Next-generation sequencing combined with serological tests based pathogen analysis for a neurocysticercosis patient with a 20-year history:a case report

Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) caused by the larval stage of Taenia solium [1]. It is the most common helminthic infection of the CNS and the leading cause of death from foodborne diseases, according to the world health organization [1, 2]. Accurate and early diagnosis of the NCC is critical for the efficient treatment outcomes of the disease. The reported clinical manifestations and neuroimaging examinations remain the conventional diagnostic approaches for NCC. However, the accuracy depends on multiple factors such as the number, stage, size, and host immune responses [3]. The location of parasitic infection in the nervous system is another critical factor that affects the diagnosis. For example, the diagnosis of extra-parenchymal NCC is more challenging than parenchymal NCC, because parasites at extra-parenchymal NCC have similar signals as cerebrospinal fluid (CSF) and that signal cannot be enhanced by intravenous contrast agents due to lack of scolex [4]. Therefore, the cyst fluid may not be detected using magnetic resonance imaging (MRI). A recent study has shown improved sensitivity and specificity in a polymerase chain reaction (PCR) assay-based diagnosis of extra-parenchymal NCC [5]. Given that next-generation sequencing (NGS) of CSF has been used frequently for the clinical diagnosis of CNS infections [6‐9], we hypothesized that NGS might be an appropriate approach to diagnose parasitic infections in extra-parenchymal NCC. Here, we describe the case of a female patient with the symptoms of NCC, reportedly aggravating atypical headache for over 20 years accompanied with fever and amaurosis, suggesting the possibility of extra-parenchymal NCC without clear radiological evidence.

To the best of our knowledge, this is the first report of NGS-based diagnosis of a patient having NCC symptoms. The patient visited our hospital because of a long history of headache episodes for over 20 years. Subsequently, extensive clinical, pathological, and radiological examinations were performed to address the cause of the headache. Most patients with NCC present headache syndromes as shown in Table 2 [10‐12]. Although CSF cytological and biochemical examinations showed increased intracranial pressure, suggesting signs of inflammation, no abnormalities were revealed in her brain by cerebral MRI. Along with the aggravating headache symptoms, the patient developed paroxysmal amaurosis, likely due to the increased cranial pressure. Further cerebral MRV exhibited the right dominant type of venous drainage system. But the cerebral venous sinus thrombosis was not considered in this case because of the chronic nature of symptoms, and the left venous sinus dysplasia was likely to be congenital. Six lumbar punctures were then performed, and the test results revealed significantly increased intracranial hypertension and an inflammatory profile of CSF with elevated white cells and slightly low glucose level. In addition, the patient had subtle leptomeningeal enhancement on cerebral MRI but without intra-parenchymal or intra-ventricular lesions. Because the patient had severe symptoms of intracranial hypertension, we hypothesized that the parasite might be located in the fourth ventricle. However, in case of extra-parenchymal NCC, the parasite can lack the typical cystic structures, making it more challenging to identify on imaging examinations. Thus, no signs of parasitic infection were detected in multiple cerebral MRI examinations. Considering the fact that the patient lives in an endemic area, where people exhibits higher occurrences of fever and headache, unusual features of NCC [13], due to eating cysticercus contaminated pork, we sought to genetically screen the pathogenic microorganisms corresponding to Taenia solium, present in the non-template controls (NTCs) Interestingly, the investigation revealed highly abundant Taenia solium in our patient with NCC, exceeding other possible background or contaminating microorganisms [10]. Furthermore, the diagnosis of NCC was confirmed by a clinical CSF cysticercus antibody (IgG) assay and serological examinations. After anti-helminthic therapy, the elevated levels of serum/CSF cysticercus antibody titers and the DNA load in CSF were started to decrease as the treatment progressed. The clinical symptoms of the patient were relieved as well. One possible explanation for this phenomenon could be the subsequent release of Taenia solium DNA into the CSF when the larvae were destroyed [5, 14]. It has been found that parasitic DNA load in CSF could either increase or decrease after treatment, suggesting that serial analysis of parasitic load in CSF may not be useful, and further studies are needed [14].

The diagnosis of extra-parenchymal NCC can be challenging since the application of serological tests are very limited. In our case, the patient was initially misdiagnosed as a neuropathic headache before performing serological tests for NCC. For patients with atypical neuroimaging for subarachnoid NCC, such as chronic meningitis, the diagnosis can be more challenging as the infecting parasites often lack their typical cystic structures. Subarachnoid NCC may also cause pronounced inflammation, making it difficult to control and treat [15, 16]. The case we report here with a history of 20 years since the onset of the initial symptoms is a rare one of its kind. The pathogenesis of this case was assumed to be induced by repeated autoinfection, and a possible underlying mechanism might be the spreading of the tapeworm eggs to the whole body by intestinal blood circulation after the reflux from the small intestine to the stomach, where the eggs were digested by the gastric juice, and the larvae were then hatched. Likely, during the viable stage, the parasite somehow can evade the host defenses, causing only a minor immune response at the beginning [17]. The cyst degeneration occurs when the parasite is detected by the immune system, leading to a robust granulomatous inflammatory response accompanied by significant neurological morbidity [18, 19]. The final calcified stage contains a dead parasite that doesn’t cause an inflammatory response [1, 20]. Erin S. Beck [21] has reported the case of a 41-year-old woman with chronic relapsing meningitis. The diagnosis of NCC was confirmed with metagenomic NGS of total RNA, a clinical CSF metacestode antigen assay, and serology after 16 years since the onset, which previously had not been performed. In retrospect, lumbar spine MRI showed a cyst-like structure in the lumbosacral sac, enhancement of the nerve roots, and an extramedullary, intradural nodule. Reasons for the delayed diagnosis were lack of more typical brain cysts, as well as the larger clinical context of recurrent fever and constitutional symptoms. The case also vividly illustrates that either improvement or lack of clinical deterioration in the setting of immunosuppression does not rule out an underlying infectious etiology, even after years of treatment. The patient was eventually diagnosed with NCC using NGS of CSF, demonstrating the effectiveness of the test. The NGS is a convenient, accurate, and fast tool for detecting a broad range of bacterial, viral, fungal, or parasitic infections in clinical samples. We have demonstrated here that the NGS is efficient in accurately diagnosing the patient with NCC. Several previous studies have reported that the Taenia solium DNA in CSF can stably exist for years, even after anti-helminthic treatment. Given that the symptoms are also persistent in those cases, the larvae may not be entirely eliminated by the treatment [14]. The results may be affected by various factors, including diagnosis time, therapeutic strategies, and the location of the parasite in the host body [11]. In this case report, although the patient’s CSF cysticercus antibody test remained positive, the tapeworm was eliminated, which was further confirmed by the third NGS analysis, consistent with the negative serum cysticercus antibody reaction. The sensitivity of antibody detection in serum is comparable with CSF but with less precision. As reported previously, antibody-based detection is unable to distinguish between exposed, inactive, or active infections with low positive predictive power for the cases of viable cysticercosis [22]. Therefore, genetic analysis via NGS should be considered as a standard tool for a pathogen diagnosis under a range of clinical settings. Further studies are needed to confirm the efficiency of the CSF DNA load for assessing disease severity, stage, and monitoring the therapeutic responses.

In conclusion, this study suggests that combining CSF NGS with the cysticercus IgG test can be a promising approach for diagnosing the challenging cases of NCC. Further studies are needed to evaluate the parasitic DNA load in patients’ CSF for the diagnosis of disease severity, stage, and monitoring of therapeutic responses.