Antibody isotype responses to egg antigens in human chronic Schistosomiasis mansoni before and after treatment

In the present communication we analyzed the levels of IgG1, IgG2, IgG3, IgG4 and IgE isotypes to soluble egg antigen of Schistosoma mansoni by ELISA in individuals from an endemic area for schistosomiasis in Northeast Brazil. The analysis was performed before and after treatment to evaluate the age-dependent pattern, and to identify differences in the reactivities to antigens. Our results suggest that schistosomiasis treatment would not interfere with this sort of immune response.

The effect of treatment on the age-antibody relationship in children infected with Schistosoma mansoni and Schistosoma haematobium

The effect of praziquantel treatment on the age-antibody relationship was studied in 174 children aged between 6 and 17 years from a schistosome endemic area in Zimbabwe. The children were co-infected with Schistosoma mansoni and S. haematobium with infection prevalences of 74% and 53% respectively. Antibody levels for the isotypes IgA, IgE, IgM, IgG1, IgG2, IgG3 and IgG4, directed against soluble egg antigen were measured using an indirect ELISA assay. Treatment resulted in a significant increase in levels of IgG2 and IgG3 while levels of IgA decreased significantly. In untreated children there were significant decreases in levels of IgG4. Treatment also resulted in significant alteration in the age-antibody profiles for the isotypes IgE, IgM, IgG1 and IgG2 in treated children but not in untreated children. The results are discussed in the context of factors believed to give rise to the age-antibody relationship; i.e. age-related exposure patterns, age-related development of acquired immunity, age-related hormonal changes and age-related changes in innate susceptibility to infection.

Anti-human immunodeficiency virus-1 antibody titers in injection drug users compared to sexually infected individuals

Sera from infected injection drug users (IDU) have shown to have antibodies against synthetic human immunodeficiency virus-1 (HIV-1) envelope peptides more frequently. In this study, reactivity of 48 IDU plasma were compared to 60 plasmas obtained from sexually infected individuals (S). The overall reactivity of plasma from IDU compared to S was higher, and the reactivity titers were much higher for IDU plasma than S. IDU plasma also showed a broader antibody response. The higher reactivity titers were observed mainly for the gp41 immunodominant epitope and V3 peptides corresponding to the consensus sequences of HIV-1 subtypes/variants prevalent in Brazil (B, F, C) indicating the specificity in the higher immune response of IDU.

Perspective of a new diagnostic for human trichomonosis

Several diagnostic techniques have been employed for the detection of Trichomonas vaginalis. Microtubules constitute the cytoskeleton in eukaryotic cells and are sensitive to antimitotic drugs, such as Taxol (paclitaxel). We used FLUTAX a fluorescent taxoid - to analyze the microtubule distribution in living trophozoites of T. vaginalis in urine and in vaginal discharge. A high intensity of fluorescence was observed in living T. vaginalis, epithelial cells and leukocytes present in urine and vaginal discharge. Our preliminary results show the perspective of a new diagnostic technique for trichomonosis and will contribute to the understanding of the cytoskeleton of T. vaginalis.

A technique for preparing polytene chromosomes from Aedes aegypti (Diptera, Culicinae)

Polytene chromosome preparations were obtained from larval, pupal and adult female Malpighian tubules of Aedes aegypti. The Malpighian tubules of the pupae (0-4 h old) from larvae reared at 20ºC provided the best cytogenetic analysis. The interaction of nucleic acids and proteins that influence the spreading of the chromosomes could be reduced with the preparation technique of the sheets submitted to a stronger treatment starting with the hypotony of tissue and successive bathings with acetic acid. A simple technique should facilitate molecular cytogenetics used in the location of resistance and vector competence genes.

Antibody isotype responses in Balb/c mice immunized with the cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi

In the present report we analyzed the levels of IgG1, IgG2a, IgG2b and IgG3 isotypes from Balb/c mice immunized with cytoplasmic repetitive antigen (CRA), and flagelar repetitive antigen (FRA) of Trypanosoma cruzi. The immunization was done by subcutaneous route three times (20 days apart) and the analysis was performed 14 days after each treatment. CRA-immunized mice produced high levels of all IgG isotypes, mainly IgG3 and IgG1. FRA-immunization elicited only high levels of IgG1.

An alternative technique to reveal polysaccharides in Toxoplasma gondii tissue cysts

Ultrathin sections of tissue cysts isolated from the brain of Toxoplasma gondii infected mice were submitted to two different methodologies derived from the periodic acid - Schiff's reagent (PAS) technique. The use of osmium tetroxide vapor as a developing agent of the aldehyde oxidation to reveal polysaccharides with periodic acid resulted in positive reaction in amylopectin granules in bradyzoites, as well as in the wall and matrix of the cysts, with excellent increment of the ultrastructural morphology. This technique can be used for study of T. gondii-host cell intracellular cycle, the differentiation tachyzoite-bradyzoite, and also for the formation of cysts into the host cells.

Circulating filarial antigen in the hydrocele fluid from individuals living in a bancroftian filariasis area - Recife, Brazil: detected by the monoclonal antibody Og4C3-assay

The purpose of this study was to examine the circulating filarial antigen (CFA) detected by the monoclonal antibody (mAb) Og4C3-ELISA in paired samples of serum and hydrocele fluid from 104 men with hydrocele, living in an endemic area of Wuchereria bancrofti. Nocturnal blood specimens were filtered and examined for microfilariae (MF) and ultrasound was used in order to identify the presence of adult worms (the filaria dance sign - FDS) in the lymphatic vessels of the scrotal area. Four groups were selected according to their parasitological status: group I - 71 MF- and FDS-; group II - 21 MF+ and FDS+; group III - 10 MF- and FDS+ and group IV- 2 MF+ and FDS-. CFA was identified simultaneously (fluid and serum) in 11 (15.5%), 21 (100%), 3 (30%), and 1 (50%) in groups I, II, III, and IV, respectively. In despite of high CFA+ level (antigen Og4C3) units/ml, the Geometrical Mean (GM) = 2696) in the sera of these 36/104 paired samples, when compared to the hydrocele fluid, (GM = 1079), showed a very good correlation between the CFA level in the serum and CFA level in the fluid (r = 0.731). CFA level in the serum of the 23 microfilaremics (groups II and IV) was extremely high (GM = 4189) and was correlated with MF density (r = 0.442). These findings report for the first time the potential alternative use of the hydrocele fluid to investigate CFA using the mAb Og4C3-ELISA.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Study of the antibody response against Mycobacterium tuberculosis antigens in Warao Amerindian children in Venezuela

This study was aimed at investigating alternate methods for serodiagnosis of tuberculosis (TB), which are needed because bacteriologic diagnosis of childhood TB is difficult. A selection of 80 serum and saliva samples were tested from Warao indigenous children under 15 years of age; 34 high TB suspects (28 positive and 6 negative for the tuberculin skin test, TST) and 46 healthy contact children (32 positive and 14 negative for the TST). Several enzyme-linked immunosorbent assay (ELISA) serological tests were developed to test for Mycobacterium tuberculosis-specific antibodies, including serum IgA, IgG, IgE, and secretory IgA (sIgA) in saliva against 3 specific antigens (PPD, HSP60, 38 kDa). Of these, 2 antigens, PPD and 38 kDa, showed significantly higher reactivity. The sensitivity and specificity of these tests for diagnosis remained limited, between 26.5% and 38.2%, and 77.4% and 97%, respectively. Of all the samples studied and combinations realized between all isotypes and antigens combined with 3 isotypes (anti-PPD IgG, IgE, and anti-38kDa sIgA) managed to detect the largest number of patients, showing an improved sensitivity level of 64.7%, although specificity levels dropped to 81.8%. These results were compared with the Omega diagnostics commercial kit results. The commercial kits showed significantly lower reactivity (sensitivity of 20% and 13.33% to Myco G and Complex Plus, respectively) and a specificity of 100%. This study shows that in indigenous populations of Venezuela, where invasive procedures cannot be used to select samples but evaluation with a chest X-ray for radiological studies is available, the combination of 3 specific isotypes may be a useful tool to increase diagnostic accuracy with pulmonary TB in this population, when used together with clinical and epidemiological criteria.

Evaluation of tests based on the antibody response to keyhole limpet haemocyanin and soluble egg antigen to differentiate acute and chronic human schistosomiasis mansoni

Specific IgG and IgM responses to soluble egg antigen (SEA) and keyhole limpet haemocyanin (KLH) were measured by ELISA in patients with acute and chronic schistosomiasis. The tests based upon IgM and IgG antibodies responses to KLH presented the best diagnostic discrimination, and can be used in conjunction with clinical and epidemiological data to the differential diagnosis of acute schistosomiasis.

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Diphtheria-neutralizing antibody levels in healthy adults from Rio de Janeiro, Brazil

In Brazil, until 2004, the immunization policy against diphtheria involved childhood vaccination with no official routine booster dose administered after 15 years of age. This study assessed functional antibody levels against diphtheria among blood donors. A total of 140 blood samples were collected, and diphtheria antitoxin levels were evaluated by Vero cell neutralization test. The mean age of the population was 34 years old (range: 18-61 years); 37.8% females and 62.2% males. Overall, 30.7% (95%, CI: 23.4-38.7) individuals presented neutralizing antitoxin antibody titers < 0.01 IU/ml; 42.1% (95%, CI: 34.1-50.4) showed values between 0.01-0.09 IU/ml and, 27.1% (95%, CI: 20.2-34.9) had ³ 0.1 IU/ml. In the subgroup of individuals with history of diphtheria immunization during childhood (85%), a number of 28.5% showed unprotective levels of circulating neutralizing antibody (< 0.01 IU/ml). Despite the continuous progress of immunization programs directed to Brazilian population, currently healthy adults remain susceptible to diphtheria.

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86).