Fungal Biomarkers: Primer for Pharmacists

Article

Biomarkers are commonly used by physicians to assist in the earlier diagnosis of some of the most common fungal diseases.

Introduction

Invasive fungal diseases create a serious burden on both the health care system and quality of life for immunocompromised patients. The most common invasive fungal infections within the United States are Aspergillosis, Candida, and Histoplasmosis.

Estimated total costs after fungal disease diagnoses are estimated to be as high as $48 billion per year. The number of hospitalizations associated with fungal diseases are more than 75,000 per year, along with an estimate of close to 9 million outpatient visits. The estimated number of deaths in 2021 was >7000.

Although these numbers are startling, they are likely underestimated by statisticians due to the fact that many fungal diseases go undiagnosed.1 Fungal biomarkers (biological products from the structure of the fungi/yeasts) are being used by physicians to assist in the earlier diagnosis of some of the most common fungal diseases, which will both decrease long-term health care costs and mortality rates.

Galactomannan (GM) and (1,3)-B-D-glucan (BDG) are 2 biomarkers that used to diagnose invasive fungal infections. Antigen/antibody and/or polymerase chain reaction (PCR) testing are used when tissue cultures are inconclusive or unavailable.

Determining which test to use depends on the type of fungi, the location of the infection, patient-specific factors, and institutional preference. Some fungal infections are not responsive to fungal biomarker testing, such as Mucorales.

Depending on the location of the fungal infection, accurate results with certain routes of testing may not be likely. Patient-specific factors to take into account include cost, additional medications, concurrent infections, number of tests given, and ability to follow up.

Some institutions may not have fungal biomarker testing available within the acute setting, which would also limit options. All of these factors are important to take into consideration when diagnosing an invasive fungal infection and deciding on the conduction of a fungal biomarker test for diagnostic support.

Galactomannan (GM) Assay

GM is a polysaccharide antigen that is a critical part of Aspergillus’s cell wall structure, as well as other fungi. The antigen releases into the serum and other body fluids during the early stages of fungal growth and can survive in the body for up to 8 weeks.2

GM tests use samples from serum or bronchoalveolar lavage as an adjunct to other diagnostic criteria. Positive cutoffs for GM (using optical density index) are at least 0.5 in the serum and at least 1.0 in the bronchoalveolar lavage.2,4-7

Sensitivity and specificity also varies depending on whether the antigen was extracted from serum or bronchoalveolar lavage as well as what the diagnosis cutoff was defined as. In general, sensitivity ranges from 67% to 92% and specificity ranges from 60% to 98%.The higher the cutoff value, the higher the sensitivity and specificity is.2,3

Detection of GM can also occur in Penicillium, Paracoccidioides, Histoplasma, Fonsecaea, Blastomyces, Fusarium, Paecilomyces and Cryptococcus.4 Because of the variety of paths to detection and GM being a polysaccharide, false positives can occur.

Common causes of falsely positive tests are electrolyte solutions containing sodium gluconate, beta-lactam antibiotics, chemotherapy, and other fungal infections.5,6 Some patients can have circulating GM below the detection limit at the time of testing, which could cause false negatives.8

(1,3)-B-D-glucan (BDG) Assay

(1,3)-B-D-glucan is also a polysaccharide found in the cell walls of pathogenic fungi. It was approved by the FDA in 2004 to be used as a diagnostic test for invasive candidiasis diseases.4,9

BDG can survive in the body for up to 7 weeks after blood cultures become stable.10 Cultures of blood and other samples for BDG testing are considered the gold standard by the IDSA.

For critically ill patients, IDSA guidelines suggest serum BDG testing alone.6 The cutoff for detection in the serum is 1 colony-forming unit/mL or less. Sensitivity is ~50% to 90%; and specificity is ~75% to 95%, with higher specificity noted in hematologic malignancy patients.11

Based on this information, this test may be more useful in excluding invasive fungal infections. Serial repeat testing may be performed weekly to assess response in some patients.12

(1,3)-B-D-glucan can also be detected in Aspergillus, Pneumocystis jiroveci, Fusarium, Trichosporon, Saccharomyces, Coccidioides, Histoplasma, Sporothrix, and Acremonium.4,9 Common causes of falsely positive tests are hemodialysis with certain cellulose membranes, surgical packing, bacteremia, recent use of albumin or immunoglobulin products, beta lactam antibiotics, and other invasive fungal infections.9,11 Piperacillin/tazobactam has been listed as causing false positives in the past, but improved manufacturing processes of this antibiotic have reportedly led to this not being an issue in the United States.14

Non-culture Fungal Biomarker Testing: Antigen/Antibody and Polymerase Chain Reaction (PCR)

The best studied antigen/antibody test is the mannan/antimannan test in Europe, which can detect antigens/antibodies from invasive fungal infections prior to blood cultures. It is not specific for certain fungal infections, so it is most commonly used as an adjunct to blood/tissue cultures.

Antigen/antibody testing is most commonly used with endemic mycosis, such as Histoplasmosis, Blastomycosis, and Coccidioidomycosis. Sensitivity and specificity are about 75% to 80%.

There is no consistency with what the cutoff for positivity should be, how many positives are needed for diagnosis, or how long after a blood/tissue culture the testing should be done. Common causes of falsely positive tests are bacteremia, beta-lactam antibiotics, hemodialysis, other fungal infections, recent use of albumin or immunoglobulin products, surgical packing, and mucositis.11

PCR testing detects fungal DNA, which allows detection of both active and inactive infections.13 Because of what it detects, it is also used as an adjunct to blood/tissue culture for many invasive fungal infections on a case-by-case basis.7

PCR testing can help shorten time to diagnosis.11 Its use is studied the most in Aspergillosis, Pneumocystis pneumonia, and Candidiasis compared to other invasive fungal infections. Common causes of falsely positive tests are contamination from the environment, previously amplified PCR products, cross-reactivity of PCR primers, and probes with other organisms. Common causes of falsely negative tests are suboptimal DNA extraction, PCR inhibition, or suboptimal analysis of the sensitivity of PCRs.13

Conclusion

GM, (1,3)-B-D-Glucan, antigen/antibody, and PCR testing are options for diagnosing invasive fungal infections that pharmacists need to be familiar with. Depending on which type of fungal infection the patient is diagnosed with, the complexity level of each patient’s case, and/or the location of the patient, fungal biomarker testing can very useful in helping clinicians diagnose invasive fungal infections.

Pharmacists need to be aware of the specificity/sensitivity of the test, fungi identified and missed, and false positives associated with each test. Current IDSA Guidelines for diagnosing and treating invasive fungal infections have not been updated since 2016. With all positive factors and limitations in consideration, fungal biomarkers are a reasonable addition to diagnostic criteria for various invasive fungal infections.

References

  1. Impact of fungal diseases in the United States. Centers for Disease Control and Prevention. https://www.cdc.gov/fungal/cdc-and-fungal/burden.html. Published September 30, 2022. Accessed November 27, 2022.
  2. Zhou W, Li H, Zhang Y, et al. Diagnostic Value of Galactomannan Antigen Test in Serum and Bronchoalveolar Lavage Fluid Samples from Patients with Nonneutropenic Invasive Pulmonary Aspergillosis. J Clin Microbiol. 2017;55(7):2153-2161. doi:10.1128/JCM.00345-17
  3. Seval Özen, Halil Özdemir, Ebru Evren, Esra Çakmak Taşkın, Gül Arga, Hatice Kübra Konca, Hasan Fatih Çakmaklı, Şule Haskoloğlu, Emel Okulu, Handan Dinçaslan, Elif İnce, Talia İleri, Nurdan Taçyıldız, Figen Doğu, Ebru Us, Zeynep Ceren Karahan, Suat Fitöz, Tanıl Kendirli, Zarife Kuloğlu, Ercan Tutar, Aydan İkincioğulları, Emel Ünal, Mehmet Ertem, Erdal İnce & Ergin Çiftçi (2022) The role of galactomannan test results in the diagnosis of pediatric invasive aspergillosis, Infectious Diseases, 54:4, 269-276,DOI: 10.1080/23744235.2021.2008486
  4. Huppler AR, Fisher BT, Lehrnbecher T, Walsh TJ, Steinbach WJ. Role of Molecular Biomarkers in the Diagnosis of Invasive Fungal Diseases in Children. J Pediatric Infect Dis Soc. 2017;6(suppl_1):S32-S44. doi:10.1093/jpids/pix054
  5. Qualification of Biomarker - Galactomannan in studies of treatments of invasive: Guidance portal. Qualification of Biomarker - Galactomannan in studies of treatments of invasive | Guidance Portal. https://www.hhs.gov/guidance/document/qualification-biomarker-galactomannan-studies-treatments-invasive. Accessed November 27, 2022.
  6. Hage CA, Carmona EM, Epelbaum O, et al. Microbiological Laboratory Testing in the Diagnosis of Fungal Infections in Pulmonary and Critical Care Practice. An Official American Thoracic Society Clinical Practice Guideline [published correction appears in Am J Respir Crit Care Med. 2019 Nov 15;200(10):1326]. Am J Respir Crit Care Med. 2019;200(5):535-550. doi:10.1164/rccm.201906-1185ST
  7. Thomas F. Patterson, George R. Thompson, III, David W. Denning, Jay A. Fishman, Susan Hadley, Raoul Herbrecht, Dimitrios P. Kontoyiannis, Kieren A. Marr, Vicki A. Morrison, M. Hong Nguyen, Brahm H. Segal, William J. Steinbach, David A. Stevens, Thomas J. Walsh, John R. Wingard, Jo-Anne H. Young, John E. Bennett, Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America, Clinical Infectious Diseases, Volume 63, Issue 4, 15 August 2016, Pages e1–e60, https://doi.org/10.1093/cid/ciw326
  8. Paul E Verweij, Monique A. S. H Mennink-Kersten, Issues with galactomannan testing, Medical Mycology, Volume 44, Issue Supplement_1, September 2006, Pages S179–S183, https://doi.org/10.1080/13693780600904918
  9. The (1,3)β-D-glucan test as an aid to early diagnosis of invasive fungal infections following lung transplantation https://journals.asm.org/doi/pdf/10.1128/JCM.01183-10. Accessed November 28, 2022.
  10. Mikulska M, Furfaro E, Del Bono V, et al. Persistence of a positive (1,3)-beta-D-glucan test after clearance of candidemia in hematopoietic stem cell transplant recipients. Clin Vaccine Immunol. 2011;18(3):518-519. doi:10.1128/CVI.00513-10
  11. Peter G. Pappas, Carol A. Kauffman, David R. Andes, Cornelius J. Clancy, Kieren A. Marr, Luis Ostrosky-Zeichner, Annette C. Reboli, Mindy G. Schuster, Jose A. Vazquez, Thomas J. Walsh, Theoklis E. Zaoutis, Jack D. Sobel, Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America, Clinical Infectious Diseases, Volume 62, Issue 4, 15 February 2016, Pages e1–e50, https://doi.org/10.1093/cid/civ933
  12. Edwards CJ, Estrad BL. Basic Skills in Interpreting Laboratory Data. 7th ed., ASHP, 2022.
  13. Khot PD, Fredricks DN. PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther. 2009;7(10):1201-1221. doi:10.1586/eri.09.104
  14. Mikulska M, Furfaro E, Del Bono V, Raiola AM, Ratto S, Bacigalupo A, Viscoli C. Piperacillin/tazobactam (Tazocin™) seems to be no longer responsible for false-positive results of the galactomannan assay. J Antimicrob Chemother. 2012 Jul;67(7):1746-8. doi: 10.1093/jac/dks111. Epub 2012 Apr 11. PMID: 22499998.
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