Development and evaluation of a rapid diagnostic method for Sporothrix globosa in Asia using quantitative real-time PCR

Abstract

Background

Sporotrichosis is a chronic granulomatous infection of the skin and subcutaneous tissue that can affect any organ through lymphatic spread. The prevalence of sporotrichosis infections is increasing and its treatment is challenging as there are no unified and standard diagnostic techniques or antifungal medications. Controlling further spread requires a rapid diagnosis. Assessment of clinical symptoms, histological analysis, serological testing, and pathogen culture are all necessary for the diagnosis of sporotrichosis. However, these procedures are unable to identify the species. The development of safe, reliable, and species-specific diagnostic techniques is essential.

Objective

To establish and evaluate a new quantitative real-time PCR assay for the rapid diagnosis of sporotrichosis and to identify relevant species.

Methods

Polymorphisms in calmodulin (CAL) gene sequences and the internal transcribed spacer (ITS) were used in a quantitative real-time PCR assay to identify S. globosa, S. schenckii, and non-target species.

Results

The quantitative real-time PCR assay had 100% sensitivity and specificity. The limit of detection was 6 fg/µl. Thirty-four clinical specimens were verified to be infected with S. globosa with a 100% positive detection rate.

Conclusions

The quantitative PCR technique developed in this study is a quick, accurate, and targeted method of identifying S. globosa based on polymorphisms in CAL sequences and ITS. It can be used for a prompt clinical diagnosis to identify S. globosa in clinical specimens from patients with sporotrichosis.

Introduction

Sporotrichosis is an important fungal infection worldwide that is caused by the pathogenic dimorphic fungus Sporothrix [1]. The Genus Sporothrix comprises 53 species,, is one of the most widely distributed implantation mycose in the world. It typically spreads to the surrounding lymphatic system, subcutaneous tissue, mucosal membranes, and skin [2]. S. schenckii, S. globosa, S. brasiliensis, and S. luriei are the species that cause such infections in humans and other mammals [3]. Both sexes are affected by sporotrichosis, which has no preference for age or race. The World Health Organization classifies it as a fungal neglected tropical disease [4]. Despite sporotrichosis becoming more commonplace globally, endemic areas are primarily located in temperate and tropical regions [5, 6], with Brazil, China, and South Africa having the highest rates [7, 8]. The variety of Sporothrix species currently described have regional distribution.

The diagnosis of sporotrichosis is problematic with poor sensitivity across the most widely available laboratory tests except fungal culture. None of the traditional assays can identify Sporothrix down to the species level and the identification of the Sporothrix complex depends critically on the correlation between molecular data and phenotypic characteristics [9], highlighting the need to improve mycological diagnostic capacity and develop innovative diagnostic solutions. Several studies have shown that different species of Sporothrix complex and host populations have significant differences in pathogenicity and transmission [10,11,12].

This study aimed to determine a better method of detecting Sporothrix complex to better guide its treatment. Despite the importance of accurate identification, there remains a need to establish new diagnostic methods. Based on the quantitative real-time PCR and the phylogenetic analysis of polymorphic loci, this research used multiple sites to establish a more convenient, rapid, and objective molecular diagnostic method to diagnose the Sporothrix complex within Asia.

Materials and methods

Study population

The study included 34 patients at Shandong Provincial Hospital for Skin Diseases (Jinan, China) who were diagnosed with sporotrichosis between January 2022 and November 2023. Patients with sporotrichosis were diagnosed according to previously described criteria [2]. The inclusion criteria were as follows: meeting the diagnostic criteria for sporotrichosis and patient consent to histopathological examination or tissue culture. Informed consent was obtained from all participants under a protocol approved by the Shandong Provincial Institute of Dermatology and Venereology (Jinan, China).

Strains

The strains involved in this study comprised 35 S. globosa, one S. shenckiis, 25 other common pathogenic fungi, three bacteria, and two mycobacteria. Among them, one S. schenckii (CBS498.86 T) and one S. globosa (CBS120340T) were purchased from the Centraalbureau voor Schimmelcultures (CBS). The rest of the strains were gathered and reserved by the Shandong Provincial Hospital for Skin Diseases. Fungi and bacteria were subcultured on Sabouraud Dextrose Agar by the Hangzhou Binhe Microorganism Reagent Co., Ltd (Hangzhou, China) and bacteria were subcultured on Blood Agar Plate by the Autobio Diagnostics Co., Ltd (Zhengzhou, China) for 5 to 7 days at 28℃. All strains of fungi and bacteria were previously recognized by morphology and molecular sequencing to the species level. The molecular assay used Sanger sequencing of ITS 1 and ITS 4 for identification.

DNA extraction

DNA was extracted from fungal and bacteria colonies through Sample preparation, Cell lysis, Remove proteins, polysaccharides and other impurities, DNA washing, Resuspend DNA with the Quick-DNA Fungal/Bacterial Miniprep Kit (D6005, Zymo Research, USA), and stored at − 20℃ until used. The purity of DNA conformed to an examination standard and was tested by NanoDrop 8000 spectrophotometer (Thermo Scientific, USA). Two healthy participants provided human DNA.

Design of primers and probes

The CAL, ITS, and EF-1α sequences of the species from the CBS were compared with the Sporothrix complex in the NCBI database (NCBI seqences: CAL: KP101477.1, KC693877.1; ITS: KY387687.1; EF-1α: KP017041.1). The research also included CAL, ITS, and EF-1α sequences from related species, genera, and other pathogens to increase genetic diversity and cover most of the varieties described in the literature. Clustal Omega and DNAMAN software was used to align and select a specific sequence of S. globosa. Primer Premier 6 was used to design specific primers and probes for S. globosa, and manually adjusted to obtain a short and informative region (sequences are shown in Table 1). Melting temperatures, %CG contents, and mismatches in candidate sequences were assessed by the above software. Probes were labeled with FAM and MGB fluorescence, and synthesized by the Beijing Genomics Institute (Beijing, China).

Table 1 Primers and probes used inin the real-time PCR assay

Optimization of quantitative PCR (qPCR)

The qPCR assay was performed in a 25 μl final reaction volume including TaqMan Gene Expression Master Mix (4,369,016, Thermo Fisher, USA) 12.5 μl, forward-primer Sg-F (10 μmol/l) 1 μl, reverse-primer Sg-R (10 μmol/l) 1 μl, TaqMan probe Sg-P (10 μmol/l) 0.5 μl, DNA template 2 μl, and nuclease-free water 8 μl. The annealing temperature range was from 58 to 62 °C. The qPCR was performed using a qPCR Detection System (Thermo Fisher, USA) under the following conditions: pre-denaturation at 95 °C for 10 min, followed by 50 cycles of 95 °C for 15 s, 58/60 °C for 15 s. Data collection was enabled at the extension step. A blank control (nuclease-free water), negative control (Non S. globosa), and positive control (Standard S. globosa) were established for each test. A genuine amplification was determined by the following results: NTC ( −), NEG ( −), and POS ( +).

Specificity, sensitivity, and limit of detection (LOD)

Two standard strains and thirty-four S. globosa isolated from clinical samples had been validated by DNA sequencing or fungla culture in order to validate the sensitivity of the system. Clinically isolated 34 S. globosa validated by sequencing or fungal culture were used for system sensitivity validation. The DNA of S. globosa LOD was measured by a tenfold dilution using 6 ng/μl to 0.6 fg/μl for seven gradient concentrations.

Results

Development and optimization of the qPCR assay

To standardize the qPCR test, the primer set Sg-F and Sg-R was tested in duplicate. Four combinations of primers and probes could amplify genus Sporothrix specifically (Table 1). However, the primers and probe combination designed by using EF-1a sequences could amplify S. schenckii nonspecifically. Three combinations were used in the verification. Under the annealing temperature range of 58 to 62 °C, the optimum annealing temperature was 60 °C with the minimum Ct value with CAL sequences. The optimum annealing temperature was 58 °C with the minimum Ct value with ITS sequences.

Specificity, sensitivity, standard curve, and LOD

For the negative detection of 30 common pathogens, S. schenckii, and the human genome DNA, the qPCR specificity was 100% (Table 2). However, the sensitivity of the qPCR verification method in detecting 34 S. globosa clinical isolates was 100%. As seen in Fig. 1 (CAL1: Fig. 1a; ITS: Fig. 1b; CAL2: Fig. 1c;), a standard curve (Fig. 1d) was produced by the amplification of 6 ng/µl to 0.6 fg/µl S. globosa nucleic acid, and 2 μl of the template was applied to every gradient concentration. Three parallel samples were used for each concentration gradient, and the real-time PCR was conducted in accordance with the reaction system and conditions. The LOD outcomes are presented in Table 3.

Fig. 1

a The amplification curve of the CAL 1 gradient concentration template shows the limit of detection (LOD) of the assay is 60 fg. b The amplification curve of the ITS gradient concentration template shows the LOD of the assay is 6 fg. c The amplification curve of the CAL 2 gradient concentration template shows the LOD of the assay is 60 fg. d Standard curve of the qPCR assays. CAL1: the slope is − 3.22, the y intercept is 33.133, and the correlation coefficient is 0.9982; CAL2: the slope is − 3.3, the y intercept is 31.633, and the correlation coefficient is 0.9986; ITS: the slope is − 3.296, the y intercept is 37.367, and the correlation coefficient is 0.9963. (1: 6 ng, 2: 600 pg, 3: 60 pg, 4: 6 pg, 5: 600 fg, 6: 60 fg, 7: 6 fg)

Table 2 Strains and isolates used in the present study

Table 3 The specificity, sensitivity, LOD, and Ct value of three sets of primers and probes

Clinical samples for real-time PCR evaluation

Following the culture of the specimens, filamentous hyaline colonies began to grow and eventually took on a black hue, particularly in the center of the colonies. This is a typical example of the morphology of the Sporothrix. We discovered that the isolated pathogens were S. globose. Therefore, every specimen that was enrolled was infected by S. globosa. Real-time PCR results for 30 clinical specimens were all positive, and the positive detection rate was 100%. The Ct values of CAL 1, CAL 2, and ITS are shown in Table 3.

Discussion

Sporothrix complex causes a subacute or chronic infectious condition called sporotrichosis. Sporotrichosis may include the infection of the skin, mucosa, subcutaneous tissue, and the lymphatic system locally and multiple sites may be involved. The Sporothrix complex is present in soil, sphagnum moss, and decomposing vegetation, globally [13]. The classical transmission of Sporothrix is typically by traumatic inoculation by vegetation, wood, and soil manipulation. Therefore, the main high-risk industries are farming, mining, horticulture, and timber exploration [14]. Farming is the foundation of Asian culture, with a substantial number of gardeners, farmers, and forest rangers across the region. As a result, the incidence of sporotrichosis is increasing in Asia. Considering the range of species that are currently known, S. schenckii and S. globosa are believed to be the causative agents of endemic sporotrichosis in the Asian region [15].

A combination of clinical presentation, histological features, serology, and the culture of the causative agents is used to diagnose sporotrichosis. Isolating and identifying the sporotrichosis fungus from clinical samples, such as abscess aspirates, skin lesions, dander, etc., is the gold standard for diagnosing the disease [2, 10]. This is a straightforward and inexpensive diagnostic technique, yet its use in identifying some systemic and atypical sporotrichosis cases may be restricted [14]. Additionally, obtaining results from cultures frequently requires 2–4 weeks, making it inappropriate for a quick clinical diagnosis. Sporothrix can be observed by direct microscopy with potassium hydroxide. The sensitivity and specificity of sporotrichosis is low. The absence of fungal components in the tissue, results in less sensitive histopathological detection, and the real structure is seen only in 18% to 35.5% of cases, depending on the detection technique. However, none of the above assays could identify Sporothrix down to the species level.

Species differences in virulence, geographic distribution, and host relationships have emerged, so genetic studies are needed to identify species [12]. Thus far, molecular techniques have been used extensively in diagnosis within the Sporothrix and are more sensitive than histology. Sporothrix molecular diagnostic techniques use rapid and inexpensive genotyping methods to facilitate accurate disease diagnosis and epidemiological studies [16]. Common molecular diagnostics include nested PCR [17], rolling circle amplification (RCA) [18], duplex PCR [19], restriction fragment length polymorphism, (RFLP) and so on. A species-specific PCR was used by Rodrigues et al. [20] in 2015. This assay was successful in locating and identifying Sporothrix gene members that are clinically significant. The LOD attained was 10 fg. A real-time multiplex PCR based on Calmodulin gene sequences was established by Zhang et al. in 2019. The specificity of the test was 100% and detected 100, 10, and 100 copies of Calmodulin gene sequences from S. brasiliensis, S. schenckii, and S. globosa, respectively [21]. In 2020, Zhang et al. established a real-time PCR method involving ITS for the recognition of S. globosa and demonstrated a sensitivity and specificity of 100% and a limitation of 10 fg [22]. The same year, Della Terra et al. took advantage of the polymorphisms in the single-copy gene of β-tubulin, to distinguish S. brasiliensis and S. schenckii in a one-tube multiplex probe-based qPCR assay. The assay showed 100% selectivity, and for all species, the lower LOD was three copies of the target [23].

More and more assays could determine the Sporothrix species by species. However, qPCR is a low-cost, efficient, and robust molecular detection method of identification down to the species level. There is no high requirement for experimental conditions, and it is also convenient for diagnosis in areas where public resources are scarce.

The ITS and the CAL gene sequences are discernible fragments that can be used to synthesize primers and probes. Nowadays, partial calmodulin sequences are used to distinguish the Sporothrix complex between species [24, 25]. The CAL gene sequences of Sporothrix vary slightly in the areas that code for proteins. Variations of areas that code for proteins tend to be synonymous substitutions, which indicates strong conservation of the protein sequence [26]. In previous studies [27, 28], clinical specimen molecular characterization has made considerable use of CAL gene sequences. Therefore, selecting sparsely informative regions within the CAL gene sequence that were conserved intra-specifically was an option. The ITS is a portion of the fungal rRNA gene and is a universal marker for Sporothrix identification. It displays differences across closely related species and has a broad spectrum of polymorphisms in most eukaryotes. Within the clade derived from S. schenckii, the ITS region has been used to improve the ability to classify and recognize unique fungal genes because this commonly used barcode gene is distinctive enough to detect all four currently identified species [29]. Furthermore, with more than 200 copies of ITS in a single chromosome, ITS has a large copy number that improves diagnostic sensitivity. This study used a high concentration of nucleic acid from S. globosa as a template to test the assay and compared several CAL and ITS sequences of the Sporothrix complex published in GenBank to ensure the intra-species specificity of the approach. The length of primer required by qPCR is generally 18–30 nt with a target fragment length of 100–150 bp. Primers that can amplify precisely and probes that meet the specifications of the qPCR must be designed. Three sets of candidate primer and probe combinations were acquired through software comparison and the manual adjustment of human sequences. Following confirmation, a mixture with the TaqMan MGB probe (Table 1 was acquired. The system had excellent sensitivity and specificity. The sets of CAL 1 and CAL 2 had a LOD of 60 fg/μl and the set of ITS had an LOD of 6 fg/μl.

Overall, thirty clinical specimens were gathered to confirm the capacity of the real-time PCR to detect S. globosa infections in tissues. Additionally, we examined several DNA samples that came from non-target species, such as agents responsible for human superficial, subcutaneous, and systemic mycoses. The positive rate of detection was 100% and the specific rate was 100%. This demonstrates that there are potential uses for this technique in clinical fast diagnosis where Sporothrix could be identified down to the species level.

In the past, the use of ITS sequence has been explored for the qPCR diagnosis of sporotrichosis [22]. This study assessed the possibility of using CAL sequence in qPCR for the diagnosis of Sporothrix in Asia. The main purpose of this study was to establish a method that can distinguish and identify S. globosa found in Asia and explore the possibility that multiple sequences can become diagnostic sites. Our intention was to support the clinical diagnosis, scientific study, and epidemiological surveillance of Sporothrix.