Fasciolopsis buski Detected in Humans in Bihar and Pigs in Assam, India

The foodborne intestinal trematode Fasciolopsis buski causes the neglected zoonotic disease fasciolopsiasis. We detected F. buski infection in 14 pediatric patients in Sitamarhi, Bihar, and in pigs in Sivasagar, Assam, India. Proper diagnostic methods and surveillance are urgently needed to accurately estimate the true burden of this disease in India.

Naked eye examination of the feces revealed the presence of live parasites in 2 patients and dead parasites in 12. Two patients had mixed infection: 1 with both F. buski and A. sufrartyfex parasites and the other with F. buski worms and the roundworm Ascaris lumbricoides. On microscopic examination of feces samples, no eggs or ova of intestinal flukes were identified, except 1 sample showed fertilized roundworm ova. We isolated the recovered parasites and processed them for morphologic, anatomic, and genetic analysis. All of the patients were hospitalized and treated with praziquantel (75 mg/kg bodyweight; 3 divided doses for 2 d) and supportive measures administered for dehydration, electrolyte imbalance, and malnutrition. All patients were cured and discharged after being counseled for nutritional rehabilitation.
In 2019-2020, a survey of pigs for F. buski infection was performed in the Charaideo, Sivasagar, Lakhimpur, Biswanath, and Tezpur districts of Assam. A total of 128 pigs were examined; 3 in the Sivasagar district displayed evidence of parasite infection. The flukes were collected in 0.9% phosphate-buffered saline (pH 7.2) from the intestines of freshly slaughtered pigs in Sivasagar district, as well as from the feces and vomitus of SSL Hospital patients, and brought to the Sikkim University Department of Zoology for further analysis ( Figure 1).
We extracted and purified genomic DNA from the flukes collected from both patients and pigs using QIAGEN DNeasy Blood and Tissue Kit (https:// www.qiagen.com) according to manufacturer instructions. We performed amplification and sequencing of the complete ITS2 and partial cox1 genes using the primers 3S: 5′-GGTACCGGTGGATCACTCG-GCTCGTG-3′ (forward), A28: 5′-GGGATCCTGGT-TAGTTTCTTTTCCTCCGC-3′ (reverse) (12,13), and The ITS2 sequences of the Sitamarhi and Sivasagar isolates were genetically similar and showed the greatest sequence similarity with previously identified F. buski isolates from Uttar Pradesh (Gen-Bank accession no. KF564866) and Meghalaya, India (accession no. DQ351841), with little or no genetic variability. In contrast, F. buski sequences from China and Vietnam (GenBank accession nos. MN970005 and EF612489) had 7.7%-8.2% genetic difference from the isolates from India. However, the sequences from China and Vietnam were identical to each other. Similarly, the cox1 sequences from Sitamarhi and Sivasagar exhibited only 0.4% variation between each other but 12.1%-12.3% variation from sequences from Vietnam (accession no. MF287794) and China (accession no. KX169163). The sequences from China and Vietnam had only 0.5% variation from each other ( Table  2; Appendix 2 Figures 1, 2, https://wwwnc.cdc.gov/ EID/article/28/6/22-0171-App1.pdf).
We constructed phylogenetic trees based on the 2 gene regions using the maximum-likelihood method. Both trees clearly showed the Indian isolates forming a separate clade from the isolates from China and Vietnam (Figure 2). On the basis of our findings, we concluded that the samples collected in our study belonged to F. buski but that the isolates from China and Vietnam were separate taxa from those from India; however, F. buski samples from China and Vietnam were the same species. This discovery is consistent with the findings of a prior study in China (11).

Conclusions
Our study confirmed that the parasites obtained from both human patients in Sitamarhi and pigs in Sivasagar were of the F. buski species. However, we also corroborated that the species found in India might differ from those in China and Vietnam, and species taxonomy might need to be revised in the future (11).  In recent years, F. buski infection from humans and pigs has been documented in India in the states of Assam, Bihar, Delhi, Meghalaya, and Uttar Pradesh. According to our findings, this parasite is an increasing public health threat in India, especially in remote locations and among persons from low socioeconomic backgrounds, because of the substantial risk to human and animal health it poses. Surveys are urgently needed to determine the true burden of fasciolopsiasis in the country. A lack of effective diagnostic tools for detecting neglected foodborne trematode infections, including F. buski, means there are no prevalence data on these infections in the country. Therefore, there is a pressing need to design and develop a rapid and easy detection tool for F. buski and other neglected trematode infections.