Cytology and Genetic Analysis of Liver Echinococcosis in a Woman Came from Bolivia ()
1. Introduction
Echinococcosis is a global and zoonotic helminthic disease caused by Echinococcus sp. tapeworm larvae [1]. Intermediate hosts, such as wild herbivores, livestock and small mammals, as well as accidentally infected humans receive their infections through ingestion of contaminated food and water with Echinococcus eggs from the feces of the definitive hosts, such as dogs and foxes, etc. [2]. Nine species of Echinococcus have been reported globally, with confirmed zoonoses caused by E. granulosus, E. multilocularis, E. vogeli, E. oligarthra, E. canadensis and E. ortleppi; while no zoonotic evidence for E. equinus, E. felidis and E. shiquicus [3] [4].
Among those various Echinococcus spp., the most detected species in human echinococcosis (CE) is E. granulosus, which parasitizes in dogs and livestock globally [5]. Based on the previous molecular taxonomic findings, E. granulosus has been regarded as genotype complex, i.e. E. granulosus sensu lato (s.l.) comprising genotypes 1 to 10 (G1 - G10). The E. granulosus s.l. further split into five species as like E. granulosus sensu strico (s.s.) (G1/G2/G3 genotypes), E. equinus (G4 genotype), E. ortleppi (G5 genotype), E. canadensis group (G6/G7/G8/G10 genotypes), and E. felidis (lion strain) [6] [7] [8] [9] [10].
Most cases of liver echinococcosis in Japan have been reported Echinococcus multilocularis, and most of which were detected in Hokkaido, north of Japan [11]. Meanwhile, E. ortleppi infection was first reported in a herd of cattle in Holland; thus, it was named a cattle strain [6] [7]. Hosts of E. ortleppi have been reported from cattle, camels, pigs, goats, sheep and wild animals, and humans in many regions, Europe, Africa, the Middle East, Asia and Latin America [12] [13]. However, E. ortleppi infection has not been reported in Japan. Although the patient came from Bolivia, Latin America before two years, this is the first case report including the cytology and genetic analysis of liver echinococcosis in Japan.
2. Case Report
A 24-year-old woman, coming from Bolivia two years before, visited to our hospital complaining of high fever (37.9˚C) and general fatigue. Her laboratory data showed high CRP [14.51 (≤0.3) mg/dL] and a relatively high WBC [9.68 × 103 (4.0 - 9.0 × 103)/mL] and a relatively high ratio of eosinophilic 8.2 (0.2 - 6.8)% (Table 1). An abdominal ultrasonography (USG) (Figure 1(a)) and a plain CT showed a cystic lesion, 42 mm in diameter, in right lobe of her liver, suggesting a liver abscess. Calcification at the margins and low concentration bands were also seen (Figure 1(b)). Whole body CT and USG examinations, showed no evidence of extra-hepatic invasion or metastasis. After then, fine-needle aspiration cytology (FNAC) was performed with the help of a 22-gauge needle under USG. FNAC material was smeared on glass slides followed by wet mount, fixation in 95% ethanol for Papanicolaou stain and air-dried for May-Giemsa stain. Many protoscoleces were found (Figure 2(a)) and hooks and suckers on the tip of the scolex were also recognized in unstained wet mount smears (Figure 2(b)). Basophilic stained protoscoleces were commonly seen in May-Giemsa stain (Figure 3(a)). In Papanicolaou stain, orange and purple stained protoscoleces were also observed (Figure 3(b)). From the above findings, the patient was diagnosed as CE in her liver [14].
According to the WHO informed working group on echinococcosis international classification [15], the patient was classified as CE1, smaller than 5 cm.
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Figure 1. (a) An abdominal ultrasonography showed a monocystic lesion, measuring 42 mm in diameter with coating formation of high-intensity echoes were recognized in right lobe of her liver. (b) A 42 × 37 mm lesion at the S4/S8 boundary area in her right liver lobe was observed. Calcification at the margins and low concentration zones around it were recognized.
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Table 1. Laboratory data of the patient on admision.
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Figure 2. (a) Many protoscoleces were found in unstained wet mount smears (×200). (b) The scolex was found with growing head. Hooks and rounded suckers on the tip of the scolex were recognized in unstained wet mount smears. Many calcareous corpuscles were found in the scolex (×400).
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Figure 3. (a) Basophilic stained protoscoleces were seen in May-Giemsa stain (×400). Many calcareous corpuscles were observed. (b) Orange and purple stained protoscoleces were also observed in Papanicolaou stain (×400). Many calcareous corpuscles were recognized.
Also, modified according to the WHO informed working group on the PNM classification and staging of alveolar echinococcosis [16], the patient was diagnosed as P1N0M0, stage I.
We determined the parasite identification using molecular methods as follows. For DNA extraction, we extracted DNA by using QIAamp DNA Mini (Qiagen) from parasite protoscolexies collected from hydatid sand of the patient. We amplified approxate 450 bp of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene by using JB3: 5'-TTTTTTGGGCATCCTGAGGTTTAT-3' for forward primer, and JB4.5: 5'-TAAAGAAAGAACATAATGAAAATG-3', for reverse primer [6]. PCR was run with T100 thermal cycler (Bio-Rad Laboratories) and then we conducted cox1 gene sequencing. SimpliFi HS Mix (Meridian Bioscience) was used for amplification. PCR protocol was as follows: initial denaturation at 95˚C for 30 s, 40 cycles of denaturation at 95˚C for 15 s, Annealing at 51˚C for 15 s, extension at 72˚C for 30 s, and final extension at 72˚C for 5 min. The DNA sequence of PCR product (396 bp) was read and then used for homology search at NCBI web site (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and phylogenetic analysis with MEGA 11 software. Basic local alignment search tool (BLAST) results were shown in Table 2. The patient sample was matched over 99% to E. ortleppi. The phylogenetic trees of Echinococcus spp. based on cox1 gene showed that the isolates on the patient’s sample belonged to E. ortleppi (Figure 4).
The patient was diagnosed as stage I, P1N0M0 with CE1, smaller than 5 cm, and thus conservatively treated with albendazole (ABZ, 600 mg/day) for 28 days and the withdrawal period for 14 days, according to the WHO informed working group on echinococcosis international classification [17]. The ABZ therapy
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Figure 4. The phylogenetic trees of Echinococcus spp. based on cox1 genes showed that the isolates on this patient’s sample belonged to E. ortleppi.
was repeated for two courses, and then the laboratory data showed no abnormalities and the cystic lesion in her liver almost disappeared. Five months after the start of ABZ therapy, the patient returned to Bolivia.
3. Discussion
In diagnostic imaging, calcification findings at the margins and low concentration zones around it are recognized by her plain CT, suggesting the parasitic or tuberculotic diseases [18] [19]. Meanwhile, the unstained wet mount smears and cytological findings showed hydatid protoscolex [14]. The unstained wet mount smears were useful for diagnosis of echinococcus in the liver, although they could not be recorded permanently [14]. In diagnostic imaging, hydatid disease is similar to malignant [18] [19]. FNAC helps in eliminating the dilemma and gives conclusive diagnosis [20] [21]. Her relatively high ratio of eosinophilia was also consistent with parasitic disease infection.
CE is caused by Echinococcus granulosus s.l., which including 5 species as mentioned above. The highest homology to the DNA sequence of the PCR product of this case wase observed with E. ortleppi (99.2% - 100%), while others as follows; E. granulosus sensu stricto (96.4%), E. canadensis (95.7%), E. equinus (92.1%), and E. equinus (92.1%). In South America, main causative agent of human CE is E. granulosus sensu stricto, and only 1.9% of human cases were caused by E. ortleppi [22]. There have been no reports of echinococcosis caused by E. ortleppi in Japan. In Bolivia, although E. ortleppi in cattle was reported [23], the human case of this species has not reported also. E. ortleppi can parasitize various host mammals such as cattle, camels, pigs, goats, sheep and human [17]. This is the first case of echinococcosis caused by E. ortleppi in Japan. Considering the incubation period of echinococcosis i.e., month to years [12] [13], this case might be imported from Bolivia.
Most cases of liver echinococcosis in Japan have been reported Ehinococcus multilocularis [11], while the current cases were caused by Echinococcus granulosus, especially E. ortleppi. For the diagnosis for this case was useful for the unstained wet mount smears and cytological findings showing hydatid protoscolex [14]. As this case was diagnosed relatively early stage, stage I, P1N0M0 with CE1, smaller than 5 cm, the only ABZ therapy was useful and prognosis was good.
4. Conclusion
Echinococcosis is a global and zoonotic tapeworm disease caused by Echinococcus spp. A 24-year-old woman, who came from Bolivia, was diagnosed as CE by unstained wet mount and cytological findings through FNAC from the liver abscess. The patient was classified as CE1, smaller than 5 cm, and diagnosed as P1N0M0, stage I, and treated with albendazole therapies. The phylogenetic trees of Echinococcus spp., based on cox1 genes showed that the isolates on the patient belonged to E. ortleppi. Although the patient came from Bolivia before two years, this is the first case report of E. ortleppi in liver echinococcosis in Japan.
Acknowledgements
We thank the editor and reviewers for the constructive comments, which helped us to improve the manuscript.
Consent
Verbal consent was obtained from the patient before writing this case report.
Ethical Approval
This was obtained from the ethical committee of Gujo City Hospital before writing this case report (21102901).