Molecular diagnosis of echinococcosis in patients based on frozen paraffin tissue samples and fixed formalin and hydatid cysts isolated from livestock in a slaughterhouse

Abstract

Background:

Various genotypes of Echinococcus granulosus have been studied in high-disease-risk areas and identified as causative agents of cystic echinococcosis (CE). This study was performed to examine and identify the molecular hydatid cyst in the dissected human specimens in paraffin tissue, and the dissected animal cyst was characterized using the DNA polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 1 (ITS1).

Materials and Methods:

To determine the molecular properties of E. granulosus, 20 hydatid cyst samples (including 6 sheep samples, 9 camel samples, and 10 human paraffin samples) were collected from Zahedan and Zabol cities. After DNA extraction, molecular PCR was performed, and RFLP was evaluated. In this study, the Taq1 endonuclease cleavage enzyme was used.

Results:

The patterns of DNA bands found in the isolates from human CE and animal bladder cysts were the same, as indicated by the results of ribosomal DNA-ITS1 amplification from E. granulosus. Two nested primer pairs were used. The rough size of the enhanced ITS1 piece was 444 and 391 base pairs (bp), individually. After cutting the PCR product with the Taq1 enzyme, the patterns of the fragments revealed that the samples had two identical RFLP patterns. The aftereffects of this study showed that the parasite genotypes confined to sheep, camels, and people had hereditary changes.

Conclusion:

The transcendent type of E. granulosus sensu lato in the area is E. granulosus sensu stricto, which featured the meaning of the sheep/canine cycle in human transmission. Albeit the band profile in the camel is now and again like the sheep strain, RLFP can be recognized utilizing the PCR strategy, and two differentiating band profiles using the chemical were found in this review.

INTRODUCTION

The taxonomy of the genus Echinococcus is well established and includes 8–10 species according to the latest studies, 6 of which are described as pathogenic to humans: Echinococcus multilocularis, Echinococcus granulosus sensu stricto, Echinococcus canadensis, Echinococcus ortleppi, Echinococcus vogelii, and Echinococcus oligarthrus.[1]

Cystic echinococcosis (CE) is a global zoonotic disease caused by the larval stage, the metacestode stages of E. granulosus.[2,3] CE is caused by three species of the E. granulosus complex (E. granulosus sensu stricto, E. canadensis, and E. ortleppi).[1] The adult worm lives in the dog’s intestine, and the dog is the definitive host. Its larva or bladder cyst is formed in the herbicide body as the intermediate host. People accidentally develop hydatid cysts from eating contaminated vegetables, dog contact, and dust. The treatment of the disease is important in humans because it affects vital organs (such as the liver and lungs), and in herbivorous animals, it causes significant livestock damage.[3,4] The disease is widespread in most areas, especially in rural areas where slaughterhouse waste is unhealthy and unsuitable. Compared to other Echinococcus species, E. granulosus is more important in Iran.[5] The adult worm of this parasite is separated from dogs, jackals, and wolves in Iran.[5,6] Various studies conducted in Iran and other countries have shown the diversity of hydatid cysts with a frequency of 1.5%–70%.[7,8] In recent years, there is evidence that not only is the prevalence of the disease increasing worldwide but also is severity in humans and animals in many countries.[9,10] Further, human cases are constantly reported from different parts of Iran.[11] In areas where the disease is biologically endemic, there is usually a relatively large amount of genetic variation in E. granulosus, with many genetic strains adapted to various intermediate hosts.[12]

Most human isolates belong to sheep genotype G1, the most common genotype worldwide. Ten genotypes have been identified in various intermediate and definitive hosts. E. granulosus was subdivided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10).[13] However, some studies suggest that some strains are more pathogenic to humans than others, and some may be neurotrophic, as the observations showed that human cases of cerebral hydatid disease showed genotype G6 in the brains of Iranian patients. These results suggest that genotype G6 tends to infect the brain in human infections.[14]

More studies on the genetic characterization of Echinococcus cysts with different anatomical localizations are needed to determine whether other E. granulosus genotypes have preferential localization in humans or other hosts.[15] Polymerase chain reaction (PCR)-based methods can be used to detect E. granulosus DNA in various samples, including cyst fluid, blood, and feces. The specific primers used for PCR amplification can target various regions of the parasite’s genome, such as the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene, the 18S ribosomal RNA gene, or the internal transcribed spacer (ITS) region.

Identifying strains is important to control and prevent the disease.[16] Therefore, accurate identification of genotypes of E. granulosus in endemic areas includes gene sequence, restriction fragment length polymorphism (RFLP) of ITS1 ribosomal DNA fragment, and comparative analysis of homologous DNA sequences.[17] The study of molecular characteristics of strains and genotypes of E. granulosus causing paraffin-fixed human hydatid cysts using the RFLP method is one of the new methods used in this study. Sistan and Baluchestan Province is a high-risk area for hydatid cyst disease due to its special climatic conditions, livestock rearing, especially camel and sheep rearing as one of the most important and common occupations of the people in the region, and dog keeping by shepherds and villagers.

MATERIALS AND METHODS

Specimens collected in the hospital during the decade (1990–1999), in which the cyst diagnosis was established, were brought to the laboratory for molecular analysis. Human surgical samples were also collected. These cysts were taken from Imam Ali (AS) Hospital in Zabol and Zahedan, Katam Al-Nabie Hospital in Zahedan, and Amir Al-Momenin Hospital in Zabul. Furthermore, ten animal samples (including six sheep and four camels) were collected from the industrial slaughterhouses of Zahedan and Zabol cities and brought to the laboratory (Zabol University of Medical Sciences).

Deparaffinization of paraffin samples

In the pathology laboratory, two 10-μm-thick sections were prepared from the tissue blocks for each patient, and the extra paraffin was removed. We used disposable blades and carefully cleaned what might have come into contact with the samples between the different tissue blocks.[18,19] The collection consisted of postoperative frozen and FFPE samples (ten samples). The diagnosis of echinococcosis was initially made using imaging techniques and histopathology. The year of surgery or the date of paraffin encapsulation was noted in this study. In the pathology laboratory, 6–92-μm sections of paraffin blocks of each patient were placed in 1.5-μL tubes. To deparaffinized add 9 mL xylene to the tube to cover the tissue sections to dissolve the paraffin and stored at 37°C for 10 min. The samples were then centrifuged at 15,000 g for 5 min, and the liquid was discarded. After the samples were completely deparaffinized by repeating the xylene washing step. Rehydrate the tissue sections by immersing the slides in a series of graded ethanol solutions (100%, 95%, 70%, and 50% ethanol) for 2–3 min each vortexing, and centrifugation at 14,000 rpm for 5 min; by washing the slides in distilled water for 5 min to remove any remaining ethanol finally, the sediments were air-dried for 5 min and was blotted them gently with a clean paper towel.[20]

DNA extraction

For DNA extraction, the hydatid cyst samples kept in 72% alcohol were washed with phosphate-buffered saline at a pH of 0.7. Then, DNA extraction was performed using DNA extraction kits (Azmaelixir Pajooh company, Taiwan). We analyzed DNA cases using the PCR, followed by RFLP.

For amplification of the ITS1 fragment, in this study, two pairs of primers were used as nested primers. The primers we provided are external, EGF1, and EGR2, designed to amplify a larger DNA fragment from the target DNA in the first round of PCR.. The nested primers, BD1 and 4S, are designed to amplify a smaller region within the first PCR product in the second round of PCR. The first primers were: forward, EGF1 (5 CCA AAC TTG ATC ATT TAG AGG AAG3) and reverse, EGR2 (5 TAT GGG CCA AAT TCA CTC ATT ACC 3) as forward and reverse outer oligonucleotide primers. Prepare the first round of PCR by combining the reagents in a PCR tube (1 μL of extracted DNA, 1 μL of each outer primer [EGF1 and EGR2], 25 μL of PCR master mix [Taq polymerase, dNTPs, buffer, etc.], and 23 μL of nuclease-free water). We applied the first round of protocol PCR with the following cycling conditions: initial denaturation 95°C for 5 min, 95°C for 30 s, annealing 55°C for 30 s, extension 72°C for 2 min, and final extension 72°C for 10 min.

We kept it at 4°C. The first PCR product was diluted 1:100 in nuclease-free water to reduce nonspecific amplification. In the second round of PCR by combining the reagents in a PCR tube (1-2 μL of the first PCR product [as template DNA], 1 μL of each nested primer [BD1 and 4S], 12.5 μL of PCR master mix [Taq polymerase, dNTPs, buffer, etc.), and 35.5–36.5 μL of nuclease-free water). We used inner primer BD1 (5 GTC GTA ACA AGG TTT CCG TA 3) and 4S (5 TCT AGA TGC GTT CGA ART GTC GAT G 3) as forward and backward.[21,22,23]

Protocol second cycling conditions round were as initial denaturation 95°C for 5 min, denaturation 95°C for 30 s, annealing 55°C for 30 s, extension: 72°C for 1 min, and final extension 72°C for 10 min. After the second round of PCR is complete, we used electrophoresis. Ethidium bromide staining was used to visualize the product after running it on an agarose gel. The bands were clearly visible on the agarose gel under UV light.

Enzymatic digestion

The DNA bands were observed using a computer which was connected to Gel Doc apparatus. For enzymatic digestion of the PCR product (according to the kit’s protocol), two separate endonuclease-degrading enzymes Msp1 (which cut-point in DNA base is CGC/C) and Taq1 (which cut-point in DNA base is CGA/T) with the following properties and concentrations were used for each isolation in X10 buffer, prepared at 37°C for 6 h–10 h (as construction of kit). For enzymatic digestion of PCR products, enzyme digestion was done in a total reaction of 20 μL, including 2 μL of buffer, 10 μL of PCR products, and 1 μL of the enzyme (10 units). The temperature was 62°C as a protocol procedure for Taq1 and incubation time was 1 h–2 h. For MspI incubation, the temperature was 37°C and incubation time was 1 h–2 h. We do not have any digestion with MspI.

After enzymatic digestion, the RFLP product was run on a 3% agarose gel in a TBE buffer at a voltage of 50–100 mV. DNA bands were observed, and images were generated using a channel gel instrument connected to a computer.

RESULTS

Polymerase chain reaction results

In the first step of the molecular analysis, DNA was extracted from the germ layers of hydatid cysts using extraction kits (Azma Alksir Pajooh Company, Taiwan), and the concentration of the extracted DNA was measured using a spectrometer.

First, the ITS1 region of E. granulosus was amplified by PCR and then compared to RFLP templates using two endonuclease enzymes. The liver, lung, femoral, and renal cyst specimens were operated on in both males and females. The mean age of the patients was between 14 and 66 years.

The next step was to perform PCR on the device according to the given program, where DNA amplification was performed by specific primers designed for the ITS1 gene. The PCR product obtained from ITS1 amplification was patterned in number and size of a single-stranded DNA fragment in human and animal isolates. The pattern of PCR product bands was observed in terms of band size depending on the type of primer. The band size with EGF1and EGR2 primers was 444 base pairs (bp), and a band of 391 bp was observed with BD1 and 4S primers, indicating a genotypic difference in the pattern of amplified DNA fragments from hydatid cysts [Table 1]. After performing PCR, the samples on the gel were taken to ensure the amplification of the desired fragment [Figures 1 and 2].

Table 1.

Approximate size of Echinococcus granulosus DNA fragments after polymerase chain reaction amplification

Primer	Fragment size of isolated human DNA (BP)
EGF1/EGR2	444
BD1/4S	391

BP: Base pairs, EGF: Epidermal growth factor, EGR: Exhaust gas recirculation

Figure 1.

Electrophoresis of polymerase chain reaction product of Echinococcus granulosus parasites amplified with BD1 and 4S primers based on the internal transcribed spacer 1 ribosomal DNA gene on 1% agarose gel, M: DNA marker (leader) 100 bp. Line C; standard sample, line 1-4 patient samples (444 bp); N; negative sample M: Marker 100 bp

Figure 2.

Electrophoresis of polymerase chain reaction product of Echinococcus granulosus parasites, which are amplified with 4S/BD1 DNA primer based on internal transcribed spacer 1 rDNA gene on 1% agarose gel. DNA marker (leader) 100 bp, Line N: negative sample (without DNA), Line 1 standard sample, line 2 samples of the patient (391 bp)

In summary, while the size of the PCR product obtained using the outer primers EGF1 and EGR2 is smaller than expected, the size of the PCR product obtained using the nested primers BD1 and 4S is within the expected range, indicating successful amplification of the target DNA.[24,25]

Polymerase chain reaction–restriction fragment length polymorphism results

The third step was to perform PCR-RFLP. As mentioned above, the desired fragment was amplified by PCR. The limiting enzyme Taq1 with different nucleotide sequences was used for the digestion of E. granulosus DNA endonuclease by the PCR of hydatid cyst samples. The enzymatic digestion of E. granulosus DNA from human and animal isolates on agarose gel and the pattern of enzymatic digestion with the above enzyme was as follows [Table 2]:

Table 2.

Approximate number and size of DNA fragments Echinococcus granulosus by the nested PCR method after enzymatic digestion with restriction enzyme

DNA bands and enzyme digestion	Primer size of DNA band fragments with EGF1/EGR2 (BP)	Primer size of DNA band fragments with BD1/4S (BP)
Primary band	444	391
Taq1	294–150	281–110

BP: Base pairs, EGF: Epidermal growth factor, EGR: Exhaust gas recirculation

Taq1 enzyme

Amplification of E. granulosus DNA from humans and animals yielded a band of size 444 bp with EGR2/EGF1 (as primers), and digestion with Taq1 enzyme showed two band patterns of sizes 150 and 294 bp [Figure 3, Line 6 and 7]. When E. granulosus DNA was primed with 4S/BD1 primer, a 391 bp band resulted. After enzymatic digestion with Taq1, two DNA bands of 110 and 281 bp were observed [Figure 3, Line 1-3]. In human and animal isolates [Figure 3].

Figure 3.

The pattern of Echinococcus granulosus DNA bands in human and animal isolates after enzymatic digestion of internal transcribed spacer 1 fragments by the RFLP method with the Taq1 enzyme. Lin1: the size marker. Lines 2, 3, and 4: DNA amplified with 4S/BD1 DNA primer (391-bp band) digested by the Taq1-restricted enzyme. Line 5 is a gap, Line 6 and 7 DNA bands amplified with EGR2/EGF1 as outer primers (444 bp band) break to 150-294 bp bands, digested by Taq1-restricted enzyme

DISCUSSION

The amplicon size generated by PCR amplification using specific primers can vary depending on the target region and the primers used. For example, primers targeting the COX1 gene may generate an amplicon of around 450–550 bp, whereas primers targeting the ITS region may generate amplicons of around 200–400 bp.[26] Amplicons of 444 and 310 bp were generated in the present research applying two primer pairs as outer and inner primers, respectively.

The study and identification of isolates of human and sheep hydatid cysts using the mitochondrial Co × 1 gene in Sistan and Baluchestan Province have been studied limitedly.[27] On the other hand, studying the molecular properties of E. granulosus genotypes obtained from paraffin-fixed human hydatid cysts compared with animal strains using the RFLP method is one of the new methods used in this study.

Sistan and Baluchestan Province is a high-risk area for hydatid cyst disease due to its particular climatic conditions, livestock farming, especially camel and sheep farming as one of the important and widespread occupations of the people of the region, and dog keeping by shepherds and villagers. A lot of camel meat is consumed in Sistan and Baluchestan Province. This has led to increased demand and slaughter, resulting in increased contact between hydatid cyst-infected organs and definitive hosts, thus continuing the parasite’s evolutionary cycle and increasing environmental pollution. The presence of E. granulosus eggs on camel skin may also pose a risk of contamination for humans and camels themselves.

It should be noted that Sistan and Baluchestan Province border neighboring countries such as Afghanistan and Pakistan and there is no control over the introduction of diseases into the country of Iran. Further, the prevalence of hydatidosis is very high in these countries.[28] Other reasons, such as differences in environmental conditions critical to the survival of the parasite in the environment, the number and severity of infection of definitive hosts, the number of wild carnivores, and the condition of wildlife in the area, are related to the severity of the disease.[29] Studies conducted on intermediate hosts in different parts of Iran have revealed that two strains are present in Iran: camel–dog G6 and dog–sheep G1. The predominant strain in livestock in Iran is G1. In addition, 11% of G1 cases have been reported in humans.[19,30,31] This indicates that farm animals are an important source of human infection and underscore the importance of control and prevention programs. Further, previous molecular studies have shown that the most important and predominant strain infecting Iranian cows is the G1 strain,[31] which agrees with the results of the present study. Most isolates from sheep, cattle, goats, and humans produced patterns comparable to those acquired with the standard sheep strain, including those from Afghanistan.[4] In the present study, Taq1 enzyme with limited activity and different nucleotide sequences were used to digest E. granulosus endonuclease DNA in human and animal isolates by the RFLP method. According to previous studies in this area, the E. granulosus strains determined by the number and weight of the bands are G1-G3 in sheep and human hydatid cyst samples and G6-G7 in camel hydatid cyst samples; the subtraction of these strains in each grouping from each other is only by the sequencing method possible. In the first study by Zhang et al. in 1998, four cases of a bovine hydatid cyst from four isolates were identified by sequencing in different regions of Iran.

Fasihi Harandi et al. conducted a study on 32 bovine samples in different parts of Iran in 2002.[4] Using the PCR-RFLP method, they reconfirmed the existence of two strains in sheep and camels in Iran. However, restriction enzyme shows that there may be subspecies in this region [Figure 3].

In another 2007 study from northern and western Iran, Rostami Nejad et al. collected three bovine hydatid cyst samples and then extracted DNA. As previous studies have confirmed the presence of two strains in Iran, new specific primers on 12S RNA for G1 and G6 were designed. Amplification of this gene fragment revealed a band of 259 bp for G1 and 676 bp for G6. Primer G1 failed to replicate G6 and vice versa. All samples belonged to strain G1. After sequencing and comparing the data, the results were similar to previous studies, and the sequences were consistent with the GenBank database.[7] Sharbatkhori et al. examined 92 isolates from sheep and cattle in different regions of Iran in 2009. The Single-Strand Conformational Polymorphism (SSCP) technique was performed on CO1 (cox 1 gene) and ND1 (Nad1 gene) genes from the mitochondrial genome to determine the strain. E. granulosus 5 and 9 were detected by electrophoresis. Their results showed that 100% of the bovine samples belonged to G1-G3 or sensu stricto.[22]

Shahnazi et al. in Isfahan identified G1 and G6 strains in 94 cows using the PCR-RFLP technique on the ITS1 gene and sequencing of CO1 and ND1 genes. Of the 94 cows, 1 had a sheep genotype, and 5 had a camel genotype.[32]

Gholami et al. examined fertile bladder cyst protoscolices from 99 bovine and ovine liver isolates using PCR-RFLP on the ITS1 gene, with 5 endonuclease restriction enzymatic digests of Alu1, Msp1, EcoR1, Hha1, and Taq1. in Sari in 2009 and Gorleston in 2012 only the G1 strain was reported as a sheep strain.[33] In a study by Parsa et al. in 2010, a G1 strain was detected in 27 samples of hydatid cysts in Lorestan Province by PCR for ITS1-RFLP genes with four enzymes: TaqI, AluI, HpaII, and RsaI.[34] The results of the present study and previous studies conducted in Iran and other countries indicate the importance of sheep in hydatid cyst infections. However, it should be remembered that the number of studies on camel hydatid cysts is not comparable to the number of studies on sheep, but the importance of camels in hydatid infections cannot be ignored.

CONCLUSION

In Sistan and Baluchestan Province, great importance should be attached to camels regarding infections, and more studies should be conducted in this regard. To substantiate the importance of the role of ovine strains in the development of human hydatidosis, similar molecular studies on the occurrence of the parasite in humans should be conducted. If the role of the ovine strain in human infection is confirmed, practical steps can be taken to prevent and control infection in humans and animals, including the production of appropriate recombinant vaccines against hydatidosis. Education and reconnaissance in the area of unscrupulous slaughtering of livestock without subsequent monitoring of sanitary facilities are also considered important. Genotypic differences and similarities between the sizes of E. granulosus DNA band fragments by the PCR-RFLP method indicate the presence of more distinct parasite genotype variations than expected in Sistan and Baluchestan Province.

Ethical consideration

Since we are working with a harvested cyst that was sent to the pathology laboratory for human samples and the animals at the slaughterhouse, there were no unethical practices. The study was approved by the University Research and Ethics Committee (code: IR. ZBMU. REC.1399.014), and all procedures were performed following institutional guidelines for the care and use of humans and animals.

Limitation

As we occasionally work with human samples, we lack DNA due to the need for paraffin extraction; on the other hand, we could not obtain samples from some camels.

Financial support and sponsorship

The study was supported by grants from the deputy of Research of Zabol Medical University.

Conflicts of interest

There are no conflicts of interest.