Laboratory diagnosis of Taenia asiatica in humans and animals.

Taenia asiatica is a recently described species known to cause intestinal teniasis in humans and cysticercosis in animals. This species has close morphological resemblance to Taenia saginata and has a life cycle resembling Taenia solium, hence has been posing diagnostic dilemma and had been the reason for its comparatively late discovery. Recent diagnostic tools such as serological and molecular techniques have thrown light on its exact prevalence in the endemic countries. Hence introduction of utilization of these techniques in addition to the routine morphological analysis would be helpful in diagnosis of T. asiatica infections and early implementation of preventive measures.

INTRODUCTION

The taeenid tapeworms known to cause zoonotic infections in humans are Taenia solium, Taenia saginata and Taenia asiatica. The first two species are well known agents causing cysticercosis and intestinal taeniasis respectively.[1] T. asiatica was discovered when epidemiological inconsistency was observed between the species ratio of tapeworms and the eating habits of hosts in South Korea.[2] T. asiatica has equivocal features of T. saginata but recent deoxyribonucleic acid (DNA) studies, molecular and epidemiologic characteristics had made the taxonomic conversion of T. saginata subsp. asiatica to the current separate species T. asiatica. T. asiatica has been identified to parasitise human intestinal lumen in eight Asian countries i.e. China, Korea, Indonesia, Philippines, Taiwan, Thailand, Vietnam and Japan, with the prevalence being as high as 21% causing significant economic burden.[3] This unique species exhibits a morphology resembling T. saginata and life cycle resembling T. solium infection, pigs being the main intermediate host.[4] Although T. asiatica is well-known to cause intestinal teniasis, less is known about its role in causation of cysticercosis in humans.

LABORATORY DIAGNOSIS OF T. ASIATICA TAENIA ASIATICA

The laboratory diagnosis of human intestinal taeniasis for decades had been based on the detection of bile stained eggs and the gravid proglottids of the adult worm passed in the feces. Recent studies reveal that microscopic examination of eggs or the gravid proglottids alone is not a reliable method of differentiating T. saginata from T. asiatica because of their similarities.[5] Hence it is hypothesized that the exact epidemiologic prevalence of T. asiatica might be underestimated. Newer methods of reliable detection and differentiation of these parasites include serological and molecular methods.

MORPHOLOGICAL METHODS

Species-specific detection of Taenia plays an important role in public health importance and understanding the epidemiology of the Taenia spp. The role in public health importance being the different consequences of Taenia infections since detection of T. solium cases can prevent human cysticercosis cases. Routine stool examination from cases of intestinal taeniasis reveals bile stained eggs measuring 30-35 μm in diameter, radially striated with an internal oncosphere containing sin refractile hooklets.[5] The eggs of Taenia spp. are indistinguishable from each other yet studies report utilization of Ziehl-Neelsen staining in less-sensitive differentiation of T. solium and T. saginata.[6] This staining has not yet been studied in T. asiatica. Hence for all practical purposes, it is implied that examination of Taenia eggs cannot be used for species differentiation. All tapeworm eggs and proglottids should be handled with care, especially if the species has not been determined since the eggs of T. solium and Hymenolepis nana are infectious to humans. Another method of differentiation between species is the examination of scolices and gravid proglottids passed in feces. Evacuation of these gravid proglottids are usually uncommon and hence medications used for evacuation. Traditionally ricine-oil purgation was utilized. Recently, anti-Taenia drugs such as niclosamide and in niclosamide-resistant cases, praziquantel and mepacrine are utilized along with purgatives like electrolyte-polyethyleneglycol salt for expulsion of scolices and proglottids.[789] Examination of the expulsed scolices and gravid proglottids helps in differentiation of Taenia spp.[10] These structures after isolation from fecal debris are stored in 10% buffered formalin or 70% ethyl alcohol for long-term storage. The scolex or the gravid proglottid is stained with India ink or carmine dye by passing a 25 gauge needle through the central uterine stem or the uterine pore and is pressed between glass slides, fixed with alcohol-formalin-acetic acid, dehydrated with ethyl alcohol and mounted.[510] Another alternative for examination of the morphology is hematoxylin and eosin (H and E) staining. Here the gravid proglottids after fixation in neutral buffered 10% formalin is paraffin-mounted and longitudinal sections of 6 μm performed. These sections are subjected to H and E staining for visualization of the morphological features.[11] The morphological features used in differentiation of species are shown in Table 1.

Table 1

Morphological differences between Taenia spp. causing human infections

In animals, characteristically the larval stage or the cysticercus of T. asiatica is observed in the liver of pigs (viscerotropic), whereas in T. solium and T. saginata the larvae are seen in the muscle of pigs and cattle respectively.[5] Examination of cysticercus of T. asiatica reveals the characteristic wart-like outgrowths on its surface [Table 1].[1012]

SEROLOGICAL DIAGNOSIS

Recent advances and research in immunodiagnosis of taeniasis have improved the diagnostic sensitivity and specificity. These techniques are particularly well developed for detection of cases of human cysticercosis, which include specific antibody detection or circulating parasite antigen from serum and cerebrospinal fluid samples.[13] Excretory-secretory antigens derived from adult T. solium are utilized in enzyme-linked immunoelectrotransfer blot (EITB) to detect molecules ranging between 32.7 and 41.2 kDa which are highly specific for T. solium taeniosis.[14] Coproantigen detection by enzyme-linked immunosorbent assays (ELISA) has been reported to be highly sensitive and specific in diagnosis of T. solium taeniosis.[1516] For diagnosis of cysticercosis, crude or purified glycoproteins from cyst fluid and wall of T. solium or Taenia crassiceps have been utilized in EITB with high sensitivity and specificity.[17181920] Antigen-based ELISA are helpful in detection of active cysticercosis. Immunoblot patterns of sera from T. asiatica-infected humans had been extensively studied using adult and egg antigens of Taenia and were compared with other cestode infections by Jeon and Eom.[21] In their study, 38 proteins between 121 and 21.5 kDa were observed reactive in sera of T. asiatica infected individuals when tested against T. asiatica adult antigens and when tested with egg antigens reactive bands were observed between 120 and 8 kDa. Bands of size 120, 110-100, 98-96, 68-66, 58-56 and 37 kDa were found to be shared by other cestode infections, whereas 21.5 kDa band was highly specific to T. asiatica. When adult T. saginata crude antigens were blotted with sera infected with T. saginata, major bands detected were 110-100, 86 and 67 kDa. Whereas sera from T. asiatica and T. solium-infected individuals, bands were detected at 110-100, 66, 58 and 46 kDa, indicating that 86 kDa band is specific for T. saginata when adult T. saginata crude antigens are used. Hence EITB utilizing highly these highly specific protein bands helps in species differentiation of Taenia. Two other promising immunodominant candidate antigens which has been identified and expressed are the recombinant T. asiatica Lactate dehydrogenase (rTaLDH) and the recombinant T. asiatica enolase (rTaENO).[2223] These immunodoninant proteins have been localized to their presence in the tegument of adult T. asiatica and embryonic membrane of oncosphere. Coproantigen ELISA is currently widely used in differentiation of taeniosis due to T. solium and T. saginata.[24] Introduction of coproantigen ELISA utilizing the above mentioned recombinant immunodominant epitopes highly specific for T. asiatica would unmask the true prevalence of this infection in humans and animals.

MOLECULAR DIAGNOSIS

Molecular methods have been widely used in the diagnosis of various infectious diseases including various parasities changing the trend of microbiology to rapid and sensitive diagnosis.[2526] These methods also have been developed in the accurate detection and differentiation of Taenia species. For any molecular method to be successful in detection, the prerequisite is obtaining DNA of good quality and quantity. Samples amenable to DNA extraction are adult proglottids or scolices obtained naturally from human carriers/expelled after chemotherapy, eggs passed in the feces and cysticercus (metacestodes) obtained from animals. These samples should be freshly collected, rinsed in physiological saline and immediately frozen/fixed in 75% ethanol. Although DNA has been detected from formalin-fixed specimens stored in museums and laboratories, formalin is known to render the quality of the DNA substandard due to degradation and hence not recommended for fixation and long-term storage.[13] Conventionally, DNA can be extracted using proteinase K digestion, phenol-chloroform-isoamyl alcohol extraction and precipitation of DNA with ethyl alcohol. This method has been successfully used widely for extraction of DNA from various parasites.[25] Alternate methods of extraction of DNA include commercial kits like DNeasy Tissue kit and QIAamp DNA stool mini kit (Qiagen, Hilden, Germany).

The molecular approaches which have been tried in the detection and differentiation of Taenia species are Restriction fragment length polymorphism-Polymerase polymerase chain reaction (RFLP-PCR), Random amplified polymorphic DNA-PCR (RAPD-PCR), Single single strand conformation polymorphism (SSCP), multiplex PCR, Looploop-mediated isothermal amplification (LAMP), DNA probes and nucleotide sequencing.

RFLP of nuclear ribosomal DNA (rDNA) and mitochondrial DNA had been studied to differentiate Taenia species.[2728] Here the extracted DNA is digested with restriction enzymes, electrophoretically separated on agarose gel, transferred to nitrocellulose membrane and hybridized with specific DNA probe. In RFLP-PCR, the target sequence is amplified initially and then subjected to RFLP procedure, increasing the sensitivity and simplifying the procedure. The amplicon spanning the internal transcribed spacer 1 (ITS1) as target sequence mainly containing the 5.8S gene, is subjected to RFLP producing characteristic patterns for each Taenia species.[27] Other targets utilized successfully in RFLP-PCR analysis are mitochondrial cytochrome c oxidase subunit 1 (cox 1), mitochondrial 12S rDNA etc.[2930] SSCP display of amplicons targeting mitochondrial cox 1 and NADH dehydrogenase subunit (nad1) genes have been used to differentiate Taenia species.[31] Similarly DNA sequencing of mitochondrial cox 1 and nad1, cytochrome b, 12S rDNA, nuclear 28S rDNA and ITS1/ITS2 rDNA genes have been valuable in differentiating mainly T. saginata from T. solium by comparing the obtained sequence with already published DNA sequences.[31323334] Targets other than mitochondrial cox 1 have been less studied in differentiation of T. saginata from T. asiatica. Yamasaki et al., reported differential detection of taeniid cestodes by base excision sequence scanning thymine-base reader analysis of mitochondrial genes, cox 1 and cytochrome b. The T-base peak profiles obtained were distinct for the three Taenia species causing human intestinal taeniosis. T-base peak appeared at nucleotide position 153 in T. asiatica, position 174 and 189 for T. saginata, position 189 and 195 for American/African genotype of T. solium and position 195 only for Asian genotype of T. solium.[35]

Multiplex PCR employing genus-specific and species-specific primers have been reported in literature to differentiate Taenia species. Multiplex PCR based on mitochondrial cox 1 as target gene, produced specific amplicon size of 827 and 269 bp for T. saginata and T. asiatica respectively. For T. solium of American/African and Asian genotype, specific amplicon size of 720 and 984 bp were reported.[3637] González et al., designed HDP-2 gene polymorphism-based PCR protocol to differentiate species of Taenia. This species-specific identification was based on the size difference in amplicons generated: Fragments of 1300 bp, 600 bp and 300 bp for T. asiatica, amplicons of 1300 bp and 300 bp for T. saginata and one amplicon of 600 bp for T. solium samples were produced with the HDP2-PCR protocol.[38] Isothermal amplification methods like LAMP have also been utilized in diagnosis and differentiation of Taenia species with higher sensitivity and specificity than multiplex PCR.[39] Nkouawa et al., have evaluated LAMP essay targeting cathepsin L-like cysteine peptidase (clp) and cox 1 genes. LAMP with primers targeting the cox 1 could differentiate between the three species, whereas he primers targeting clp gene couldn’t differentiate T. saginata from T. asiatica.[4041] This LAMP assay evaluated was able to detect one copy of the target gene or five eggs of T. asiatica/T. saginata per gram of feces, showing sensitivity similar to other PCR methods. The advantages of these molecular methods are that it can be used for large scale screening to detect worm carriers and cases of intestinal taeniosis in humans and cysticercosis in animals.

CONCLUSION

T. asiatica is a recently identified zoonotic human parasite causing intestinal taeniosis, which closely resembles T. saginata. Although slight differences in morphology isslight differences in morphology are noted between the species, they cannot be used solely for the diagnosis and differentiation of species. Hence, supplementation of serological and more promising molecular methods in addition to morphological study would help in differentiation of the species accurately. T. asiatica has been identified only from few regions of the world and introduction of diagnostic protocols in countries endemic to Taenia can reveal the true prevalence of T. asiatica world-wide.