Molecular Strategies to Diagnose Mucormycosis

Abstract

Molecular techniques have provided a new understanding of the epidemiology of mucormycosis and improved the diagnosis and therapeutic management of this life-threatening disease. PCR amplification and sequencing were first applied to better identify isolates that were grown from cultures of biopsies or bronchalveolar lavage samples that were collected in patients with Mucorales infection. Subsequently, molecular techniques were used to identify the fungus directly from the infected tissues or from bronchalveolar lavage, and they helped to accurately identify Mucorales fungi in tissue samples when the cultures were negative. However, these tools require invasive sampling (biospsy, bronchalveolar lavage), which is not feasible in patients in poor condition in Hematology or Intensive Care units. Very recently, PCR-based procedures to detect Mucorales DNA in non-invasive samples, such as plasma or serum, have proved successful in diagnosing mucormycosis early in all patients, whatever the clinical status, and these procedures are becoming essential to improving patient outcome.

The Brief Case: Cutaneous Fungal Infection in a Pediatric Patient with Newly Diagnosed Acute Lymphocytic Leukemia

“…Recent studies have demonstrated the application of molecular techniques on formalin-fixed and paraffin-embedded (FFPE) tissues for the diagnosis of mucormycosis in culture-negative cases (6). 18S ribosomal DNA (rDNA) PCR followed by sequencing had a higher accuracy than culture.…”

“…Also, many other Mucorales-specific PCRs with different targets were demonstrated for tissue samples, such as a multiplex real-time quantitative PCR (qPCR) targeting the internal transcribed spacer 1 and 2 (ITS1/ITS2) region and a specific qPCR targeting the 28S rDNA. However, all of the molecular methods in FFPE tissues are limited by the compromised quality of DNA after tissue fixation and embedding (6).…”

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

“…The order Mucorales is assigned to the subphylum Mucoromycotina, one of the most ancient, early divergent groups of fungi [1]. In recent decades, the incidence of mucormycosis has increased due to: (a) a growing population of immunocompromised patients [2], (b) increased awareness [3], (c) the impact of mold-active prophylaxis [4], and (d) increasing numbers of patients with uncontrolled diabetes mellitus [5].…”

Special Issue: Mucorales and Mucormycosis

“…The fungi responsible for this disease, the Mucorales, are evolutionary diverse, belong to several distantly related genera [3] and are characterized by their intrinsic resistance to short-tailed azoles [4] and the whole echinocandin substance class [5]. A better knowledge of the biology of mucormycetes and the disease entities caused by them is of prime importance for an accurate and fast diagnosis [6][7][8] and subsequent targeted treatment strategy [9] for patients suffering from mucormycosis.…”

“…In clinics, diagnosis of mucormycosis is based on culture and direct detection of the causative agent in patient material (biopsy, bronchoalveolar lavage (BALs), blood, serum, plasma, urine) by molecular methods. In critically ill patients, non-invasive samples, such as blood, plasma or serum, have been successfully evaluated for diagnosing mucormycosis [8]. For tissue samples, the major conclusion, as highlighted by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) and the European Confederation of Medical Mycology (ECMM) joint clinical guidelines [21], was that fresh/frozen samples performed better than FFPE samples, independent of the PCR format (a PCR with panfungal primers or Mucorales-specific primers, or a combination of specific quantitative PCRs) used.…”