First report and molecular identification of Opisthorchis viverrini infection in human communities from Lower Myanmar

Abstract

Opisthorchis viverrini is endemic in the South East Asian region, especially in Cambodia, Lao People's Democratic Republic, Vietnam and Thailand, but there have been no previous records from Myanmar. During stool surveys of rural populations in three regions of Lower Myanmar, Opisthorchis-like eggs were found in 34 out of 364 (9.3%) participants by stool microscopy after using the modified formalin-ether concentration technique. DNA was extracted from these positive stool samples and a portion of the mitochondrial cytochrome c oxidase subunit I (cox1) gene was amplified using the polymerase chain reaction and then sequenced. DNA sequences, successfully obtained from 18 of 34 positive samples (Bago Region, n = 13; Mon State, n = 3; Yangon Region, n = 2), confirmed that the eggs were of O. viverrini. Sequences showed 99.7% identity with O. viverrini mitochondrial cox1 (GenBank accession no. JF739555) but 95%, 88.7%, 82.6% and 81.4% identities with those of Opisthorchis lobatus from Lao People's Democratic Republic (GenBank accession nos. HQ328539-HQ328541), Metorchis orientalis from China (KT239342), Clonorchis sinensis from China (JF729303) and Opisthorchis felineus from Russia (EU921260), respectively. When alignement with other Opisthorchiidae trematodes, 81% similarity with Metorchis bilis from Czech Republic (GenBank accession nos. KT740966, KT740969, KT740970) and Slovakia (GenBank accession nos. KT740971-KT740973), 84.6% similarity with Metorchis xanthosomus from Czech Republic (GenBank accession no. KT740974), 78.6% similarity with M. xanthosomus from Poland (GenBank accession no. KT740968) and 82.2% similarity with Euamphimerus pancreaticus from Czech Republic (GenBank accession no. KT740975) were revealed. This study demonstrated, for the first time, O. viverrini from rural people in Myanmar using molecular methods and is an urgent call for surveillance and control activities against opisthorchiasis in Myanmar.

Fig 1. Map of the survey areas in Myanmar.

Prevalences (in parentheses) of Opisthorchis-like eggs in Bago, Mon and Yangon Regions in the years 2015–2016. M = Male; F = Female. The Myanmar map was modified from a map in The World Factbook, published by the Central Intelligence Agency [22].

Fig 2. Showing O. viverrini egg, fish dish and cyprinoid fish.

A) Opisthorchis viverrini egg from stool microscopy. B) The undercooked or fermented fish dish (small raw fish fermented with cooked rice and salt) named “Ngar Lay Chin”. C) Cyprinoid fish (Henicorhynchus sp.) “Nga Khone Ma” (intermediate host of O. viverrini).

Fig 3. Alignment of O. viverrini cox1 nucleotide sequences.

The multiple sequences alignment of representative O. viverrini partial mitochondrial cytochrome c oxidase subunit I (cox1) sequence from Bago Region of Lower Myanmar (KX922693; bold letters) and other Opisthorchiidae trematodes. A, T, G, C nucleotides represent in green, red, black and blue, respectively. Incomplete sequences are represented by dashed lines (-). ISO 3166 Countries Codes are presented (THA, Thailand; LAO, Lao People's Democratic Republic, MMR; Myanmar, VNM; Vietnam, RUS; Russian Federation, CHN; China, CZE; Czech Republic, SVK; Slovakia and POL; Poland).