1887

Abstract

Invasive mucormycosis (IM) is a life-threatening infection caused by fungi belonging to the order Mucorales. Histopathology, culture and radiology are the mainstay of diagnosis but lack sensitivity, leading to a delay in timely diagnosis and intervention. Recently, PCR-based approaches have been shown to be a promising method in diagnosing IM.

Molecular-based approaches may be a valuable adjunct to standard conventional methods for diagnosing IM, especially among culture negatives and patients on antifungal therapy.

In the present study we aimed to evaluate the clinical utility of panfungal and Mucorales-specific PCR for diagnosing IM from various clinical specimens.

This was a prospective study in which 239 clinically suspected cases of IM attending our tertiary care hospital from August 2015 to March 2018 were enrolled. All the cases were defined as ‘proven’, ‘probable’ or ‘possible’ based on EORTC/MSGERC guidelines. In addition to conventional diagnostics (KOH-calcofluor stain and culture), panfungal and Mucorales-specific PCR assays were also performed. The amplified products were sequenced for species identification. antifungal susceptibility was performed on all the culture-positive isolates.

Among 239 clinically suspected cases of IM, only 140 cases were diagnosed by the demonstration of aseptate ribbon-like hyphae on direct microscopy. Culture was positive in 35.7 % (54/140) of direct microscopy-positive samples. Among the proven cases (=11), the sensitivity for both Mucorales-specific nested PCR and panfungal PCR was 100 %, but specificity was 91.9 and 73.7% respectively. In probable cases (=129), the sensitivity of both the PCRs was 98.5 % and specificity for panfungal PCR was 73.7 and 91.9 % for Mucorales-specific PCR.

Pan fungal PCR in combination with Mucorales-specific PCR, followed by sequencing, may play a significant role in IM diagnosis especially among those negative for both direct microscopy and culture.

Funding
This study was supported by the:
  • Indian Council of Medical Research (Award OMI/15/2014/ECD-I. dated 14.03.2017)
    • Principle Award Recipient: ImmaculataXess
  • Indian Council of Medical Research (Award File No. 80/955/2015-ECD-I.)
    • Principle Award Recipient: MragnayaniPandey
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.001370
2021-05-26
2022-01-24
Loading full text...

Full text loading...

References

  1. Chakrabarti A, Dhaliwal M. Epidemiology of mucormycosis in India. Curr Fungal Infect Rep 2013; 7:287–292 [View Article]
    [Google Scholar]
  2. Richardson M, Lass‐Flörl C. Changing epidemiology of systemic fungal infections. Clin Microbiol Infect 2008; 14:5–24
    [Google Scholar]
  3. Chakrabarti A, Das A, Mandal J, Shivaprakash M, George VK et al. The rising trend of invasive zygomycosis in patients with uncontrolled diabetes mellitus. Sabouraudia 2006; 44:335–342
    [Google Scholar]
  4. Skiada A, Pagano L, Groll A, Zimmerli S, Dupont B et al. Zygomycosis in Europe: analysis of 230 cases accrued by the registry of the European Confederation of Medical Mycology (ECMM) Working Group on Zygomycosis between 2005 and 2007. Clin Microbiol Infect 2011; 17:1859–1867 [View Article][PubMed]
    [Google Scholar]
  5. Arvanitis M, Anagnostou T, Fuchs BB, Caliendo AM, Mylonakis E. Molecular and nonmolecular diagnostic methods for invasive fungal infections. Clin Microbiol Rev 2014; 27:490–526 [View Article][PubMed]
    [Google Scholar]
  6. Trubiano J, Dennison A, Morrissey C, Chua K, Halliday C et al. Clinical utility of panfungal polymerase chain reaction for the diagnosis of invasive fungal disease: a single center experience. Sabouraudia 2015; 54:138–146
    [Google Scholar]
  7. Bialek R, Konrad F, Kern J, Aepinus C, Cecenas L et al. PCR based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue. J Clin Pathol 2005; 58:1180–1184 [View Article][PubMed]
    [Google Scholar]
  8. Dannaoui E, Schwarz P, Slany M, Loeffler J, Jorde AT 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 Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation. J Clin Microbiol 2010; 48:2043–2046 [View Article][PubMed]
    [Google Scholar]
  9. Kasai M, Harrington SM, Francesconi A, Petraitis V, Petraitiene R 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; 46:3690–3702 [View Article][PubMed]
    [Google Scholar]
  10. Rickerts V, Just-Nübling G, Konrad F, Kern J, Lambrecht E et al. Diagnosis of invasive aspergillosis and mucormycosis in immunocompromised patients by seminested PCR assay of tissue samples. Eur J Clin Microbiol Infect Dis 2006; 25:8–13 [View Article][PubMed]
    [Google Scholar]
  11. Rickerts V, Mousset S, Lambrecht E, Tintelnot K, Schwerdtfeger R et al. Comparison of histopathological analysis, culture, and polymerase chain reaction assays to detect invasive mold infections from biopsy specimens. Clin Infect Dis 2007; 44:1078–1083 [View Article][PubMed]
    [Google Scholar]
  12. Cao GJ, Xing ZF, Hua L, YH J, Sun JB et al. Evaluation of the diagnostic performance of panfungal polymerase chain reaction assay in invasive fungal diseases. Exp Ther Med 2017; 14:4208–4214
    [Google Scholar]
  13. Lass-Flörl C, Mutschlechner W, Aigner M, Grif K, Marth C et al. Utility of PCR in diagnosis of invasive fungal infections: real-life data from a multicenter study. J Clin Microbiol 2013; 51:863–868 [View Article][PubMed]
    [Google Scholar]
  14. Guegan H, Iriart X, Bougnoux ME, Berry A, Robert-Gangneux F et al. Evaluation of MucorGenius mucorales PCR assay for the diagnosis of pulmonary mucormycosis. J Infect 2020; 81:311–317 [View Article]
    [Google Scholar]
  15. Zhao Z, Li L, Wan Z, Chen W, Liu H et al. Simultaneous detection and identification of Aspergillus and mucorales species in tissues collected from patients with fungal rhinosinusitis. J Clin Microbiol 2011; 49:1501–1507 [View Article][PubMed]
    [Google Scholar]
  16. Muñoz-Cadavid C, Rudd S, Zaki S, Patel M, Moser S et al. Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J Clin Microbiol 2010; 48:2147–2153 [View Article][PubMed]
    [Google Scholar]
  17. Lau A, Chen S, Sorrell T, Carter D, Malik R et al. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J Clin Microbiol 2007; 45:380–385 [View Article][PubMed]
    [Google Scholar]
  18. Donnelly JP, Chen SC, Kauffman CA, Steinbach WJ, Baddley JW et al. Revision and update of the consensus definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin Infect Dis 2019
    [Google Scholar]
  19. Hammond SP, Bialek R, Milner DA, Petschnigg EM, Baden LR et al. Molecular methods to improve diagnosis and identification of mucormycosis. J Clin Microbiol 2011; 49:2151–2153 [View Article][PubMed]
    [Google Scholar]
  20. Zaman K, Rudramurthy SM, Das A, Panda N, Honnavar P et al. Molecular diagnosis of rhino-orbito-cerebral mucormycosis from fresh tissue samples. J Med Microbiol 2017; 66:1124–1129 [View Article][PubMed]
    [Google Scholar]
  21. Gholinejad‐Ghadi N, Shokohi T, Seifi Z, Aghili SR, Roilides E et al. Identification of Mucorales in patients with proven invasive mucormycosis by polymerase chain reaction in tissue samples. Mycoses 2018; 61:909–915 [View Article]
    [Google Scholar]
  22. Millon L, Herbrecht R, Grenouillet F, Morio F, Alanio A et al. Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF. Clin Microbiol Infect 2016; 22:e1–e8 [View Article]
    [Google Scholar]
  23. Millon L, Larosa F, Lepiller Q, Legrand F, Rocchi S 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–e101 [View Article]
    [Google Scholar]
  24. Scherer E, Iriart X, Bellanger AP, Dupont D, Guitard J et al. Quantitative PCR (qPCR) detection of Mucorales DNA in bronchoalveolar lavage fluid to diagnose pulmonary mucormycosis. J Clin Microbiol 2018; 56: [View Article][PubMed]
    [Google Scholar]
  25. Baldin C, Soliman S, Jeon H, Gebremariam T, Alkhazraji S et al. PCR-based Diagnosis of Mucormycosis Targeting Mucorales-specific Genes. Open Forum Infectious Diseases US: Oxford University Press; 2017
    [Google Scholar]
  26. Bialek R, Feucht A, Aepinus C, Just-Nübling G, Robertson VJ et al. Evaluation of two nested PCR assays for detection of Histoplasma capsulatum DNA in human tissue. J Clin Microbiol 2002; 40:1644–1647 [View Article][PubMed]
    [Google Scholar]
  27. White TJ, Bruns T, Lee S, Taylor J. Amplification and Direct Sequencing of Fungal ribosomal RNA Genes for Phylogenetics. PCR protocols: a Guide to methods and Applications 1990 pp 315–322
    [Google Scholar]
  28. Rampini SK, Zbinden A, Speck RF, Bloemberg GV. Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients. BMC Microbiol 2016; 16:1–8 [View Article]
    [Google Scholar]
  29. Sugita C, Makimura K, Uchida K, Yamaguchi H, Nagai A. PCR identification system for the genus Aspergillus and three major pathogenic species: Aspergillus fumigatus Aspergillus flavus and Aspergillus niger. Med Mycol Open Access 2004; 42:433–437
    [Google Scholar]
  30. CLSI 2017 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi CLSI Standard M38, 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2017
    [Google Scholar]
  31. Prakash H, Ghosh AK, Rudramurthy SM, Singh P, Xess I et al. A prospective multicenter study on mucormycosis in India: epidemiology, diagnosis, and treatment. Med Mycol Open Access 2019; 57:395–402 [View Article]
    [Google Scholar]
  32. Lass-Florl C, Resch G, Nachbaur D, Mayr A, Gastl G et al. The value of computed tomography-guided percutaneous lung biopsy for diagnosis of invasive fungal infection in immunocompromised patients. Clin Infect Dis 2007; 45:e101–e104 [View Article]
    [Google Scholar]
  33. Badiee P, Arastefar A, Jafarian H. Comparison of histopathological analysis, culture and polymerase chain reaction assays to detect mucormycosis in biopsy and blood specimens. Iran J Microbiol 2013; 5:406–410[PubMed]
    [Google Scholar]
  34. Kontoyiannis DP, Wessel VC, Bodey GP, Rolston KV. Zygomycosis in the 1990s in a tertiary-care cancer center. Clin Infect Dis 2000; 30:851–856 [View Article][PubMed]
    [Google Scholar]
  35. Spellberg B, Edwards J, Ibrahim A. Novel perspectives on mucormycosis: pathophysiology, presentation, and management. Clin Microbiol Rev 2005; 18:556–569 [View Article][PubMed]
    [Google Scholar]
  36. Drogari-Apiranthitou M, Panayiotides I, Galani I, Konstantoudakis S, Arvanitidis G et al. Diagnostic value of a semi-nested PCR for the diagnosis of mucormycosis and aspergillosis from paraffin-embedded tissue: A single center experience. Pathol Res Pract 2016; 212:393–397 [View Article]
    [Google Scholar]
  37. Pandey M, Singh G, Agarwal R, Dabas Y, Jyotsna VP et al. Emerging Rhizopus microsporus infections in India. J Clin Microbiol 2018; 56: [View Article][PubMed]
    [Google Scholar]
  38. Herrera DA, Dublin AB, Ormsby EL, Aminpour S, Howell LP. Imaging findings of rhinocerebral mucormycosis. Skull Base 2009; 19:117–125 [View Article][PubMed]
    [Google Scholar]
  39. McAdams HP, Rosado de Christenson M, Strollo DC, Patz Jr EF. Pulmonary mucormycosis: radiologic findings in 32 cases. Am J Roentgenol 1997; 168:1541–1548 [View Article]
    [Google Scholar]
  40. Walsh TJ, Gamaletsou MN, McGinnis MR, Hayden RT, Kontoyiannis DP. Early clinical and laboratory diagnosis of invasive pulmonary, extrapulmonary, and disseminated mucormycosis (zygomycosis. Clin Infect Dis 2012; 54:S60–S55
    [Google Scholar]
  41. Espinel-Ingroff A, Chakrabarti A, Chowdhary A, Cordoba S, Dannaoui E et al. Multicenter evaluation of MIC distributions for epidemiologic cutoff value definition to detect amphotericin B, posaconazole, and itraconazole resistance among the most clinically relevant species of Mucorales. Antimicrob Agents Chemother (Bethesda) 2015; 59:1745–1750
    [Google Scholar]
  42. Bala K, Chander J, Handa U, Punia RS, Attri AK. A prospective study of mucormycosis in north India: experience from a tertiary care hospital. Med Mycol Open Access 2015; 53:248–257 [View Article]
    [Google Scholar]
  43. Diwakar A, Dewan R, Chowdhary A, Randhawa H, Khanna G et al. Zygomycosis–a case report and overview of the disease in India. Mycoses 2007; 50:247–254 [View Article][PubMed]
    [Google Scholar]
  44. Dorgan E, Denning DW, McMullan R. Burden of fungal disease in Ireland. J Med Microbiol 2015; 64:423–426 [View Article][PubMed]
    [Google Scholar]
  45. Lin E, Moua T, Limper AH. Pulmonary mucormycosis: clinical features and outcomes. Infection 2017; 45:443–448 [View Article][PubMed]
    [Google Scholar]
  46. Si-bu W, Ruo-yu L, Jin Y. Epidemiology of Zygomycosis in mainland China. Chinese J Mycol 2013; 2013:13
    [Google Scholar]
  47. Kontoyiannis DP, Yang H, Song J, Kelkar SS, Yang X et al. Prevalence, clinical and economic burden of mucormycosis-related hospitalizations in the United States: A retrospective study. BMC Infect Dis 2016; 16:730 [View Article][PubMed]
    [Google Scholar]
  48. Camara-Lemarroy CR, González-Moreno EI, Rodríguez-Gutiérrez R, Rendón-Ramírez EJ, Ayala-Cortés AS et al. Clinical features and outcome of mucormycosis. Interdiscip Perspect Infect Dis 2014; 2014:562610 [View Article][PubMed]
    [Google Scholar]
  49. Petrikkos G, Skiada A, Lortholary O, Roilides E, Walsh TJ et al. Epidemiology and clinical manifestations of mucormycosis. Clin Infect Dis 2012; 54:S23–S34 [View Article]
    [Google Scholar]
  50. Springer J, Lackner M, Ensinger C, Risslegger B, Morton CO et al. Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples. J Med Microbiol 2016; 65:1414–1421 [View Article][PubMed]
    [Google Scholar]
  51. Nithyanandam S, Jacob MS, Battu RR, Thomas RK, Correa MA et al. Rhino-orbito-cerebral mucormycosis. A retrospective analysis of clinical features and treatment outcomes. Indian J Ophthalmol (Poona City) 2003; 51:231
    [Google Scholar]
  52. Reed C, Bryant R, Ibrahim AS, Edwards Jr J, Filler SG et al. Combination polyene-caspofungin treatment of rhino-orbital-cerebral mucormycosis. Clin Infect Dis 2008; 47:364–371
    [Google Scholar]
  53. Asai K, Suzuki K, Takahashi T, Ito Y, Kazui T et al. Pulmonary resection with chest wall removal and reconstruction for invasive pulmonary mucormycosis during antileukemia chemotherapy. Jpn J Thorac Cardiovasc Surg 2003; 51:163–166 [View Article][PubMed]
    [Google Scholar]
  54. Pavie J, Lafaurie M, Lacroix C, Zagdanski AM, Debrosse D et al. Successful treatment of pulmonary mucormycosis in an allogenic bone-marrow transplant recipient with combined medical and surgical therapy. Scand J Infect Dis 2004; 36:767–769 [View Article]
    [Google Scholar]
  55. Peterson KL, Wang M, Canalis RF, Abemayor E. Rhinocerebral mucormycosis: evolution of the disease and treatment options. Laryngoscope 1997; 107:855–862 [View Article][PubMed]
    [Google Scholar]
  56. Petrikkos G, Skiada A, Sambatakou H, Toskas A, Vaiopoulos G et al. Mucormycosis: ten-year experience at a tertiary-care center in Greece. Eur J Clin Microbiol Infect Dis 2003; 22:753–756 [View Article][PubMed]
    [Google Scholar]
  57. Reid VJ, Solnik DL, Daskalakis T, Sheka KP. Management of bronchovascular mucormycosis in a diabetic: a surgical success. Ann Thorac Surg 2004; 78:1449–1451 [View Article][PubMed]
    [Google Scholar]
  58. Tedder M, Spratt JA, Anstadt MP, Hegde SS, Tedder SD et al. Pulmonary mucormycosis: results of medical and surgical therapy. Ann Thorac Surg 1994; 57:1044–1050 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.001370
Loading
/content/journal/jmm/10.1099/jmm.0.001370
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error