Distinguishing neurocysticercosis epilepsy from epilepsy of unknown etiology using a minimal serum mass profiling platform

Abstract

Neurocysticercosis is associated with epilepsy in pig-raising communities with poor sanitation. Current internationally recognized diagnostic guidelines for neurocysticercosis rely on brain imaging, a technology that is frequently not available or not accessible in areas endemic for neurocysticercosis. Minimally invasive and low-cost aids for diagnosing neurocysticercosis epilepsy could improve treatment of neurocysticercosis. The goal of this study was to test the extent to which patients with neurocysticercosis epilepsy, epilepsy of unknown etiology, idiopathic headaches and among different types of neurocysticercosis lesions could be distinguished from each other based on serum mass profiling. For this, we collected sera from patients with neurocysticercosis-associated epilepsy, epilepsy of unknown etiology, recovered neurocysticercosis, and idiopathic headaches then performed binary group comparisons among them using electrospray ionization mass spectrometry. A leave one [serum sample] out cross validation procedure was employed to analyze spectral data. Sera from neurocysticercosis patients was distinguished from epilepsy of unknown etiology patients with a p-value of 10^-28. This distinction was lost when samples were randomized to either group (p-value = 0.22). Similarly, binary comparisons of patients with neurocysticercosis who has different types of lesions showed that different forms of this disease were also distinguishable from one another. These results suggest neurocysticercosis epilepsy can be distinguished from epilepsy of unknown etiology based on biomolecular differences in sera detected by mass profiling.

Keywords: Diagnosis; Electrospray mass spectrometry; Epilepsy; India; Neurocysticercosis; Serum.

Figure 1.

A, Flowchart of the steps taken for an electrospray ionizing mass spectrometry analysis using the comparison between neurocysticercosis (NCC) and epilepsy of unknown etiology (EUE) as an example. B, Example of statistically significant different LOOCV means in normalized spectral mass peaks seen between 650 and 750 m/Z when data from 75 NCC and 29 EUE are left-in while 1 NCC case is left out. Legend: * indicates that the difference in a normalized spectral mass peak mean between the NCC and EUE left-in subjects is statistically significant. The horizontal bar indicated the median between the two means and corresponds to the LOOCV peak classification value.

Abbreviations: LOOCV: Leave One Out Cross Validation; NCC: neurocysticercosis; EUE: epilepsy of unknown etiology.

Figure 2.

Percent total (randomized) NCC LOOCV classified mass peaks of each study subject and of randomized subjects in relation to the cut-off thresholds used to classify subjects into one of two groups and with the p-values corresponding to the difference in the means of the two groups being compared. A, Comparison of subjects with neurocysticercosis (NCC) and with epilepsy of unknown etiology (EUE). B, Comparison of subjects with NCC and headaches. C, Comparison of subjects randomized to either the NCC or EUE groups. D, Comparison of subjects randomized to the NCC or headache groups.</p>Abbreviations: NCC, neurocysticercosis; MNCC: multiple neurocysticercosis; SCG: single cysticercus granuloma; SCC: single calcified cyst; LOOCV: leave one out cross validation; SD: standard deviation.

Figure 3.

Percent total group LOOCV classified mass peaks of each subject in the three NCC subgroups in relation to the cut-off thresholds used to classify subjects into one of two groups, the p-value corresponding to the difference in the means of the two groups being compared, and the p-value obtained with the corresponding randomized database. A, Comparison of subjects with multiple neurocysticercosis (MNCC) and with single cysticercus granuloma (SCG). B, Comparison of subjects with SCG and single calcified cyst (SCC). C, Comparison of subjects with SCC and MNCC. Abbreviations: NCC, neurocysticercosis; MNCC: multiple neurocysticercosis; SCG: single,cysticercus granuloma; SCC: single calcified cyst; LOOCV: leave one out cross validation; SD: standard deviation

Figure 4.

Percent total group LOOCV classified mass peaks of each subject with calcified neurocysticercosis (NCC), active NCC, NCC with or without edema or epilepsy of unknown etiology (EUE) in relation to the cut-off thresholds used to classify subjects into one of two groups, the p-value corresponding to the difference in the means of the two groups being compared, and the p-value obtained with the corresponding randomized database. A, Comparison of subjects with calcified NCC and with EUE. B, Comparison of subjects with active NCC and with EUE. C, Comparison of subjects with active NCC and calcified NCC. D, Comparison of NCC subjects with edema* and without edema** Abbreviations: NCC, neurocysticercosis; LOOCV: leave one out cross validation; SD: standard deviation. Legend: NCC with edema*: There were a total of 48 NCC subjects with edema. Among these, 6/6 multiple NCC patients with mixed lesions, 2/2 multiple NCC patients with calcified cysts only, 1/10 multiple NCC patient with active cysts only, 4/4 single calcified cyst patients, 7/26 single cysticercus granuloma patients were included in this analysis. NCC without edema**: There were a total of 28 NCC subjects without edema. Among these, 1/1 multiple NCC patient with active cysts only, 8/8 multiple NCC patients with calcified cysts only, 8/16 single calcified cyst patients, 3/3 single cysticercus granuloma patients were included in this analysis.

Figure 5.

Classification of patients with recovered NCC (RNCC) according to their percent total group LOOCV classified mass peaks using the data on the percent total group LOOCV classified mass peaks and cut-off thresholds using from subjects in other groups. A, Classification of RNCC patients using data from the idiopathic headache (headache) and epilepsy of unknown etiology (EUE) patients. B, Classification of the RNCC patients using data from the epilepsy of unknown etiology (EUE) and the NCC patients. C, Classification of the RNCC patients using data from the single cysticercus granuloma (SCG) and single calcified cyst (SCC) patients. D, Classification of the RNCC patients using data from patients with active and calcified NCC. Abbreviations: EUE: epilepsy of unknown etiology; NCC, neurocysticercosis; RNCC: recovered neurocysticercosis; SCG, single cysticercus granuloma; SCC: single calcified cyst (SCC); LOOCV: leave one out cross validation; SD: standard deviation.

Figure 6.

Classification of 28 neurocysticercosis (NCC), five epilepsy of unknown etiology (EUE), five active NCC and five calcified NCC samples taken out of the original database (blind samples) according to their percent Total group LOOCV classified mass peaks using data from the remaining samples (training set) to determine the group cut-off thresholds. A, Percent Total NCC LOOCV classification mass peaks and group cut-off threshold for classifying subjects into the NCC or EUE groups using the data from 48 NCC and 24 EUE subjects in the training dataset. B, Classification of 28 NCC and five EUE subjects not included in the training dataset according to their % Total NCC LOOCV classification mass peaks compared to the group cut-off threshold obtained in (A). C, Percent Total active NCC classification mass peaks and group cut-off threshold for classifying subjects into the active NCC or calcified NCC groups using the data from 35 active NCC and 25 calcified NCC subjects in the training dataset. D, Classification of five active NCC and five calcified NCC subjects not included in the training dataset according to their % Total NCC LOOCV classification mass peaks compared to the group cut-off threshold obtained in (C).