Paragonimus Westermani Research Articles

Development of a polymerase chain reaction applicable to rapid and sensitive detection of Clonorchis sinensis eggs in human stool samples

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity.

Gene structure of the two-domain taurocyamine kinase from Paragonimus westermani: Evidence for a distinct lineage of trematode phosphagen kinases

Taurocyamine kinase (TK) is an enzyme that catalyzes the reversible transfer of a phosphate between ATP and taurocyamine. Annelid TKs were suggested to have evolved from a CK ancestor. However, TKs from the lung fluke Paragonimus westermani comprised another lineage. Construction of phylogenetic tree and comparison of exon/intron organization showed that P. westermani TK and other trematode TKs evolved from a molluscan arginine kinase (AK) gene. Exon shuffling probably caused the changes in amino acid sequence thereby changing the affinity from AK to TK. The present study provides new insights on the evolution of phosphagen kinases found in trematodes.

P235 A report on 2 cases of Paragonimus westermani infection

P235 A report on 2 cases of Paragonimus westermani infection

Serological Diagnosis of North American Paragonimiasis by Western Blot Using Paragonimus kellicotti Adult Worm Antigen

Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

Genetic variability among Dicrocoelium dendriticum isolates from different regions in Shaanxi Province, China revealed by sequences of three mitochondrial genes

The genetic variations in three mitochondrial DNA (mtDNA) regions, namely portion of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and cytochrome b (pcytb), were examined for Dicrocoelium dendriticum samples isolated from different origins in Shaanxi Province, northwestern China. The intra-specific sequence differences within D. dendriticum samples were 0–0.52% for pcox1, 0–0.73% for pnad1 and 0–0.58% for pcytb. Phylogenetic analyses based on combined sequences of three mtDNA showed that all D. dendriticum samples were clustered together in same clade of Paragonimus westermani. But the phylogenetic trees could not reveal geographically genetic relationships of D. dendriticum isolates in this province. These findings will provide basic information for further study of molecular epidemiology and control of D. dendriticum infection in this province as well as in China.

PUBLIC HEALTH THREAT CAUSED BY ZOONOTIC DISEASES IN BANGLADESH

Diseases and infections shared between animals and humans are mainly called zoonotic diseases which may be categorized as emerging, reemerging and neglected. All types of these diseases occur t hroughout the world including Bangladesh. The results on the prevalence and effects of zoonotic diseases in humans and animals of Bangladesh are analyzed from the published literatures and presented in this report. It appears from the literature that there are about 1415 human pathogens of which 61% are zoonotic and nearly half of all human pathogens can be classified as emerging, of which 75% of these are caused by zoonotic pathogens. The major zoonotic bacterial diseases recorded in Bangladesh are Anthrax, Tuberculosis, Brucellosis, Salmonellosis, Campylobacteriosis and Leptospirosis, of which only Anthrax has been reported as clinical outbreaks form in both the humans and cattle. During the period 2009 to 2012, anthrax caused death of hundreds of cattle and more than 650 cases of cutaneous anthrax in humans including fatalities in two humans associated with anthrax. The major reported viral zoonotic diseases in Bangladesh include Avian influenza, Rabies, Nipah virus infection, Japanese encephalitis, Rotavirus and Dengue fever. Avian influenza caused by highly pathogenic H5N1 in humans and poultry in Bangladesh and about six humans affected with H5N1 but all of them have recovered. Since 27 March 2007 when Avian influenza was reported to have occurred for the first time in Bangladesh, this virus spread in 51 out of 64 districts with more than 480 outbreaks, culled more than two million poultry birds and cost Tk. 55 billion (US $ 757.9 million) in Bangladesh. Rabies is considered as a priority zoonosis in Bangladesh and it is mainly transmitted to humans and food animals mainly through dog bite nearly 100,000 people and at least 2000 died of rabies in 2009 in Bangladesh. Nipah virus infection is an important emerging infectious disease has been recognized since 2001 in Bangladesh and up to February 2011, 173 humans cases have been identified, of which 110 (63.58%) died. Japanese encephalitis (JE) is a vector borne zoonotic disease, first recognized in Bangladesh since an outbreak in 1977 and 12.38% encephalitis patients had JE virus infection which was associated with mortality, physical disability and cognitive difficulties. Rotavirus is a worldwide distributed zoonotic disease affecting mammals and birds and it has been reported from Bangladesh in humans (23.75%), animals (12 to 43.78%) and broiler birds (13.15%) associated with diarrheic syndrome. Dengue fever was first reported in Bangladesh in 1964 and outbreak that began in 2000 predominantly caused by DENV-3 in which 5551 cases recovered and 93 Dengue related deaths were reported. The dermatomycosis has been reported in 9.3% cattle, 18.6% goats and 25.2% in contact humans. The major zoonotic parasites recorded in cats in Bangladesh include Paragonimus westermani (9.09%), Ancyclostoma tubaeforme (36.36%), Dirofilaria immitis (9.09%), Toxocara cati (45.45%) and Toxoplasma gondii (9.09%), in dogs include Diphyllobothrium latum (13.3%), Diphylidium caninum (16.69%), Echinococcus granulosus (9.17%), Ancylostomum caninum (9-100%), Gnathostoma spingerum (40.0%), Toxocara canis (23.3%), Trichuris vulpis (13.8%) and in pigs include Entaemebea histolytica (17.24%) and Balantidium coli (60.34%). The prevalence of zoonotic protozoan diseases recorded in humans in Bangladesh include amebiasis (E. histolytica 4.71% & E. dispar 12.6%), giardiasis (21% in children & 51% in malnourished children), cryptosporidiasis (1.4 to 3.5% in diarrheic patients) and visceral leishmaniasis (kala-azar) which was first reported in Jassore in 1824 but still sporadic outbreaks are occurring in the different districts in Bangladesh. Sero-surveillance studies showed 16 to 17% cattle, 17.65 to 53.6% sheep, 12.09 to 35.4% goats and 15.89% women had T. gondii antibodies in Bangladesh. It appears from these results that all types of emerging, reemerging and neglected zoonotic diseases are widely prevalent and pose a great threat to human health in Bangladesh. Neglected Veterinary medical profession and its extension services, poor people without any knowledge of zoonotic diseases who are in close contact with livestock and their products, and unhygienic processing, maintaining and marketing the livestock and livestock products have made the situation graver in Bangladesh.DOI: http://dx.doi.org/10.3329/bjvm.v9i2.13451

Pleuropulmonary paragonimiasis: mimicker of tuberculosis

Infection caused by the lung fluke is endemic in north eastern parts of India. Paragonimus westermani and Paragonimus heterotremus are known to be endemic in eastern Indian states of Manipur and Nagaland. The infection is related to eating habits of the locals and is acquired by ingestion of raw, inadequately cooked crabs or crayfish containing encysted metacercariae which act as second intermediate hosts during the life cycle of the lung fluke. Diagnosis is generally delayed due to lack of suspicion and presentation similar to tuberculosis which is endemic in the population. We report pleuropulmonary paragonimiasis in a soldier from eastern India who presented with chest pain, haemoptysis, and eosinophilia. He gave history of consumption of raw crabs while on leave at his native village in Nagaland. Ova morphologically resembling Paragonimus heterotremus were detected in sputum and bronchoalveolar lavage specimen. Symptoms resolved with praziquantel treatment.

A Case of Paragonimus westermani Infection with Metachronous Bilateral Pleural Effusion

A Case of Paragonimus westermani Infection with Metachronous Bilateral Pleural Effusion

PREVALENCE OF <i>PARAGONIMUS</i> INFECTION

Paragonimiasis (human infections with the lung fluke Paragonimus westermani) is an important public health problem in parts of Africa. This study was aimed at assessing the prevalence of Paragonimus infection in Ebonyi State. Deep sputum samples from 3600 individuals and stool samples from 900 individuals in nine Local Government Areas in Ebonyi State, Nigeria were examined for Paragonimus ova using concentration technique. The overall prevalence of pulmonary Paragonimus infection in the area was 16.30%. Six foci of the infection were identified in Ebonyi North and Ebonyi Central but none in Ebonyi South. The intensity of the infection was generally moderate. Of the 720 individuals examined, 16 (12.12%) had less than 40 ova of Paragonimus in 5 mL sputum and 114 (86.36%) had between 40 and 79 ova of Paragonimus in 5 mL sputum. While 2 individuals (1.52%) had over 79 ova of Paragonimus in 5 mL Sputum. Furthermore, there was higher prevalence of paragonimiasis in rainy season than in dry season. The results of this study indicated the growing public health threat posed by paragonimiasis in Ebonyi North and Ebonyi Central. A combination of chemotherapy, to bring relief to persons already infected by the disease and public health education related to paragonimiasis transmission to increase awareness of the infection in the areas is recommended.

Molecular identification of Paragonimus species by DNA pyrosequencing technology

DNA pyrosequencing for PCR amplicons is an attractive strategy for the identification of microorganisms because of its short time performance for large number of samples. In this study, the primers targeting the fragment of ITS2 region of nuclear ribosomal RNA gene were newly developed for pyrosequencing-based identification of 6 Paragonimus species, Paragonimus bangkokensis, Paragonimus harinasutai, Paragonimus heterotremus, Paragonimus macrorchis, Paragonimus siamensis and Paragonimus westermani. Pyrosequencing determination of 39 nucleotides of partial ITS2 region could discriminate 6 Paragonimus species, and could also detect intra-species genetic variation of P. macrorchis. This DNA pyrosequencing-based identification can be a valuable tool to improve species-level identification of Paragonimus in the endemic areas.

Characterization of MicroRNAs in Paragonimus westermani by Solexa Deep Sequencing and Bioinformatics Analysis

Characterization of MicroRNAs in Paragonimus westermani by Solexa Deep Sequencing and Bioinformatics Analysis

A Protein Microarray for the Rapid Screening of Patients Suspected of Infection with Various Food-Borne Helminthiases

BackgroundFood-borne helminthiases (FBHs) have become increasingly important due to frequent occurrence and worldwide distribution. There is increasing demand for developing more sensitive, high-throughput techniques for the simultaneous detection of multiple parasitic diseases due to limitations in differential clinical diagnosis of FBHs with similar symptoms. These infections are difficult to diagnose correctly by conventional diagnostic approaches including serological approaches.Methodology/Principal FindingsIn this study, antigens obtained from 5 parasite species, namely Cysticercus cellulosae, Angiostrongylus cantonensis, Paragonimus westermani, Trichinella spiralis and Spirometra sp., were semi-purified after immunoblotting. Sera from 365 human cases of helminthiasis and 80 healthy individuals were assayed with semi-purified antigens by both a protein microarray and the enzyme-linked immunosorbent assay (ELISA). The sensitivity, specificity and simplicity of each test for the end-user were evaluated. The specificity of the tests ranged from 97.0% (95% confidence interval (CI): 95.3–98.7%) to 100.0% (95% CI: 100.0%) in the protein microarray and from 97.7% (95% CI: 96.2–99.2%) to 100.0% (95% CI: 100.0%) in ELISA. The sensitivity varied from 85.7% (95% CI: 75.1–96.3%) to 92.1% (95% CI: 83.5–100.0%) in the protein microarray, while the corresponding values for ELISA were 82.0% (95% CI: 71.4–92.6%) to 92.1% (95% CI: 83.5–100.0%). Furthermore, the Youden index spanned from 0.83 to 0.92 in the protein microarray and from 0.80 to 0.92 in ELISA. For each parasite, the Youden index from the protein microarray was often slightly higher than the one from ELISA even though the same antigen was used.Conclusions/SignificanceThe protein microarray platform is a convenient, versatile, high-throughput method that can easily be adapted to massive FBH screening.

Presence of three distinct genotypes within the Paragonimus westermani complex in northeastern India

The name Paragonimus westermani (Kerbert, 1878) is commonly applied to members of a species complex that includes the well-known Asian lung fluke of medical and veterinary importance. Unambiguous molecular and morphological evidence showing the presence of a member of the complex in India has recently been published. In the present study we report the occurrence of 2 more members of the P. westermani complex in northeastern (NE) India. Surveys of the freshwater crabs Maydelliatelphusa lugubris in NE India revealed 2 morphologically distinct types of lung fluke metacercariae. Phylogenetic analyses, using DNA sequences from ITS2, 28S and cox1 gene regions indicate that these lung metacercariae belong to P. westermani complex. Type 1 metacercariae have a more basal position within the complex whereas type 2 metacercariae are closely related to the relatively derived forms of P. westermani from NE Asia (Japan, Korea, China) and Vietnam. A third type of metacercaria (type 3), detected in another crab host, Sartoriana spinigera in Assam, was phylogenetically close to P. siamensis, also a member of the P. westermani group. Molecular evidence has demonstrated the existence of 3 genotypes of lung flukes within the Paragonimus westermani complex in NE India. Two of these were previously unknown.

Pleuropulmonary and abdominal paragonimiasis: CT and ultrasound findings

The purpose of this study was to review radiological images of patients with Paragonimus westermani (PW) that simultaneously involved the chest and abdomen. Our study included four patients with serologically and histopathologically confirmed paragonimiasis. Abdomen CT (n=3) and chest CT (n=3) scans were available, and abdominal wall ultrasonography was performed in all patients. We retrospectively reviewed the clinical, radiological and histopathological findings of these patients. The most common abdominal CT findings were ascites and intraperitoneal or abdominal wall nodules. Low-attenuated serpentine lesions of the liver were another common and relatively specific feature. Radiologists should consider the possibility of PW when these abdominal CT findings are noted, especially with pleural effusion or subpleural nodules in patients with initial abdominal symptoms.

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

During our epidemiological surveys for Paragonimus species in central Viet Nam, we found four morphologically different Paragonimus metacercariae in mountainous crabs. They were identified as metacercariae of Paragonimus westermani, P. bangkokensis, P. proliferus, and P. harinasutai in the order of their prevalence in crab hosts. This is the first discovery of P. harinasutai in Viet Nam, co-inhabiting with P. bangkokensis and other species. Metacercariae of P. harinasutai were given orally to a cat to obtain adult worms. Then, ITS2 and CO1 sequences of metacercariae and adults of P. harinasutai, and metacercariae of P. bangkokensis collected from the same place were determined for analyses of phylogenetic relationships to other P. harinasutai and P. bangkokensis populations as well as related species. The results of molecular analyses showed that P. harinasutai from Quang Binh province of central Viet Nam was almost completely identical with those from Vientiane, Lao PDR; P. bangkokensis from Quang Binh, Viet Nam was also almost completely identical with those from Lao PDR and from Quang Ninh province, Viet Nam. Except for one P. harinasutai isolate from China, all populations of P. harinasutai and P. bangkokensis from Thailand, Lao and Viet Nam make a single clade in both ITS2 and CO1 trees. In ITS2 sequences, AT deletion and ATC insertion were observed in some isolates of both species, indicating recent gene flow between P. harinasutai and P. bangkokensis. Moreover, because of their extremely high genetic similarities and their co-inhabitation in the same crab hosts found in Thailand, Lao PDR and Viet Nam, they should be considered as the sister species at the early stage of divergence. In addition, P. microrchis previously described from Yunnan, China should be placed as the synonym of P. harinasutai, because of their morphological and molecular similarities.

Loop-mediated isothermal amplification (LAMP): Early detection of Toxoplasma gondii infection in mice

BackgroundToxoplasmosis is a widespread zoonotic parasitic disease that occurs in both animals and humans. Traditional molecular assays are often difficult to perform, especially for the early diagnosis of Toxoplasma gondii infections. Here, we established a novel loop-mediated isothermal amplification targeting the 529 bp repeat element (529 bp-LAMP) to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain.FindingsThe assay was performed with Bst DNA polymerase at 65°C for 1 h. The detection limit of the 529 bp-LAMP assay was as low as 0.6 fg of T. gondii DNA. The sensitivity of this assay was 100 and 1000 fold higher than that of the LAMP targeting B1 gene (B1-LAMP) and nested PCR targeting 529 bp repeat element (529 bp-nested PCR), respectively. The specificity of the 529 bp-LAMP assay was determined using the DNA samples of Trypanosoma evansi, Plasmodium falciparum, Paragonimus westermani, Schistosoma japonicum, Fasciola hepatica and Angiostrongylus cantonensis. No cross-reactivity with the DNA of any parasites was found. The assay was able to detect T. gondii DNA in all mouse blood samples at one day post infection (dpi).ConclusionsWe report the following findings: (i) The detection limit of the 529 bp-LAMP assay is 0.6 fg of T. gondii DNA; (ii) The assay does not involve any cross-reactivity with the DNA of other parasites; (iii) This is the first report on the application of the LAMP assay for early diagnosis of toxoplasmosis in blood samples from experimentally infected mice. Due to its simplicity, sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis.

A Report on 2 Cases of Paragonimus westermani Infection Caused by Eating Raw Boar Meat

A Report on 2 Cases of Paragonimus westermani Infection Caused by Eating Raw Boar Meat

Characterization and differential expression of a ferritin protein from Fasciola hepatica

Ferritins are proteins that play a central role in maintaining intracellular iron balance. A cDNA clone of Fasciola hepatica (687bp long) encoding a putative 228-amino acid polypeptide (FhFtn-1) homologous with ferritins of vertebrates and invertebrates was identified. FhFtn-1 contains a conserved motif of the ferroxidase center typical of vertebrate ferritins. Phylogenetic tree analysis showed that FhFtn-1 clusters with two ferritins of Paragonimus westermani, which suggests a common ancestry for the ferritins of these two trematodes. Recombinant FhFtn-1 protein expressed and purified from an Escherichia coli system showed iron-uptake ability. Moreover, FhFtn-1 showed strong reactivity with sera from rabbits infected with F. hepatica for 2–12 weeks, which suggests that this protein could be a potential antigen for immunodiagnosis of fascioliasis. qPCR analysis demonstrated that FhFtn-1-mRNA is expressed at significantly higher levels in adults and unembryonated eggs than in juveniles or miracidia. These results represent the first characterization of a ferritin protein from the liver fluke F. hepatica.

Serological diagnosis of clonorchiasis: using a recombinant propeptide of cathepsin L proteinase from Clonorchis sinensis as a candidate antigen

Clonorchiasis is a common zoonosis in southern and northeastern parts of China, especially in Guangdong, Guangxi and Jilin province. Anti-Clonorchis sinensis antibody detection by enzyme-linked immunosorbent assay (ELISA) has been used for epidemiological surveys of clonorchiasis for its convenience and celerity, but it is still a meaningful work to screen ideal diagnostic antigen or antibody subtype for improvement of diagnostic sensitivity and specificity and for judgement of curative effect. In the present study, recombinant CsCatL-propeptide (rCsCatL-propeptide) was highly expressed in form of inclusion body in Escherichia coli. Soluble rCsCatL-propeptide with high purity were obtained after purification in denatured condition by using His Bind Purification kit, and then renatured. The major antibody subtypes responding to rCsCatL-propeptide in sera from clonorchiasis patients were IgG1 and IgG4, but the level of IgG4 was more predominant (P < 0.05). The sensitivity of specific IgG4 detection (91.7%) was statistically significantly higher than that of IgG1 (25.0%) with rCsCatL-propeptide (P < 0.01). The specificities of IgG1 and IgG4 detection with rCsCatL-propeptide were 83.3% and 88.5%, respectively, and the difference between them was not statistically significant (P > 0.05). Cross-reactions took place when we detected IgG1 of sera from patients infected with Schistosoma japonicum, Paragonimus westermani, hookworm, Trichuris trichiura and Ascaris lumbricoides with rCsCatL-propeptide, while cross-reactions only took place in sera from patients infected with S. japonicum and P. westermani when we detected specific IgG4. The positive rate of IgG4 detection in sera from clonorchiasis patients with <1,000, 1,000-4,999, 5,000-9,999, and ≥10,000 eggs per gram faeces (EPG) were 76.9%, 89.3%, 95.6%, and 100.0%, respectively. The positive rates of serodiagnosis correlated well with the EPG (r = 0.93). Overall, rCsCatL-propeptide is a valuable candidate for specific IgG4 detection in sera from clonorchiasis patients by the method of ELISA for its few cross-reaction and acceptable sensitivity. In addition, specific IgG4 detection can be used to valuate infected degree and therapeutic effect of clonorchiasis patients.

Loop-mediated isothermal amplification: rapid detection of Angiostrongylus cantonensis infection in Pomacea canaliculata

BackgroundAngiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans. The most common source of infection with A. cantonensis is the consumption of raw or undercooked mollusks (e.g., snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify in snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails.FindingsWe used a loop-mediated isothermal amplification (LAMP) assay, which was performed using Bst DNA polymerase. Reactions amplified the A. cantonensis 18S rRNA gene and demonstrated high sensitivity; as little as 1 fg of DNA was detected in the samples. Furthermore, no cross-reactivity was found with other parasites such as Toxoplasma gondii, Plasmodium falciparum, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani and Anisakis. Pomacea canaliculata snails were exposed to A. cantonensis first-stage larvae (L1) in the laboratory, and L3 were observed in the snails thirty-five days after infection. All nine samples were positive as determined by the LAMP assay for A. cantonensis, which was identified as positive by using PCR and microscopy, this demonstrates that LAMP is sensitive and effective for diagnosis.ConclusionsLAMP is an appropriate diagnostic method for the routine identification of A. cantonensis within its intermediate host snail P. canaliculata because of its simplicity, sensitivity, and specificity. It holds great promise as a useful monitoring tool for A. cantonensis in endemic regions.