An intricate history of human dispersal and geographic colonization has strongly affected the distribution of human pathogens. The pig tapeworm Taenia solium occurs throughout the world as the causative agent of cysticercosis, one of the most serious neglected tropical diseases. Discrete genetic lineages of T. solium in Asia and Africa/Latin America are geographically disjunct; only in Madagascar are they sympatric. Linguistic, archaeological and genetic evidence has indicated that the people in Madagascar have mixed ancestry from Island Southeast Asia and East Africa. Hence, anthropogenic introduction of the tapeworm from Southeast Asia and Africa had been postulated. This study shows that the major mitochondrial haplotype of T. solium in Madagascar is closely related to those from the Indian Subcontinent. Parasitological evidence presented here, and human genetics previously reported, support the hypothesis of an Indian influence on Malagasy culture coinciding with periods of early human migration onto the island. We also found evidence of nuclear-mitochondrial discordance in single tapeworms, indicating unexpected cross-fertilization between the two lineages of T. solium. Analyses of genetic and geographic populations of T. solium in Madagascar will shed light on apparently rapid evolution of this organism driven by recent (<2,000 yr) human migrations, following tens of thousands of years of geographic isolation.
During 2012–2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.
We confirmed infection of 2 patients with Borrelia miyamotoi in Japan by retrospective surveillance of Lyme disease patients and detection of B. miyamotoi DNA in serum samples. One patient also showed seroconversion for antibody against recombinant glycerophosphodiester phosphodiesterase of B. miyamotoi. Indigenous relapsing fever should be considered a health concern in Japan.
Borrelia miyamotoi; bacteria; relapsing fever; Lyme disease; Ixodes persulcatus; ticks; vector-borne infections; Japan
Cystic echinococcosis (CE) is a globally distributed cestode zoonosis that causes hepatic cysts. Although Echinococcus granulosus sensu stricto (s.s.) is the major causative agent of CE worldwide, recent molecular epidemiological studies have revealed that E. canadensis is common in countries where camels are present. One such country is Mongolia.
Forty-three human hepatic CE cases that were confirmed histopathologically at the National Center of Pathology (NCP) in Ulaanbaatar (UB) were identified by analysis of mitochondrial cox 1 gene as being caused by either E. canadensis (n = 31, 72.1%) or E. granulosus s.s. (n = 12, 27.9%). The majority of the E. canadensis cases were strain G6/7 (29/31, 93.5%). Twenty three haplotypes were identified. Sixteen of 39 CE cases with data on age, sex and province of residence were citizens of UB (41.0%), with 13 of the 16 cases from UB caused by E. canadensis (G6/7) (81.3%). Among these 13 cases, nine were children (69.2%). All pediatric cases (n = 18) were due to E. canadensis with 17 of the 18 cases (94.4%) due to strain G6/7. Serum samples were available for 31 of the 43 CE cases, with 22 (71.0%) samples positive by ELISA to recombinant Antigen B8/1 (rAgB). Nine of 10 CE cases caused by E. granulosus s.s. (90.0%) and 13 of 20 CE cases by E. canadensis (G6/7) (65.0%) were seropositive. The one CE case caused by E. canadensis (G10) was seronegative. CE cases caused by E. granulosus s.s. showed higher absorbance values (median value 1.131) than those caused by E. canadensis (G6/7) (median value 0.106) (p = 0.0137).
The main species/strains in the study population were E. canadenis and E. granulossus s.s. with E. canadensis the predominant species identified in children. The reason why E. canadensis appears to be so common in children is unknown.
Cystic echinococcosis (CE) is a parasitic zoonosis with a cosmopolitan distribution. Molecular analysis was carried out on 43 hepatic CE cysts from 43 cases confirmed histopathologically at the NCP, Mongolia. Molecular analysis revealed two species, Echinococcus canadensis and Echinococcus granulosus s.s. Twenty three haplotypes of the cox1 gene were identified. All pediatric cases (n = 18) were by E. canadensis. Sixteen of 39 CE cases with data on age, sex and province of residence were from UB (41.0%), and 13 of these 16 cases were caused by E. canadensis (81.3%). Among the 13 cases from UB, nine were children (69.2%). A total of 31 serum samples from these 43 cases were analyzed for antibody response to rAgB with 22 (71.0%) samples positive by ELISA to rAgB. Thirteen of 20 E. canadensis (G6/7) (65%) and nine of 10 E. granulosus s.s. (90%) were seropositive. CE cases by E. granulosus s.s. showed a higher absorbance value than cases by E. canadensis (p = 0.0137). This is the first study to evaluate age distribution of and antibody responses to rAgB in CE cases caused by the two species in Mongolia. It remains unknown why E. canadensis appears to be more common in pediatric cases.
The species Babesia microti, commonly found in rodents, demonstrates a high degree of genetic diversity. Three lineages, U.S., Kobe, and Hobetsu, are known to have zoonotic potential, but their tick vector(s) in Japan remains to be elucidated. We conducted a field investigation at Nemuro on Hokkaido Island and at Sumoto on Awaji Island, where up to two of the three lineages occur with similar frequencies in reservoirs. By flagging vegetation at these spots and surrounding areas, 4,010 ticks, comprising six species, were collected. A nested PCR that detects the 18S rRNA gene of Babesia species revealed that Ixodes ovatus and I. persulcatus alone were positive. Lineage-specific PCR for rRNA-positive samples demonstrated that I. ovatus and I. persulcatus carried, respectively, the Hobetsu and U.S. parasites. No Kobe-specific DNA was detected. Infected I. ovatus ticks were found at multiple sites, including Nemuro and Sumoto, with minimum infection rates (MIR) of ∼12.3%. However, all I. persulcatus ticks collected within the same regions, a total of 535, were negative for the Hobetsu lineage, indicating that I. ovatus, but not I. persulcatus, was the vector for the lineage. At Nemuro, U.S. lineage was detected in 2 of 139 adult I. persulcatus ticks (MIR, 1.4%), for the first time, while 48 of I. ovatus ticks were negative for that lineage. Laboratory experiments confirmed the transmission of Hobetsu and U.S. parasites to hamsters via I. ovatus and I. persulcatus, respectively. Differences in vector capacity shown by MIRs at Nemuro, where the two species were equally likely to acquire either lineage of parasite, may explain the difference in distribution of Hobetsu throughout Japan and U.S. taxa in Nemuro. These findings are of importance in the assessment of the regional risk for babesiosis in humans.
Taenia solium is a zoonotic cestode that causes taeniasis and cysticercosis in humans. The parasite is traditionally found in developing countries where undercooked pork is consumed under poor sanitary conditions and/or as part of traditional food cultures. However, the recent increase in international tourism and immigration is spreading the disease into non-endemic developed countries such as the United States. Although there has been concern that the number of cysticercosis cases is increasing in Japan, the current situation is not clear. This is largely because taeniasis and cysticercosis are not notifiable conditions in Japan and because there have been no comprehensive reviews of T. solium infections in Japan conducted in the last 15 years. Herein, we provide an overview of the status of T. solium infection in Japan over the past 35 years and point out the potential risks to Japanese society.
Taenia solium; Cysticercosis; Taeniasis; Japan
Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.
The first workshop towards the control of cestode zoonoses in Asia and Africa was held in Asahikawa Medical University, Japan on 15 and 16 Feb 2011. This meeting was fully supported by the Asian Science and Technology Strategic Cooperation Promotion Programs sponsored by the Special Coordination Funds for Promoting Science and Technology, the Ministry of Education Japan (MEXT) for 3 years from 2010 to Akira Ito. A total of 24 researchers from 9 countries joined together and discussed the present situation and problems towards the control of cestode zoonoses. As the meeting was simultaneously for the establishment of joint international, either bilateral or multilateral collaboration projects, the main purposes were directed to 1) how to detect taeniasis/cysticercosis infected patients, 2) how to differentiate Taenia solium from two other human Taenia species, T. saginata and T. asiatica, 3) how to evaluate T. asiatica based on the evidence of hybrid and hybrid-derived adult tapeworms from Thailand and China, 4) how to evaluate T. solium and T. hyaenae and other Taenia species from the wild animals in Ethiopia, and 5) how to detect echinococcosis patients and 6) how to differentiate Echinococcus species worldwide. Such important topics are summarized in this meeting report.
The genetic polymorphisms of Echinococcus spp. in the eastern Tibetan Plateau and the Xinjiang Uyghur Autonomous Region were evaluated by DNA sequencing analyses of genes for mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear elongation factor-1 alpha (ef1a). We collected 68 isolates of Echinococcus granulosus sensu stricto (s.s.) from Xinjiang and 113 isolates of E. granulosus s. s., 49 isolates of Echinococcus multilocularis and 34 isolates of Echinococcus shiquicus from the Tibetan Plateau. The results of molecular identification by mitochondrial and nuclear markers were identical, suggesting the infrequency of introgressive hybridization. A considerable intraspecific variation was detected in mitochondrial cox1 sequences. The parsimonious network of cox1 haplotypes showed star-like features in E. granulosus s. s. and E. multilocularis, but a divergent feature in E. shiquicus. The cox1 neutrality indexes computed by Tajima's D and Fu's Fs tests showed high negative values in E. granulosus s. s. and E. multilocularis, indicating significant deviations from neutrality. In contrast, the low positive values of both tests were obtained in E. shiquicus. These results suggest the following hypotheses: (i) recent founder effects arose in E. granulosus and E. multilocularis after introducing particular individuals into the endemic areas by anthropogenic movement or natural migration of host mammals, and (ii) the ancestor of E. shiquicus was segregated into the Tibetan Plateau by colonizing alpine mammals and its mitochondrial locus has evolved without bottleneck effects.
Echinococcus; Mitochondrial DNA; Genetic diversity; Population genetic structure; China
Cystic echinococcosis (CE) or hydatid disease is known to be cosmopolitan in its global distribution, while alveolar echinococcosis (AE) is a much rarer though more pathogenic hepatic parasitic disease restricted to the northern hemisphere. Both forms of human echinococcosis are known to occur on the Tibetan Plateau, but the epidemiological characteristics remain poorly understood. In our current study, abdominal ultrasound screening programs for echinococcosis were conducted in thirty-one Tibetan townships in Ganze and Aba Tibetan Autonomous Prefectures of northwest Sichuan Province during 2001-2008. Hospital records (1992-2006) in a major regional treatment centre for echinococcosis in Sichuan Province were also reviewed. Of 10,186 local residents examined by portable ultrasound scan, 645 (6.3%) were diagnosed with echinococcosis: a prevalence of 3.2% for CE, 3.1% for AE and 0.04% for dual infection (both CE and AE). Human cystic and alveolar echinococcosis in pastoral areas was highly co-endemic, in comparison to much lower prevalences in semi-pastoral or farming regions. The high ultrasound prevalence in these co-endemic areas in northwest Sichuan Province was also reflected in the hospital study, and hospital records furthermore indicated another possible highly co-endemic focus in Guoluo Prefecture of Qinghai Province, located at the border of northwest Sichuan. These chronic cestode zoonoses constitute an unparalleled major public health problem for pastoral Tibetan communities, and pose great difficulties for adequate treatment access and effective transmission control in such remote regions.
Cystic echinococcosis; Alveolar echinococcosis; Ultrasound; Prevalence; Tibetan; Sichuan Province; Qinghai Province
Alveolar echinococcosis cases diagnosed histopathologically in 2002, 2006, 2007, and 2009 in Ulaanbaatar, Mongolia were reconfirmed by evaluating the cytochrome c oxidase subunit I gene of mitochondrial DNA. The most recent three cases using paraffin-embedded and ethanol-fixed specimens revealed that one was of the “Asian” haplotype, whereas two others were of the “Inner Mongolian” type. All patients were born in the western provinces of Mongolia, they never resided outside of Mongolia, and they were given a preliminary diagnosis of malignant hepatic tumor or abscess. The most recent two cases were also confirmed serologically to be active alveolar echinococcosis.
An understanding of the correlation of the specific antibody responses and the disease phase is essential in evaluating diagnostic values of immunological tests in human echinococcosis. In this study, 422 echinococcosis patients diagnosed by ultrasonography, including 246 with cystic echinococcosis (CE), 173 with alveolar echinococcosis (AE), and 3 with dual infection, were tested for specific IgG in sera against recombinant AgB (rAgB) and recombinant Em18 (rEm18) in an enzyme-linked immunosorbent assay. As a result, rAgB-specific antibody was detected in 77.6% of CE and 86.1% of AE patients, while rEm18-specific antibody was present in 28.9% of CE and 87.3% of AE patients. Additionally, all three patients with dual infection exhibited specific antibodies responding to rAgB and rEm18. Further analysis revealed that rAgB-specific antibody was elevated in a significantly greater proportion (87.3%) of CE patients with cysts at active or transitional stages (CE1, CE2, or CE3), compared to 54.8% of other patients with cysts at an early or an inactive stage (CL or CE4 or CE5). Furthermore, rAgB-specific antibody was detected in 95.6% of CE2 cases, which was statistically greater than that (73.7%) in CE1 patients. Although rEm18-specific antibody was elevated in 28.9% of CE patients, the positive reaction was much weaker in CE than in AE cases. Serum levels and concentrations of rEm18-specific antibody were further indicated to be strongly disease phase correlated in AE patients, with positive rates of 97.4% in cases with alveolar lesions containing central necrosis and 66.7% in patients with early alveolar lesions that measured ≤5 cm.
We compared the performance of loop-mediated isothermal amplification (LAMP) with that of a multiplex PCR method for differential detection of human Taenia parasites in fecal specimens from taeniasis patients. The LAMP method, with no false positives, showed a higher sensitivity (88.4%) than the multiplex PCR (37.2%). Thus, it is expected that the LAMP method has a high value for molecular diagnosis of taeniasis.
Both epilepsy and paragonimiasis had been known to be endemic in Southwest Cameroon. A total of 188 people (168 and 20 with and without symptoms confirmed by clinicians, respectively, 84.6% under 20 years old) were selected on a voluntary basis. Among 14 people (8.3%) with history of epilepsy, only one suffered from paragonimiasis. Therefore, we challenged to check antibody responses to highly specific diagnostic recombinant antigens for two other helminthic diseases, cysticercosis and toxocariasis, expected to be involved in neurological diseases. Soil-transmitted helminthic infections were also examined.
Fecal samples were collected exclusively from the 168 people. Eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms were found from 56 (33.3%), 72 (42.8%), and 19 (11.3%) persons, respectively. Serology revealed that 61 (36.3%), 25 (14.9%) and 2 (1.2%) of 168 persons showed specific antibody responses to toxocariasis, paragonimiasis and cysticercosis, respectively. By contrast, 20 people without any symptoms as well as additional 20 people from Japan showed no antibody responses. Among the 14 persons with epilepsy, 5 persons were seropositive to the antigen specific to Toxocara, and one of them was simultaneously positive to the antigens of Paragonimus. The fact that 2 children with no history of epilepsy were serologically confirmed to have cysticercosis strongly suggests that serological survey for cysticercosis in children is expected to be useful for early detection of asymptomatic cysticercosis in endemic areas.
Among persons surveyed, toxocariasis was more common than paragonimiasis, but cysticercosis was very rare. However, the fact that 2 children were serologically confirmed to have cysticercosis was very important, since it strongly suggests that serology for cysticercosis is useful and feasible for detection of asymptomatic cysticercotic children in endemic areas for the early treatment.
A total of 188 people (168 and 20 with and without symptoms confirmed by clinicians, respectively, 84.6% under 20 years old) were selected on a voluntary basis in Cameroon. Soil transmitted helminthic infections were prevalent among persons surveyed as is common in developing countries, since eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms were found from 56 (33.3%), 72 (42.8%) and 19 (11.3%) persons, respectively. Serological analyses revealed that 61 (36.3%), 25 (14.9%) and 2 (1.2%) persons were positive to the diagnostic antigens specific for toxocariasis, paragonimiasis and cysticercosis, respectively. Among 14 people with epilepsy, 5 persons were seropositive to the antigen of Toxocara and one of them was simultaneously positive to the antigens of Paragonimus. Serological confirmation of cysticercosis in two children is very important, and we suggest that further serologic surveys of cysticercosis be carried out in both children and adults in this area for the promotion of a better quality of life including control and early treatment.
Two cases of alveolar echinococcosis (AE) with multiple-organ involvement (the liver, lungs, and bone) were monitored by imaging and serology for 20 years. Resection of the bone lesion was complete in one case but incomplete in the other case. Albendazole treatment was markedly to moderately effective against hepatic and pulmonary AE lesions in both cases, whereas it had almost no effect against the bone lesion in one case. The results of the serological tests with recombinant Em18 antigen coincided with the clinical findings in each case. An enzyme-linked immunosorbent assay for the detection of immunoglobulin G (IgG) responses, especially IgG4 responses, is expected to be a real-time indicator of the dynamics of active AE.
Echinococcus vogeli infection in a hunter from the rain forest of French Guiana was confirmed by imaging and mitochondrial DNA sequence analysis. Serologic examination showed typical patterns for both alveolar and cystic echinococcosis. Polycystic echinococcis caused by E. vogeli may be an emerging parasitic disease in Central and South America.
zoonosis; helminthic infection; parasites; Echinococcus vogeli; French Guiana; dispatch
Echinococcus granulosus is usually transmitted between canid definitive hosts and ungulate intermediate hosts.
Lesions found in the livers of ground squirrels, Spermophilus dauricus/alashanicus, trapped in Ningxia Hui Autonomous Region, an area in China co-endemic for both E. granulosus and E. multilocularis, were subjected to molecular genotyping for Echinococcus spp. DNA. One of the lesions was shown to be caused by E. granulosus and subsequently by histology to contain viable protoscoleces.
This is the first report of a natural infection of the ground squirrel with E. granulosus. This does not provide definitive proof of a cycle involving ground squirrels and dogs or foxes, but it is clear that there is active E. granulosus transmission occurring in this area, despite a recent past decline in the dog population in southern Ningxia.
Echinococcus granulosus and E. multilocularis are important zoonotic pathogens that cause serious disease in humans. E. granulosus can be transmitted through sylvatic cycles, involving wild carnivores and ungulates; or via domestic cycles, usually involving dogs and farm livestock. E. multilocularis is primarily maintained in a sylvatic life-cycle between foxes and rodents. As part of extensive investigations that we undertook to update available epidemiological data and to monitor the transmission patterns of both E. granulosus and E. mulilocularis in Ningxia Hui Autonomous Region (NHAR) in northwest China, we captured small mammals on the southern slopes of Yueliang Mountain, Xiji, an area co-endemic for human alveolar echinococcosis and cystic echinococcosis. Of 500 trapped small mammals (mainly ground squirrels; Spermophilus dauricus/alashanicus), macroscopic cyst-like lesions (size range 1–10 mm) were found on the liver surface of approximately 10% animals. One of the lesions was shown by DNA analysis to be caused by E. granulosus and by histology to contain viable protoscoleces. This is the first report of a natural infection of the ground squirrel with E. granulosus. We have no definitive proof of a cycle involving ground squirrels and dogs/foxes but it is evident that there is active E. granulosus transmission occurring in this area.
Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.
Human cystic echinococcosis, caused by infection with the larval stage of Echinococcus granulosus, and alveolar echinococcosis, caused by the larval form of E. multilocularis, are known to be important public health problems in western China. Echinococcus shiquicus is a new species of Echinococcus recently described in wildlife hosts from the eastern Tibetan plateau and its infectivity and/or pathogenicity in humans remain unknown. In the current study, parasite tissues from various organs were collected post-operatively from 68 echinococcosis patients from Sichuan and Qinghai provinces in eastern China. The tissues were examined by histopathology and genotyped using DNA sequencing and PCR-RFLP. Histopathologically, 38 human isolates were confirmed as E. granulosus and 30 as E. multilocularis. Mitochondrial cob gene sequencing and PCR-RFLP with rrnL as the target gene confirmed 33 of 53 of the isolates to have the G1 genotype of sheep/dog strain of E. granulosus as the only source of infection, while the remaining 20 isolates were identified as E. multilocularis. No infections were found to be caused by E. shiquicus. Additionally, 5 of 20 alveolar echinococcosis patients were confirmed to have intracranial metastases from primary hepatic alveolar echinococcosis lesions. All these cases originated from four provinces or autonomous regions but most were distributed in Sichuan and Qinghai provinces, where high prevalence rates of human alveolar echinococcosis and cystic echinococcosis were previously documented.
Alveolar echinococcosis; Cystic echinococcosis; Echinococcus shiquicus; Histopathology; PCR; Tibet
We confirmed sympatric occurrence of Taenia solium, T. saginata, and T. asiatica in western Thailand. DNA analysis of morphologically identified T. saginata, in a dual infection with T. solium, indicated it was T. asiatica. To our knowledge, this report is the first of T. asiatica and a dual Taenia infection from Thailand.
Taenia asiatica; Taenia saginata; Taenia solium; sympatric distribution; dual infection; mitochondrial DNA analysis; Kanchanaburi; Thailand; dispatch
A Japanese woman presenting with neurologic symptoms was presumptively diagnosed with neurocysticercosis based on imaging findings. Hooklets in the scolex of the resected lesion were not confirmed through histopathological observation. However, the illness was confirmed by mitochondrial DNA analysis to be a solitary neurocysticercosis case caused by the Asian genotype of Taenia solium.
Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH2-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.
Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections.
To further evaluate recombinant Em18 antigen (rEm18) for immunodiagnosis of human alveolar echinococcosis, 208 serum samples were examined by enzyme-linked immunosorbent assay (ELISA). To comparatively assess the results of rEm18-ELISA, ELISA and immunoblot analysis with two affinity-purified native antigens were also performed with 45 selected serum samples. The results indicate that rEm18 is highly useful for serodiagnosis.
Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out “blindly” using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.