Co-existence of Paragonimus harinasutai and Paragonimus bangkokensis metacercariae in fresh water crab hosts in central Viet Nam with special emphasis on their close phylogenetic relationship

Doanh, Pham Ngoc; Hien, Hoang Van; Nonaka, Nariaki; Horii, Yoichiro; Nawa, Yukifumi

doi:10.1016/j.parint.2012.01.011

Cited by 12 publications

(14 citation statements)

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“…This grouping is in agreement with the molecular phylogenetic tree analysis using ITS2 sequences performed in a previous study, with P. siamensis being grouped in the P. westermani complex 2 and P. bangkokensis and P. harinasutai being grouped in the same species complex. 4 The present results show that the Tm value can differentiate Paragonimus species at the species complex level. The method can be used not only for the detection of P. heterotremus eggs in cat feces but also for the differential detection of P. heterotremus and P. macrorchis among all 6 other Paragonimus spp.…”

supporting

confidence: 54%

“…were obtained by conventional PCR using the PhITS2_F and PhITS2_R primers. This PCR was performed using 1 μl of template DNA, 0.2 μM of each primer, 2.5 μl of 10× PCR buffer (1.5 mM MgSO 4 and 0.2 mM of each deoxyribonucleotide triphosphate), and 0.5 U Taq DNA polymerase. The amplification procedure was 95°C for 5 min; 35 cycles of 95°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec; and 72°C for 7 min.…”

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confidence: 99%

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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

et al. 2013

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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...

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confidence: 54%

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confidence: 99%

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

et al. 2013

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“…However, morphology-based classification often produces difficulties and complexity in identification, particularly in species that exhibit only minor morphological differences. Fortunately, the application of DNA sequence analysis to taxonomy has helped to clarify the phylogenetic relationship between species and their taxonomic status (Blair et al, 1999(Blair et al, , 2005Doanh et al, 2008Doanh et al, , 2012. However, the use of molecular differences for taxonomy has brought about a trend to describe new species based on differences of only a few nucleotides in their ribosomal spacer DNA sequences (Kunz, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Recent molecular data, however, placed some species as synonyms or subspecies of others, and closely related species in a single clade form a species complex, such as P. westermani, P. skrjabini and P. bangkokensis/P. harinasutai complexes (Blair et al, 1999(Blair et al, , 2005Doanh et al, 2008Doanh et al, , 2012. On the other hand, new Paragonimus species, such as P. vietnamensis from Vietnam (Doanh et al, 2007a) and P. pseudoheterotremus from Thailand (Waikagul, 2007), have been described recently.…”

Section: Introductionmentioning

confidence: 99%

Metacercarial polymorphism and genetic variation of Paragonimus heterotremus (Digenea: Paragonimidae), and a re-appraisal of the taxonomic status of Paragonimus pseudoheterotremus

Doanh

Thaenkham

et al. 2013

J. Helminthol.

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Paragonimus heterotremus, which is an important pathogen for human paragonimiasis in Asia, is recognized as having the smallest metacercariae (maximum diameter < 300 μm) of any previously reported Paragonimus species. Recently, P. pseudoheterotremus has been described from Thailand as a new species having metacercariae (about 200 μm) slightly smaller than those of Thai P. heterotremus. In fact, the small size of P. pseudoheterotremus metacercariae is compatible with those of P. heterotremus from India and China. In this study in Vietnam, we found variably sized small metacercariae which are expected to consist of both P. heterotremus and P. pseudoheterotremus. Contrary to expectation, the adult flukes obtained by separate infection of experimental cats with different sized metacercariae were all identified as P. heterotremus, using both morphological and molecular characteristics. The molecular analyses of an extensive collection of P. heterotremus/P. pseudoheterotremus isolates from Asian countries also indicated that genetic distances between different populations of P. heterotremus are even larger than that between P. pseudoheterotremus and P. heterotremus. The haplotype network showed that all P. heterotremus and P. pseudoheterotremus isolates formed a P. heterotremus complex consisting of three groups with strong geographical origins. In addition, the Indian P. heterotremus group is the root of the other P. heterotremus and P. pseudoheterotremus populations. Based on the observed metacercarial polymorphisms and genetic variation in P. heterotremus, P. pseudoheterotremus should be considered a geographically isolated population of the P. heterotremus complex.

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“…Metacercariae may occur in the same individual crab host [18,32-34]. Recent molecular studies provided strong evidence of their close phylogenetic relationship [16,18].…”

Section: Inter- and Intra-species Variation And Molecular Phylogenetimentioning

confidence: 99%

Paragonimus and Paragonimiasis in Vietnam: an Update

2013

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Paragonimiasis is a food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Vietnam, research on Paragonimus and paragonimiasis has been conducted in northern and central regions of the country. Using a combination of morphological and molecular methods, 7 Paragonimus species, namely P. heterotremus, P. westermani, P. skrjabini, P. vietnamensis, P. proliferus, P. bangkokenis and P. harinasutai, have been identified in Vietnam. Of these, the first 3, P. heterotremus, P. westermani and P. skrjabini, are known to infect humans in other countries. However, in Vietnam, only P. heterotremus, found in some northern provinces, has been shown to infect humans. Even nowadays, local people in some northern provinces, such as Lai Chau and Yen Bai, are still suffering from P. heterotremus infection. In some provinces of central Vietnam, the prevalence and infection intensity of P. westermani metacercariae in freshwater crabs (the second intermediate hosts) are extremely high, but human cases have not been reported. Likewise, although P. skrjabini was found in Thanh Hoa Province, its pathogenicity to humans in Vietnam still remains uncertain. The results of molecular phylogenetic analyses of Vietnamese Paragonimus species provides new insights on the phylogeny and taxonomy of the genus Paragonimus. Comprehensive molecular epidemiological and geobiological studies on the genus in Vietnam and adjacent countries are needed to clarify the biodiversity and public health significance of the lung flukes.

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Co-existence of Paragonimus harinasutai and Paragonimus bangkokensis metacercariae in fresh water crab hosts in central Viet Nam with special emphasis on their close phylogenetic relationship

Cited by 12 publications

References 16 publications

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

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

Metacercarial polymorphism and genetic variation of Paragonimus heterotremus (Digenea: Paragonimidae), and a re-appraisal of the taxonomic status of Paragonimus pseudoheterotremus

Paragonimus and Paragonimiasis in Vietnam: an Update

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