Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin ®, Larp33 was expressed in Escherichia coli after cloning of a 2,2 kb Sau3A digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L.(L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-g, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis

Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and Western-blot

Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM) were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8) and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P. brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus. Before treatment, 81.5% (Dot-blot) and 84% (ELISA) of the patients presented elevated IgG anti-P. brasiliensis antibody titers which dropped slightly with treatment. On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (5l.9% and 5l.8%: Dot-blot; 16.5 and 36%: ELISA, respectively) but the titers tended to become negative more frequently with treatment. Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively. Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively. The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies. During the period of study, the patients responded well to treatment. The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P. brasiliensis antibodies

Cryo-Microtome sections of coproculture larvae of Strongyloides stercoralis and Strongyloides ratti as antigen sources for the immunodiagnosis of human strongyloidiasis

Cryo-microtome sections of larvae of Strongyloides stercoralis and S. ratti respectively obtained from human and rat feces cultures, were used as antigens. Fluoresceinate conjugates against human IgG were employed at the ideal titer of 10 for S. stercoralis and 100 for S. ratti. The sensitivity of the indirect immunofluorescence reaction (IIF) was 94.4% and 92.5% and the specificity 94.2% and 97.1% for the two specific larval antigens, respectively. Sera from 123 persons (54 from carriers of S. stercoralis infections and 69 from controls) were submitted to the reaction. The titers of different sera varied from 20 to 2560. There was a significant linear correlation (r = 0.85 p £ 0.001) between the antibodies from the two species of larval antigens. We conclude that both antigens may be used in the IIF reaction for the diagnosis of human strongyloidiasis. Due to the feasibility of safe and low-cost mass production of S. ratti larvae in the laboratory with a considerable economy of conjugate, their utilization in the serum diagnosis of human strongyloidiasis is recommended

DETECTION OF HTLV-I ANTIBODIES AND DNA IN BLOOD SAMPLE OF A PATIENT WITH MYELOPATHY IN NIGERIA

We describe a case of human T-lymphotropic virus type I associated myelopathy in a 50-year old woman in Nigeria. The patient presented with progressive loss of tone to the two lower limbs and later inability to walk. The HTLV-I antibody presence in the plasma collected from the patient was repeatedly detected by enzyme immunoassays (Abbott HTLV-I EIA and Coulter SELECT-HTLV I/II) and confirmed by Western blot technique. In addition, HTLV-I DNA was amplified from the genomic DNA isolated from the peripheral blood mononuclear cells of the patient by the polymerase chain reaction technique. This finding is significant being the first report of association of HTLV-I with myelopathy in Nigeria.

Evolution of IgG antibody response against Toxoplasma gondii tissue cyst in acute and chronic human infections

The recognition profile of the tissue cysts antigens by IgG antibodies was studied during acute and chronic human toxoplasmic infection. Thus the IgG response against Toxoplasma gondii was investigated by immunoblotting in two patients accidentally infected with the RH strain as well as in group of naturally infected patients at acute and chronic phase. There was an overall coincidence of molecular mass among antigens of tachyzoites and tissue cysts recognized by these sera, however, they appear not to be the same molecules. The response against tissue cysts starts early during acute infection, and the reactivity of antibodies is strong against a wide range of antigens. Six bands (between 82 and 151 kDa) were exclusively recognized by chronic phase sera but only the 132 kDa band was positive in more than 50% of the sera analysed. A mixture of these antigens could be used to discriminate between the two infection phases. The most important antigens recognized by the acute and the chronic phase sera were 4 clusters in the ranges 20-24 kDa, 34-39 kDa, 58-80 kDa and 105-130 kDa as well as two additional antigens of 18 and 29 kDa. Both accidentally infected patients and some of the naturally infected patients showed a weak specific response against tissue cyst antigens.

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

Evaluation of anti-Schistosoma mansoni igG antibodies in patients with chronic schistosomiasis mansoni before and after specific treatment

The circumoval precipitin test (COPT), enzyme-linked immunosorbent assay (ELISA) and the immunoblotting anti-adult worm antigen (AWA) and soluble egg antigen (SEA) tests were applied to 17 chronically schistosome-infected patients for the detection of anti-Schistosoma mansoni antibodies before and on four occasions after oxamniquine administration over a period of six months. Compared to a control group, schistosomiasis patients showed high levels of IgG antibodies in AWA and SEA-ELISA. A decrease in IgG levels was observed six months after treatment, although negative reactions were not obtained. Significant decreases in IgG1, IgG3 and, mainly, IgG4, but not anti-SEA IgG2 levels were observed six months after treatment, again without negativity. Analysis of anti-AWA IgG antibodies by immunoblotting before treatment showed a 31 kDa strand in 14 patients (82%) which disappeared in three cases up to six months after treatment; furthermore, anti-SEA IgG antibodies showed the same band in nine patients (53%) before treatment, which disappeared in only four cases up to six months after treatment.

Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.