Assessment of antibody responses to antigens of Mycobacterium tuberculosis and Cysticercus cellulosae in cerebrospinal fluid of chronic meningitis patients for definitive diagnosis as TBM/NCC by passive hemagglutination and immunoblot assays

Tanned sheep erythrocytes stabilized with pyruvic aldehyde and glutaraldehyde, called double-aldehyde-stabilized cells, were used to standardize passive hemagglutination assay (PHA) for detection of antibody responses to sonicate extract of Mycobacterium tuberculosis and Cysticercus cellulosae soluble antigens. PHA was performed in the following groups of cerebrospinal fluid (CSF) samples: group I - chronic infections of the central nervous system with the possible diagnosis of tuberculous meningitis (TBM), tuberculoma and neurocysticercosis (NCC) (n=88), and group II - controls which included (a) non-infectious non-neurological conditions (n=30), (b) infectious neurological conditions (n=21) and (c) non-infectious neurological conditions (n=133). PHA could detect anti-mycobacterial antibodies at the sensitivity level of 80.76% with a specificity of 92.4% and anti-cysticercal antibodies with a sensitivity of 100% and specificity of 92.94%. However, in 6.33% (i.e. 14/221) of group I and group II (c) CSFs both anti-mycobacterial and anti-cysticercal antibodies were detected. Immunoblot analysis of CSFs derived from TBM patients reacted predominantly to 120-kDa, 96-kDa, 65-kDa, 38-kDa, 26-kDa, 23-kDa, 19-kDa and 12-14-kDa and 4-6-kDa antigens of M. tuberculosis sonicate extract (MTSE), whilst CSFs of proven NCC reacted to >110-kDa, 96-kDa, 80-kDa, 66-68-kDa, 52-kDa and 26-28-kDa antigens of porcine whole cyst sonicate extract (PCSE). On immunoblot analysis, some of the CSFs of TBM patients were PHA positive for both MTSE and PCSE showed antibody reactivity to 70-kDa and 10-kDa antigens of C. cellulosae. Similarly CSF antibody of some Guillain Barre syndrome and myeloradiculopathy patients reacted with cysticercal antigens. But per se no cross-reactivity between MTSE and anti-cysticercal antibodies and vice-versa were observed. However, findings of this study should alert laboratory personnel especially in endemic areas to be extra careful in interpretation of antibody detection results.     

Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis

Neurocysticercosis (NCC) is caused by the larval form of the pork tapeworm Taenia solium when lodged in the central nervous system (CNS). Clinical diagnosis of NCC is complicated due to its polymorphic manifestations with no specific signs or symptoms. A wide range of serological assays and neuroimaging modalities are used for its diagnosis. The aim of the present study was to evaluate the MTT assay for the diagnosis of NCC and to determine its sensitivity, specificity and accuracy. MTT assay was based upon the cellular reduction of the tetrazolium salt by the proliferating cells and quantification of the colored product. Total 59 patients with NCC-related active epilepsy (AE), 30 with AE other than NCC (disease controls) and 64 healthy volunteers were enrolled for the study. Lymphocytes were freshly isolated from the enrolled subjects and cultured on cyst fluid antigen coated tissue culture plates. MTT assay was performed according to the standard protocol. The mean values of proliferation index (PI) with cyst fluid antigens were 2.13 ± 0.72, 0.622 ± 0.31 and 0.71 ± 0.36 for NCC patients, disease controls and healthy volunteers respectively. PI values for NCC patients were higher than the cut-off value (mean of controls + 2 standard deviations; 1.31). The sensitivity, specificity and accuracy of the MTT assay for the diagnosis of NCC were 87.93%, 94.68% and 91.5% respectively. For single cyst infection the sensitivity of the assay was found to be 86.4%. The present study shows that MTT is an adaptable technique which can be used for diagnosis of NCC.     

Eradication of Taenia solium cysticercosis: A role for vaccination of pigs

Taenia solium is the causative agent of neurocysticercosis, a disease responsible for substantial human morbidity and mortality. It is a zoonotic parasite, involving pigs as intermediate hosts. The parasite’s full life cycle is restricted to poor people in developing countries. Attempts to date to control transmission of the parasite have been relatively poorly effective and not sustainable. Over the past decade research has been undertaken to develop practical vaccines for use in pigs to prevent transmission of T. solium. The most effective of these vaccines in controlled experimental trials has been the TSOL18 vaccine. More recently, TSOL18 has been proven to be highly effective against naturally acquired infection with T. solium in pigs. Application of TSOL18 together with a single treatment of pigs with oxfendazole achieved the complete elimination of transmission of the parasite by pigs involved in the field trial. This strategy may provide a relatively low cost and sustainable control tool which could assist towards the goal of achieving eradication of the parasite. An assessment is made of the potential value of various control measures that are available for T. solium, and two options are suggested as potential parasite control programs.     

Mimotope peptides selected from phage display combinatorial library by serum antibodies of pigs experimentally infected with Taenia solium as leads to developing diagnostic antigens for human neurocysticercosis

Neurocysticercosis is caused by penetration of the tapeworm Taenia solium larvae into the central nervous system resulting in a diverse range of neurologic complications including epilepsy in endemic areas that globalization spreads worldwide. Sensitive and specific immunodiagnosis is needed for the early detection and elimination of the parasite, but the lack of standardized, readily obtainable antigens is a challenge. Here, we used the phage display for resolving the problem. The rationale of the strategy rests on the concept that the screening of combinatorial libraries with polyclonal serum to pathogens reveals families of peptides mimicking the pathogen most immunodominant epitopes indispensable for the successful diagnosis. The screening of a 7mer library with serum IgG of four pigs experimentally infected with parasite followed by computer aided segregation of the selected sequences resulted in the discovery of four clusters of homologous sequences of which one presented a family of ten mimotopes selected by three infected pig serum IgGs; the common motif sequence LSPF carried by the family was considered to be the core of an immunodominant epitope of the parasite critical for the binding with the antibody that selected the mimotopes. The immunoassay testing permitted to select a mimotope whose synthetic peptide free of the phage with the amino acid sequence Leu-Ser-Fen-Pro-Ser-Val-Val that distinguished well a panel of 21 cerebrospinal fluids of neurocysticercosis patients from the fluids of individuals with neurological complications of other etiology. This peptide is proposed as a lead for developing a novel molecularly defined diagnostic antigen(s) for the neurocysticercosis.     

Immune response to different fractions of Taenia solium cyst fluid antigens in patients with neurocysticercosis

The immunopathogenesis of neurocysticercosis (NCC) largely remains unknown. We analyzed the immune response to different fractions of Taenia solium cyst fluid antigens in patients with NCC. Lymphocytes were separated from 48 patients with NCC-related active epilepsy and 30 healthy controls. T. solium (isolated from pig muscles) antigens (crude lysate, CL; cyst wall, CW and cyst fluid, CF) at 20 μg/well concentrations were used to stimulate the cells in a lymphocyte transformation test (LTT). Only CF antigen stimulated cell proliferation significantly greater than control (p < 0.001), hence cyst fluid antigens were further studied. The CF antigens were electro-blotted on nitrocellulose membrane (NC), cut at 0.5 cm distance and particulate antigens were prepared. A total of 12 fractions, designated F1 to F12 according to molecular weight were tested in-vitro for LTT. After 72 h of stimulation by the different fractions, Th1 (IL-1β, TNF-α, IL-2) and Th2 (IL-4, IL-10) cytokine responses were determined in culture supernatants by ELISA. Low molecular weight fractions F1 through F4 (Mol. wt. < 25 kDa) were found to be potent inducers of cytokines. Fractions F1, F3 and F4 induced the production of Th1 (IL-1β, TNF-α, IL-2), whereas F2 induced the production of Th2 (IL-4 and IL-10) cytokine. The study shows that the low molecular weight fractions of CF antigens are immuno-dominant. Most of these fractions (F1, F3, F4) induce strong Th1 immune response except F2 which induces Th2 response. Further studies are needed to identify the different antigens present in these fractions to determine the molecules responsible for the immune response.     

Taenia solium: characterization of a small heat shock protein (Tsol-sHSP35.6) and its possible relevance to the diagnosis and pathogenesis of neurocysticercosis

A cDNA encoding for a predicted small heat shock protein (sHSP), Tsol-sfISP35.6, has been isolated by antibody screening of a Taenia solium c-DNA library. The clone was a full-length sequence (1172 bp) with an open reading frame of 945 bp and encoded for a 314 amino acid protein with deduced molecular mass of 35.6 kDa, isoelectric point of 5.6 arid the characteristic HSP20/alpha-crystallin domain duplicated. It was highly conserved, with a high sequence similarity with other platyhelminth sHSPs. Western blot analysis, using serum from neurocysticercosis patients (NCC), indicated that the purified Tsol-sHSP35.6 expression product was immunogenic, while in indirect ELISA, using the purified Tsol-sHSP35.6 expression product as antigen and serum samples from pigs and humans, 80% of T. solium infected pigs and 84% of patients with active, or 71% of patients with inactive NCC were sero-positive. The possible relevance of Tsol-sHSP35.6 in the diagnosis and pathogenesis of NCC is discussed.     

Prediction of viability of porcine neurocysticercosis with proton magnetic resonance spectroscopy: correlation with histopathology

Neurocysticercosis (NCC) is the most frequent parasitic disease of central nervous system. In our earlier study, we had observed creatine [(creatine + phosphocreatine); (tCr)] on ex vivo proton MR spectroscopy (1H MRS) in some of the cysticercus cyst fluid samples obtained from swine's brain parenchyma. In current study, swine brains of freshly slaughtered animals naturally infected with NCC were subjected to ex vivo magnetic resonance (MR) imaging on a 1.5Tesla MR system. Cysticercus cysts (n = 12) were removed from these brains and were labeled depending upon presence or absence of edema around cysts as observed on imaging. Cysticercus cyst fluid (100 microl) was subjected to different 1H MRS experiments and results were compared with histopathological examinations to look for any relationship between tCr and parameters like quantification of musculature, and cellular infiltration in wall of the parasite. Histopathology of cyst wall was categorized into two groups based on cellular characteristics and the amount of musculature. Grade I cysts (n = 5) with no or minimal inflammation and large amount of musculature showed tCr on 1H MRS. However, grade II cysts (n = 7) with profuse inflammation and less amount of musculature in the cyst wall lacked tCr. Higher amount of musculature in grade I cysts was associated with higher concentration of tCr in the cyst fluid (r2 = 0.93, P = 0.007). Creatine appears to be a marker of innocuous and viable NCC.     

What triggers seizures in neurocysticercosis? A MRI-based study in pig farming community from a district of North India

Colloidal and calcified cysts are considered responsible for seizure in neurocysticercosis (NCC); however, calcified cysts have also been reported in asymptomatic individuals. We carried out a MRI-based study in a rural pig farming community of North India to detect the various stages, locations and numbers of the cyst in asymptomatic individuals and compared them with symptomatic NCC cases to see its association with occurrence of seizures. A total of 107 asymptomatic family members of 29 symptomatic NCC confirmed cases were evaluated clinically, immunologically and by neuroimaging for NCC. MRI-based staging of the parasite was done in both groups, and compared to look for the differences, if any. Thirty-one (29.0%) asymptomatic family members of symptomatic cases were diagnosed to have NCC. There was no difference in proportion of colloidal/degenerating and calcified stages of the parasite between symptomatic and asymptomatic groups; however, significantly higher proportion of the asymptomatic populations had vesicular stage of the parasite (P = 0.029). Our findings show that a large number of individuals harboring different stages of cysticerci in their brain remain symptoms free and question the belief that the degenerating/calcified stages of the parasite are primarily responsible for seizure occurrence in NCC.     

Taenia crassiceps antigens induce a Th2 immune response and attenuate injuries experimentally induced by neurotoxoplasmosis in BALB/c mice

Toxoplasma gondii is a pathogenic agent responsible for causing both systemic and local disease which elicits a typically pro-inflammatory, Th1 immune response. Taenia crassiceps antigen induces a Th2 immune response that immunomodulates Th1 based infections. Therefore the aim of this study was to evaluate whether T. crassiceps cysticerci antigens are able to modulate the inflammatory response triggered in experimental neurotoxoplasmosis (NT). BALB/c mice were inoculated with T. gondii cysts and/or cysticerci antigens and euthanized at 60 and 90 days after inoculation (DAI). The histopathology of the brains and cytokines produced by spleen cells culture were performed. The animals from the NT group, 90DAI (NT90), presented greater intensity of lesions such as vasculitis, meningitis and microgliosis and cytokines from Th1 profile characterized by high levels of IFN-gamma. While in the T. crassiceps antigens group, 60DAI, there were more discrete lesions and high levels of IL-4, a Th2 cytokine. In the NT co-inoculated with cysticerci antigens group the parenchyma lesions were more discrete with lower levels of IFN-gamma and higher levels of IL-4 when compared to NT90. Therefore the inoculation of T. crassiceps antigens attenuated the brain lesions caused by T. gondii inducing a Th2 immune response.