Advances in the Diagnosis of Cysticercosis

Abstract

Human cysticercosis is one of the most pathogenic and lethal diseases. It is caused by the accidental ingestion of Taenia solium eggs. All Taenia species lead to cysticercosis in animals; T. solium and Torenia asiatica are responsible for cysticercosis in pigs, while T. saginata causes bovine cysticercosis. Cysticercosis in humans is considered a neglected tropical disease. Diagnosing taeniasis—an infection with the adult parasite—poses challenges. The clinical manifestations of the disease are nonspecific, and no easy method is available to confirm the diagnosis. The diagnosis of cysticercosis is mainly based on imaging techniques, including computed tomography and magnetic resonance. These techniques are valuable and accurate but sometimes limited due to atypical images that are difficult to distinguish from neoplasms. Therefore, sensitive and specific methods, such as immunological tests and molecular methods, are essential to confirm clinical findings and differentiate cysticercosis from other diseases.

1. Introduction

Taeniasis and cysticercosis (T/C) are zoonotic infections caused by tapeworms of the genus Taenia. Humans are definitive hosts for three Taenia species that can cause human taeniasis: Theridion solium, Theridion saginata, and Torenia asiatica [1]. Cysticercosis refers to the tissue infection of the intermediate host: bovines for T. saginata and pigs for T. solium and T. asiatica—the first one is endemic to many developing regions, while the latter is restricted to some Asian countries [2]. Humans can get infected by ingesting raw or undercooked meat contaminated with cysticerci (larval cysts). However, humans can get infected with T. solium by consuming food or water contaminated with parasite eggs and act as dead-end intermediate hosts, developing cysticercosis. This infection often leads to neurocysticercosis (NCC), a major cause of epilepsy associated with considerable morbidity and mortality [3].

Neurocysticercosis is currently the most prevalent helminthic infection of the central nervous system (CNS). Although its prevalence is unknown, millions of people are likely infected with this parasite, and many eventually develop clinical symptoms. Most nations in Latin America, sub-Saharan Africa, and parts of Asia have endemic NCC. However, it is not typical in Northern Europe, the US, Canada, Australia, Japan, or New Zealand—except among immigrant communities [4].

T. solium and T. saginata are flat, segmented, and hermaphrodite parasites measuring 2–10 m. Adult parasites are located in the small intestine. They comprise a head—or scolex—with a diameter of ~1 mm bearing four muscular suckers for fixation and some form of locomotion. Unlike T. saginata, T. solium has an armed rostellum bearing 22–36 hooks ordered in two rows. A thin neck measuring ~5–10 mm constitutes the portion with the most biokinetic activity; the entire body—or strobila—is formed from this part [5]. The adult T. asiatica worm measures 3.41 m in length and 9.5 mm in width and comprises 712 segments. The scolex of adult or larva T. asiatica has four suckers of 0.24–0.29 mm in diameter and a cuspidal rostellum with a maximal width of up to 0.81 mm. The maximum width of the T. asiatica scolex is 1.5 times smaller than that of T. saginata [6]. The strobila of Taenia species comprises 800–4000 proglottids—or segments—divided into immature, mature, and gravid segments. Immature segments are wider transversely than longitudinally, whereas mature segments are square, with primary sexual organs fully developed. Finally, gravid segments are rectangular, with the longest axis running lengthwise; most primary genital organs are atrophied, while the uterus is almost entirely branched and filled with oncospheres.

Oncospheres, or spherical eggs, are found in the uterus and range from 29 to 77 μm for T. solium, 39 to 50 μm in T. saginata, and 33.8 to 40 μm in T. saginata. The eggs of Taenia species cannot be distinguished by conventional light microscopy [7]. A cysticercus is an ovoid vesicle with a translucent membrane and a 5–15 mm diameter. It is filled with a colorless liquid and has an invaginated scolex. The larval stage is called Cysticercus spp., more appropriately called a “metacestode” of Taenia spp. The term “cysticercus racemosus” is frequently used to describe a wild-growing T. solium cysticercus in humans. This metacestode has a degenerative form and is found in the meninges and ventricular system of the brain. Cysticerci can persist in the brain for many years before patients experience symptoms; in some instances, they may never. Pleomorphic symptoms and signs are frequently brought on by the degradation of cysticerci and the accompanying host immunological response. Epileptic seizures, headaches, focal neurological impairments, and indicators of elevated intracranial pressure are the most typical symptoms. Neurological manifestations depend on the number—single or multiple—size, location (e.g., intraparenchymal or extraparenchymal), stage of the cysticerci—viable or calcified—and the host’s immune response [8].

Researchers have been trying to develop diagnostic techniques to detect the presence of parasites within bodily tissues since the early 20th century. Counting white blood cells, specifically eosinophils, was an initial nonspecific test that raised the possibility of infections [9]. Serological tests for circulating antibodies (Abs) were created very quickly. Although unable to distinguish between active and dormant infections, antibody detection proved to be a better attempt at diagnosis, with greater predictive values than the earlier eosinophil counting [10]. The enzyme-linked immunosorbent test (ELISA), introduced in 1971, quickly gained popularity as the preferred method for detecting antibodies; it was the most sensitive method available at the time to process multiple samples simultaneously [11]. Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), developed in 1977 and 1986, respectively, significantly improved the classification of NCC features [12]. These scans made it easier for clinicians to make definitive diagnoses based on the size, type, stage, and location of cysticerci in the patient’s CNS. Neuroimaging is still the reference standard today.

2. Diagnosis

2.1 Clinical diagnosis

Although clinical history alone is insufficient for NCC diagnosis, healthcare professionals should become familiar with some of the nonspecific clinical manifestations, particularly in endemic regions [13]. NCC symptoms vary depending on the location, number, and size of the infecting worm(s), the duration of the infection, the evolutionary stage of the lesions, and the presence or absence of a cephalic budding cysticercus [14].

Patients with symptomatic NCC frequently appear with nonspecific signs that may not allow diagnosis. Seizures are the most prevalent manifestation, accounting for 70% of symptomatic cases, and can happen at any age [15]. Headaches, focal neurologic impairments, intracranial pressure, and cognitive deterioration are other clinical signs of the illness [13]. Focal neurological signs are uncommon and depend on where parasites are located in the nervous system or when a cysticercosis-related stroke occurs. Intracranial hypertension is mainly limited to people with hydrocephalus, cysticercotic encephalitis, and large subarachnoid or ventricular cysts [16].

Health professionals must gather epidemiological information about patients, such as travel history, birthplace, address, and awareness of past or present intestinal tapeworm infection in oneself or household members, in addition to clinical assessments [13, 17]. Based on clinical suspicion, neuroimaging and serological tests will be required to confirm or rule out a diagnosis. The target sites for cysticercosis detection are illustrated in Figure 1.

Figure 1.

Taeniasis/cysticercosis diagnostic tests (dx). Human: Adult tapeworm (taeniasis detection), larval cysts ([neuro]cysticercosis detection); pigs: Cysticerci in live pig or pork (cysticercosis detection).

2.2 Neuroimaging methods

Imaging techniques, such as CT and MRI, are essential because they can reveal the parasite’s presence, number, location, size and stage, and the host’s immune response, which manifests as diffuse or perilesional inflammation and blood-brain barrier dysfunction visible by focal contrast enhancement. Additionally, those techniques might indicate additional related diseases, such as hydrocephalus or a stroke [3, 18].

Both CT and MRI should be performed in patients with suspected NCC. MRI offers better imaging of tiny lesions, particularly those close to the skull and in the posterior fossa and provides more information on parenchymal inflammation or periventricular effusion in hydrocephalus [15, 19]. However, CT is considerably better at detecting calcifications and quantifying lesions and is more available in hospitals in endemic regions [20]. In addition to imaging studies, electroencephalography is useful in diagnosing NCC. It can provide a helpful map and the source of abnormal brain activity consistent with the regional distribution of lesions observed on CT scans [21, 22].

2.3 Laboratory diagnosis

2.3.1 Serologic diagnosis

Immunodiagnostic techniques are essential to support clinical results and help with diagnosis. Two immunological assays are available: antibody detection of past and current infections and antigen detection of recent infections. T. solium infection triggers the formation of a specific immunoglobulin G antibody that can be detected in serum and cerebrospinal fluid (CSF) [23].

Lentil lectin-purified glycoproteins are used in an enzyme-linked immunoelectrotransfer blot format in the serodiagnostic assay for cysticercosis and neurocysticercosis. They are approved by the World Health Organization and the Pan-American Health Organization [24]. Despite this assay’s excellent sensitivity and specificity, antigen purification requires advanced techniques and specialized knowledge. It is not a quantitative assay and is challenging to apply in field research [25]. In addition, multiple types of antigens have been used for the immunodiagnosis of cysticercosis, including low molecular mass antigens, excretory/secretory antigens, crude soluble extract, total saline extract, antigen B, vesicular fluid, membrane and scolex extracts, somatic antigens, recombinant proteins, and synthetic peptides [26].

The major drawback of this approach is the possibility of false positives because antibodies do not always indicate an active infection with viable metacestodes but a resolved infection or exposure to the parasite [27]. Another disadvantage is the possibility of cross-reactivity with other parasitic diseases, such as the one caused by Echinococcus granulosus. However, cross-reactivity has also been reported with other diseases, including hymenolepiasis, fascioliasis, toxocariasis [28], toxoplasmosis, malaria, amoebiasis, cerebral tuberculosis [29], syphilis, and hepatitis [30].

There has been an increased interest in diagnosing NCC using new alternative antigenic sources because the presence or absence of antibodies cannot distinguish between different stages of the disease. A 2017 study by Nunes et al. used protein purification and gel filtration chromatography to identify potential heterologous antigens in a T. solium metacestode [31]. The study sought to discover specific polypeptides of interest and B cell epitopes for diagnosing NCC using gel filtration fractions and mass spectroscopy. Precursors of enolase and the calcium-binding protein calreticulin unique to the metacestode were discovered to have particular B cell epitopes indicative of NCC patients. Identifying these markers in serological samples is crucial and could be a reliable diagnostic tool for identifying NCC patients.

Detecting the specific circulating parasitic antigens can confirm the presence of viable parasites and overcome potential limitations. Several antigen detection techniques utilizing polyclonal or monoclonal antibodies have been studied. Two monoclonal antibody-based tests are standardized: B158/B60 Ag-ELISA and HP10. Antigen detection assays can also be used to monitor the efficacy of anthelminthic drugs and differentiate between viable parasites and locate them in the central nervous system [32]. A 2020 study by Kabululu et al. observed that the B158/B60 monoclonal-based sandwich enzyme-linked immunosorbent assay (Ag-ELISA) was more reliable in ruling out T. solium cysticercosis in pigs [33].

3. Histological study of the parasite from biopsy

Visualization of histopathological characteristics of cysticerci in biopsy material, including the spinal canal, the rostellum with its four suckers, and the double crown of hooks, can confirm NCC diagnosis. In many subarachnoid cysts, the scolex cannot be identified, but the typical three-layered membrane wall often allows the correct identification of the parasite. However, a problem arises when biopsy material comes from calcified or granular cysticerci since the scolex and membranes may not be present in these involution stages of the parasite. In such cases, the presence of the calcareous corpuscles may help identify the lesion’s parasitic (cestode) nature [13].

3.1 Molecular techniques

PCR has been used for the amplification of DNA sequences. Since its creation by Mullis in 1983, PCR has developed into a crucial diagnostic tool. Several molecular assays have been described for the detection and differentiation of parasites, including Taenia species, using genomic or mitochondrial DNA: multiplex-PCR, nested PCR, quantitative real-time PCR, PCR-Restriction Fragment Length Polymorphism, a base excision sequence scanning thymine-base method (Yamasaki et al., 2002) and random amplified polymorphic DNA (RAPD) [34, 35, 36, 37, 38].

The most significant contribution of molecular methods has been in the genotyping of the genus Taenia, which has served to determine the phylogeny and taxonomy of the species and to understand the level of genetic diversity within the genus [38]. Another essential contribution of molecular biology was the identification and production of antigenic molecules used as candidates for vaccines or serological tests. Direct use of molecular techniques for NCC diagnosis was first reported in 2006. It identified T. solium DNA in the CSF of 29 out of 30 consecutive patients by PCR with primers against pTsol9, specific to T. solium [39, 40]. Another study found parasite DNA in human CSF, using primers against HDP2, based on a noncoding sequence of T. saginata, which cross-reacts with T. solium. The study also reported different sensitivities depending on the NCC type (10/14 for extraparenchymal NCC cases compared to 4/24 for intraparenchymal, degenerating NCC cases) [41].

MicroRNAs (miRNA) have recently been investigated as potential biomarkers. They are single-stranded, short, noncoding, endogenous RNAs with a role in post-transcriptional gene regulation. Although miRNAs have been linked to many parasites, little is known regarding T. solium. Possibly, during chronic infection, T. solium manipulates the host’s mRNA with miRNA to control the host-parasite interaction. Although these miRNAs have never been employed as biomarkers for infection diagnosis, they are increasingly recognized as novel disease regulators. To help with disease diagnosis and treatment, more investigation into the host-parasite interaction and miRNA is necessary [42].

Finally, neurocysticercosis remains a significant public health concern and financial burden in endemic areas. Numerous neurological symptoms caused by this parasite require expensive diagnostic methods. Despite efforts to create diagnostic techniques, serological analyses combined with neuroimaging currently serve as the primary diagnostic method. Neuroimaging is pricey and cannot detect isolated cysts or extraparenchymal infections. Additionally, the ideal serological biomarker remains elusive. Creating novel, trustworthy, and reasonably priced diagnostic techniques is crucial for the therapeutic management of this underappreciated tropical disease and for determining the actual global health burden it poses.