**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) [34–38].

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.
