Abstract
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 (Tm) 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. 1. All HRM analysis results were confirmed by sequencing. None of the nonmucormycete fungi were positively tested. The results are summarized in Table 1.
FIG. 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 analysisa
| 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 |
CCM, 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 2, and representative HRM analysis curves are shown in Fig. 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 |
The 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.
Nucleotide sequence accession numbers.
Sequences of products from the internal round of PCR were deposited in GenBank under the following accession numbers: for Rhizopus microsporus, HM234125; for Rhizopus oryzae, HM234126; for Mucor racemosus, HM234127; for Rhizomucor pusillus, HM234128; for Lichtheimia corymbifera, HM234129; and for Mucor circinelloides, HM234130.
Acknowledgments
This study was supported by grants from the Ministry of Health of the Czech Republic (NS10442-3/2009 and NS10441-3/2009).
Footnotes
Published ahead of print on 30 June 2010.
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