High-resolution melting analysis: A novel approach for clade differentiation in Pythium insidiosum and pythiosis

High-resolution melting analysis: A novel approach for clade differentiation in Pythium... Abstract Pythium insidiosum causes life-threatening human pythiosis. Based on phylogenetic analysis using internal transcribed spacer (ITS) region, mitochondrial cytochrome C oxidase II (COX2) gene, intergenic spacer (IGS) region and exo-1,3-β-glucanase gene (exo1), P. insidiosum is classified into clade ATH, BTH, and CTH related to geographic distribution. At present, polymerase chain reaction in any of these specific regions with DNA sequencing is the only technique to provide clade diagnosis. In this study, P. insidiosum-specific primers targeting COX2 gene were designed and used in real-time quantitative polymerase chain reaction (qPCR) with subsequent high-resolution melting (HRM) to provide rapid identification as well as clade classification for P. insidiosum. Based on the qPCR-HRM method, 15 P. insidiosum isolates could be differentiated from 28 related organisms with 100% specificity and 1 pg limit of detection. This technique was, in addition, directly tested on clinical samples from proved human pythiosis cases: nine corneal scrapes and six arterial clots. The qPCR-HRM results of all nine corneal samples were a 100% match with the results from the conventional PCR at clade level. However, the qPCR-HRM results of arterial clot samples were only matched with the nucleotide sequencing results from the conventional PCR at species level. In conclusion, the qPCR-HRM is a simple one closed tube, inexpensive and user-friendly method to identify P. insidiosum into clade level. Pythium insidiosum, pythiosis, high-resolution melting, clade differentiation Introduction Pythium insidiosum, is an aquatic oomycete fungus-like organism, which causes pythiosis in humans, mammals, and birds.1 This organism is widely found in its natural habitat, specifically agricultural swamp areas and stagnant fresh water. It is known as a soil borne saprophyte as well as a plant pathogen throughout the world.2,3 Recently, this organism was ubiquitously found in urban and suburban lakes in North Central Florida, USA, and this was probably the first report of natural habitat in the United States.4P. insidiosum is classified into 3 clades: clade I, II, and III based on phylogenetic tree analysis using internal transcribed spacer (ITS) region or intergenic spacer (IGS) region or exo-1,3-β-glucanase gene (exo1) or clade ATH, BTH, CTH by using mitochondrial cytochrome C oxidase II (COX2). Clade ATH or clade I is mostly found in American continent; Clade BTH or clade II is usually found in Asia and Australia; Clade CTH or clade III is found in Thailand and the United States.5–8 With regards to human diseases, the majority of the cases have been reported from Thailand.9,10 Human pythiosis is classified into four groups based on clinical presentations: cutaneous/subcutaneous, ocular, vascular, and disseminated disease.9,10 The combination of aggressive surgery, antifungal agents, and immunotherapy has been used as salvage treatment in several institutions; however, the effective treatment has not yet been established. It is known that surgery with adequate surgical margins is the main factor for patients’ survival in vascular disease regardless of antifungal agents or immunotherapy administration.10 Given this reason, early diagnosis and treatment is a very crucial determinant to decrease morbidity and mortality. The inefficacy of immunotherapy in the previous series was thought to be due to administration of immunotherapy from a discordant pathogen clade.11 Currently, immunotherapy is only available for P. insidiosum ATH; however, none of the Thai patients were infected with clade ATH. Chindamporn et al. found that serum antibodies from P. insidiosum infected patients recognized various immunogens in geographically divergent P. insidiosum strains by Western blot, but dominant antigen-antibody reactions were observed in the same pathogen clade.11 This implied the efficacy of immunotherapy may require antigens belonging to the same clade of the pathogens. Presently, at least two diagnostic methods including histopathological staining, culture, serology, and polymerase chain reaction (conventional PCR) with DNA sequencing are required to provide definitive diagnosis for human pythiosis in patients with compatible clinical contexts.2,10 At our medical center, the PCR with DNA sequencing by ITS region and COX2 gene have been used for clade differentiation. The phylogenetic tree using COX2 gene has showed higher discriminatory power than using ITS for sister-group classification.5,7 The alignment of P. insidiosum’s partial COX2 nucleic acid sequences presented the clade specific single-nucleotide polymorphisms (SNPs). These mutations are significant points particularly for clade disclosure. In addition to DNA sequencing, real-time quantitative polymerase chain reaction (qPCR) with subsequent High Resolution Melting (HRM) is another method to detect the SNPs.12 The qPCR can amplify nucleotide at the specific target of interest. The SNPs in amplicons, covered by designed primers, are used for the following HRM analysis steps. By theory, HRM is one of high sensitivity assays to detect the different melting temperature (Tm) caused by A/T or C/G bonding in each SNP pattern. Thus, the SNPs can be detected accurately without sequencing process. In the past few years, this novel technique has been investigated, and it has been successfully applied for diagnostic purposes in various research areas including detection and analysis of cancer-related mutations in humans, identification and genotyping of parasites, bacteria, and fungi.13–22 Despite its wide use in clinical medicine and agriculture, this technique has not been studied for P. insidiosum identification yet. In this study, the qPCR-HRM technique was developed for P. insidiosum identification as well as clade differentiation from clinical and environmental isolates. Methods The study protocol was approved by the Chulalongkorn University Institutional Review Board based on the international guidelines for human research protection including Declaration of Helsinki, the Belmont Report, The Council for International Organizations of Medical Sciences (CIOMS) Guidelines, and International Conferences on Harmonization in Good Clinical Practice (ICH-GCP) (certificate of authenticity; COA No.086/2016, IRB No. 015/59). Organisms and clinical specimens A total of 58 samples were tested in this study. Forty-three isolates of P. insidiosum (n = 15), Pythium spp. (n = 2), pathogens with similar morphology in kingdom Stramenopila (n = 3) and kingdom Fungi (n = 23) (Table 1), and 15 clinical specimens from proved human pythiosis, nine corneal scrapes and six arterial clots, were included in this study (Table 2). All isolates’ genus and species were proved by the conventional PCR with sequencing of ITS region and/or COX2 gene. Similar to those isolates, all recruited specimens with P. insidiosum infection were proved by positive conventional PCR with sequencing of ITS region and COX2 gene directly from specimens and/or positive culture. Sera from all patients, whose arterial clots were collected for this study, were tested for specific P. insidiosum antibody by in-house enzyme-linked immunosorbent assay (ELISA). Traditionally, P. insidiosum and other related fungi were differentiated by growth rate, presence of sterile hyphae, zoospore production, P. insidiosum specific antibody detection by ELISA, and conventional PCR with DNA sequencing technique. These methods were used as control in this study. Table 1. Strains, Genbank number, isolation sources and similarity percentage of P. insidiosum compared to reference strains base on qPCR with HRM analysis. No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - $Random isolates which were run the test in triplicate to evaluate the reproducibility and consistency of the qPCR-HRM protocols. * Confidence percentage, compared with P. insidiosum Tm curve profile. #Isolates which can be amplified by CoxII primers aCBS 574.85, contributed from Dr. L. Mendoza, MSU, USA. bPlant pathogens, identified by ITS sequences, from Ministry of Agriculture and Cooperative, Thailand. cGenomic DNA from Supabandhu J, Prince of Songkla University, Thailand. dContributed from Postgraduate Institute of Medical Education and Research (PGIMER),Chandigarh, India. View Large Table 1. Strains, Genbank number, isolation sources and similarity percentage of P. insidiosum compared to reference strains base on qPCR with HRM analysis. No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - $Random isolates which were run the test in triplicate to evaluate the reproducibility and consistency of the qPCR-HRM protocols. * Confidence percentage, compared with P. insidiosum Tm curve profile. #Isolates which can be amplified by CoxII primers aCBS 574.85, contributed from Dr. L. Mendoza, MSU, USA. bPlant pathogens, identified by ITS sequences, from Ministry of Agriculture and Cooperative, Thailand. cGenomic DNA from Supabandhu J, Prince of Songkla University, Thailand. dContributed from Postgraduate Institute of Medical Education and Research (PGIMER),Chandigarh, India. View Large Table 2. Comparison among the isolates here evaluated with previous study of Kammarnjessadakul et al.5 according to clade level and clinical samples tested in this study: corneal scraping (n = 9) and tissue biopsy (n = 6). P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH *Arterial clot after DNA extraction/Corneal directly from specimens. NA, not available; ND, not done. + = positive for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. − = negative for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. View Large Table 2. Comparison among the isolates here evaluated with previous study of Kammarnjessadakul et al.5 according to clade level and clinical samples tested in this study: corneal scraping (n = 9) and tissue biopsy (n = 6). P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH *Arterial clot after DNA extraction/Corneal directly from specimens. NA, not available; ND, not done. + = positive for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. − = negative for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. View Large DNA preparation The genomic DNAs (gDNAs) of all tested isolates were prepared by QIAamp DNA Mini kit (Cat. 51306, QIAGEN, Germantown, MD, USA). In brief, isolates were lysed by 180 μl ATL buffer and 20 μl proteinase K at 56 °C for 1 h. Then, protein denaturation step was performed by 200 μl AL buffer. After centrifugation, the upper part was purified and precipitated with 200 μl absolute ethanol. The mixer was filtered through the QIAamp Mini Spin column and washed again by 500 μl AW1 and AW2 buffer. Finally, a ratio of 1.8 to 2.0, based on A260/A280 ratio by NanoDrop (NanoDropTM 1000 Spectrophotometer Thermo scientific, Skokie, IL, USA), was used to determine DNA quality and quantity. Real-time quantitative polymerase chain reaction (qPCR) and post-HRM analysis The qPCR amplification of 91 bp in COX2 gene was performed by the newly designed primers: Cox_Pi_5 (5’-TAA TTT GGA CTA CTA TTC CAG C-3’) and Cox_Pi_6 (5’-GGA TCA ATG TAT TTC ATC CAT AG-3’) (Fig. 1). These specific primers can amplify SNP regions in all three clades of P. insidiosum, and other related organisms: Pythium aphanidermatum (P. aphanidermatum), P. catenulatum, Phytophthora parasitica (Phy. parasitica), Phy. sojae, Lagenidium albertoi, Basidiobolus meristosporus, and Conidiobolus coronatus. The reactions of qPCR (Rotor-Gene™ 6000, Corbett Research, Austria) were performed in 20 μl volume containing of 10 μl high-resolution melting master mix (Cat. 22655, Roche, Indianapolis, IN, USA), 2.5 mM MgCl2, 0.125 μM of each primer, and 10 ng of gDNA. The amplification steps consisted of an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 25 s, and extension at 72°C for 1 min. In this study, the interval created by the average crossing point value (Cp value) ± 2 cycles was accepted. To identify the genus and species level from the qPCR amplicons, the melt curve genotyping analysis program was used. In addition, the HRM program was further processed to separate into clade level. Both programs were included in the Rotor-Gene™ 6000, software version 1.7, Corbett Research, Austria. The reference gDNA templates of all three clades were optimized to obtain unique different curve of each clade. In every run, these templates were used as reference curves. The melt curve genotyping was plotted based on melting temperatures of the amplicons. Regarding post-HRM genotyping analysis, the amplification products were denatured at 95°C for 1 min and were cooled at 40°C for 1 min to complete double-stranded amplicons. The HRM melting curves were generated by ramping from 65 to 90°C, and the curves were monitored fluorescence at the rate of 0.1°C/s, taking 25 fluorescence acquisitions per 1°C. These curves were plotted by using the automated grouping option in the program (Fig. 2B and C). More than 95% confidence of similarity compared to reference gDNA templates by HRM analysis was accepted for clade classification (Fig. 2A). To evaluate the limit of detection (LOD) of qPCR and post-HRM, 1 pg to 10 ng of gDNA were tested. The gDNA of all isolates were tested in triplicate blinded experiments. The gDNA of Staphylococcus spp., human and molecular grade water were used as negative controls. The specificity test was challenged by using other fungal gDNAs (n = 28) as follows: P. aphanidermatum, P. catenulatum, Phy. parasitica, Phy. sojae, Lagenidium albertoi, Basidiobolus meristosporus, Conidiobolus coronatus, Lichtheimia corymbifera, Apophysomyces elegans, Cunninghamella bertholletiae, Rhizomucor pusillus, Rhizopus arrhizus, Rhizopus arrhizus, Saksenaea vasiformis, Syncephalastrum racemosum, Cladosporium spp., Fonseccaea pedrosoi, Phialophora verrucosa, Aureobasidium spp., Aspergillus flavus, Penicillium marneffei, Scedosporium apiospermum, Trichophyton rubrum, Trichophyton tonsurans, Histoplasma capsulatum, Sporothrix schenckii, Candida albicans, and Cryptococcus neoformans under the same condition. The cut-off for the same clade was more than 95% confidence of similarity calculated by HRM analysis. Finally, the results of the qPCR-HRM analysis of the tested isolates were compared to the sequences of ITS region and COX2 gene which was the standard method in this study. Figure 1. View largeDownload slide The 91 base pairs amplicon bearing SNPs of P. insidiosum clade ATH, BTH and CTH by Cox_Pi_5_forward and Cox_Pi_6_reverse primer. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide The 91 base pairs amplicon bearing SNPs of P. insidiosum clade ATH, BTH and CTH by Cox_Pi_5_forward and Cox_Pi_6_reverse primer. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Normalized graph (A) and difference graph (B): comparison among Clade ATH, Clade BTH, and CTHP. insidiosum (―) and other related fungi (—) which were amplified COX2 gene: L. albertoi (1); P. aphanidermatum (2); P. catenulatum (3); C. coronatus (4); Phy. sojae (5); B. meristosporus (6); Phy. parasitica (7). This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Normalized graph (A) and difference graph (B): comparison among Clade ATH, Clade BTH, and CTHP. insidiosum (―) and other related fungi (—) which were amplified COX2 gene: L. albertoi (1); P. aphanidermatum (2); P. catenulatum (3); C. coronatus (4); Phy. sojae (5); B. meristosporus (6); Phy. parasitica (7). This Figure is reproduced in color in the online version of Medical Mycology. The qPCR-HRM for clinical samples To shorten the processes and avoid complicated gDNA extraction steps, we applied this qPCR-HRM analysis directly to clinical specimens, arterial clots, and corneal scrapes. All clinical specimens were cut into small pieces (approximately 1 mm × 1 mm in length and width) and directly mixed in the qPCR reactions (Table 2). This mixture was processed by the qPCR-HRM as same as the fungal isolates described in previous section. The additional DNA extraction was required in arterial clot samples to improve the efficiency of the qPCR-HRM. Their gDNA were prepared by QIAamp DNA Mini kit (Cat. 51306, QIAGEN, USA) prior to being used as templates in the qPCR-HRM reactions. Results The qPCR-HRM results: fungal isolates Twenty-two out of 43 tested isolates could be amplified by COX2 specific primers (Table 1) with Cp value between cycles 14 and 18 (cycles 16 in average) in the qPCR process. Twenty isolates were from phylum Oomycota, and two isolates were from order Entomophthorales, phylum Glomeromycota (former Zygomycota).23 No PCR products were detected from the negative controls. Based on the melt curve genotyping, there were no significant differences in melting temperatures (Tm) among those 22 amplifiable isolates. The 22 amplifiable isolates were classified into four groups based on the curve patterns from HRM analysis-clade ATH pattern, clade BTH pattern, clade CTH pattern, and non-ATHBTHCTH pattern compared to the reference P. insidiosum curve patterns. Fifteen out of 22 amplifiable isolates met the criteria of ≥95% confidence of similarity (Cp 16.15 ± 0.94): clade ATH (1/15 isolate, 99.3% confidence of similarity; Cp 15.3 ± 0.58), clade BTH (8/15 isolates, 98.1–99.6% confidence of similarity; Cp 15.95 ± 0.92), and clade CTH (6/15 isolates, 97.7–99.0% confidence of similarity; Cp 16.41 ± 0.98) (Fig. 2A). The three clades achieved from HRM analysis were correlated with the clustering based on DNA sequences. The non–ATHBTHCTH pattern was produced from seven out of 22 amplifiable isolates, and this pattern did not meet the criteria of ≥95% confidence of similarity. This result indicated that these seven isolates were not P. insidiosum, correlated with the identification results by conventional PCR with DNA sequencing. The non–ATHBTHCTH pattern was generated by P. aphanidermatum, P. catenulatum, Phy. parasitica, Phy. sojae, Lagenidium albertoi, Basidiobolus meristosporus, and Conidiobolus coronatus. The qPCR-HRM results: clinical specimens A total of nine corneal scrapes and six arterial clots were examined (Table 2). Only 11/15 clinical samples, nine corneal tissues, and two arterial clots were amplified. None of them met the criteria of ≥95% confidence of similarity compared to reference P. insidiosum isolates. All samples from corneal scrapes showed ≥85% confidence of similarity; Cp 16.27 ± 0.68. Two clades were identified: clade BTH (5/9 samples, 87.2–89.4% confidence of similarity) and clade CTH (4/9 samples, 85.1–89.0% confidence of similarity). None of arterial clots can be classified into clade level without an additional gDNA extraction. However, with the additional gDNA extraction from the specimens, all 6 arterial clots were successfully amplified and can be classified into clade level by qPCR-HRM with Cp of 16.39 ± 0.8. Specificity and limit of detection The specificity of the qPCR-HRM was evaluated by using other fungal gDNAs as templates for qPCR-HRM reactions under the same condition as P. indiosum’s gDNA. There were no false positive or false negative results. These results revealed 100% specificity of the qPCR-HRM. With regards to the limit of detection, different concentrations of P. insidiosum’s gDNA ranging from 1 pg to 10 ng were examined. One picogram of gDNA was the lowest detectable amount by this qPCR-HRM. Reproducibility of the qPCR-HRM The reproducibility and consistency of qPCR-HRM protocols were evaluated by running HRM in triplicate from six random isolates: three from clade BTH and three from clade CTH. Since there was the only one isolate for clade ATH, the reproducibility and consistency of these protocols were tested by extracting gDNA in triplicate from three different subcultures of the isolate. The HRM analysis was performed in each lot of gDNA preparation. Our protocols were found to have very high consistency and reproducibility (Table 1). Among the control reactions, very small variation with Cp of 15.4 ± 0.4 was observed. Discussion In this study, the qPCR-HRM analysis was first developed to provide rapid clade specific diagnosis for patients with pythiosis. The results from conventional PCR with DNA sequencing for clade identification were similar to the results from the qPCR-HRM. Several advantages of the qPCR-HRM are as follows; first, P. insidiosum can be differentiated from other true fungi and be classified into the clade level with turnaround time of four hours while at least 18 hours are required for conventional approach. Second, this technique was first implemented to identify P. insidiosum directly clinical samples, and it was successful for corneal scrapes. Third, the qPCR-HRM is convenient because only one closed tube system is required and this will significantly reduce risk of contamination during multistep processes of conventional PCR. Lastly, the qPCR-HRM is approximately 5 times cheaper than the conventional PCR with DNA sequencing, and this technology will be widely accessible especially among developing countries. Several techniques for P. insidiosum identification such as thermophilic helicase DNA amplification with restriction fragment length polymorphism (tHDA-RFLP) targeting COX2 gene,23 real-time PCR targeting exo-1,3-beta-glucanase gene (PinsEXO1),245 and screening method based on the growth of P. insidiosum mycelia around minocycline disks (30 μg)25 have been reported. All identification methods have mainly used gDNA template from cultures or non-clinical specimens; however, they are only able to provide diagnosis into the species level23-25. In this study, both species and clade diagnosis can be obtained by nonsequencing based qPCR-HRM with 100% specificity. All the clade classification results from 15 P. insidiosum gDNA template tested samples obtained from both conventional PCR-sequencing and the new qPCR-HRM methods are 100% match. The confidence percentage of similarity results from our qPCR-HRM for clade identification are as high as the previous reported results in the literature for species identification in other organisms.14,26–28 This indicates that our developed assay is highly accurate and applicable for P. insidiosum identification. Direct amplification from clinical specimens is the ultimate goal. The main advantage of amplification directly from clinical specimens is to increase detection yield of P. insidiosum in case of cultivation failure. The intolerance to low temperature of P. insidiosum during transportation and sample preparation is a major barrier causing false negative results in real-world practice.29 In this study, we successfully identified P. insidiosum from all corneal scrapes samples corresponding to the DNA sequence base assay. Ocular pythiosis can be diagnosed within a working day by the qPCR-HRM, but it is almost impossible by conventional PCR with DNA sequencing and standard cultivation method. In case of arterial clots, low success rate was observed by the qPCR-HRM. We hypothesize that clot samples may not have been completely lysed by typical denatured temperature, and it led to impurity of DNA extracts.30 To prove this hypothesis, we extracted the gDNA from these samples and processed the qPCR-HRM. As expected, all six arterial clots were amplified and P. insidiosum was identified. Although a few extra hours were required for DNA extraction from arterial clots, the qPCR-HRM could still be considered as a rapid diagnosis compared to the conventional PCR with DNA sequencing. In terms of clade classification by directly using clinical specimens, all of the results were 100% match to the nucleotide sequence assay, even though only 85.1–89.4% similarity by HRM were achieved from the corneal scrapes. The lower percentage of similarity in clinical specimens was probably due to unpurified samples, inhibitors in DNA extracts, or different concentrations of magnesium salt.32 To increase higher percentage of similarity for the qPCR-HRM, additional steps may be required. These steps are pre-amplification step (gDNA extraction and purification) and pre-HRM analysis step (PCR purification).31 However, we decided not to pursue the additional DNA extraction and purification because we had limited specimens and DNA would be lost during these processes. In conclusion, this new qPCR-HRM technique is a novel tool for conclusive P. insidiosum identification and clade classification. Owing to the rapid, user-friendly, and inexpensive process, this technique is another option to help improve patient care by facilitating early diagnosis and guiding appropriate immunotherapy in the future. With clade identification, we also hope this technique will provide more epidemiologic data in different geographic areas. Acknowledgments This work was supported by (1) the 90th Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) year 2009 and (2) Chulalongkorn's Research Repository (CU-57-008-FW). We would like to extend our sincere gratitude toward Drs. Chakrabarti, Shivaprakash, and Ms. Sunita Gupta from Postgraduate Institute of Medical Education and Research, Chandigarh, India, for providing the fungal isolates of Zygomycetes group. Finally, we would like to sincerely thank all members in Mycology Unit, Department of Microbiology, King Chulalongkorn Memorial Hospital and Mycology research unit, Faculty of Medicine, Chulalongkorn University for all supports. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. Other related information This intervention (1) is being applied for the patent (patent application no. 1201006141 requested on 27 November 2012) under the authorization of Chulalongkorn University, Thailand, by Dr. Ariya Chindamporn and Ms. Navaporn Worasilchai as researchers, (2) was presented as a poster presentation in “19th Congress International Society for Human and Animal Mycology (ISHAM) 2015” Melbourne, Australia, by Ms. Navaporn Worasilchai. References 1. Pesavento PA , Barr B , Riggs SM , Eigenheer AL , Pamma R , Walker RL . Cutaneous pythiosis in a nestling white-faced ibis . 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Keeratijarut A , Lohnoo T , Yingyong W et al. Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-β-glucanase . J Med Microbiol . 2015 ; 64 : 971 – 977 . Google Scholar Crossref Search ADS PubMed 26. Tondolo JS LE , Denardi LB , Mario DA , Alves SH , Santurio JM . A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum . J Microbiol Method . 2013 ; 93 : 52 – 54 . Google Scholar Crossref Search ADS 27. Shi M LX , Feng J , Jia S , Xiao X , Chen C , Fransisca C , Xi L , Zhang J . High-resolution melting analysis assay for identification of Fonsecaea species . J Clin Lab Anal . 2017 . 28. Felipe Jilbertoa CA , María Angélica Larraín. High resolution melting analysis for identification of commercially-important Mytilus species . Food Chemistry . 2017 ; 229 : 716 – 720 . Google Scholar Crossref Search ADS PubMed 29. Fehlberg HF , Maciel BM , Albuquerque GR Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis . PLoS ONE . 2017 ; 12 . 30. Schurko AM , Mendoza L , de Cock AW , Bedard JE , Klassen GR . Development of a species-specific probe for Pythium insidiosum and the diagnosis of pythiosis . J Clin Microbiol . 2004 ; 42 : 2411 – 2418 . Google Scholar Crossref Search ADS PubMed 31. Wittwer CT. High-resolution DNA melting analysis: advancements and limitations . Hum Mutat . 2009 ; 30 : 857 – 859 . Google Scholar Crossref Search ADS PubMed 32. Garritano S , Gemignani F , Voegele C et al. Determining the effectiveness of high rResolution melting analysis for SNP genotyping and mutation scanning at the TP53 locus . BMC Genet . 2009 ; 10 : 5 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

High-resolution melting analysis: A novel approach for clade differentiation in Pythium insidiosum and pythiosis

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Oxford University Press
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© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
ISSN
1369-3786
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1460-2709
D.O.I.
10.1093/mmy/myx123
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Abstract

Abstract Pythium insidiosum causes life-threatening human pythiosis. Based on phylogenetic analysis using internal transcribed spacer (ITS) region, mitochondrial cytochrome C oxidase II (COX2) gene, intergenic spacer (IGS) region and exo-1,3-β-glucanase gene (exo1), P. insidiosum is classified into clade ATH, BTH, and CTH related to geographic distribution. At present, polymerase chain reaction in any of these specific regions with DNA sequencing is the only technique to provide clade diagnosis. In this study, P. insidiosum-specific primers targeting COX2 gene were designed and used in real-time quantitative polymerase chain reaction (qPCR) with subsequent high-resolution melting (HRM) to provide rapid identification as well as clade classification for P. insidiosum. Based on the qPCR-HRM method, 15 P. insidiosum isolates could be differentiated from 28 related organisms with 100% specificity and 1 pg limit of detection. This technique was, in addition, directly tested on clinical samples from proved human pythiosis cases: nine corneal scrapes and six arterial clots. The qPCR-HRM results of all nine corneal samples were a 100% match with the results from the conventional PCR at clade level. However, the qPCR-HRM results of arterial clot samples were only matched with the nucleotide sequencing results from the conventional PCR at species level. In conclusion, the qPCR-HRM is a simple one closed tube, inexpensive and user-friendly method to identify P. insidiosum into clade level. Pythium insidiosum, pythiosis, high-resolution melting, clade differentiation Introduction Pythium insidiosum, is an aquatic oomycete fungus-like organism, which causes pythiosis in humans, mammals, and birds.1 This organism is widely found in its natural habitat, specifically agricultural swamp areas and stagnant fresh water. It is known as a soil borne saprophyte as well as a plant pathogen throughout the world.2,3 Recently, this organism was ubiquitously found in urban and suburban lakes in North Central Florida, USA, and this was probably the first report of natural habitat in the United States.4P. insidiosum is classified into 3 clades: clade I, II, and III based on phylogenetic tree analysis using internal transcribed spacer (ITS) region or intergenic spacer (IGS) region or exo-1,3-β-glucanase gene (exo1) or clade ATH, BTH, CTH by using mitochondrial cytochrome C oxidase II (COX2). Clade ATH or clade I is mostly found in American continent; Clade BTH or clade II is usually found in Asia and Australia; Clade CTH or clade III is found in Thailand and the United States.5–8 With regards to human diseases, the majority of the cases have been reported from Thailand.9,10 Human pythiosis is classified into four groups based on clinical presentations: cutaneous/subcutaneous, ocular, vascular, and disseminated disease.9,10 The combination of aggressive surgery, antifungal agents, and immunotherapy has been used as salvage treatment in several institutions; however, the effective treatment has not yet been established. It is known that surgery with adequate surgical margins is the main factor for patients’ survival in vascular disease regardless of antifungal agents or immunotherapy administration.10 Given this reason, early diagnosis and treatment is a very crucial determinant to decrease morbidity and mortality. The inefficacy of immunotherapy in the previous series was thought to be due to administration of immunotherapy from a discordant pathogen clade.11 Currently, immunotherapy is only available for P. insidiosum ATH; however, none of the Thai patients were infected with clade ATH. Chindamporn et al. found that serum antibodies from P. insidiosum infected patients recognized various immunogens in geographically divergent P. insidiosum strains by Western blot, but dominant antigen-antibody reactions were observed in the same pathogen clade.11 This implied the efficacy of immunotherapy may require antigens belonging to the same clade of the pathogens. Presently, at least two diagnostic methods including histopathological staining, culture, serology, and polymerase chain reaction (conventional PCR) with DNA sequencing are required to provide definitive diagnosis for human pythiosis in patients with compatible clinical contexts.2,10 At our medical center, the PCR with DNA sequencing by ITS region and COX2 gene have been used for clade differentiation. The phylogenetic tree using COX2 gene has showed higher discriminatory power than using ITS for sister-group classification.5,7 The alignment of P. insidiosum’s partial COX2 nucleic acid sequences presented the clade specific single-nucleotide polymorphisms (SNPs). These mutations are significant points particularly for clade disclosure. In addition to DNA sequencing, real-time quantitative polymerase chain reaction (qPCR) with subsequent High Resolution Melting (HRM) is another method to detect the SNPs.12 The qPCR can amplify nucleotide at the specific target of interest. The SNPs in amplicons, covered by designed primers, are used for the following HRM analysis steps. By theory, HRM is one of high sensitivity assays to detect the different melting temperature (Tm) caused by A/T or C/G bonding in each SNP pattern. Thus, the SNPs can be detected accurately without sequencing process. In the past few years, this novel technique has been investigated, and it has been successfully applied for diagnostic purposes in various research areas including detection and analysis of cancer-related mutations in humans, identification and genotyping of parasites, bacteria, and fungi.13–22 Despite its wide use in clinical medicine and agriculture, this technique has not been studied for P. insidiosum identification yet. In this study, the qPCR-HRM technique was developed for P. insidiosum identification as well as clade differentiation from clinical and environmental isolates. Methods The study protocol was approved by the Chulalongkorn University Institutional Review Board based on the international guidelines for human research protection including Declaration of Helsinki, the Belmont Report, The Council for International Organizations of Medical Sciences (CIOMS) Guidelines, and International Conferences on Harmonization in Good Clinical Practice (ICH-GCP) (certificate of authenticity; COA No.086/2016, IRB No. 015/59). Organisms and clinical specimens A total of 58 samples were tested in this study. Forty-three isolates of P. insidiosum (n = 15), Pythium spp. (n = 2), pathogens with similar morphology in kingdom Stramenopila (n = 3) and kingdom Fungi (n = 23) (Table 1), and 15 clinical specimens from proved human pythiosis, nine corneal scrapes and six arterial clots, were included in this study (Table 2). All isolates’ genus and species were proved by the conventional PCR with sequencing of ITS region and/or COX2 gene. Similar to those isolates, all recruited specimens with P. insidiosum infection were proved by positive conventional PCR with sequencing of ITS region and COX2 gene directly from specimens and/or positive culture. Sera from all patients, whose arterial clots were collected for this study, were tested for specific P. insidiosum antibody by in-house enzyme-linked immunosorbent assay (ELISA). Traditionally, P. insidiosum and other related fungi were differentiated by growth rate, presence of sterile hyphae, zoospore production, P. insidiosum specific antibody detection by ELISA, and conventional PCR with DNA sequencing technique. These methods were used as control in this study. Table 1. Strains, Genbank number, isolation sources and similarity percentage of P. insidiosum compared to reference strains base on qPCR with HRM analysis. No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - $Random isolates which were run the test in triplicate to evaluate the reproducibility and consistency of the qPCR-HRM protocols. * Confidence percentage, compared with P. insidiosum Tm curve profile. #Isolates which can be amplified by CoxII primers aCBS 574.85, contributed from Dr. L. Mendoza, MSU, USA. bPlant pathogens, identified by ITS sequences, from Ministry of Agriculture and Cooperative, Thailand. cGenomic DNA from Supabandhu J, Prince of Songkla University, Thailand. dContributed from Postgraduate Institute of Medical Education and Research (PGIMER),Chandigarh, India. View Large Table 1. Strains, Genbank number, isolation sources and similarity percentage of P. insidiosum compared to reference strains base on qPCR with HRM analysis. No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - No. Organisms Genbank no. Source of isolate; clade % Confidence* Kingdom Stramenopila,    Phylum Oomycota, Order Peronosporales 1 P. insidiosum# GQ451569 Equine, Costa-Ricaa: ATH    gDNA lot. 1$ 99.26%, 99.19%, 99.26%    gDNA lot. 2$ 99.22%, 99.35%, 99.31%    gDNA lot. 3$ 99.24%, 99.29%, 99.27% 2 GQ451575 Human (vascular pythiosis), BTH$ 99.03%, 99.15%, 99.11% 3 GQ451585 Human (vascular pythiosis), BTH$ 99.61%, 99.75%, 99.59% 4 GQ451586 Human (vascular pythiosis), BTH$ 99.90%. 99.81%, 99.89% 5 GQ451587 Human (vascular pythiosis), BTH 98.78% 6 JQ409331 Human (ocular pythiosis), BTH 98.45% 7 GQ451573 Human (vascular pythiosis), CTH$ 99.12%, 99.45%, 99.39% 8 GQ451574 Human (ocular pythiosis), CTH$ 97.95%, 99.81%, 99.84% 9 GQ451588 Human (vascular pythiosis), CTH$ 99.14%, 99.67%, 99.75% 10 GQ451589 Human (vascular pythiosis), CTH 99.09% 11 GQ451590 Human (ocular pythiosis), CTH 98.80% 12 KX371896 Human (vascular pythiosis), CTH 98.23% 13 KX389264 Human (cerebral pythiosis), CTH 99.09% 14 GQ451581 Field reservoir, Northern part, BTH 97.65% 15 GQ451576 Pasak Chonlasith Dam, Central part, CTH 98.12% 16 P. aphanidermatumb,# KX371903 Leave of Areca catechu tree 57.31% 17 P. catenulatumc,# JQ794534 Environment, Thailand 54.79% 18 Phy. parasiticab,# - Leaves of Citrus reticulate Blanco 5.78% 19 Phy. sojaeb,# KX371904 Leaves of Citrus Japonica Thunb 6.57% Order Lagenidiales 20 Lagenidium albertoi# KX389265 Human (Ocular lagenidiosis) 65.35% Kingdom Fungi, Phylum Glomeromycota, Order Entomophthorales 21 Basidiobolus meristosporus# Gecko's droppings, Thailand 1.02% 22 Conidiobolus coronatusb,# Environment, Thailand 1.22% Order Mucorales 23 Lichtheimia corymbiferad Environment, Thailand - 24 Apophysomyces elegansd Human - 25 Cunninghamella bertholletiaed Human - 26 Rhizomucor pusillusd Human - 27 Rhizopus arrhizusd Human - 28 Rhizopus microsporesd Human - 29 Saksenaea vasiformisd Human - 30 Syncephalastrum racemosumd Human - Phylum Ascomycota, Order Capnodoalea 31 Cladosporium spp. Human - Order Chaetothyriales 32 Fonseccaea pedrosoid Human - 33 Phialophora verrucosa Human - Order Dothideales 34 Aureobasidium spp. Human - Order Eurotiales 35 Aspergillus flavus Human - 36 Talaromyces marneffei Human - Order Microascales 37 Scedosporium apiospermumd Human - Order Onygenales 38 Trichophyton rubrum Human - 39 Trichophyton tonsuransd Human - 40 Histoplasma capsulatum Human - Order Ophiostomatales 41 Sporothrix schenckiid Human - Order Saccharomycetales 42 Candida albicans Human - Phylum Basidiomycota, Order Tremellales 43 Cryptococcus neoformans Human - $Random isolates which were run the test in triplicate to evaluate the reproducibility and consistency of the qPCR-HRM protocols. * Confidence percentage, compared with P. insidiosum Tm curve profile. #Isolates which can be amplified by CoxII primers aCBS 574.85, contributed from Dr. L. Mendoza, MSU, USA. bPlant pathogens, identified by ITS sequences, from Ministry of Agriculture and Cooperative, Thailand. cGenomic DNA from Supabandhu J, Prince of Songkla University, Thailand. dContributed from Postgraduate Institute of Medical Education and Research (PGIMER),Chandigarh, India. View Large Table 2. Comparison among the isolates here evaluated with previous study of Kammarnjessadakul et al.5 according to clade level and clinical samples tested in this study: corneal scraping (n = 9) and tissue biopsy (n = 6). P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH *Arterial clot after DNA extraction/Corneal directly from specimens. NA, not available; ND, not done. + = positive for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. − = negative for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. View Large Table 2. Comparison among the isolates here evaluated with previous study of Kammarnjessadakul et al.5 according to clade level and clinical samples tested in this study: corneal scraping (n = 9) and tissue biopsy (n = 6). P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH P. insidiosum isolates Clinical samples used in this study Pythiosis confirmation Clade identification No. Genbank no. Kammarnjessadakul HRM qPCR Specimen- PCR with Culture ELISA from by HRM qPCR* et al. study isolate; (Average Cp sample no. sequencing/ serum sample (Average Cp clade value ± SD/clade) clade value ± SD/clade) 1 GQ451569 MTPI19; ATH 15.3 ± 0.58 / ATH NA ND ND ND ND 2 GQ451575 PC10; BTH 16.3 ± 0.58 / BTH Arterial clots-S2 + / BTH + + 16.0 ± 0.00 / BTH 3 GQ451585 ND 16.7 ± 0.58 / BTH Arterial clots-S3 + / BTH + + 17.0 ± 0.00 / BTH 4 GQ451586 PMS1; BTH 17.0 ± 1.00/ BTH Arterial clots-S4 + / BTH + + 16.3 ± 0.58 / BTH 5 GQ451587 PCM1; BTH 15.0 ± 1.00 / BTH NA ND ND + ND 6 JQ409331 ND 14.7 ± 0.58 / BTH Corneal scraping-S6 + / BTH + ND 17.0 ± 0.00 / BTH 7 GQ451573 PC3; CTH 15.0 ± 1.00 / CTH Arterial clots-S7 + / CTH + + 17.0 ± 0.00 / CTH 8 GQ451574 PC6; CTH 16.7 ± 0.58 / CTH Corneal scraping-S8 + / CTH + ND 16.3 ± 0.58 / CTH 9 GQ451588 PC7; CTH 15.0 ± 1.00 / CTH Arterial clots-S9 + / CTH + + 17.0 ± 0.00 / CTH 10 GQ451589 PC2; CTH 17.3 ± 0.58 / CTH NA ND ND + ND 11 GQ451590 PC5; CTH 17.3 ± 0.58 / CTH Corneal scraping-S11 + / CTH + ND 17.0 ± 0.00 / CTH 12 KX371896 ND 17.3 ± 0.58 / CTH Arterial clots-S12 + / CTH + + 15.0 ± 0.00 / CTH 13 KX389264 ND 16.0 ± 0.00 / CTH NA ND ND ND ND 14 GQ451581 PECM8; BTH 16.0 ± 1.00 / BTH NA ND ND ND ND 15 GQ451576 PEC1; CTH 16.7 ± 0.58 / CTH NA ND ND ND ND 16 NA ND ND Corneal scraping-S16 + / BTH - ND 15.7 ± 0.58 / BTH 17 NA ND ND Corneal scraping-S17 + / BTH - ND 15.7 ± 0.58 / BTH 18 NA ND ND Corneal scraping-S18 + / BTH - ND 16.7 ± 0.58 / BTH 19 NA ND ND Corneal scraping-S19 + / CTH - ND 15.0 ± 0.00 / CTH 20 NA ND ND Corneal scraping-S20 + / CTH - ND 16.7 ± 0.58 / CTH 21 NA ND ND Corneal scraping-S21 + / CTH - ND 16.3 ± 0.58 / CTH *Arterial clot after DNA extraction/Corneal directly from specimens. NA, not available; ND, not done. + = positive for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. − = negative for P. insidiosum by molecular detection with sequencing or cultivation or ELISA test. View Large DNA preparation The genomic DNAs (gDNAs) of all tested isolates were prepared by QIAamp DNA Mini kit (Cat. 51306, QIAGEN, Germantown, MD, USA). In brief, isolates were lysed by 180 μl ATL buffer and 20 μl proteinase K at 56 °C for 1 h. Then, protein denaturation step was performed by 200 μl AL buffer. After centrifugation, the upper part was purified and precipitated with 200 μl absolute ethanol. The mixer was filtered through the QIAamp Mini Spin column and washed again by 500 μl AW1 and AW2 buffer. Finally, a ratio of 1.8 to 2.0, based on A260/A280 ratio by NanoDrop (NanoDropTM 1000 Spectrophotometer Thermo scientific, Skokie, IL, USA), was used to determine DNA quality and quantity. Real-time quantitative polymerase chain reaction (qPCR) and post-HRM analysis The qPCR amplification of 91 bp in COX2 gene was performed by the newly designed primers: Cox_Pi_5 (5’-TAA TTT GGA CTA CTA TTC CAG C-3’) and Cox_Pi_6 (5’-GGA TCA ATG TAT TTC ATC CAT AG-3’) (Fig. 1). These specific primers can amplify SNP regions in all three clades of P. insidiosum, and other related organisms: Pythium aphanidermatum (P. aphanidermatum), P. catenulatum, Phytophthora parasitica (Phy. parasitica), Phy. sojae, Lagenidium albertoi, Basidiobolus meristosporus, and Conidiobolus coronatus. The reactions of qPCR (Rotor-Gene™ 6000, Corbett Research, Austria) were performed in 20 μl volume containing of 10 μl high-resolution melting master mix (Cat. 22655, Roche, Indianapolis, IN, USA), 2.5 mM MgCl2, 0.125 μM of each primer, and 10 ng of gDNA. The amplification steps consisted of an initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 55°C for 25 s, and extension at 72°C for 1 min. In this study, the interval created by the average crossing point value (Cp value) ± 2 cycles was accepted. To identify the genus and species level from the qPCR amplicons, the melt curve genotyping analysis program was used. In addition, the HRM program was further processed to separate into clade level. Both programs were included in the Rotor-Gene™ 6000, software version 1.7, Corbett Research, Austria. The reference gDNA templates of all three clades were optimized to obtain unique different curve of each clade. In every run, these templates were used as reference curves. The melt curve genotyping was plotted based on melting temperatures of the amplicons. Regarding post-HRM genotyping analysis, the amplification products were denatured at 95°C for 1 min and were cooled at 40°C for 1 min to complete double-stranded amplicons. The HRM melting curves were generated by ramping from 65 to 90°C, and the curves were monitored fluorescence at the rate of 0.1°C/s, taking 25 fluorescence acquisitions per 1°C. These curves were plotted by using the automated grouping option in the program (Fig. 2B and C). More than 95% confidence of similarity compared to reference gDNA templates by HRM analysis was accepted for clade classification (Fig. 2A). To evaluate the limit of detection (LOD) of qPCR and post-HRM, 1 pg to 10 ng of gDNA were tested. The gDNA of all isolates were tested in triplicate blinded experiments. The gDNA of Staphylococcus spp., human and molecular grade water were used as negative controls. The specificity test was challenged by using other fungal gDNAs (n = 28) as follows: P. aphanidermatum, P. catenulatum, Phy. parasitica, Phy. sojae, Lagenidium albertoi, Basidiobolus meristosporus, Conidiobolus coronatus, Lichtheimia corymbifera, Apophysomyces elegans, Cunninghamella bertholletiae, Rhizomucor pusillus, Rhizopus arrhizus, Rhizopus arrhizus, Saksenaea vasiformis, Syncephalastrum racemosum, Cladosporium spp., Fonseccaea pedrosoi, Phialophora verrucosa, Aureobasidium spp., Aspergillus flavus, Penicillium marneffei, Scedosporium apiospermum, Trichophyton rubrum, Trichophyton tonsurans, Histoplasma capsulatum, Sporothrix schenckii, Candida albicans, and Cryptococcus neoformans under the same condition. The cut-off for the same clade was more than 95% confidence of similarity calculated by HRM analysis. Finally, the results of the qPCR-HRM analysis of the tested isolates were compared to the sequences of ITS region and COX2 gene which was the standard method in this study. Figure 1. View largeDownload slide The 91 base pairs amplicon bearing SNPs of P. insidiosum clade ATH, BTH and CTH by Cox_Pi_5_forward and Cox_Pi_6_reverse primer. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide The 91 base pairs amplicon bearing SNPs of P. insidiosum clade ATH, BTH and CTH by Cox_Pi_5_forward and Cox_Pi_6_reverse primer. This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Normalized graph (A) and difference graph (B): comparison among Clade ATH, Clade BTH, and CTHP. insidiosum (―) and other related fungi (—) which were amplified COX2 gene: L. albertoi (1); P. aphanidermatum (2); P. catenulatum (3); C. coronatus (4); Phy. sojae (5); B. meristosporus (6); Phy. parasitica (7). This Figure is reproduced in color in the online version of Medical Mycology. Figure 2. View largeDownload slide Normalized graph (A) and difference graph (B): comparison among Clade ATH, Clade BTH, and CTHP. insidiosum (―) and other related fungi (—) which were amplified COX2 gene: L. albertoi (1); P. aphanidermatum (2); P. catenulatum (3); C. coronatus (4); Phy. sojae (5); B. meristosporus (6); Phy. parasitica (7). This Figure is reproduced in color in the online version of Medical Mycology. The qPCR-HRM for clinical samples To shorten the processes and avoid complicated gDNA extraction steps, we applied this qPCR-HRM analysis directly to clinical specimens, arterial clots, and corneal scrapes. All clinical specimens were cut into small pieces (approximately 1 mm × 1 mm in length and width) and directly mixed in the qPCR reactions (Table 2). This mixture was processed by the qPCR-HRM as same as the fungal isolates described in previous section. The additional DNA extraction was required in arterial clot samples to improve the efficiency of the qPCR-HRM. Their gDNA were prepared by QIAamp DNA Mini kit (Cat. 51306, QIAGEN, USA) prior to being used as templates in the qPCR-HRM reactions. Results The qPCR-HRM results: fungal isolates Twenty-two out of 43 tested isolates could be amplified by COX2 specific primers (Table 1) with Cp value between cycles 14 and 18 (cycles 16 in average) in the qPCR process. Twenty isolates were from phylum Oomycota, and two isolates were from order Entomophthorales, phylum Glomeromycota (former Zygomycota).23 No PCR products were detected from the negative controls. Based on the melt curve genotyping, there were no significant differences in melting temperatures (Tm) among those 22 amplifiable isolates. The 22 amplifiable isolates were classified into four groups based on the curve patterns from HRM analysis-clade ATH pattern, clade BTH pattern, clade CTH pattern, and non-ATHBTHCTH pattern compared to the reference P. insidiosum curve patterns. Fifteen out of 22 amplifiable isolates met the criteria of ≥95% confidence of similarity (Cp 16.15 ± 0.94): clade ATH (1/15 isolate, 99.3% confidence of similarity; Cp 15.3 ± 0.58), clade BTH (8/15 isolates, 98.1–99.6% confidence of similarity; Cp 15.95 ± 0.92), and clade CTH (6/15 isolates, 97.7–99.0% confidence of similarity; Cp 16.41 ± 0.98) (Fig. 2A). The three clades achieved from HRM analysis were correlated with the clustering based on DNA sequences. The non–ATHBTHCTH pattern was produced from seven out of 22 amplifiable isolates, and this pattern did not meet the criteria of ≥95% confidence of similarity. This result indicated that these seven isolates were not P. insidiosum, correlated with the identification results by conventional PCR with DNA sequencing. The non–ATHBTHCTH pattern was generated by P. aphanidermatum, P. catenulatum, Phy. parasitica, Phy. sojae, Lagenidium albertoi, Basidiobolus meristosporus, and Conidiobolus coronatus. The qPCR-HRM results: clinical specimens A total of nine corneal scrapes and six arterial clots were examined (Table 2). Only 11/15 clinical samples, nine corneal tissues, and two arterial clots were amplified. None of them met the criteria of ≥95% confidence of similarity compared to reference P. insidiosum isolates. All samples from corneal scrapes showed ≥85% confidence of similarity; Cp 16.27 ± 0.68. Two clades were identified: clade BTH (5/9 samples, 87.2–89.4% confidence of similarity) and clade CTH (4/9 samples, 85.1–89.0% confidence of similarity). None of arterial clots can be classified into clade level without an additional gDNA extraction. However, with the additional gDNA extraction from the specimens, all 6 arterial clots were successfully amplified and can be classified into clade level by qPCR-HRM with Cp of 16.39 ± 0.8. Specificity and limit of detection The specificity of the qPCR-HRM was evaluated by using other fungal gDNAs as templates for qPCR-HRM reactions under the same condition as P. indiosum’s gDNA. There were no false positive or false negative results. These results revealed 100% specificity of the qPCR-HRM. With regards to the limit of detection, different concentrations of P. insidiosum’s gDNA ranging from 1 pg to 10 ng were examined. One picogram of gDNA was the lowest detectable amount by this qPCR-HRM. Reproducibility of the qPCR-HRM The reproducibility and consistency of qPCR-HRM protocols were evaluated by running HRM in triplicate from six random isolates: three from clade BTH and three from clade CTH. Since there was the only one isolate for clade ATH, the reproducibility and consistency of these protocols were tested by extracting gDNA in triplicate from three different subcultures of the isolate. The HRM analysis was performed in each lot of gDNA preparation. Our protocols were found to have very high consistency and reproducibility (Table 1). Among the control reactions, very small variation with Cp of 15.4 ± 0.4 was observed. Discussion In this study, the qPCR-HRM analysis was first developed to provide rapid clade specific diagnosis for patients with pythiosis. The results from conventional PCR with DNA sequencing for clade identification were similar to the results from the qPCR-HRM. Several advantages of the qPCR-HRM are as follows; first, P. insidiosum can be differentiated from other true fungi and be classified into the clade level with turnaround time of four hours while at least 18 hours are required for conventional approach. Second, this technique was first implemented to identify P. insidiosum directly clinical samples, and it was successful for corneal scrapes. Third, the qPCR-HRM is convenient because only one closed tube system is required and this will significantly reduce risk of contamination during multistep processes of conventional PCR. Lastly, the qPCR-HRM is approximately 5 times cheaper than the conventional PCR with DNA sequencing, and this technology will be widely accessible especially among developing countries. Several techniques for P. insidiosum identification such as thermophilic helicase DNA amplification with restriction fragment length polymorphism (tHDA-RFLP) targeting COX2 gene,23 real-time PCR targeting exo-1,3-beta-glucanase gene (PinsEXO1),245 and screening method based on the growth of P. insidiosum mycelia around minocycline disks (30 μg)25 have been reported. All identification methods have mainly used gDNA template from cultures or non-clinical specimens; however, they are only able to provide diagnosis into the species level23-25. In this study, both species and clade diagnosis can be obtained by nonsequencing based qPCR-HRM with 100% specificity. All the clade classification results from 15 P. insidiosum gDNA template tested samples obtained from both conventional PCR-sequencing and the new qPCR-HRM methods are 100% match. The confidence percentage of similarity results from our qPCR-HRM for clade identification are as high as the previous reported results in the literature for species identification in other organisms.14,26–28 This indicates that our developed assay is highly accurate and applicable for P. insidiosum identification. Direct amplification from clinical specimens is the ultimate goal. The main advantage of amplification directly from clinical specimens is to increase detection yield of P. insidiosum in case of cultivation failure. The intolerance to low temperature of P. insidiosum during transportation and sample preparation is a major barrier causing false negative results in real-world practice.29 In this study, we successfully identified P. insidiosum from all corneal scrapes samples corresponding to the DNA sequence base assay. Ocular pythiosis can be diagnosed within a working day by the qPCR-HRM, but it is almost impossible by conventional PCR with DNA sequencing and standard cultivation method. In case of arterial clots, low success rate was observed by the qPCR-HRM. We hypothesize that clot samples may not have been completely lysed by typical denatured temperature, and it led to impurity of DNA extracts.30 To prove this hypothesis, we extracted the gDNA from these samples and processed the qPCR-HRM. As expected, all six arterial clots were amplified and P. insidiosum was identified. Although a few extra hours were required for DNA extraction from arterial clots, the qPCR-HRM could still be considered as a rapid diagnosis compared to the conventional PCR with DNA sequencing. In terms of clade classification by directly using clinical specimens, all of the results were 100% match to the nucleotide sequence assay, even though only 85.1–89.4% similarity by HRM were achieved from the corneal scrapes. The lower percentage of similarity in clinical specimens was probably due to unpurified samples, inhibitors in DNA extracts, or different concentrations of magnesium salt.32 To increase higher percentage of similarity for the qPCR-HRM, additional steps may be required. These steps are pre-amplification step (gDNA extraction and purification) and pre-HRM analysis step (PCR purification).31 However, we decided not to pursue the additional DNA extraction and purification because we had limited specimens and DNA would be lost during these processes. In conclusion, this new qPCR-HRM technique is a novel tool for conclusive P. insidiosum identification and clade classification. Owing to the rapid, user-friendly, and inexpensive process, this technique is another option to help improve patient care by facilitating early diagnosis and guiding appropriate immunotherapy in the future. With clade identification, we also hope this technique will provide more epidemiologic data in different geographic areas. Acknowledgments This work was supported by (1) the 90th Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) year 2009 and (2) Chulalongkorn's Research Repository (CU-57-008-FW). We would like to extend our sincere gratitude toward Drs. Chakrabarti, Shivaprakash, and Ms. Sunita Gupta from Postgraduate Institute of Medical Education and Research, Chandigarh, India, for providing the fungal isolates of Zygomycetes group. Finally, we would like to sincerely thank all members in Mycology Unit, Department of Microbiology, King Chulalongkorn Memorial Hospital and Mycology research unit, Faculty of Medicine, Chulalongkorn University for all supports. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. Other related information This intervention (1) is being applied for the patent (patent application no. 1201006141 requested on 27 November 2012) under the authorization of Chulalongkorn University, Thailand, by Dr. Ariya Chindamporn and Ms. Navaporn Worasilchai as researchers, (2) was presented as a poster presentation in “19th Congress International Society for Human and Animal Mycology (ISHAM) 2015” Melbourne, Australia, by Ms. Navaporn Worasilchai. References 1. Pesavento PA , Barr B , Riggs SM , Eigenheer AL , Pamma R , Walker RL . Cutaneous pythiosis in a nestling white-faced ibis . 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Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Medical MycologyOxford University Press

Published: Oct 1, 2018

References

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