Detection and subtyping of Blastocystis sp. in human and animal stool samples using high-resolution melting analysis

Abstract

Objective

This study aimed to investigate the prevalence and subtype distribution of the genus Blastocystis using high-resolution melting curve analysis (HRM), a novel molecular technique. Blastocystis sp., a unicellular, anaerobic, and zoonotic parasite, is commonly found in the digestive tracts of humans and animals. Transmission occurs via the fecal-oral route, with prevalence rates ranging from 20% in developed countries to over 60% in developing regions. Although the pathogenicity of Blastocystis remains debated, it has been detected in both symptomatic and asymptomatic individuals. Traditionally, direct microscopy with wet mounts is used for the rapid identification of Blastocystis in stool samples. However, molecular diagnostics have become essential for the accurate detection and differentiation of Blastocystis subtypes

Materials and methods

We collected 730 stool samples from humans and domestic animals. Initial screening for Blastocystis sp. was performed using direct microscopy. Negative samples were cultured in a two-phase culture medium and re-examined after 2–3 days. HRM analysis was then employed to identify and differentiate Blastocystis subtypes using specific primers via real-time PCR.

Results

Six subtypes were identified, with ST7 (30%) being the most prevalent, followed by ST3 (28%), ST2 (16%), ST1 (14%), ST5 (6%), and ST14 (6%). In humans, ST3 was the most common subtype, also found in poultry and sheep, whereas ST7 was mainly detected in domestic animals. Notably, ST1-ST3 were identified in domesticated animals, indicating a pattern of cross-species transmission.

Conclusion

ST3 (28%) and ST7 (30%) were the most prevalent subtypes across all samples, with distinct distributions in human and animal hosts. The HRM technique demonstrated efficiency and cost-effectiveness, providing a rapid and accurate method for Blastocystis subtype identification in developing countries, which can expedite diagnostic responses and reduce the need for sequencing. This supports the potential for HRM to improve epidemiological surveillance and understanding of cross-species transmission.

Introduction

Blastocystis sp. is a gastrointestinal protozoan commonly found in various human and animal populations. It is a polymorphic, unicellular parasite, with the vacuolar form being the most frequently observed under the light microscope, while the cystic form is the primary agent responsible for transmission [1]. This parasite is distributed globally, with reported prevalence rates ranging from 0.5 to 24% in industrialized countries and 30–60% in developing nations [2]. One key mode of transmission is the fecal-oral route, leading to a range of symptoms from mild to severe. However, the pathogenicity of Blastocystis remains unclear. Symptoms associated with this parasite include nausea, abdominal pain, diarrhea, and gastric distension [3]. Several forms of the parasite have been identified, including vacuolar, granular, cyst, and amoeboid forms [4].

Rapid identification of Blastocystis sp. in stool samples can be achieved using various methods, such as microscopic examination and concentration techniques. Direct examination of a wet mount under a light microscope is considered the most cost-effective and simplest diagnostic method. Additionally, Lugol’s iodine solution and permanent dyes, such as trichrome, are commonly used to enhance diagnostic accuracy [5]. Concentration methods, such as formalin-ether sedimentation, improve the sensitivity and precision of microscopy. Culturing Blastocystis has been reported to be five times more sensitive than direct smear methods, although it requires 24 to 48 h [5].

Molecular techniques have emerged as valuable tools for diagnosing and differentiating Blastocystis subtypes. Methods such as PCR/sequencing of the SSU rRNA gene, PCR-RFLP, and conventional PCR are widely used. However, these techniques have limitations, such as difficulty in detecting mixed subtypes and point mutations. Furthermore, they are limited to identifying only subtypes 1–7 [5, 6].

The High-Resolution Melting Curve Analysis (HRM) technique, introduced in 1997, has proven to be highly sensitive, accurate, and efficient for mutation detection and disease diagnosis [7, 8]. HRM works by determining the melting temperatures of amplicons, which allows for unique subtype identification. Advances in fluorescent diagnostics, such as HRM, have improved subtype differentiation, making this technique valuable in both medical and veterinary applications. While sequencing is often needed for confirmation, HRM is increasingly recognized as a reliable method for subtype identification in parasitology [9, 10].

Currently, HRM has been successfully applied to the diagnosis of cutaneous leishmaniasis [11], echinococcosis [12], asymptomatic malaria [13], liver flukes like Fasciola sp [14]., and Giardia sp [15].

Gene analysis of the SSU rRNA has suggested at least 44 subtypes (ST1-ST44), although the validity of ST18-ST20 and ST22 remains controversial [16, 17]. Among humans, the most prevalent subtypes (over 90%) are ST1-ST4, with ST3 being the most common [18, 19]. ST4 is typically found in rodents, and ST5 in pigs [19, 20]. In birds, ST6 and ST7 are frequently detected [21]. Subtypes like ST1, ST3, and ST7 have been linked to gastrointestinal disorders, while ST4 is primarily found in rodents, indicating varying zoonotic potentials [19].

The clinical significance of Blastocystis subtypes is still under investigation. ST1, ST2, and ST4 have been associated with gastrointestinal symptoms, and their higher prevalence in symptomatic patients has been noted [17]. ST1 is particularly linked to irritable bowel syndrome (IBS), and ST2 with gastrointestinal issues, urticaria, and diarrhea, although it is also found in asymptomatic cases [4, 22, 23]. The anthroponotic subtype ST3 is associated with gastrointestinal symptoms and urticaria, while ST4 is commonly detected in diarrhea patients [23–25]. The transition from the vacuolar to amoeboid form has been suggested to correlate with clinical symptoms [26]. ST3 has been implicated in causing gastrointestinal symptoms and urticaria [22].

Khorasan Province, a region with significant human-animal interaction due to pastoral activities, provides a unique setting to study Blastocystis transmission dynamics and subtype distribution. This study aims to use HRM techniques to detect and differentiate Blastocystis subtypes. To the best of our knowledge, this is the first study in the region employing HRM for subtype differentiation.

Materials and methods

This study was approved by the Ethical Committee of Mashhad University of Medical Sciences (IR.MUMS.MEDICAL.REC.1400.328) in accordance with the Helsinki Declaration and guidelines.

Study design and sample collection

This cross-sectional descriptive study was conducted between July 2021 and 2023 in the southern part of Razavi Khorasan province, specifically in Torbat-Heydarieh city and its surrounding rural areas in the southwest region of Iran. A total of 730 stool samples were collected, comprising 207 human samples and 523 animal samples. Human samples were collected from individuals both with and without gastrointestinal symptoms, spanning a range of educational levels, clinical symptoms, and age groups from both urban and rural areas. Additionally, some participants had direct contact with domestic animals, while others did not. The region is characterized by widespread pastoral activities, and individuals living on the outskirts of the city often engage in livestock farming (unpublished data). The sample size was intentionally chosen to ensure comprehensive representation across diverse host groups and environmental conditions, providing robust statistical power for analyzing subtype distribution and potential risk factors.

Animal samples were collected from a variety of domestic and farm animals, including livestock (sheep, goats, and cattle) and companion animals (poultry, turkey, quail, and pigeon), which are prevalent in the region due to extensive pastoral and agricultural activities. Sampling was carried out at farms, households, and local veterinary clinics in both urban and rural areas. Stool samples were collected from animals under diverse conditions, including those with gastrointestinal symptoms and those appearing asymptomatic, to capture a broad spectrum of potential carriers.

The selection of animal subjects aimed to reflect their close interaction with human populations and the environment. Ethical guidelines and biosafety measures were followed during sample collection to minimize stress to the animals and prevent contamination of the samples. Including animals with varying health statuses, species, and living conditions strengthens the study’s ability to assess zoonotic and environmental transmission dynamics of the studied subtypes.

Stool examination

Human participants received stool containers and instructions for safe sample collection, while samples from domestic animals were collected using sterile gloves. Initially, the stool samples were subjected to macroscopic examination to assess consistency and detect visible parasites. Microscopic examination was performed using the wet mount method with normal saline and Lugol’s iodine solution. While these methods are reliable, they may have limitations in detecting all viable Blastocystis forms, especially at low parasite loads or when atypical morphologies are present.

Culture method

Negative stool samples for Blastocystis sp. were cultured in a two-phase culture medium designed to solidify deactivated human serum at 75 °C, along with a liquid phase comprising Ringer’s solution, homogenized egg albumin, rice starch, and streptomycin. This medium was specifically developed to enhance Blastocystis detection sensitivity. Stool samples were inoculated, and the supernatant was examined microscopically after 2 to 3 days. Positive cultures were stored at -70 °C for molecular analysis. While effective, the culture method’s limitations include the risk of missing certain viable forms if optimal growth conditions are not maintained.

DNA extraction

While culture methods were used to identify Blastocystis, we acknowledge the limitations of relying solely on culture-positive samples. To enhance detection sensitivity and include non-viable forms, future research should consider direct DNA isolation from stool samples as a complementary approach. DNA extraction was performed using the FavorPrep™ Stool DNA Isolation Mini Kit. Briefly, 200 mg of stool sample was transferred to a bead tube on ice. Samples were mixed with lysis buffer and proteinase K, incubated at 60 °C for 20 min, and centrifuged. The supernatant was then loaded onto a silica column, washed, and the DNA was eluted in 50 to 200 µL of elution buffer or deionized water. Extracted DNA samples were stored at -20 °C until further use.

Real-time PCR and HRM analysis

The partial SSU rRNA gene of Blastocystis sp. was amplified using specific primers (forward 5’-CGAATGGCTCATTATATCAGTT-3’ and reverse 5’-AAGCTGATAGGGCAGAAACT-3’), as described by Hamed Mirjalali et al. [1]. The choice of primers was based on prior validation, ensuring high amplification efficiency and the ability to detect various Blastocystis subtypes. Real-time PCR and HRM analysis were performed using a 20 µL reaction volume, consisting of 4 µL HOT FIREPol EvaGreen HRM Mix (Solis BioDyne Co, Estonia), 10.2 µL DNase/RNase-free water (SinaClon BioScience Co, Iran), 0.4 µL of HPLC-purified primers at a concentration of 10 µM, and 5 µL of DNA template.

The PCR cycling conditions included an initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 25 s, 57 °C for 30 s, and 72 °C for 30 s, with a final extension at 72 °C for 5 min. HRM analysis involved heating at 95 °C for 15 s, with the temperature gradually increasing from 75 °C to 95 °C at a rate of 0.3 °C/second. Changes in fluorescence were recorded at each temperature increment. The HRM assay was optimized for precision, with PCR replicates performed three times for each sample to assess intra-assay and inter-assay variability. Coefficients of variation (CV) were calculated to ensure reproducibility. Positive controls and non-template controls were included in every run to validate the HRM assay.

Temperature uniformity across the cycler block was verified by reamplifying samples in different positions during the same cycle. The methodology ensured reliable and consistent detection of Blastocystis sp., with HRM analysis providing high-resolution differentiation of genetic variations.

Result

A total of 730 stool samples were analyzed, comprising 203 human samples (116 males, 57.1%; 87 females, 42.9%) and 527 animal samples, including those from livestock and companion animals. The culture method demonstrated higher sensitivity compared to the direct examination method. Out of the total 730 stool samples, the culture method identified 68 (9.3%) positive samples, while the direct examination method detected 44 (6%) positive samples. Of the 68 positive samples obtained through the culture method, 50 were randomly selected for further analysis using the High-Resolution Melting (HRM) technique.

Human samples

Of the 50 randomly selected samples for HRM analysis, 21 (42.0%) were human samples. Among these, ST3 was the most frequently identified (10 cases, 47.6%), followed by ST2 (5 cases, 23.8%), ST1 (4 cases, 19%), and ST5 and ST7, each detected in 1 sample (4.8%). ST14 was not detected in human samples. Notably, no significant relationship was found between gender and infection rate (p = 0.056).

Animal samples

The 29 animal samples analyzed by HRM included samples from sheep, poultry, pigeons, turkeys, and quails. The results revealed a high prevalence of ST7 in poultry (13/14), whereas ST14 was the most common subtype in sheep (3/9). Notably, ST5 and ST14 were found exclusively in sheep.

Combined analysis

Overall, six subtypes were identified across human and animal samples: ST1, ST2, ST3, ST5, ST7, and ST14. ST3 and ST7 exhibited the highest prevalence in both humans and animals, suggesting potential zoonotic transmission (Table 1). The HRM analysis consistently produced reliable results, with melting temperature (Tm) values showing consistent differentiation between subtypes.

Table 1.

Characteristics of identified subtypes based on HRM analysis

Subtype	Mean Tm (°C)	SD	Intra-assay CV (%)	Inter-assay CV (%)
ST5	79.6	0.14	0.08	0.15
ST14	80.3	0.10	0.07	0.12
ST2	80.9	0.12	0.09	0.14
ST3	81.4	0.12	0.08	0.11
ST1	81.9	0.13	0.07	0.12
ST7	82.6	0.11	0.05	0.11

The HRM analysis consistently produced reliable results, with the melting temperature (Tm) values for each subtype remaining within the specified range (Tm ± SD), indicating good repeatability of the HRM technique.

Among the six identified subtypes, ST7 was the most prevalent (30%), while ST5 and ST14 each had a frequency of 6%. Human samples exhibited the greatest diversity of subtypes, with ST3 being the most frequent (10 cases). Notably, ST14 was not detected in human samples. In contrast, the lowest diversity was observed in samples from turkeys, quails, and pigeons, where only one subtype was identified per species. Due to the limited number of positive samples in these species, making conclusive statements about subtype diversity is challenging. Poultry samples displayed three different subtypes, with ST7 being the most common (13 cases). Sheep samples included four subtypes, with ST14 being the most frequent (three cases) (Table 2).

Table 2.

Distribution of identified subtypes among different hosts

Host	ST1	ST2	ST3	ST5	ST7	ST14	Total (Percentage)
Human	4	5	10	1	1	0	21 (42%)
Poultry	0	1	2	0	13	0	16 (32%)
Sheep	0	2	2	2	0	3	9 (18%)
Turkey	1	0	0	0	0	0	1 (2%)
Quail	0	0	0	0	1	0	1 (2%)
Pigeon	2	0	0	0	0	0	2 (4%)
Total	7	8	14	3	15	3	50 (100%)

The HRM analysis demonstrated high repeatability, with the melting temperature (Tm) values for all samples within each subtype remaining consistent. Specifically, the Tm values for different samples of a particular subtype were within the expected range (Tm ± SD), showing no significant variation. This highlights the reliability of the HRM technique in accurately identifying Blastocystis subtypes. Notably, although our study did not detect subtype 6, a previous investigation using PCR on similar samples did report its presence (Fig. 1).

Fig. 1.

This derivative melt curve compares the melting temperatures (Tm) of amplicons identified by the HRM technique for various Blastocystis sp. subtypes. The distinct points on the curves clearly highlight structural differences in the DNA sequences among the subtypes. Lower melting temperatures (e.g., ST5) are associated with sequences containing weaker bonds (higher A-T content), while higher melting temperatures (e.g., ST7) correspond to sequences with stronger bonds (higher G-C content). This data is essential for the rapid and accurate identification of Blastocystis subtypes

Discussion

Blastocystis remains a controversial parasite, with ongoing challenges in its detection, characterization, and pathogenicity in both human and animal stool samples. In developing countries, traditional microscopic methods, such as wet mount and diluted Lugol’s solution, are primarily used for detection [26]. However, in developed countries, molecular techniques have become the preferred approach due to their enhanced sensitivity and specificity. These techniques typically rely on specific primers to amplify target genes, allowing for more precise identification [27].

The current study revealed the presence of Blastocystis subtypes ST1, ST2, ST3, ST5, ST7, and ST14, determined through melting temperature (Tm) analysis following amplification. Notably, no co-infections with multiple subtypes were observed, which may be due to the sensitivity of the detection methodology or the specific characteristics of the samples studied. Subtypes ST7 (30%) and ST3 (28%) were identified as the most predominant.

Comparison with previous studies

Previous research has extensively investigated Blastocystis and its identification methods, primarily using molecular techniques such as PCR and sequencing. However, studies employing High-Resolution Melting (HRM) for subtype identification remain limited. For example, Rahimi et al. [1]. analyzed 72 stool samples from humans and animals in Iran using HRM, identifying subtypes ST1, ST2, ST3, ST6, ST7, ST10, and ST14, with ST3 being the most common. While ST3 was also the predominant subtype in their study, our research incorporated a larger sample size and broader geographic sampling, which could explain the difference in the predominance of subtypes, likely reflecting regional variations in the samples’ origin.

Similarly, Hussein et al. [28] studied the genetic diversity of Blastocystis subtypes in precancerous colons in Egypt, identifying ST3 (54.7%) as the most common. Our study, however, found ST7 to be more prevalent than ST3, suggesting potential regional differences in subtype distribution. In Hussein’s study, which focused on patients with gastrointestinal symptoms, subtypes associated with clinical symptoms and anthroponotic transmission (ST1, ST2, and ST3) were identified, while animal-related subtypes were absent. In contrast, our study included several subtypes from both human and domesticated animal sources, reflecting close human-animal interactions in Khorasan Province.

Cian et al. [29]. analyzed animal samples from French zoos and found ST3 to be the most prevalent subtype (60%). Differences in geographic location and the source of the samples likely account for the variation in identified subtypes. Their study identified subtypes ST1-ST5, ST7, ST8, and ST15, with ST1-ST3 accounting for about 67% of isolates. Our study, on the other hand, found ST1-ST3 represented approximately 59% of the total subtype distribution, supporting the idea of significant transmission between humans and animals, a pattern consistent across both studies.

In contrast, a large epidemiological study by Khaled et al. [30] in northern Senegal reported a high prevalence of Blastocystis (80.4%), with ST2, ST1, and ST3 being the most common subtypes. Additionally, Naguib et al. [31] studied domestic animals in Egypt and found the highest prevalence of Blastocystis in chickens, predominantly comprising ST7. These studies highlight the importance of considering geographical and host-specific factors when assessing subtype prevalence.

In Naguib et al. [21] study benefits a large-scale molecular epidemiological survey included 825 symptomatic/asymptomatic patients. Among 597 positive stool samples that shown a high level of infection rate of Blastocystis, in contrast to our study, 39.4% presented mixed infection while in the present study, no mixed infection was observed. ST3 (48.3%) was predominant subtypes followed by ST1 (39.5%), ST2 (10.8%), ST14 (1.1%), and ST10 (0.3%). High level presence of ST1-ST3 similar to our study in human samples, confirms this fact that these subtypes belong to human samples.

Zoonotic potential and human-animal interaction

Our study identified ST2 and ST3 among sheep fecal samples, alongside ST5, ST7, and ST14, reinforcing the notion of higher zoonotic transmission due to close human-animal interactions in Khorasan Province. This is in line with reports from Shahrekord, southwest Iran [4], where ST5 was the predominant subtype in ruminants, including sheep, and other studies in Egypt [32] that found similar subtype distributions.

Furthermore, studies conducted in Ilam, Iran [33], also reported a prevalence rate of Blastocystis in human stool samples (8.1%) identified by direct microscopy. Although our study employed more sensitive and accurate HRM techniques for detection, the overall infection rate and patterns in males (57.1%) and females (42.9%) showed no significant difference in gender, similar to findings from studies in Vietnam [19], where no gender-based variation was observed. The predominance of infection in males in some studies may be attributed to their higher levels of contact with domesticated animals, possibly due to pastoral activities and poorer hygiene practices.

Limitations and future directions

Although our study provides valuable insights into the prevalence and distribution of Blastocystis subtypes in both human and animal populations, there are some limitations. HRM proved to be a reliable method for subtype identification, but the absence of subtype 6 in our study, which was reported in other studies, suggests that further research with larger sample sizes is needed to comprehensively understand the full spectrum of Blastocystis subtypes. The lack of mixed infections also warrants further investigation to explore potential co-infections that might be missed with the current diagnostic techniques.

Conclusion

In conclusion, Blastocystis remains a challenging parasitic organism, detected in both humans and a variety of animal hosts. HRM proves to be an efficient and cost-effective method for rapid detection and differentiation of subtypes, especially in regions where access to advanced sequencing methods is limited. Our study identifies ST3 as the predominant subtype in humans and ST7 in animals, with poultry recognized as a key vector for transmission. Larger-scale studies with a One Health approach should be conducted to further evaluate the strengths and limitations of emerging techniques like HRM and to enhance our understanding of Blastocystis transmission dynamics across diverse environments.

Author contributions

A: Ghodratollah Salehi Sangani > B: Ahmad Hosseini-Safa > C: Bibi Razieh Hosseini Farash > D: Pooria Salehi Sangani > E: Mehdi Zarean > F: Elham Moghaddas > G: Saied Zoghdar Moghdam > H: Lida Jarahi > I: Ali Gholizadeh > J: Fariba Berenji ( A.B. and J suggested the topic and G. collected the samples and A.B. and C.E. and F. specified the method and D.I. Analyzed the samples and B. prepared Figs. 1 and 2 and H. performed the statistical analysis and I. wrote the main manuscript. All authors reviewed the manuscript.)

Fig. 2.

The normal melting curves for the subtypes identified are presented, with ST5 (Curve A) having the lowest melting temperature (Tm) at 79.6 °C, and ST7 (Curve F) exhibiting the highest Tm at 82.6 °C. The subtypes are arranged in order of their Tm values, starting with ST5 at 79.6 °C and increasing to ST7 at 82.6 °C. This arrangement highlights the differentiation of subtypes based on their unique melting points

Funding

This study received financial support from Mashhad University of Medical Sciences under Project No: 992196.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethical approval

This study was approved by the Ethical Committee of Mashhad University of Medical Sciences (IR.MUMS.MEDICAL.REC.1400.328), in accordance with the Helsinki Declaration and ethical guidelines. The project was part of the thesis titled “Thesis number: 992196.” During the study, we only accessed anonymized samples, with all personal identifying information, including names, addresses, and phone numbers, removed before the samples were provided to the research team. Consequently, we did not have access to any personally identifiable information either during or after data collection. The research adhered to strict ethical standards to ensure the privacy and confidentiality of all participants.

Consent to participate

In this study, we utilized daily and weekly archived stool samples from patients who attended our medical centers. These samples were provided with a coding system, and we did not have access to any identifying information, such as the patients’ names, addresses, or phone numbers. As a result, we did not obtain informed consent directly from the patients, as their personal identities were not involved in the research process. The ethical committee reviewed and approved the use of these anonymized samples, waiving the requirement for informed consent due to the absence of identifiable information.

Consent for publication

All participants and institutions involved in this study provided consent for the publication of the anonymized results.

Clinical trail

Not applicable.

Competing interests

The authors declare no competing interests.