Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats

Abstract

ArticleParagonimiasis is an important food-borne parasitic zoonosis caused by a lung fluke belonging to the genus Paragonimus. 2,10 It is estimated that more than 20 million people are infected worldwide, 21 and it has been calculated that 293 million people are at risk. 9 Human beings and other mammals are infected by ingesting raw crustaceans containing metacercariae 22 and usually present with signs and symptoms in the lower respiratory tract (i.e., cough and hemoptysis).2 Paragonimus westermani is the most widely distributed species in Asia and the most important human pathogen in China, Korea, and Japan. 2,10 In Southeast Asia, however, P. heterotremus is the most important pathogen, and confirmed human cases have been found. 2,10 To date, at least 7 lung fluke species have been documented in Thailand: Paragonimus heterotremus, P. siamensis, P. westermani, P. bangkokensis, P. macrorchis, P. harinasutai, and P. pseudoheterotremus. 3,19,20 However, only P. heterotremus and P. pseudoheterotremus are proven human pathogens. 6 The standard diagnosis of paragonimiasis is based on the demonstration of the presence of the eggs of Paragonimus spp. in the sputum (by alkaline decontamination and centrifuge sedimentation technique) and/or feces (by formalin-ether concentration technique).1,22 However, species identification 497944V DIXXX10.1177/1040638713497944Real-time PCR for the differential detection of ParagonimusTantrawatpan et al. research-article2013From the Research and Diagnostic Center for Emerging Infectious Diseases (Tantrawatpan, Intapan, Thanchomnang, Sanpool, Janwan, Lulitanond, Maleewong), the Departments of Parasitology (Intapan, Sanpool, Janwan, Maleewong) Abstract. Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 × 10 2 copies of the positive control plasmid and 10 -3 ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus-infected cats and showed a negative relationship (r = -0.852, P < 0.001) between the number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus, P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can be useful for the detection of, a...

Laboratory diagnostic methods

“…A number of molecular-based detection methods targeting ribosomal, mitochondrial, microsatellite, repetitive elements, and mobile elements within the genome of liver, lung, and intestinal flukes have been developed for their specific detection from clinical samples and host tissue (Petney et al, 2013;Tantrawatpan et al, 2013). Realtime PCRs have been developed for the detection of O. viverrine metacercarial DNA in fish and are capable of detecting as little as one metacercaria inoculated in a pool of 30 noninfected caudal fins of 100 mg each (Intapan et al, 2008).…”