Mucorales Real-time PCR

Test Code: 3200

Real-time PCR detection of Mucorales (also referred to as Zygomycetes) DNA in samples may aid in diagnosis of invasive fungal infection (IFI) in high risk populations. The Mucorales PCR detects the most common causative agents of mucormycosis including 32 strains encompassing eight Mucorales genera.

Patients at risk of infection with Mucorales include those with neutropenia, solid organ transplant, hematopoietic stem cell transplant, cancer, diabetes and skin trauma. Difficult to differentiate from other filamentous fungi, mucormycosis has been found to have mortality rates in excess of 50%, depending on patient population and presentation.⁷ Further, Mucorales demonstrates limited susceptibility to a variety of antifungal therapies including Voriconazole.¹⁰ Early diagnosis of Mucorales has been associated with improved patient outcomes.¹

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Clinical and Procedure

Clinical Utility

The use of Mucorales PCR has shown superior sensitivity over culture samples from animal models of infection and various publications indicate performance in human specimens (e.g. 100% sensitivity in biopsy tissue, 100% sensitivity established in fresh tissue samples that were culture negative, and 100% specificity in control tissue samples).^1-5 The number of published studies utilizing BAL (15 studies) and biopsy (>1300) are significantly higher than any other specimen type, the next most often cited being upper respiratory specimens (n=3 published studies).⁶

Detection of target Mucorales fungal sources. Thirty-two target strain isolates representing 18 species from the order Mucorales were tested in duplicate for detection by the pan Mucorales 18S real-time PCR assay. Fungal DNA from 1 or more isolates of the following target species were evaluated: Absidia coerulea, Lichtheimia corymbifera (formerly Absidia corymbifera), Absidia glauca, Apophysomyces elegans, Cunninghamella echinulata, Cunninghamella elegans, Mucor circinelloides (5 isolates), Mucor flavus, Mucor hiemalis, Mucor indicus (2 isolates), Mucor mucedo, Mucor racemosus (3 isolates), Mucor ramosissimus, Rhizomucor pusillus (2 isolates), Rhizopus microsporus (4 isolates), Rhizopus oryzae (4 isolates), Rhizopus stolonifer and Saksenaea vasiformis. DNA from > 1000 spores was added to each reaction well. Detection was as expected for all target strains and were proportional to the number of spores extracted.

Procedure

Extraction of Mucorales DNA from serum followed by PCR amplification of targets using real-time PCR methods. An internal control is added to ensure that extraction was performed correctly and that the PCR reaction was not inhibited.

Specificity

The Mucorales PCR detects the most common causative agents of mucormycosis including 32 strains encompassing eight Mucorales genera. Additionally, no cross reactivity was detected from the most common non-Mucorales fungal pathogens with the exception of high levels of Trichosporon spp.

Turnaround Time

Same day (within 12 - 24 hours from receipt of specimen), Monday through Saturday.

Specimen Information

Specimen Type	Test Code	CPT Code	NY Approved	Volume	Assay Range	Special Instructions
Tissue	3205	87798	Yes	5 mg fresh tissue (approximately ½ of a pencil eraser size)	Detected/Not Detected	Place fresh tissue in a sterile, screw top container. The preferred handling is to not add water, saline or other fluid media to the tissue container; however, fluid media filled containers will be accepted for qualitative results. Store frozen and ship on dry ice for overnight delivery. Formalin fixed, paraffin embedded tissue will not be accepted.
BAL	3209	87798	Yes	2 mL (min. 0.5 mL)	Detected/Not Detected	Collect in a sterile, screw top tube. Specimen should be stored at 2 to 8°C or frozen in a non-self-defrosting freezer. Ship with frozen gel packs or dry ice for overnight delivery. If storage longer than 2 days is needed, specimens should be frozen at -70° C prior to shipment.
serum	3210	87798	Yes	2 mL (min. 0.5 mL)	Quantitative LOD = 181 target copies/mL LLOQ = 253 target copies/mL	Collect 4-5 mL whole blood in red top tube. Avoid using gel separator tubes; samples collected in gel separator tubes have increased rate of PCR inhibition. Centrifuge and transfer 2 mL serum to sterile, screw top tube. Can be shipped at frozen temperature Monday through Friday. Stability: 30 days frozen at -20C, 30 days at -80C. Ambient and refrigerated; 4 days
bronch wash	3226	87798	Yes	2 mL (min. 0.5 mL)	Detected/Not Detected	Collect in a sterile, screw top tube. Can be shipped at ambient or frozen temperature Monday through Friday. Specimens shipped at ambient temperature must be received within 4 days of collection.

Shipping

Ship Monday through Friday. Friday shipments must be labeled for Saturday delivery. All specimens must be labeled with patient's name and collection date. A Eurofins Viracor test requisition form must accompany each specimen. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight^® to: Eurofins Viracor, 18000 W 99th St. Ste, #10, Lenexa, KS 66219.

Causes for Rejection

Specimens beyond their acceptable length of time from collection as listed in the specimen handling, grossly hemolyzed serum specimens, or specimen types other than those listed.

Disclaimer

Specimens are approved for testing in New York only when indicated in the Specimen Information field above.

The CPT codes provided are based on Eurofins Viracor’s interpretation of the American Medical Association's Current Procedural Terminology (CPT) codes and are provided for informational purposes only. CPT coding is the sole responsibility of the billing party. Questions regarding coding should be addressed to your local Medicare carrier. Eurofins Viracor assumes no responsibility for billing errors due to reliance on the CPT codes illustrated in this material.

References

(1)CDC Web site. http://www.cdc.gov/fungal/mucormycosis/definition.html Last updated Jan 5, 2012.
(2)Kasai M., Harrington SM, Francesconi A, et al. Detection of a molecular biomarker for Zygomycetes by quantitative PCR assays of plasma, bronchoalveolar lavage and lung tissue in a rabbit model of experimental pulmonary zygomycosis. J Clin Microbiol. 2008 Nov;46(11);3690-702.
(3)Dannoui E, Schwarz P, Slany M, et al. Molecular detection and identification of Zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Disease (ESCMID) fungal infection study group (EFISG) evaluation. J Clin Microbiol. 2010 June;48(6):2043-6.
(4)Bernal-Martinzez L, Buitrago MJ, Castelli MV, Rodriquez-Tudela JL and Cuenca-Estrella M. Development of a single tube multiplex PCR to detect the most clinically relevant Mucormycetes species. Clin Microbiol Infect. 2013 Jan;19(1):E1-7.
(5)Hammond SP, Bialek R. Milner DA, Petschnigg EM, Baden LR, Marty FM. Molecular methods to improve diagnosis and identification of mucormycosis. J Clin Microbiol. 2011 June;49(6):2151-3.
(6)Hata DJ, Buckwalter SP, Pritt BS, Roberts GD, Wengenack NL (2008) Real-time PCR method for detection of zygomycetes .J Clin Microbiol. 2008 Jul;46(7):2353-8.
(7) Laurence Millon,DenisCaillot,AnaBerceanu,StéphaneBretagne,FannyLanternier,etal..Evaluation ofserumMucoralesPCRforthediagnosisofMucormycoses:TheMODIMUCORprospective trial. ClinicalInfectiousDiseases,OxfordUniversityPress(OUP),Inpress, 10.1093/cid/ciab1066 . hal-03528144.
(8) Millon L, Larosa F, Lepiller Q, et al. Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients. Clin Infect Dis 2013; 56:e95–101.
(9) Million, L, Caillot, Denis, et al. Evaluation of Serum Mucorales Polymerase Chain Reaction (PCR) for the Diagnosis of Mucormycoses: The MODIMUCOR Prospective Trial. Clin Infect Dis 2022;75(5):777–85.
(10)Data on file at Eurofins Viracor.

Mucorales Real-time PCR

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