you. 21

22 22

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