Humoral and T cell-mediated immune response against trichomoniasis.

Trichomonas vaginalis (T. vaginalis) infection leads to the synthesis of specific antibodies in the serum and local secretions. The profile of T. vaginalis-specific antibodies and T cell-mediated immune responses may influence the outcome of infection, towards parasite elimination, persistence or pathological reactions. Studies have indicated that Th1-, Th17- and Th22 cell-related cytokines may be protective or pathogenic, whereas Th2- and Treg cell-related cytokines can exert anti-inflammatory effects during T. vaginalis infection. A number of T. vaginalis-related components such as lipophosphoglycan (TvLPG), α-actinin, migration inhibitory factor (TvMIF), pyruvate:ferredoxin oxidoreductase (PFO), legumain-1 (TvLEGU-1), adhesins and cysteine proteases lead to the induction of specific antibodies. T. vaginalis has acquired several strategies to evade the humoral immune responses such as degradation of immunoglobulins by cysteine proteases, antigenic variation and killing of antibody-producing B cells. The characterization of the T. vaginalis-specific antibodies to significant immunogenic molecules and formulation of strategies to promote their induction in vaginal mucosa may reveal their potential protective effects against trichomoniasis. In this review, we discuss the current understanding of antibody and T cell-mediated immune responses to T. vaginalis and highlight novel insights into the possible role of immune responses in protection against parasite.

Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Immunity in urogenital protozoa.

Innate and adaptive immunity play a significant role in urogenital infections. Innate immunity is provided by the epithelial cells and mucus lining along with acidic pH, which forms a strong physical barrier against the pathogens in female reproductive tract. Cells of innate immune system, antimicrobial peptides, cytokines, chemokines and adaptive immunity in the reproductive tract are evolved during infection, and a pro-inflammatory response is generated to fight against the invading pathogen Trichomonas vaginalis, a primary urogenital protozoa, the etiological agent of human trichomoniasis, a curable sexually transmitted infection. The involvement of the urogenital tract by other protozoal infections such as P. falciparum, Trypanosoma, Leishmania, Toxoplasma, Entamoeba histolytica and Acanthamoeba infection is rarely reported. Trichomonas induce pro-inflammatory and immunosuppressive responses in infected subjects. Multifactorial pathogenic mechanisms including parasite adherence, cysteine proteases, lipophosphoglycan, free radical, cytokine generation and Toll-like receptors appear to interplay with the induction of local and systemic immune responses that ultimately determine the outcome of the infection. However, the involvement of urogenital pathogen-specific immune mechanisms and effect of normal local resident flora on the outcome (symptomatic vs. asymptomatic) of infection are poorly understood. Moreover, immune interactions in trichomoniasis subjects co-infected with bacterial and viral pathogens need to be elucidated.

Evaluation of excretory secretory and 10-30 kDa antigens of Taenia solium Cysticerci by EITB assay for the diagnosis of neurocysticercosis.

Neurocysticercosis (NCC), caused by the presence of Taenia solium Cysticerci in the Central Nervous System is the most common neurological disease of parasite aetiology. The serodiagnostic methods available at present have variable sensitivity and specificity depending upon the antigen and technique used. The present study was aimed to assess the efficacy of T. solium Cysticerci excretory secretory (ES) and lower molecular mass (LMM) 10-30 kDa antigenic fractions for antibody detection in serum and urine samples by enzyme-linked immunoelectrotransfer blot (EITB) for the diagnosis of NCC. Serum and urine samples were collected from 125 clinically suspected and radiologically proven NCC children (111 patients with single lesion and 14 with multiple lesions) and 125 control subjects. With the use of ES and LMM antigenic fractions, the sensitivity of the EITB assay was 85.6% and 80.8% with serum and 76.8% and 50.4% with urine, respectively. The specificity was 64% and 61.6% with serum and 48% and 33.6% with urine samples, respectively. The study suggests that antibody detection to ES antigen in serum by EITB assay may serve better purpose for the serodiagnosis of human NCC.

Polymorphism in merozoite surface protein-1 gene in north & northwest Indian field isolates of Plasmodium vivax.

BACKGROUND & OBJECTIVE: Merozoite surface protein-1 of Plasmodium vivax (Pvmsp-1) is a strong vaccine candidate against asexual blood stages. Extensive polymorphism in msp-1 gene has been reported in P. vivax isolates from different geographical regions which is necessary before a field trial of any malaria vaccine based on msp-1 is undertaken. There are only a few reports available on polymorphism in msp-1 gene in Indian field isolates of P. vivax. The aim of the present study was therefore to investigate the polymorphism in Pvmsp-1 gene in 25 isolates of P. vivax collected from malaria patients from regions of north and northwest India. METHODS: DNA was extracted from whole blood samples collected in citrated anticoagulant. The polymorphic region-5, the most variable region of the Pvmsp-1 gene was amplified by PCR. The PCR products were further analyzed by restriction fragment length polymorphism (RFLP) using Mva-1 restriction enzyme. The DNA fragments obtained on PCR and RFLP were analyzed by agarose gel electrophoresis. RESULTS: On the basis of PCR, significant size polymorphism was seen and 4 allelic types were observed amongst the 25 isolates. Further analysis by RFLP discriminated these 4 allelic types into 9 sub-allelic types indicating that PCR-RFLP can be a good tool to study polymorphism in msp-1 gene of Plasmodium. INTERPRETATION & CONCLUSION: Marked genetic polymorphism was observed in msp-1 gene among the isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax.

Genetic polymorphism in msp-2, ama-1 and csp genes in Plasmodium falciparum field isolates from north and north-western India.

BACKGROUND & OBJECTIVES: Malaria is a major public health problem in tropical and sub-tropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphism in vaccine candidate antigens might be a hurdle in developing an effective vaccine. Merozoite surface protein-2, apical membrane antigen-1 and circumsporozoite protein of Plasmodium falciparum are vaccine candidate antigens. The aim of this study was to detect extent of genetic polymorphism in potential vaccine candidate antigen genes, i.e. msp-2, ama-1 and csp of P. falciparum isolates prevalent in northern and north-western parts of India. METHODS: Overall 88 parasite isolates of P. falciparum were collected during July 1998-March 2002 from different parts of northern and north-western India. DNA was extracted and analyzed for genetic polymorphism by PCR-RFLP method. For msp-2 gene, family-specific (FC-27 and 3D7) nested PCR was also performed. RESULTS: PCR showed size polymorphism in all the target genes. Three alleles were observed in msp-2 and ama-1, while only two in csp. RFLP of ama-1 and csp with Dra-1 and Ssp-1 endonucleases respectively, failed to differentiate isolates in sub-allelic types, while Hinf-I digestion of msp-2 amplicons differentiated three alleles into two distinct allelic families, i.e. FC-27 and 3D7. The allelic family-specific PCR generally confirmed the results of PCR-RFLP except in a few isolates, which showed mixed (two) clones of msp-2 gene. INTERPRETATION & CONCLUSION: There was extensive polymorphism in msp-2 gene while ama-1 and csp genes showed low polymorphism which may be due to the functional constraints of these proteins. The low level transmission of malaria in the study area may also be a factor for low polymorphism.

Concomitant intestinal parasitism and non-cholera vibrio infection.

Concomitant parasitism is not uncommon especially in tropical countries with low socioeconomic status. Here we report an unusual combination of intestinal infection due to Strongyloides stercoralis, Blastomyces hominis and non-cholera Vibrio in a patient suffering from acute gastroenteritis and hypoalbuminemia. Early recognition and accurate treatment of gastrointestinal infections and infestations before the patient develops complications is important.

Antibody response to Toxoplasma gondii in saliva samples from human immunodeficiency virus-infected patients.

Toxoplasma gondii is an important opportunistic infection among human immunodeficiency virus (HIV)-infected patients as it causes fatal encephalitis. In the present study, antibody response to T. gondii is assessed in saliva samples from 100 HIV-seropositive patients and 25 HIV-negative healthy controls by indirect enzyme-linked immunosorbent assay (ELISA). Sensitivity and specificity for detection of IgG and IgM in saliva is calculated using a positive antibody response in serum samples (from an earlier study) as the gold standard. IgG and IgM antibodies were found in 20% and 25% patients, respectively. One control subject showed the presence of IgM antibody. Sensitivity for IgG and IgM antibodies was 64% and 81.25%, respectively, while specificity was 94.67% and 85.71%, respectively. This study indicates that saliva samples can be used as an alternative to serum samples to detect anti-toxoplasma antibodies, particularly IgM, for the diagnosis of toxoplasma encephalitis in HIV/acquired immune deficiency syndrome patients.

Antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus-infected patients.

Toxoplasma encephalitis in immunocompromised patients results from reactivation of previously acquired (latent) infection. The aim of the study is to assess the antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected patients to determine the best marker for early diagnosis of toxoplasmosis in such patients. Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG, IgM and IgA anti-toxoplasma antibodies and double-sandwich ELISA for toxoplasma antigen is carried out in serum samples collected from 100 HIV seropositive patients and 75 controls. Toxoplasma-specific IgG, IgM and IgA antibody response and antigenaemia were detected in 12%, 6%, 7% and 14% of HIV-infected patients, respectively. On retrospective analysis of 14 patients with antigenaemia only one had central nervous system (CNS) symptoms attributable to toxoplasma infection. In this patient, the CD4+ cell count was below 50/microL and none of the specific immunoglobulin isotype responses could be detected. The patient showed clinical improvement following specific chemotherapy for toxoplasmosis. In 25 HIV-negative and anti-toxoplasma IgG antibody-positive controls, IgM was detected in two (8%), IgA in five (20%) and antigenaemia in 10 (40%), while 50 HIV seronegative healthy controls were negative for both antigen and antibody responses. The study indicates that detection of toxoplasma antigen in addition to IgG antibody response may prove to be a useful indicator in the early diagnosis of reactivated toxoplasmosis in HIV/AIDS patients.

Genetic polymorphism in Plasmodium falciparum vaccine candidate antigens.

Malaria is still a major public health problem in many tropical and subtropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphisms in malaria vaccine candidates antigens might be a hurdle in developing an effective vaccine. The present article reviews the genetic polymorphism in several antigens expressed on the parasite surface, which are targets for immunological responses of the host and are good candidates for vaccine development against P. falciparum. Variable regions of most genes are generally dimorphic probably as a result of intragenic recombinations. Each allele in turn shows polymorphism resulting from point mutations, or other mechanisms. Several antigens like merozoite surface protein-1 and 2 (MSP-1 and MSP-2) and S antigen show high polymorphism while in others like circumsporozoite protein (CSP), apical membrane antigen-1 (AMA-1) and erythrocyte binding antigen-175 (EBA-175) functional constraints limit the degree of polymorphism. Polymorphism reported in these genes is discussed.

An ocular cysticercosis case: Caused by Asian genotype of Taenia solium.

An ocular cysticercosis case of a 42-year-old male, who presented with anterior uveitis is being reported. Microscopical examination of the cyst revealed presence of only one hooklet suggestive of T. solium cysticercus. Mitochondrial DNA analysis confirmed it to be T. solium cysticercus of Asian genotype. This is the first report on molecular typing of cysticercus isolate from ocular cysticercosis patient in India. The study suggests that the molecular analysis of cox1 gene may be a useful diagnostic tool in cases where microscopic examination is not confirmatory.

Evaluation of the efficacy of albendazole against the larvae of Taenia solium in experimentally infected pigs, and kinetics of the immune response.

Cysticercosis, a disease of economic and public health importance, is caused by Cysticercus cellulosae, the metacestode stage of Taenia solium. Experimental induction of cysticercosis was achieved in young pigs by feeding an optimum dose of 20,000 T. solium (Indian strain) eggs after immunosuppression, to assess the effect of albendazole and development of the immune response to cysticercus antigens before and after treatment. Histopathological studies revealed the presence of cysticerei in liver, lungs and muscles. Treatment with albendazole at 15 mg kg-1 body weight daily for 30 days starting from day 0 or 15 days post-infection resulted in 100% cure rates. Increases in antibody titre to crude soluble extract and a Sephadek G-200 purified antigenic fraction of Cysticercus cellulosae were found on days 25, 40 and 55 post-infection in untreated pigs and those in which treatment started on day 15 post-infection, whereas no increase in antibody response was observed in pigs in which treatment started on day 0.

Microbicidal mechanisms of liver macrophages in experimental visceral leishmaniasis.

To study the differential microbicidal potentials of liver macrophages, the oxygen-dependent and oxygen-independent pathways in Kupffer cells and immigrant macrophages of Leishmania donovani-infected BALB/c mice were investigated. Hydrogen peroxide assay was performed using horse radish peroxidase-dependent oxidation of phenol red to quantitate the reactive oxygen species produced. To examine the oxygen-independent pathway, the enzymes N-acetyl-beta-glucosaminidase (NAG) and beta-glucuronidase (beta G) were investigated after exposure of cells to lipopolysaccharide. Hydrogen peroxide release by Kupffer cells was significantly decreased only at 21 days postinfection, whereas hydrogen peroxide release by immigrant macrophages was significantly increased on all postinfection days with a maximum at 21 days postinfection. The pattern of release of NAG and beta G was similar in both cell populations with a peak at 21 days postinfection. The present study therefore suggests that Kupffer cells and immigrant macrophages adopt different pathways to cope with this infection.

Evaluation of a 200-kDa amastigote-specific antigen of L. donovani by enzyme-linked immunosorbent assay (ELISA) for the diagnosis of visceral leishmaniasis.

A purified 200-kDa antigenic fraction from Leishmania donovani axenic amastigotes was evaluated by ELISA for the detection of antibody response in visceral leishmaniasis (VL) patients, post kala-azar dermal leishmaniasis (PKDL) patients and controls, for the diagnosis of visceral leishmaniasis. A positive antibody response to the 200-kDa amastigote fraction and to Leishmania amastigote soluble antigen (LASA) was found in 29 (96.6%) and 30 ((100%) confirmed VL patients, respectively, by the use of ELISA. However, only 1 (10%) out of 10 PKDL patients had detectable antibody response to 200-kDa fraction while all the 10 (100%) PKDL patients exhibited an immune response to LASA. Therefore, use of the 200-kDa antigenic fraction for the detection of antibody response in an ELISA follow-up (post treatment) of VL patients may have prognostic significance, and it may also be useful for differentiating active VL and PKDL.

Diagnostic accuracy of rapid enzyme linked immunosorbent assay for the diagnosis of human hydatidosis.

Rapid enzyme linked immunosorbent assay (ELISA) was compared with the standard ELISA and indirect haemagglutination (IHA) techniques for the diagnosis of human hydatidosis. Eighty nine serum samples including 17 from hydatidosis patients (10 surgically confirmed and 7 clinically suspected), 50 from patients with other parasitic diseases and 22 samples from normal healthy individuals were analysed for anti-hydatid IgG antibodies using sheep hydatid cyst fluid antigen. The sensitivity and specificity respectively was found to be 82.3 and 100 per cent by rapid ELISA; 88.23 and 90.27 per cent by standard ELISA and 70.58 and 100 per cent by IHA technique. No cross reactions were observed with rapid ELISA technique using samples from cysticercosis and amoebiasis patients and normal healthy controls. The present study indicates that rapid ELISA can easily be performed in place of the standard ELISA for the serodiagnosis of human hydatidosis with the advantage of minimising reporting time and manpower hours.

Evaluation of RNA rich fraction of Litmosoides carinii in induction of protection in infected rats.

The RNA rich fraction of adult L. carinii worms was evaluated in evoking a protective response in infected rats. The RNA immunization was seen to be effective in limiting the microfilaraemia in peripheral blood as well as the adult worm burden. The antibodies to both RNA antigen and adult worm antigen were high in this group of animals at the peak of infection. The RNA immunization was seen to evoke hyperresponsiveness in lymphocytes to mitogens like adult worm antigen, PHA and Con A.

Role of calcium influx & energy metabolism in histamine release by filarial antigen in Mastomys natalensis experimentally infected with Brugia malayi.

The present work was undertaken to study the biochemical pathways involved in terms of role of calcium influx and status of energy metabolism in the activation of mast cells obtained from Mastomys natalensis infected with Brugia malayi when challenged in vitro with a potentially allergenic antigen (60 kDa) of Brugia malayi. It was observed that histamine release from sensitized lung and peritoneal mast cells was associated with intracellular influx of radioactive Ca2+, thus establishing the role of calcium in histamine release. The process of activation of mast cells by 60 kDA antigen was an energy requiring process as it utilized the energy phosphates in the form of ATP and the cells followed the aerobic respiratory pathway for the generation of energy molecules.

Dot enzyme linked immunosorbent assay for immunodiagnosis of amoebiasis.

Dot-enzyme linked immunosorbent assay (Dot-ELISA) used for the serodiagnosis of both intestinal and extraintestinal amoebiasis, showed positivity in 94 per cent patients of amoebic liver abscess and 87.5 per cent of intestinal amoebiasis. Only one of the 18 normal controls was positive by Dot-ELISA test. This test showed good correlation with indirect haemagglutination test. As it is simple and easy to perform, the test appears to have great potential for the specific diagnosis of amoebiasis in peripheral hospitals and also at the field level where sophisticated immunodiagnostic facilities are not available.

Effect of sodium stibogluconate on the status of interleukin-I production in normal & Leishmania donovani infected BALB/c mice.

Sodium stibogluconate, did not bring about significant increase in the production of IL-1, when both specific leishmanial antigen, or non specific Staphylococcus epidermidis was used as stimulus in normal uninfected animals. However, Staph. epidermidis was found to be a better stimulus as it brought about a significant increase (P less than 0.001) in IL-1 production when compared with leishmania antigen. In BALB/c mice infected with L. donovani there was a significant reduction (P less than 0.001) in IL-1 levels on various post infection days irrespective whether Staph. epidermidis or leishmanial antigen was used as stimulus when compared with controls. IL-1 levels were significantly increased (P less than 0.05) when L. donovani infected animals were treated with SSG, after 14 days post infection, irrespective of the stimuli used.