Lactose hydrogen breath test in Giardia lamblia-positive patients.

The study was conducted in 54 adult patients with Giardia lamblia infection and 54 adult controls to detect lactose maldigestion employing the noninvasive lactose hydrogen breath test. Forty of 54 (74%) patients with Giardia lamblia and 24 of 54 (44.4%) controls showed lactose maldigestion (P < 0.01). In conclusion, this study shows that the frequency of lactose maldigestion is significantly higher in adult Indians suffering from Giardia lamblia infection compared to healthy individuals.

Cellular and serological markers of disease activity in Indian patients with HIV/AIDS.

There has been an exponential rise of HIV positive patients as observed at the surveillance center of Nehru Hospital. Most patients are poor and cannot afford repeated viral load assays. Therefore, there is a need to identify cost effective and reliable surrogate markers of disease activity. In the present study absolute number of CD4 cells, beta2 micro-globulin, circulating nucleosomes were studied in 30 patients of AIDS, 30 seropositives and 30 healthy controls. In addition viral load, P-24 assay, and TNFR-II assays were done in seropositive and AIDS patients. The mean CD4 cells in patients with AIDS were 69.66 +/- 68.25 mm3 while in seropositives values was 370 +/- 201.29 mm3. The mean CD4 cells in healthy controls were however 690 +/- 198 mm3. The differences in all the groups were highly significant (p<0.001). The mean CD4 values in Indians are significantly lower than reported from the west. The lower number of CD4 cells in healthy population is interpreted to be due to immune activation. The CD8 cell number in controls was 650 +/- 207 mm3 this figure is also higher than that observed in the west. P-24 assay failed to delineate between seropositives and patients with AIDS. Although, beta2 microglobulin levels were significantly higher in AIDS than in seropositives and higher in seropositives than in controls yet with the best possible cut off, it had a sensitivity of only 70% in delineating the two conditions. The correlation between CD4 cells and viral load was more significant when the CD4 cells were below 200 mm3. Five out of 30 patients with a CD4 of 300-600 mm3 had a viral load of over 1 x 10(5) cop/ml. The difference in TNF R-II levels between seropositives and AIDS was however more impressive. With a cut off of 550 pg/ml it had a sensitivity of 95% in delineating HIV from AIDS. It is concluded that a combination of absolute number of CD4 cells and TNF R-II assay along with clinical evaluation may be used to monitor therapy in resource poor countries where frequent viral load assay is unaffordable.

Identification and characterization of an excretory-secretory product from Giardia lamblia.

A 58 kDa excretory-secretory product (ESP) of Giardia lamblia has been characterized. The ESP was purified over 508-fold by a combination of ammonium sulphate precipitation and sequential chromatography on affinity matrix and a gel filtration column. The homogeneity of the purified protein was established by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Mr, 58 kDa) and analytical isoelectrophoresis (pI 4.75). The purified protein was recognized by the pooled sera of G. lamblia-positive patients as well as an antiserum raised against crude Giardia extract, thus indicating it to be an immunodominant parasite product. The ESP was found to agglutinate rabbit erythrocytes. The haemagglutinating activity of this protein was inhibited strongly by thyroglobulin, fetuin, asialofetuin and monosialoganglioside but not by simple sugars. The purified protein was characterized immunochemically and was found to be heat stable as well as protease sensitive. Lectin-binding studies of the purified ESP and its sensitivity to periodic-acid silver staining as well as to metaperiodate treatment clearly indicated its glycoprotein nature. The major localization site of the ESP was found to be on the surface of the parasite as revealed by flow cytometric analysis. Further, this glycoprotein induced fluid accumulation in ligated rabbit ileal loops and revealed a positive skin permeability reaction in the rabbit.

Partial characterization of a 36-kDa antigen of Entamoeba histolytica and its recognition by sera from patients with amoebiasis.

A 36-kDa antigen of axenically grown pathogenic Entamoeba histolytica (HM1-IMSS) was eluted from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-resolved crude amoebic extract antigens. The immunoreactivity of this partially purified 36-kDa antigen with monoclonal antibody (MoAb) 3D(10) altered significantly (P<0.01) after heat and trypsin treatment but remained unaltered after treatment with sodium metaperiodate (P0.5), thereby indicating the protein nature of the epitope recognized by MoAb 3D(10). The epitope was found to be localized on the surface as well as in the cytoplasm of the E. histolytica trophozoites with the majority of it in the cytoplasm. In addition, this epitope was also found to be present on the cyst form of the parasite. The 36-kDa molecule was recognized by the sera from 29 (85%) of the 34 patients with amoebic liver abscess and five (83%) of the six patients with amoebic colitis. No serum samples from asymptomatic cyst passers, from patients with non-amoebic hepatic or intestinal disorders and apparently healthy subjects had antibodies that reacted with this 36-kDa molecule. The immune responses in man to this 36-kDa amoebic molecule indicate a potential specific role for this molecule in invasive amoebiasis.

Effect of Giardia lamblia on duodenal disaccharidase levels in humans.

The study was conducted to detect the effect of giardiasis on human disaccharidase levels. Forty patients attending the medical outpatient department of PGIMER, Chandigarh were enrolled. Twenty patients, positive for Giardia lamblia comprised the study group while 20 patients negative for Giardia lamblia were taken as controls. Upper gastrointestinal endoscopy was performed in all patients. Estimation of lactase, sucrase, maltase and trehalase was done in biopsies. Histopathological investigation was carried out in all biopsy specimens after Haematoxylin and Eosin staining. Complaints of pain abdomen and bloating occurred commonly in giardiasis. Four biopsy samples in study group showed mild increase in lymphomononuclear infiltrate. Giardia lamblia was detected in 7 biopsies. Lactase levels were decreased significantly (p < 0.05) in giardiasis. Rest of the enzymes were comparable to the controls. No differences in the enzyme activities were observed between males and females in either group and with the duration of symptoms.

A monoclonal antibody-based test system for detection of Entamoeba histolytica-specific coproantigen.

BACKGROUND: Diagnosis of amebiasis based on stool microscopy or demonstration of anti-amebic antibodies has limitations. A diagnostic system based on demonstration of the parasite product in clinical specimens holds promise. METHODS: Murine monoclonal antibodies were developed against an Entamoeba histolytica-specific coproantigen. A monoclonal antibody (MoAb) 3D10 was employed in a double-antibody sandwich microELISA system for the detection of amebic coproantigen in fecal specimens. The system was evaluated in three groups of subjects: 63 patients with intestinal amebae, 27 with non-amebic parasitosis, and 57 apparently healthy controls. RESULTS: The MoAb 3D10 belonged to IgG1 isotype and recognized three antigens, with mol. wt. 36, 25 and 17 kDa in the crude extract of E. histolytica (HM1-IMSS), and an amebic coproantigen with MW 36 kDa in the stool supernatant from patients with intestinal amebae. The coproantigen was detected in the stool eluates of 56 (89%) patients with intestinal amebae and in none of the stool eluates from other subjects, thereby giving this system a sensitivity of 89% and specificity of 100% for the detection of intestinal amebae. CONCLUSIONS: This monoclonal antibody recognizes as intact epitope on the E. histolytica-specific coproantigen. The validity of the MoAb-based microELISA system needs to be established.

Immune effector responses to an excretory-secretory product of Giardia lamblia.

The prior immunisation of mice with purified excretory-secretory product (ESP) led to a complete failure of Giardia lamblia colonisation following challenge inoculation of these animals with trophozoites. The prior immunisation of mice with ESP resulted in a significant stimulation of local immunity as evidenced by a significant enhancement of T helper/inducer activity along with a significant increase in immunoglobulin A-bearing cells. Further, the presence of anti-ESP antibodies in the serum of immunised as well as immunised-challenged animals indicated the stimulation of the systemic lymphoid system. This suggests that the ESP is highly immunogenic and it could be one of the major antigens of G. lamblia responsible for protection against the infection.

Differential tropism of EB rotavirus (serotype 3) to small intestine of homologous murine model.

The 4-5 days-old NMRI strain infant mice were orally inoculated with EB rotavirus (serotype 3). The intestinal disaccharidases activity was studied separately in three segments of the small intestine i.e. duodenum, jejunum and ileum on day 1 to 7 post inoculation (p.i.). The severity of EB rotavirus infection correlated with a significant decrease of small intestinal lactase, maltase and sucrase on day 3 p.i. The level of maltase after the initial decline increased in all the three segments of small intestine of infected mice. However, the lactase activity remained suppressed for a relatively longer period in ileum of infected mice than in controls. These enzymes began to approach to normal value by day 5 p.i., but in ileum, lactase activity continued to be severely depressed even on day 7 p.i. Rotavirus was consistently detected in intestinal contents by ELISA on days 1 to 7 p.i. The infected mice showed a significant increase in rotavirus (serotype 3)-specific serum IgG and IgM antibody level during the declining (days 5-7 p.i.) phase of infection. Diarrhoea was noted up to day 6 p.i. The protracted suppression of the lactase activity in ileum in comparison to duodenum and jejunum showed a differential tropism of EB rotavirus (serotype 3) strain to the small intestine of homologous murine model.

Significance of detection of immune-complexed 8 kDa hydatid-specific antigen for immunodiagnosis of hydatidosis.

Three micro-enzyme-linked immunosorbent assay (micro-ELISA) systems were developed and evaluated for detection of specific free circulating antigen and circulating immune-complexes (CICs) of 8 kDa antigen in the sera of patients with hydatidosis. All (100%) the sera of 30 confirmed positive cases of hydatidosis had detectable levels of antigen in the acid-treated sera. However, 23 (77%) and 26 (87%) sera of 30 confirmed cases had free as well as CICs of 8 kDa antigen in the untreated and in the polyethylene glycol (PEG) precipitated sera, respectively. None of the sera from other patients with parasitic infections or viral hepatitis had any detectable levels of 8 kDa antigen in the untreated, acid-treated or PEG-precipitated serum samples. The investigations, therefore, suggested that the demonstration of circulating antigen employing monospecific antibodies to affinity purified 8 kDa antigen in acid-treated sera is more efficient as compared to detection of free circulating antigen or CICs in the untreated or in the PEG-precipitated sera which could provide a specific immunodiagnostic tool for ongoing hydatid infection.

Leishmania donovani flagellum-specific epitopes mediating host-parasite interactions.

Monoclonal antibodies were developed against flagellar components of promastigotes of Leishmania donovani. The monoclonal antibody produced by clone A11 (mAb A11) recognised epitopes in the polypeptides with molecular weights of 86, 66 and weakly 53 kDa. These epitopes were found to be distributed along the flagellum and at the anterior end of promastigotes. The mAb A11 of IgG1 isotype strongly agglutinated the promastigotes of L. donovani. The prior treatment of promastigotes of L. donovani with mAb A11 resulted in a significant (P < 0.001) reduction in the attachment of promastigotes to cultured mouse peritoneal macrophages of line J774G8. The affinity-purified epitopes identified by mAb A11 were recognised by human sera of cases of visceral leishmaniasis. The present study suggest that flagellar-specific epitopes mediate host-parasite interactions and, therefore, the role of these epitopes in the disease process is speculated.

Anti-66 kDa antileishmanial antibodies as specific immunodiagnostic probe for visceral leishmaniasis.

A 66 kDa plasma membrane associated molecule of promastigotes of Leishmania donovani (MHOM/IN/1978/UR6) was affinity purified under acidic conditions. Employing purified 66 kDa antigen in micro ELISA, 36 (97.3%) of the 37 patients of visceral leishmaniasis (bone marrow aspirates positive for Leishman Donovan bodies) had detectable levels of anti 66 kDa anti leishmanial antibodies. The sera of the patients confirmed to have visceral leishmaniasis had significantly (P < 0.001) higher optical density values (0.636 +/- 0.230) as compared to sera (OD 0.185 +/- 0.131) from patients clinically suspected to have visceral leishmaniasis (bone marrow aspirates negative for Leishman Donovan bodies). None of the 35 sera from apparently healthy subjects from non endemic area had anti 66 kDa antibodies. However, sera from one (8.3%) of the 12 healthy subjects, who was a first degree relative of a patient of visceral leishmaniasis and residing in an area endemic for visceral leishmaniasis, had anti 66 kDa antibodies. It is felt that detection of anti 66 kDa antibodies in a micro ELISA assay provides a highly sensitive and specific tool for confirming ongoing visceral leishmaniasis.

Inhibition of hepatic extramedullary haemopoiesis by nucleoprotein of heterologous rotavirus strain in infant mice.

Infant mice (NMRI strain) showed the inhibition of hepatic extramedullary haemopoiesis by oral inoculation of a 100 ID50 dose of EB rotavirus and nucleoprotein of SA-11 rotavirus (serotype 3). The extramedullary haemopoiesis was observed by oral inoculation of surface protein VP7 of SA-11 rotavirus and in control (placebo administered) mice.

Evaluation of commercially available antacid tablets.

Antacids are commonly used for the treatment of acid peptic disease. There is a need to study the relative merits of various brands of antacids available commercially. Twenty three brands of antacid tablet preparations were evaluated with regard to their composition, cost, dispersion time, pH, neutralising capacity and time taken for neutralisation. The cost of various tablets ranged from Rs 0.13 to Rs. 1.48 per tablet and the dispersion time from 20 to 90 minutes. The pH of the dispersed tablet solution ranged from 5.7 to 9.5. The neutralising capacity varied between 8 to 169 meq/tablet and the neutralizing time between 20 and 45 minutes. The cost: neutralizing ratio was calculated and ranged from 102 to 9867 x 10(-3) Rs/meq. A scoring system with a maximum score of 12 has been devised. The study provides a guide for choosing a more potent, quick neutralising and low cost antacid preparation.

Detection of Entamoeba histolytica antigens in stool in amebiasis.

BACKGROUND: Stool microscopy, the conventional method of diagnosing intestinal amebiasis, fails to detect Entamoeba histolytica in more than 30-40% of clinically suspected cases. Demonstration of parasite products in clinical specimens has been suggested as an alternative. However, the usefulness of demonstrating amebic antigen in the stools of clinical cases needs to be assessed. METHODS: A double-antibody sandwich enzyme linked immunosorbent assay (ELISA) using anti-trophozoite antibodies to capture E histolytica specific coproantigen(s) was carried out on stools obtained from 31 patients with microscopically confirmed non-dysenteric amebic colitis, 18 patients with intestinal parasites other than E histolytica and 41 apparently healthy subjects. RESULTS: The assay detected E histolytica specific coproantigen(s) in stools of 23 (74.2%) of 31 subjects with non-dysenteric amebic colitis, none of 18 with other parasitic infections and 1 (2.4%) of 41 apparently healthy subjects. CONCLUSION: Our results provide evidence for the presence of E histolytica specific coproantigen(s) in stool eluates from patients with amebic infection; this finding can be exploited for confirming ongoing amebic infection. However, the sensitivity of the assay needs to be improved by the use of relevant monospecific/monoclonal antibodies.

Evaluation of the direct agglutination test as an immunodiagnostic tool for kala-azar in India.

The direct agglutination test (DAT) has been assessed as a diagnostic procedure for visceral leishmaniasis. Fifty-six of 58 sera (96.5%) from confirmed cases of visceral leishmaniasis, whose bone marrow aspirates contained Leishmania donovani amastigotes, had agglutinating antibodies above the cut-off titre of 1:800. None of the sera from healthy control subjects from non-endemic or endemic areas had anti-leishmanial antibodies. Similarly, none of the sera obtained from cases of malaria or tuberculosis had agglutinating antibodies above the cut-off titre. A significant decline in agglutinating antibody titre in 3 cases following antileishmanial chemotherapy appeared to correlate with regression of clinical symptoms and the absence of amastigotes from bone marrow aspirates. One of 3 cases developed post-kala-azar dermal lesions and sera from this subject had an elevated agglutinating antibody titre. It is concluded that the DAT is a sensitive and specific test to confirm visceral leishmaniasis. As the formalin-fixed promastigotes, stained with Coomassie blue, which are used as antigen could be stored at 4 degrees C for 6 months without any loss of ability to detect anti-leishmanial antibodies, the DAT is recommended for use under field conditions.

Isolation & immunochemical characterization of diagnostically relevant antigens of Echinococcus granulosus.

Two hydatid specific polypeptides with molecular masses of 8 kDa and 116 kDa have been successfully isolated from E. granulosus hydatid cyst fluid using affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblot analysis under reducing and denaturing conditions indicated the 116 kDa purified antigen to be a hetero-tetramer consisting of 45 kDa, 66 kDa, 75 kDa and 116 kDa subunits linked by disulphide bonds while the 8 kDa purified antigen was found to be a monomer polypeptide. Affinity purified 116 kDa molecule was heat-labile, sensitive to treatment with pronase, trypsin and pepsin and its immunoreactivity as assessed in enzyme linked immunosorbent assay remained unaltered on treatment with sodium metaperiodate. The affinity purified 8 kDa molecule was heat-stable, sensitive to proteolytic enzymes and also sodium metaperiodate oxidation. Lectin binding studies revealed that the 8 kDa molecule specifically bound Concanavalin A and Triticum vulgaris, and thus had varies; is directly proportional to-D-glucose and N-acetyl D-glucosamine sugar moieties. The immunoreactivity of both the antigens remained unaltered on treatment with lipase. However, biochemical estimation of total lipid content revealed the affinity purified 116 kDa antigen to contain 6.25 per cent total lipids suggesting it to be lipoproteinic in nature. The 8 kDa antigen had no detectable total lipids biochemically. All sera from patients confirmed to have hydatidosis recognised the 8 kDa and 116 kDa polypeptides. However, sera from seven subjects with other parasitic infections also recognised the 116 kDa antigen though not the 8 kDa antigen. The data suggested that the recognition of 8 kDa antigen of E. granulosus has potential for specific immunodiagnosis of hydatidosis.

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis.

Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia-specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.

Host response to Mycobacterial cell wall subunit during experimental tuberculosis in mice.

The cell-wall protein-peptidoglycan complex (CW-PPC) of Mycobacterium tuberculosis, an immunologically potent component, was used to study the correlation between immune response and in vivo bacterial multiplication in the course of experimental tuberculosis infection in mice. Antibodies to CW-PPC were detected only after seven weeks of infection with M. tuberculosis H37Rv and afterwards no significant change was seen throughout the experiment. Delayed type hypersensitivity (DTH) to CW-PPC showed a gradual increase from the fifth week onward with a maximum during the 12th week after infection (p.in.) which did not change significantly afterward. The increased immune response in the course of infection correlated well with the multiplication rate of bacilli in the lungs. These results indicate a role of CW-PPC in antituberculous immunity.

The significance of free and immune-complexed hydatid-specific antigen(s) as an immunodiagnostic tool for human hydatidosis.

Micro-enzyme-linked immunosorbent assay (Micro-ELISA) systems were developed and evaluated for the detection of circulating (free or immune-complexed) hydatid antigens in the sera of patients with hydatidosis, by employing monospecific antibodies to hydatid-specific antigens of 8-kDa and 116-kDa. Fifteen (75%) of 20 sera from patients with hydatidosis had both 8-kDa and 116-kDa antigens freely circulating in their sera while three and two samples, respectively, had only 8-kDa or 116-kDa antigen. All the surgically confirmed cases of hydatidosis had detectable levels of both 8-kDa and 116-kDa circulating immune complexes in glycine HCl-treated sera. However, none of the sera from control subjects (patients with cysticercosis, ascariasis, ancylostomiasis, hymenolepiasis, amoebic liver abscess or viral hepatitis) had any detectable level of either type of circulating specific antigen. These results suggest that the demonstration of either 8- or 116-kDa antigen(s) in free or immune-complex form could confirm the diagnosis of hydatidosis.

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence.

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.