Molecular characterization of Strongyloides stercoralis in Mazandaran Province, North of Iran

1. Introduction

Strongyloides stercoralis, a parasitic nematode in humans, lives inside the mucosa of the small intestine and causes severe clinical manifestations of strongyloidiasis (1). Most infected individuals are asymptomatic, while some patients have a variety of cutaneous, gastrointestinal, or pulmonary symptoms (2). Clinical symptoms of strongyloidiasis include acute, chronic, and disseminated infections (3). The most severe complications of S. stercoralis infection occur in patients with immune deficiency and, as a result, infect and spread the larva to multiple organs, including the brain (4). This parasite has infected 100-200 million people in 70 countries (5). So far, the prevalence has been reported between 4% and 50% of several regions worldwide (6). A total of 347 deaths due to strongyloidiasis were reported in the United States from 1991-2006 (7). The definitive diagnosis of strongyloidiasis is usually based on observation of the larvae in the stool exam (8). However, in most uncomplicated cases, the amount of parasite in the intestine is very low (9). For detecting S. stercoralis larvae in stool samples, various techniques have been used. These methods include Baermann concentration, Harada-Mori culture, agar plate culture (APC), and Ethyl acetate formalin concentration (10). So far, different immunological diagnostic methods have been used with variable specificities and sensitivities. Although in endemic areas, serologic responses are well-established after treatment, they are not particularly specific due to the interaction with other parasites, including Filariasis, Ascariasis, and Schistosomiasis (11). The PCR techniques have progressed greatly and have been used to detect various intestinal parasites in fecal specimens (12). Recently, the detection of parasite DNA in fecal specimens using Real-Time PCR has proven that this method is specific and sensitive for the detection of S. stercoralis infections (13). Assessing such techniques is critical to overcome the limitations of current diagnostic methods. Strongyloidiasis is still endemic in some Iranian provinces, including Mazandaran. In 2007, the infection rate in the Mazandaran province was 4.9% (14). In a study conducted in Gilan province, 42% of the people with eosinophilia were reported positive for S. stercoralis infection (15). The present study was conducted to determine the molecular characteristics of S. stercoralis collected from residents of Mazandaran, the endemic region of the parasite in Iran.

2. Materials and Methods

2.1. Sample collection

From April 2017 to September 2017, a total of 2,195 human stool samples were collected from various regions of Mazandaran province, North of Iran. In this survey, individuals profile was as follows: 1,170 (53.3%) women and 1,025 (46.7%) men, 763 (34.8%) living in urban and 1,432 (65.2%) in rural areas, hospitalized people were 680 (31%), and outpatients referred to the hospital were 1,515 (69%).

2.2. Microscopic study

All stool samples were analyzed using the formalin ether method (16). After performing parasitic experiments, the obtained larvae were washed with distilled water several times and stored in 70% ethanol at 4°C for molecular analysis. The larvae of S. stercoralis were detected based on morphological characteristics with optical microscope and classification keys of nematodes (17).

2.3. DNA extraction

The positive fecal samples of S. stercoralis and 300 random samples were selected for DNA extraction. About 5 g of each fecal sample stored in 70% ethanol was dissolved in 4% acetic acid, and the suspensions were transferred to a tube through two layers of sieve, and then 3 ml of ether was added to it. Then, the tube was shaken slowly and centrifuged at 1500 g for 2 min. The pellet was washed three to four times with distilled water. The DNA of stool specimens was extracted by DNA purification kit (YTA Genomic DNA Extraction Mini kit for Tissue). In summary, the first 200ul of buffer TG1 and proteinase k was added to the samples. Then, incubated at 60°C for overnight. This method is carried out according to the manufacturer's instructions. Finally, the extracted DNA was eluted with 50μl Elution Buffer and was kept at -20°C for molecular study.

2.4. Conventional PCR

A set of primer pairs was used for conventional PCR to activate the amplification of the 509-bp target in a PCR reaction in the mitochondrial cytochrome c oxidase subunit 1 (Cox1) gene. For amplification of Cox 1 of S. stercoralis, primers CoxF (5′-TGG TTT GGG TAC TAG TTG-3′) and CoxR(5′-GATGAG CTC AAA CTA CAC A-3′) were used (18). The PCR reaction was done using the following reaction mixtures: 10 uL Taq DNA Polymerase 2X RED PCR Master Mix (Ampliqon, 2mM MgCl2), 10 pmol/µL of each primer, 250ng/µL of DNA template, and distilled water up to a final volume of 20 µL. The reaction was done for 35 cycles: (94°C for 30s (denaturation), 55°C for 30s (annealing), and 72°C for 30s (extension)), with an initial denaturation (1cycle at 94°C for 4 min) and a final extension (1cycle at 72°C for 4 min). To confirm the optimization of the reaction process, the extracted DNA from larvae of the filariform was used as a positive control, and three samples that were negative by the formalin ether method were used as a negative control. A 5 uL of PCR product was electrophoresed on a 1% agarose gel in Tris-Acetic acid EDTA (TAE) buffer, and the band was visualized using an ultraviolet illuminator. To confirm the results of PCR, four positive products randomly were sent for two-way sequencing and sequenced by the ABI3730XL sequence analyzer (Macrogen, Korea). Sequences obtained edited and aligned with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and compared with reference sequences from GenBank. Then, the phylogenetic tree was built for S. stercoralis with the maximum likelihood algorithm using molecular evolutionary genetics analysis software (MEGA), including sequences of demonstrator species of Strongyloides infecting humans from the GenBank.

2.5. Data analysis

All the collected data was analyzed using SPSS (version 18.0) to determine the significant statistical difference between observations using the χ² test.

3. Results

3.1. Microscopic study

A total of 2,195 stool specimens were examined in this study using optical microscopy, and 183 of them were found to be infected with different parasites. Twenty-one (0.95%) stool samples were found to be positive for S. stercoralis, and 162 (7.38%) samples were positive for other parasites (Table 1). Among these parasites, Entamoeba histolytica/dispar, Giardia lamblia, and S. stercoralis were the most frequent. Among the patients who had S. stercoralis, 15 (71.43%) people were male, 6 (28.57%) people were women, 3 (14.29%) patients were living in urban, 18 (85.71%) patients were from rural areas, hospitalized people were 6 (28.58%), and outpatients referred to the hospital were 15 (71.42%).

3.2. Molecular analysis

The PCR product showed a band of ̴ 500bp in gel electrophorese (Figure 1). All 21 samples that confirmed S. stercoralis in fecal samples were found to be positive by PCR method. In addition, among 300 stool samples that were randomly selected, which were negative for S. stercoralis by formalin ether method, rechecked using the molecular method, and all of them were found to be negative.

The sequence of the samples had 99% overlap with S. stercoralis in GenBank. The sequences isolated in this study are registered in the GenBank and are available with an Accession number of MH921561-64.

Phylogenetic relationships of the mitochondrial cytochrome c oxidase subunit 1 (Cox1) gene of S. stercoralis inferred by the maximum likelihood algorithm, are shown in Figure 2.

4. Discussion

Strongyloidiasis is very important in patients with immune deficiency, which causes hyperinfections and disseminated syndromes and can be fatal if not well treated (9, 10). This parasite is rife in the tropical and subtropical regions of the world, and about 100 million people in 70 countries have been infected with this parasite (19).

In Iran, in the Northern and Southern coastal provinces where there is sufficient moisture, this parasite is reported endemic (20). During the last decades, due to improvements in the level of health and increasing public awareness, the prevalence of soil-transmitted parasites has declined substantially in the country. However, due to the fact that S. stercoralis causes autoinfection in the host body, it is still prevalent in the endemic areas. The sensitivity and specificity of various methods for the diagnosis of S. stercoralis infection have been reported differently in various studies. For this reason, the actual prevalence of the methods is often underestimated (14). The APC in stool specimens is among the best methods for diagnosing S. stercoralis (13). However, this procedure requires multiple samples of fresh stool and an experienced person for diagnosis. Therefore, PCR-dependent methods can be used as an appropriate alternative method for detecting S. stercoralis parasites. Various studies have been performed based on the PCR technique, and each has shown different results in detecting S. stercoralis DNA in stool samples (11). In this study, a molecular method was selected for the validation and verification of S. stercoralis specimens, and contributed to insight into its prevalence in the studied specimens. The prevalence (0.95%) for S. stercoralis in this study is almost similar in comparison to other studies

conducted in Iran. Although, there are studies that are not consistent with our research. In 2016, among people who were mentally impaired in Gilan province, 1.2% of them were found infected with this parasite (21). Ghasemikhah et al. (2017) studied 1,800 patients in Tabriz and reported the prevalence of S. stercoralis to be 0.3%, which is lower than our study (22). In other studies conducted in Iran, the rate of infection of this parasite varies with respect to the target population and diagnostic methods. In a study conducted by Sharifdini et al. (2018), based on nested PCR, 9.7% of people in the Northern province of Khuzestan were infected with S. stercoralis (23). Kia et al. (2007), with parasitological methods, revealed that 4.9% of rural residents in Mazandaran province were infected with S. stercoralis (13). Among those who were in  mentally retarded institutions in southern Iran in 2012, the prevalence of S. stercoralis was reported at 3.17%, using the formalin ether concentration method (24). In addition, the rate of this parasite was reported 2.1% in the people of the rehabilitation center of Mazandaran province in 2015 (25). The present study showed that males are more infected with S. stercoralis than women (71.5% versus 28.5%; P<0.05). Several studies have shown that men are more infected than women (19). This can be due to outdoor activities and the work of man in agricultural and horticultural land. The selection of an appropriate method for detecting strongyloidiasis to determine its prevalence can help us in the control and prevention program. The PCR technique is a highly sensitive and specific method for detecting protozoans and parasitic infections in fecal specimens (11). The results of this study showed that Single-PCR could detect all specimens that microscopically were positive. In addition, a suitable and cost-effective method can be used to detect S. stercoralis. The study of Moghaddassani et al. (2011) showed that Single-PCR was more effective than Nested-PCR in detecting S. stercoralis in stool samples (26). The differentiation of Hookworms from S. stercoralis larvae is somewhat difficult in the parasitological methods, including the Baermann method. However, molecular diagnostics and PCR solves this problem well. In conclusion, the results showed that conventional PCR could detect all specimens that microscopically were positive.