Rapid Detection and Identification of Mucormycetes from Culture and Tissue Samples by Use of High-Resolution Melt Analysis

We present a method for rapid and simple detection of clinically relevant mucormycetes of the Mucorales order in cultures and clinical samples. This seminested real-time PCR uses mucormycete-specific primers and is followed by species identification using high-resolution melt (HRM) analysis. The method is highly suitable for routine clinical diagnostics.Invasive infections caused by mucormycetes started to occur more frequently in the last decade and are connected with rapid progression and high mortality rates. Early diagnostics and targeted treatment are crucial. Most mucormycosis cases (over 90%) are caused by Rhizopus spp., followed by Mucor spp., Lichtheimia spp., Rhizomucor pusillus, and, rarely, some other species (2, 9, 11, 16).Definitive diagnosis of mucormycosis is usually made after histopathological proof of mucormycete-like hyphae in involved tissue; the causative agent can be determined only by culture (13). So far, no serological test is available and radiological methods are nonspecific.Molecular detection of mucormycetes is complicated by several factors, and we still do not have any standard protocol. Few methods for the detection of mucormycetes have been published, and only some have been evaluated using clinical samples (1, 5, 10, 14, 15, 17) or samples from animal models (6, 7).The aim of this study was to develop a rapid and sensitive technique for the detection and identification of clinically important mucormycetes. We adopted primers from a qualitative method previously published by Bialek et al. (1) that is specific for members of the order Mucorales targeting 18S ribosomal DNA (rDNA). We modified it to seminested real-time PCR with EvaGreen dye, followed by species distinction by high-resolution melt (HRM) analysis. HRM analysis uses amplification of DNA in the presence of intercalation dye. Fluorescence is measured during a controlled melting of PCR product that results in a melt curve that depends mainly on GC content, length, and sequence of the PCR product. This simple method can be used for genotyping or mutation scanning without the need for time-consuming sequencing (4, 12).DNA was isolated from 50 μl of fungal culture (inoculum was prepared by covering sporulating colonies with approximately 2 ml of sterile 0.85% saline) or a piece of fresh tissue (2 by 1 mm) using the ZR fungal/bacterial DNA kit (Zymo Research). Tissue samples were incubated in lysis buffer overnight, and cultures were immediately processed according to the manufacturer''s protocol. Disruption was extended to 15 min (Disruptor Genie; Scientific Industries). DNA from formalin-fixed, paraffin wax-embedded (FFPE) tissue samples was isolated from 2 or 3 scrolls (5 to 10 μm each) of paraffin block using a DNeasy blood and tissue kit (Qiagen). Paraffin was dissolved in 1 ml of xylene, and then the tissue was washed two times using 1 ml of 96% ethanol and incubated in 180 μl of ATL buffer (Qiagen) and 20 μl of proteinase K (600 mAU/ml solution, where one mAU represents the activity of proteinase K that releases folin-positive amino acids and peptides corresponding to 1 μmol of tyrosine per min) at 55°C overnight and then at 90°C for 1 h. The next steps were done in accordance with the manufacturer''s protocol. DNA isolation from clinical samples was done in a biological safety cabinet. An aliquot of sterile water was processed with each set of samples as a control of potential contamination during the isolation process.Five microliters of DNA was amplified in 25 μl of amplification mixture that contained a 0.2 μM concentration each of primers ZM1 and ZM2 (1), 120 μM deoxynucleoside triphosphates (dNTPs; Roche, Germany), 2.5 mM MgCl₂, 1× GeneAmp PCR Gold buffer, and 1.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems). The cycling conditions were 10 min at 95°C, 16 cycles of 30 s at 94°C, 30 s at 50°C, and 60 s at 72°C, and 7 min at 72°C. One microliter of PCR product from the external round was then amplified in duplicate using Rotorgene 6000 (Corbett Research, Australia). Twenty-five microliters of the amplification mixture contained a 0.4 μM concentration each of primers ZM1 and ZM3 (1), 12.5 μl of SensiMix HRM, and 1 μl of EvaGreen (both from a SensiMix HRM kit; Quantace, United Kingdom). The cycling conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C (acquired on the green channel), followed by HRM analysis (ramp from 74°C to 79.5°C, rising by 0.1°C each cycle, acquired on the HRM channel). Rotorgene 6000 series software (version 1.7) was used for analysis of the results. All positive results were confirmed by sequencing of the PCR product. DNA was purified using a QIAquick PCR purification kit (Qiagen, Germany) and sequenced using a BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) on an ABI Prism 310 genetic analyzer (Applied Biosystems). Sequences were analyzed using the BLAST alignment program of the GenBank database.We used DNA extracted from five mucormycete cultures diluted in Tris-EDTA (TE) buffer as positive controls in every run. A DNA isolation control (sterile water processed with clinical samples) and a negative control of PCR (sterile water) were added to each run as well.In this study, we tested 31 fungal isolates, comprising 10 mucormycete isolates and 21 isolates from other filamentous fungal groups (Department of Clinical Microbiology, University Hospital Brno and Czech Collection of Microorganisms, Czech Republic). All mucormycete isolates were correctly identified. The melting temperatures (T_m) for each species were as follows: for Rhizopus microsporus, 76.46°C; for Rhizopus oryzae, 76.59°C; for Mucor racemosus, 76.78°C; for Mucor circinelloides, 76.98°C; for Rhizomucor pusillus, 77.87°C; and for Lichtheimia corymbifera, 78.56°C. Representative HRM curves for six different mucormycetes are shown in Fig. ​Fig.1.1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table ​Table11.Open in a separate windowFIG. 1.Representative result of high-resolution melt (HRM) analysis. Shown are HRM curves for six mucormycete isolates (black curves) and one negative and one positive tissue sample (gray curves).

TABLE 1.

List of fungal isolates used in this study and results of HRM analysis^a

Organism	Accession no. or source	Result of zygomycete HRM analysis
Mucormycetes
Rhizopus oryzae	Clinical isolate; DCM	Rhizopus oryzae
	CCM 8075	Rhizopus oryzae
Rhizopus sp.	Clinical isolate; DCM	Rhizopus oryzae
Rhizopus microsporus	Clinical isolate; DCM	Rhizopus microsporus
Rhizomucor pusillus	CCM F-211	Rhizomucor pusillus
Mucor racemosus	CCM 8190	Mucor racemosus
Mucor circinelloides	Clinical isolate; DCM	Mucor circinelloides
Lichtheimia corymbifera	CCM 8077	Lichtheimia corymbifera
	Clinical isolate; DCM	Lichtheimia corymbifera
	Clinical isolate; DCM	Lichtheimia corymbifera
Other filamentous fungi
Fusarium oxysporum	Clinical isolate; DCM	Negative
	Clinical isolate; DCM	Negative
Fusarium proliferatum	Clinical isolate; DCM	Negative
Fusarium solani	CCM 8014	Negative
Aspergillus fumigatus	Clinical isolate; DCM	Negative
	Clinical isolate; DCM	Negative
Aspergillus niger	Clinical isolate; DCM	Negative
	CCM 8155	Negative
Aspergillus flavus	CCM 8363	Negative
	CCM F-171	Negative
Aspergillus terreus	CCM 8082	Negative
Aspergillus ustus	CCM F-414	Negative
Aspergillus nidulellus (nidulans)	CCM F-266	Negative
Aspergillus sydowii	Environment; DCM	Negative
Scedosporium apiospermum	Clinical isolate; DCM	Negative
Cladosporium cladosporioides	Environment; DCM	Negative
Cladosporium cladosporioides f. sp. pisicola	CCM F-348	Negative
Penicillium commune	CCM F-327	Negative
Penicillium brevicompactum	CCM 8040	Negative
	Environment; DCM	Negative
Penicillium chrysogenum	Environment; DCM	Negative

Open in a separate window^aCCM, Czech Collection of Microorganisms, Czech Republic; DCM, Department of Clinical Microbiology, University Hospital Brno, Czech Republic.We also tested 12 tissue samples, 7 (6 fresh and 1 FFPE) from patients with histopathologically or culture-proven mucormycosis and 5 (3 fresh and 2 FFPE) from patients without mucormycosis (obtained from hemato-oncological patients from University Hospital Brno, Czech Republic). All seven tissue samples from patients with proven mucormycosis were PCR positive, and in all cases, we were able to directly determine the mucormycete species: R. microsporus (n = 4), L. corymbifera (n = 2), and R. pusillus/miehei (these two species have 100% sequence homology in the target region and therefore cannot be distinguished; n = 1). All five tissue samples from patients without mucormycosis were negative. Results are summarized in Table ​Table2,2, and representative HRM analysis curves are shown in Fig. ​Fig.1.1. Amplification of fragmented DNA from FFPE samples can be problematic (8). In this study, we tested one FFPE tissue from a patient with proven mucormycosis, and the result was positive.

TABLE 2.

List of tissue samples used in this study and results of HRM analysis

Patient	Tissue sample	Histopathology result	Culture result	HRM analysis result
1	Lung	Positive	Negative	Rhizopus microsporus
2	Lung (FFPE)	Positive	Negative	Rhizomucor pusillus/miehei
3	Oral cavity	Positive	Lichtheimia corymbifera	Lichtheimia corymbifera
4	Lung	Positive	Rhizopus microsporus	Rhizopus microsporus
5	Lung	Positive	Lichtheimia corymbifera	Lichtheimia corymbifera
6	Oral cavity 1	Positive	Rhizopus microsporus	Rhizopus microsporus
	Oral cavity 2	Positive	Rhizopus microsporus	Rhizopus microsporus
7	Lung	Negative	Negative	Negative
8	Lung	Negative	Negative	Negative
9	Lung (FFPE)	Negative	Negative	Negative
10	Lung	Negative	Negative	Negative
11	Lung (FFPE)	Negative	Negative	Negative

Open in a separate windowThe sensitivity of the method was assessed by amplification of dilutions (2 × 10⁷ to 2 × 10⁰ copies/5 μl) of plasmid DNA (external PCR products of R. pusillus and L. corymbifera cloned into the pCR2.1 vector; Invitrogen). Reproducible melt curves were obtained for concentrations up to 0.1 fg of plasmid DNA, the detection limit corresponding to the original qualitative method (1), in both species.To assess potential PCR inhibition, human albumin gene was detected by real-time PCR (3) in all tissue samples. No inhibition was observed.In conclusion, the HRM assay presented is very simple and enables rapid and accurate detection and identification of mucormycetes in tissue samples and culture isolates. It is able to distinguish the main clinically relevant mucormycetes and shows no cross-reactivity with nonmucormycete filamentous fungi. It is highly sensitive and specific and is suitable for routine clinical diagnostics. Its potential for use in diagnostics with other clinical materials, such as bronchoalveolar lavage fluid, sputum, etc., needs further study but is evident.