Aloukick K. Singh

Human cysticercosis and Indian scenario: a review

Cysticercosis, caused by Taenia solium larva is a major public health problem, especially in the developing world and neurocysticercosis (NCC) is considered to be the most common parasitic infestation of the central nervous system. NCC is identified as the single most common cause of community acquired active epilepsy; 26.3% to 53.8% active epilepsy cases in the developing world including India and Latin America are due to NCC. It is also becoming more common in the developed world because of increased migration of people with the disease or Taenia solium carriers and frequent travel to the endemic countries. It is estimated that three quarters of the estimated 50 million people with active epilepsy live in the poor countries of the world. Recent Indian studies using neuroimaging techniques suggest that the disease burden in India surpasses many other developing countries. Hence it is important to know the epidemiology, pathogenesis and diagnostic criteria so as to assess the disease burden and adopt interventional strategies for its control. Literature search was done for this review with special emphasis on Indian studies to create awareness about the disease in India, since cysticercosis is preventable and potentially eradicable.

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.

Toll-Like Receptor 4 Polymorphism and Its Association with Symptomatic Neurocysticercosis

BACKGROUND Symptoms and signs of neurocysticercosis (NCC) are nonspecific and depend upon several factors, including the host immune response to the parasite. Toll-like receptors (TLRs) play an important role in innate immunity. Susceptibility of humans to NCC in relation to TLR polymorphism is unknown. The present study examines TLR4 polymorphism in human NCC and its role in symptomatic disease. METHODS A total of 140 patients with NCC (82 symptomatic [ie, with active epilepsy] and 58 asymptomatic) and 150 healthy control subjects were examined for TLR4 Asp299Gly and Thr399Ile polymorphisms by means of polymerase chain reaction and restriction fragment-length polymorphism. RESULTS TLR4 Asp299Gly and Thr399Ile were significantly associated with the occurrence of NCC (P < .001 for Asp299Gly; P = .003 for Thr399Ile) and progression to symptomatic NCC, compared with control subjects (P < .001 for Asp299Gly; P < .001 for Thr399Ile) or asymptomatic NCC (P < .001 for Asp299Gly; P = .002 for Thr399Ile). Frequency of haplotype Gly/Thr (P <.001) was observed to be a risk factor for susceptibility to NCC. Gly and Ile carriers had a statistically significant association with NCC (P < .001 for Gly; P = .003 for Ile) and symptomatic NCC (P < .001 for Gly; P <or= .001 for Ile), compared with control subjects. Both carriers were also associated with symptomatic NCC (P < .001 for Gly; P < .004 for Ile), when compared with asymptomatic NCC.

Association of TLR4 Asp299Gly and Thr399Ile polymorphisms with Guillain-Barré syndrome in Northern Indian population

Molecular mimicry between Campylobacter jejuni lipopolysaccharide and host gangliosides induces an immune response leading to axonal damage and Guillain-Barré syndrome (GBS). TLR polymorphisms are associated with many autoimmune diseases. The role of the TLR4 gene in GBS susceptibility largely remains unknown. We investigated TLR4 polymorphism in GBS. One hundred and twenty GBS patients and 150 healthy controls were included. TLR4 (Asp299Gly and Thr399Ile) genes were studied by PCR-RFLP. TLR4 (Asp299Gly) polymorphism was significantly associated with GBS (p, 0.045; OR, 8.75; 95% CI, 1.05-72.88); only acute motor axonal neuropathy (AMAN) was associated with Gly299Gly homozygote (p, 0.027; OR, 12.40; 95% CI, 1.33-115.77) and Thr399Ile (p, 0.019; OR, 3.42; 95% CI, 1.22-9.54) heterozygote, and TLR4-399Ile allele (p, 0.045; OR, 2.63; 95% CI, 1.02-6.75) compared to controls. In conclusion, TLR4 (Asp299Gly) polymorphism is associated with an increased susceptibility to GBS. Besides Asp299Gly, AMAN subtype is also associated with Thr399Ile polymorphism.

Association of MMP-2 and MMP-9 with clinical outcome of neurocysticercosis

Matrix metalloproteinases (MMPs) are the major endopeptidases involved in proteolysis of blood brain barrier (BBB) during central nervous system (CNS) infections. The present study detected serum levels and activities of MMP-2 and MMP-9 in patients with neurocysticercosis (NCC) and their association with symptomatic disease. In total, 68 individuals with NCC (36 symptomatic patients with active seizures and 32 asymptomatic individuals) and 37 healthy controls were enrolled for the study. Serum MMP-2 and MMP-9 levels and their activities were measured by ELISA and gel zymography respectively. Mean serum MMP-2 levels (ng/ml) were higher both in asymptomatic and symptomatic NCC cases compared to healthy controls. However, significantly higher levels of serum MMP-9 (ng/ml) were detected only in symptomatic NCC patients compared to asymptomatic NCC cases and healthy controls. Levels of both MMPs positively correlated with symptomatic NCC. Serum MMP-2 activities were significantly higher in symptomatic and asymptomatic NCC compared to healthy controls whereas serum MMP-9 activity was significantly associated with symptomatic NCC compared to healthy controls and asymptomatic NCC. In conclusion, the elevated level of MMP-9 in serum appears to play an important role in the development of symptoms i.e. active seizures in patients with NCC. However, further studies are needed to elucidate its precise role in disease pathogenesis.

Immune responses to viable and degenerative metacestodes of Taenia solium in naturally infected swine.

Neurocysticercosis, caused by the larvae of the pork tapeworm Taenia solium, is the most common helminth infection of the CNS in humans worldwide. There is no existing animal model of neurocysticercosis that resembles human infection. To overcome this limitation, swine (the natural intermediate host of the parasite) may be a suitable model. The immune response associated with different stages of the parasite larva (metacestode) has not yet been explored. Therefore, we investigated the immune response to various stages of the metacestode (cyst) in the brain and muscles of naturally infected swine. Swine with neurocysticercosis (n = 10) and healthy controls (n = 10), as confirmed by magnetic resonance imaging, were included in this study. The animals were sacrificed, and the tissues containing viable or degenerative metacestods in the brain and infected muscles were collected and subjected to reverse transcriptase-PCR and ELISA to determine the expression of different cytokines (IFN-γ, TNF-α, IL-1β, IL-2, IL-4 IL-6, IL-8 and IL-10). Higher expression of IL-10 was found to be associated with viable cysts. Degenerating cysts displayed significantly increased levels of IFN-γ, TNF-α, IL-1β, IL-2, IL-6 and IL-8, whereas calcified cysts had elevated levels of IL-4, IL-10, TNF-α and IL-6. The present study indicated a strong regulatory (IL-10) and Th1 cytokine response in viable and degenerating cysts, respectively, whereas calcified cysts had a mixed anti-inflammatory (IL-4), regulatory (IL-10) and pro-inflammatory (TNF-α and IL-6) response. Thus, Th1 and Th2 immune response operate in the vicinity of metacestodes and the type of immune response may be responsible for disease severity.

Association of ICAM-1 K469E polymorphism with neurocysticercosis

Neurocysticercosis (NCC), a central nervous system (CNS) disease is caused by the larval stage of Taenia solium. The disease is heterogeneous in clinical presentation; some infected individuals develop symptoms and others may remain symptom free. Impaired blood brain barrier allows recruitment of immune cells in the CNS during infection and soluble intercellular adhesion molecule-1 (sICAM-1) plays an important role in the recruitment of immune cells. We studied ICAM-1 K469E polymorphism among symptomatic and asymptomatic NCC patients. The study revealed that individuals with variant (EE) genotype were more susceptible to symptomatic NCC and also had an elevated level of sICAM-1.

Evaluation of ELISA, neck muscle, tongue and eyelid examinations for the diagnosis of swine cysticercosis in a highly endemic area of north India

Swine cysticercosis is very common in the developing countries where pigs are raised. Undercooked measly pork consumption leads to taeniasis; Taenia carriers act as source of human and swine cysticercosis and neurocysticercosis. Diagnosis of swine cysticercosis is important to break the cycle of disease transmission. The present study compared the neck muscle, tongue and eye examinations, and serum ELISA with different preparations (crude lysate, cyst fluid, scolex and cyst wall antigens) of Taenia solium cyst for the diagnosis of swine cysticercosis. Total of 24 pigs initially identified by neck muscle, tongue and eyelid examinations were purchased from local slaughter house and subjected to MRI for confirmation of cysticercosis. Sera from 20 MRI confirmed infected pigs and 50 disease free controls were subjected to ELISA with T. solium cyst antigens. Neck muscle examination was 100% sensitive and 75% specific for the diagnosis of swine cysticercosis, whereas tongue and eye examinations were 70% and 25% sensitive, respectively. ELISA with crude lysate had 85% sensitivity and 98% specificity. ELISA with cyst fluid, scolex and cyst wall antigens showed 70%, 65%, and 45% sensitivity, respectively. The present study showed that neck muscle examination was highly sensitive but less specific, while ELISA with crude antigens had reasonable sensitivity and high specificity for diagnosis of swine cysticercosis. ELISA with crude lysate can be used as a screening tool for swine infection.

Association of TNF-α -308G>A and TNF-β +252A>G genes polymorphisms with primary immune thrombocytopenia: a North Indian study.

Primary immune thrombocytopenia (ITP) is manifested by platelet autoantibodies that are not only responsible for platelet destruction by phagocytosis but also inhibit their production. Bleeding is the most common clinical manifestation of thrombocytopenia. ITP is a multifactorial disease in which both environmental and genetic factors have been implicated. It has been reported that several gene polymorphisms influence host susceptibility to ITP. This study was aimed to investigate the association of polymorphisms in tumor necrosis factor-alpha (TNF-&agr;) 308 G>A and TNF-&bgr; +252 A>G genes with primary ITP in Indian patients. Genotyping for the TNF-&agr; −308 G>A and TNF-&bgr; +252 A>G was performed in 80 ITP patients and 100 controls by polymerase chain reaction and restriction fragment length polymorphism. We found no significant difference in distribution of TNF-&agr; heterozygous variant genotype (GA) among patients and controls. Homozygous variant genotype (AA) was absent both in patients and controls. No statistical difference was observed in the distribution of heterozygous variant (AG) and homozygous variant (GG) genotypes of TNF-&bgr;, between patients and controls. Heterozygous (AG) genotype of TNF-&bgr; −308G>A was associated with persistent ITP. The study showed that heterozygous variant (AG) genotype of TNF-&bgr; was associated with persistent ITP, when compared with controls. We could not find any association of TNF-&agr; with susceptibility in developing ITP. Furthermore, no association was observed with respect to different categories of ITP. In addition, additive model showed two-fold increased susceptibility to ITP. We conclude that single nucleotide polymorphism in TNF-&bgr; +252 A>G gene may have impact on susceptibility to ITP.

Distinct cytokine pattern in response to different bacterial pathogens in human brain abscess.

Brain abscess is a focal suppurative process. Host inflammatory response in Gram type and specific bacteria has not been studied in brain abscess. A total of 57 brain abscess patients with monomicrobial infections were studied for Th1 (TNF-α, IFN-γ, IL1-β), Th2 (IL-4, IL-10) and Th17 (IL-17, IL-23) cytokine response by reverse-transcriptase PCR and ELISA. Th1 and Th17 cytokines were significantly elevated in Gram positive (Staphylococcus aureus and Streptococcus intermedius) and Th2 cytokine (IL-10) in Gram negative (Bacteroides fragilis and Escherichia coli) infections (p<0.05). Cytokine levels were significantly higher in abscess than blood (p<0.001). Elevated levels of several inflammatory cytokines (TNF-α, IFN-γ, IL1-β, IL-17 and IL-23) were associated with the duration of symptoms; predisposing factors also influenced the levels of several cytokines. The expression of inflammatory cytokines in abscess was influenced by the bacterial pathogen, duration of symptoms and predisposing factors. Local milieu of brain plays significant role in secretion of various cytokines.