Diagnosis of human taeniasis

Taenia solium, T. saginata and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. T. solium can also cause cysticercosis in humans. A number of diagnostic methods have been developed to diagnose Taenia species that infect humans. This article is aimed at providing an overview of currently available diagnostic methods for human taeniasis.

Human taeniasis is an important zoonotic health problem and is caused by adult stages of taeniid cestodes, including Taenia solium, T. saginata, and T. asiatica. These parasites have indirect life cycles where humans act as definitive hosts whereas pigs (for T. solium and T. asiatica) and cattle (T. saginata) serve as intermediate hosts. Humans become infected by ingesting parasite cysts present in raw or undercooked infected meat/liver; when the cyst reaches the human intestine, it develops into an adult tapeworm, releasing segments and/or eggs in the stools or motile segments (e.g. T. saginata) are expelled actively. Intermediate hosts such as cattle and pigs become infected when they ingest taeniid eggs via contaminated feed or water and the larval stage (cysticercus) forms in muscular and sometimes other tissues¹. The adult tapeworm stage of Taenia spp. is relatively innocuous and does not cause pathogenic effects in humans; however, the intermediate stage of T. solium can also develop in human brains causing neurocysticercosis, a major cause of neurological disease in many developing countries as well as other organs causing intramuscular, ocular, subcutaneous and spinal cysticercoses^2,3. T. saginata is endemic in Australia, whereas T. solium and T. asiatica are exotic. However, people coming and/or returning to Australia from endemic countries can be infected with T. solium, leading to the possibility that infected individuals may pass segments of the parasite in their stools, which can serve as a source of infection for human cysticercosis. Therefore, differentiation of Taenia species becomes significant for surveillance and control of human taeniasis.

A number of diagnostic methods have been used to differentiate the common human cestodes, T. saginata and T. solium; however, each method has its advantages and disadvantages, and careful attention should be paid to determining which particular test is best to use for differentiation of the two species⁴. The following sections provide a quick rundown on various diagnostic methods available to differentiate Taenia spp. that infect humans.

Traditionally, diagnosis of taeniasis has been based on the detection of eggs by microscopic examination but this method lacks sensitivity and specificity as T. solium and T. saginata eggs are morphologically identical, making species identification impossible⁵. However, morphological examination of gravid proglottids can allow the differentiation of T. solium and T. saginata provided the internal structures (i.e. uterine branches) are intact⁵. Sometimes, even the morphological examination of proglottids does not allow the differentiation of T. solium and T. saginata, thus requiring alternate methods for the differentiation of human Taenia spp.

The first method of coproantigen (parasite antigens in human stool) detection using enzyme-linked immunosorbent assay (ELISA) was developed by Allan et al.⁶. Although the test displayed a higher sensitivity and specificity than microscopic diagnosis for the detection of Taenia spp.⁷, it did not allow differentiation of T. saginata and T. solium. Recently, Guezala et al.⁸ developed another coproantigen ELISA and successfully differentiated T. solium from T. saginata. These tests were developed in individual labs using in-house reagents and have not been independently validated in different laboratories nor have they been widely used in diagnostic laboratories.

The first serological assay to detect specific antibodies against T. solium infection in humans was developed by Wilkins et al.⁹ Subsequently, a number of studies reported various immunoassays for the diagnosis of human taeniasis, primarily caused by T. solium^10,11 and these studies used either native excretory-secretory products collected from adult tapeworms⁹ or cloned and expressed excretory-secretory products of adult T. solium^10,11. Like coproantigen ELISAs, the detection of Taenia species-specific antibodies are also more specific and sensitive than microscopic techniques. However, these tests have been found to have some degree of cross-reactivity in sera from patients with cystic echinococcosis, ascariasis, and schistosomiasis¹¹. Furthermore, currently available immunodiagnostic tests may give false positive results as specific circulating antibodies in taeniasis patients could possibly remain detectable for some time either after treatment and recent past infections⁴.

A number of molecular methods using PCR-based technologies have been developed to either determine the presence of Taenia species-specific DNA in human stools or differentiate Taenia spp. (T. solium, T. saginata or T. asiatica) based on the analysis of DNA extracted directly from tapeworm¹². PCR-based methods have higher sensitivity in the detection of taeniasis cases (i.e. the detection of parasite DNA in human stools) than microscopy alone. In addition, a combined use of PCR and microscopy have been found to improve diagnostic sensitivity where Yamasaki et al.¹³ showed that some proven egg-positive cases were negative by PCR. Specificity of PCR is high with control faecal samples, including samples from patients with other parasitic infections, being almost always negative in PCR.

To date, predominantly conventional and multiplex PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP) have utilized various markers, including internal transcribed spacer, mitochondrial cytochrome c oxidase subunit I gene as well as 12S rDNA, cathepsin L-like cysteine peptidase, T. saginata-specific repetitive sequence (HDP1) and cestode-specific sequence (HDP2) to discriminate between human Taenia spp^13–21 (Table 1). Mayta et al.¹⁹ developed a nested PCR utilizing two rounds of PCR amplification of the Tso31 gene, which is more sensitive than conventional PCR but pose technical difficulties. A field DNA-based test known as loop mediated isothermal amplification (LAMP) was developed by Nkouawa et al.²⁰, which amplifies the cox1 gene. To date, only one real-time PCR to discriminate T. solium and T. saginata has been developed which targets the internal transcribed spacer 1 of the nuclear ribosomal RNA²¹. The majority of DNA-based methods have utilised DNA isolated from proglottids for Taenia species identification while relatively fewer studies isolated DNA from stool samples (Table 1). In addition, almost all studies have been tested only on small numbers of usually known positive and negative samples.

Table 1. Selected PCR-based assays for detection and characterization of human Taenia spp.

Human taeniasis is a worldwide parasitic disease and detection and discrimination of T. solium, T. saginata and T. asiatica remains a public health concern. Microscopic, immunological and molecular methods have been used to detect and differentiate Taenia spp., and a combination of two or more methods appears to provide higher sensitivity^13,21. Almost all of the immunological and molecular methods developed so far have not been independently tested and validated on controlled negative and positive samples as well as field samples. Among existing molecular methods, the nested PCR¹⁹ provides the highest sensitivity and specificity of those methods that have been developed and validated using unselected faecal samples from parasitological proven taeniasis carriers. Although the method is technically challenging and could be expected to be relatively expensive to use, the reagents required are available worldwide and laboratories capable of undertaking PCR competently can run this PCR⁴.

To the best of our knowledge, no standardised PCRs are available in Australia to discriminate human Taenia spp. Future studies should focus on independent validation of the nested PCR¹⁹ and the LAMP technique²⁰ as these two DNA-based tests offer higher sensitivity and a user-friendly option without sophisticated equipment, respectively. In addition, future studies should be aimed at extracting DNA from sodium acetate-acetic acid-formalin (SAF) fixed proglottids using different methods as the current DNA extraction protocols do not provide reliable and consistent DNA yield for PCRs (Jabbar, unpublished data). To date only one study has reported the extraction of DNA from long-term stored Taenia specimens in formalin²² and this has not been independently verified.