Critical analysis of old and new vaccines against N. meningitidis serogroup C, considering the meningococcal disease epidemiology in Brazil

Worldwide, the impact of meningococcal disease is substantial, and the potential for the introduction and spread of more virulent strains of N. meningitidis or strains with increased resistance to current antibiotics causes concern, making prevention essential. OBJECTIVES: Review the indications for meningococcal disease vaccines, considering the epidemiological status in Brazil. METHODS: A critical literature review on this issue using the Medline and Lilacs databases. RESULTS: In Brazil, MenB and MenC were the most important serogroups identified in the 1990s. Polysaccharide vaccines available against those serogroups can offer only limited protection for infants, the group at highest risk for meningococcal disease. Additionally, polysaccharide vaccines may induce a hypo-responsive state to MenC. New meningococcal C conjugate vaccines could partially solve these problems, but it is unlikely that in the next few years a vaccine against MenB that can promote good protection against multiple strains of MenB responsible for endemic and epidemic diseases will become available. CONCLUSIONS: In order to make the best decision about recommendations on immunization practices, better quality surveillance data are required. In Brazil, MenC was responsible for about 2,000 cases per year during the last 10 years. New conjugate vaccines against MenC are very effective and immunogenic, and they should be recommended, especially for children less than 5 years old. Polysaccharide vaccines should be indicated only in epidemic situations and for high-risk groups. Until new vaccines against MenC and MenB are available for routine immunization programs, the most important measure for controlling meningococcal disease is early diagnosis of these infections in order to treat patients and to offer chemoprophylaxis to contacts.

Dissecando o GEN

In thee present paper the classical concept of the corpuscular gene is dissected out in order to show the inconsistency of some genetical and cytological explanations based on it. The author begins by asking how do the genes perform their specific functions. Genetists say that colour in plants is sometimes due to the presence in the cytoplam of epidermal cells of an organic complex belonging to the anthocyanins and that this complex is produced by genes. The author then asks how can a gene produce an anthocyanin ? In accordance to Haldane's view the first product of a gene may be a free copy of the gene itself which is abandoned to the nucleus and then to the cytoplasm where it enters into reaction with other gene products. If, thus, the different substances which react in the cell for preparing the characters of the organism are copies of the genes then the chromosome must be very extravagant a thing : chain of the most diverse and heterogeneous substances (the genes) like agglutinins, precipitins, antibodies, hormones, erzyms, coenzyms, proteins, hydrocarbons, acids, bases, salts, water soluble and insoluble substances ! It would be very extrange that so a lot of chemical genes should not react with each other. remaining on the contrary, indefinitely the same in spite of the possibility of approaching and touching due to the stato of extreme distension of the chromosomes mouving within the fluid medium of the resting nucleus. If a given medium becomes acid in virtue of the presence of a free copy of an acid gene, then gene and character must be essentially the same thing and the difference between genotype and phenotype disappears, epigenesis gives up its place to preformation, and genetics goes back to its most remote beginnings. The author discusses the complete lack of arguments in support of the view that genes are corpuscular entities. To show the emharracing situation of the genetist who defends the idea of corpuscular genes, Dobzhansky's (1944) assertions that "Discrete entities like genes may be integrated into systems, the chromosomes, functioning as such. The existence of organs and tissues does not preclude their cellular organization" are discussed. In the opinion of the present writer, affirmations as such abrogate one of the most important characteristics of the genes, that is, their functional independence. Indeed, if the genes are independent, each one being capable of passing through mutational alterations or separating from its neighbours without changing them as Dobzhansky says, then the chromosome, genetically speaking, does not constitute a system. If on the other hand, theh chromosome be really a system it will suffer, as such, the influence of the alteration or suppression of the elements integrating it, and in this case the genes cannot be independent. We have therefore to decide : either the chromosome is. a system and th genes are not independent, or the genes are independent and the chromosome is not a syntem. What cannot surely exist is a system (the chromosome) formed by independent organs (the genes), as Dobzhansky admits. The parallel made by Dobzhansky between chromosomes and tissues seems to the author to be inadequate because we cannot compare heterogeneous things like a chromosome considered as a system made up by different organs (the genes), with a tissue formed, as we know, by the same organs (the cells) represented many times. The writer considers the chromosome as a true system and therefore gives no credit to the genes as independent elements. Genetists explain position effects in the following way : The products elaborated by the genes react with each other or with substances previously formed in the cell by the action of other gene products. Supposing that of two neighbouring genes A and B, the former reacts with a certain substance of the cellular medium (X) giving a product C which will suffer the action, of the latter (B). it follows that if the gene changes its position to a place far apart from A, the product it elaborates will spend more time for entering into contact with the substance C resulting from the action of A upon X, whose concentration is greater in the proximities of A. In this condition another gene produtc may anticipate the product of B in reacting with C, the normal course of reactions being altered from this time up. Let we see how many incongruencies and contradictions exist in such an explanation. Firstly, it has been established by genetists that the reaction due.to gene activities are specific and develop in a definite order, so that, each reaction prepares the medium for the following. Therefore, if the medium C resulting from the action of A upon x is the specific medium for the activity of B, it follows that no other gene, in consequence of its specificity, can work in this medium. It is only after the interference of B, changing the medium, that a new gene may enter into action. Since the genotype has not been modified by the change of the place of the gene, it is evident that the unique result we have to attend is a little delay without seious consequence in the beginning of the reaction of the product of B With its specific substratum C. This delay would be largely compensated by a greater amount of the substance C which the product of B should found already prepared. Moreover, the explanation did not take into account the fact that the genes work in the resting nucleus and that in this stage the chromosomes, very long and thin, form a network plunged into the nuclear sap. in which they are surely not still, changing from cell to cell and In the same cell from time to time, the distance separating any two genes of the same chromosome or of different ones. The idea that the genes may react directly with each other and not by means of their products, would lead to the concept of Goidschmidt and Piza, in accordance to which the chromosomes function as wholes. Really, if a gene B, accustomed to work between A and C (as for instance in the chromosome ABCDEF), passes to function differently only because an inversion has transferred it to the neighbourhood of F (as in AEDOBF), the gene F must equally be changed since we cannot almH that, of two reacting genes, only one is modified The genes E and A will be altered in the same way due to the change of place-of the former. Assuming that any modification in a gene causes a compensatory modification in its neighbour in order to re-establich the equilibrium of the reactions, we conclude that all the genes are modified in consequence of an inversion. The same would happen by mutations. The transformation of B into B' would changeA and C into A' and C respectively. The latter, reacting withD would transform it into D' and soon the whole chromosome would be modified. A localized change would therefore transform a primitive whole T into a new one T', as Piza pretends. The attraction point-to-point by the chromosomes is denied by the nresent writer. Arguments and facts favouring the view that chromosomes attract one another as wholes are presented. A fact which in the opinion of the author compromises sereously the idea of specific attraction gene-to-gene is found inthe behavior of the mutated gene. As we know, in homozygosis, the spme gene is represented twice in corresponding loci of the chromosomes. A mutation in one of them, sometimes so strong that it is capable of changing one sex into the opposite one or even killing the individual, has, notwithstading that, no effect on the previously existing mutual attraction of the corresponding loci. It seems reasonable to conclude that, if the genes A and A attract one another specifically, the attraction will disappear in consequence of the mutation. But, as in heterozygosis the genes continue to attract in the same way as before, it follows that the attraction is not specific and therefore does not be a gene attribute. Since homologous genes attract one another whatever their constitution, how do we understand the lack cf attraction between non homologous genes or between the genes of the same chromosome ? Cnromosome pairing is considered as being submitted to the same principles which govern gametes copulation or conjugation of Ciliata. Modern researches on the mating types of Ciliata offer a solid ground for such an intepretation. Chromosomes conjugate like Ciliata of the same variety, but of different mating types. In a cell there are n different sorts of chromosomes comparable to the varieties of Ciliata of the same species which do not mate. Of each sort there are in the cell only two chromosomes belonging to different mating types (homologous chromosomes). The chromosomes which will conjugate (belonging to the same "variety" but to different "mating types") produce a gamone-like substance that promotes their union, being without action upon the other chromosomes. In this simple way a single substance brings forth the same result that in the case of point-to-point attraction would be reached through the cooperation of as many different substances as the genes present in the chromosome. The chromosomes like the Ciliata, divide many times before they conjugate. (Gonial chromosomes) Like the Ciliata, when they reach maturity, they copulate. (Cyte chromosomes). Again, like the Ciliata which aggregate into clumps before mating, the chrorrasrmes join together in one side of the nucleus before pairing. (.Synizesis). Like the Ciliata which come out from the clumps paired two by two, the chromosomes leave the synizesis knot also in pairs. (Pachytene) The chromosomes, like the Ciliata, begin pairing at any part of their body. After some time the latter adjust their mouths, the former their kinetochores. During conjugation the Ciliata as well as the chromosomes exchange parts. Finally, the ones as the others separate to initiate a new cycle of divisions. It seems to the author that the analogies are to many to be overlooked. When two chemical compounds react with one another, both are transformed and new products appear at the and of the reaction. In the reaction in which the protoplasm takes place, a sharp difference is to be noted. The protoplasm, contrarily to what happens with the chemical substances, does not enter directly into reaction, but by means of products of its physiological activities. More than that while the compounds with Wich it reacts are changed, it preserves indefinitely its constitution. Here is one of the most important differences in the behavior of living and lifeless matter. Genes, accordingly, do not alter their constitution when they enter into reaction. Genetists contradict themselves when they affirm, on the one hand, that genes are entities which maintain indefinitely their chemical composition, and on the other hand, that mutation is a change in the chemica composition of the genes. They are thus conferring to the genes properties of the living and the lifeless substances. The protoplasm, as we know, without changing its composition, can synthesize different kinds of compounds as enzyms, hormones, and the like. A mutation, in the opinion of the writer would then be a new property acquired by the protoplasm without altering its chemical composition. With regard to the activities of the enzyms In the cells, the author writes : Due to the specificity of the enzyms we have that what determines the order in which they will enter into play is the chemical composition of the substances appearing in the protoplasm. Suppose that a nucleoproteln comes in relation to a protoplasm in which the following enzyms are present: a protease which breaks the nucleoproteln into protein and nucleic acid; a polynucleotidase which fragments the nucleic acid into nucleotids; a nucleotidase which decomposes the nucleotids into nucleoids and phosphoric acid; and, finally, a nucleosidase which attacs the nucleosids with production of sugar and purin or pyramidin bases. Now, it is evident that none of the enzyms which act on the nucleic acid and its products can enter into activity before the decomposition of the nucleoproteln by the protease present in the medium takes place. Leikewise, the nucleosidase cannot works without the nucleotidase previously decomposing the nucleotids, neither the latter can act before the entering into activity of the polynucleotidase for liberating the nucleotids. The number of enzyms which may work at a time depends upon the substances present m the protoplasm. The start and the end of enzym activities, the direction of the reactions toward the decomposition or the synthesis of chemical compounds, the duration of the reactions, all are in the dependence respectively o fthe nature of the substances, of the end products being left in, or retired from the medium, and of the amount of material present. The velocity of the reaction is conditioned by different factors as temperature, pH of the medium, and others. Genetists fall again into contradiction when they say that genes act like enzyms, controlling the reactions in the cells. They do not remember that to cintroll a reaction means to mark its beginning, to determine its direction, to regulate its velocity, and to stop it Enzyms, as we have seen, enjoy none of these properties improperly attributed to them. If, therefore, genes work like enzyms, they do not controll reactions, being, on the contrary, controlled by substances and conditions present in the protoplasm. A gene, like en enzym, cannot go into play, in the absence of the substance to which it is specific. Tne genes are considered as having two roles in the organism one preparing the characters attributed to them and other, preparing the medium for the activities of other genes. At the first glance it seems that only the former is specific. But, if we consider that each gene acts only when the appropriated medium is prepared for it, it follows that the medium is as specific to the gene as the gene to the medium. The author concludes from the analysis of the manner in which genes perform their function, that all the genes work at the same time anywhere in the organism, and that every character results from the activities of all the genes. A gene does therefore not await for a given medium because it is always in the appropriated medium. If the substratum in which it opperates changes, its activity changes correspondingly. Genes are permanently at work. It is true that they attend for an adequate medium to develop a certain actvity. But this does not mean that it is resting while the required cellular environment is being prepared. It never rests. While attending for certain conditions, it opperates in the previous enes It passes from medium to medium, from activity to activity, without stopping anywhere. Genetists are acquainted with situations in which the attended results do not appear. To solve these situations they use to make appeal to the interference of other genes (modifiers, suppressors, activators, intensifiers, dilutors, a. s. o.), nothing else doing in this manner than displacing the problem. To make genetcal systems function genetists confer to their hypothetical entities truly miraculous faculties. To affirm as they do w'th so great a simplicity, that a gene produces an anthocyanin, an enzym, a hormone, or the like, is attribute to the gene activities that onlv very complex structures like cells or glands would be capable of producing Genetists try to avoid this difficulty advancing that the gene works in collaboration with all the other genes as well as with the cytoplasm. Of course, such an affirmation merely means that what works at each time is not the gene, but the whole cell. Consequently, if it is the whole cell which is at work in every situation, it follows that the complete set of genes are permanently in activity, their activity changing in accordance with the part of the organism in which they are working. Transplantation experiments carried out between creeper and normal fowl embryos are discussed in order to show that there is ro local gene action, at least in some cases in which genetists use to recognize such an action. The author thinks that the pleiotropism concept should be applied only to the effects and not to the causes. A pleiotropic gene would be one that in a single actuation upon a more primitive structure were capable of producing by means of secondary influences a multiple effect This definition, however, does not preclude localized gene action, only displacing it. But, if genetics goes back to the egg and puts in it the starting point for all events which in course of development finish by producing the visible characters of the organism, this will signify a great progress. From the analysis of the results of the study of the phenocopies the author concludes that agents other than genes being also capaole of determining the same characters as the genes, these entities lose much of their credit as the unique makers of the organism. Insisting about some points already discussed, the author lays once more stress upon the manner in which the genes exercise their activities, emphasizing that the complete set of genes works jointly in collaboration with the other elements of the cell, and that this work changes with development in the different parts of the organism. To defend this point of view the author starts fron the premiss that a nerve cell is different from a muscle cell. Taking this for granted the author continues saying that those cells have been differentiated as systems, that is all their parts have been changed during development. The nucleus of the nerve cell is therefore different from the nucleus of the muscle cell not only in shape, but also in function. Though fundamentally formed by th same parts, these cells differ integrally from one another by the specialization. Without losing anyone of its essenial properties the protoplasm differentiates itself into distinct kinds of cells, as the living beings differentiate into species. The modified cells within the organism are comparable to the modified organisms within the species. A nervo and a muscle cell of the same organism are therefore like two species originated from a common ancestor : integrally distinct. Like the cytoplasm, the nucleus of a nerve cell differs from the one of a muscle cell in all pecularities and accordingly, nerve cell chromosomes are different from muscle cell chromosomes. We cannot understand differentiation of a part only of a cell. The differentiation must be of the whole cell as a system. When a cell in the course of development becomes a nerve cell or a muscle cell , it undoubtedly acquires nerve cell or muscle cell cytoplasm and nucleus respectively. It is not admissible that the cytoplasm has been changed r.lone, the nucleus remaining the same in both kinds of cells. It is therefore legitimate to conclude that nerve ceil ha.s nerve cell chromosomes and muscle cell, muscle cell chromosomes. Consequently, the genes, representing as they do, specific functions of the chromossomes, are different in different sorts of cells. After having discussed the development of the Amphibian egg on the light of modern researches, the author says : We have seen till now that the development of the egg is almost finished and the larva about to become a free-swimming tadepole and, notwithstanding this, the genes have not yet entered with their specific work. If the haed and tail position is determined without the concourse of the genes; if dorso-ventrality and bilaterality of the embryo are not due to specific gene actions; if the unequal division of the blastula cells, the different speed with which the cells multiply in each hemisphere, and the differential repartition of the substances present in the cytoplasm, all this do not depend on genes; if gastrulation, neurulation. division of the embryo body into morphogenetic fields, definitive determination of primordia, and histological differentiation of the organism go on without the specific cooperation of the genes, it is the case of asking to what then the genes serve ? Based on the mechanism of plant galls formation by gall insects and on the manner in which organizers and their products exercise their activities in the developing organism, the author interprets gene action in the following way : The genes alter structures which have been formed without their specific intervention. Working in one substratum whose existence does not depend o nthem, the genes would be capable of modelling in it the particularities which make it characteristic for a given individual. Thus, the tegument of an animal, as a fundamental structure of the organism, is not due to gene action, but the presence or absence of hair, scales, tubercles, spines, the colour or any other particularities of the skin, may be decided by the genes. The organizer decides whether a primordium will be eye or gill. The details of these organs, however, are left to the genetic potentiality of the tissue which received the induction. For instance, Urodele mouth organizer induces Anura presumptive epidermis to develop into mouth. But, this mouth will be farhioned in the Anura manner. Finalizing the author presents his own concept of the genes. The genes are not independent material particles charged with specific activities, but specific functions of the whole chromosome. To say that a given chromosome has n genes means that this chromonome, in different circumstances, may exercise n distinct activities. Thus, under the influence of a leg evocator the chromosome, as whole, develops its "leg" activity, while wbitm the field of influence of an eye evocator it will develop its "eye" activity. Translocations, deficiencies and inversions will transform more or less deeply a whole into another one, This new whole may continue to produce the same activities it had formerly in addition to those wich may have been induced by the grafted fragment, may lose some functions or acquire entirely new properties, that is, properties that none of them had previously The theoretical possibility of the chromosomes acquiring new genetical properties in consequence of an exchange of parts postulated by the present writer has been experimentally confirmed by Dobzhansky, who verified that, when any two Drosophila pseudoobscura II - chromosomes exchange parts, the chossover chromosomes show new "synthetic" genetical effects.

Standardization of the dot enzyme-lynked immunosorbent assay (dot-ELISA) for experimental plague

A dot enzyme linked immunosorbent assay (dot-ELISA) was previously developed to detect specific antibodies in rabbits sera immunized against FIA protein obtained from Yersina pestis. This antigen was covalently linked onto the surface of dacron (polyethyleneterephthalate). Here, standard conditions are described for the optimization of this procedure: an amount of 20 ng of FIA protein was fixed onto dacron; anti-rabbit IgG peroxidase conjugate diluted 1:8,000 and 30% non-fat instant milk as blocking substance were used throughout the method. This procedure was compared with that employing nitrocellulose as solid-phase which showed to be more sensitive. However, the method based on dacron did not show false positive reactions against non-immunized rabbits sera at low antigen amount and diluted anti-IgG peroxidase conjugate.

Standardization and evaluation of ELISA for the serodiagnosis of amoebic liver abscess

An ELISA test for the serological diagnosisof amoebic liver abscess (ALA) was standardized and evaluated in sera from three groups of patients: (1) three patients with diagnosis confirmed by isolation of the parasite,(2) thirty seven patients with diagnosis established by clinical findings and ultrasound studies and (3) seven patients whose diagnosis were established by clinical findings and a positive double immunodifusion test. Ninety one serum samples from healthy subjects and 22 from patients with other liver or parasitic diseases were also included in the study. the optimum concentration of Entamoeba histolytica antigen was 1.25 µg/ml and optimum dilutions of serum and anti-human IgG-alkaline phosphatase conjugate were 1:400 and 1:4000 respectively. The cut-off point of the ELISA test in this study was an absorbance value of 0.34. The test parameters were: sensitivity = 95.7 per cent, specificty = 100 per cent, positive predictive value = 100 per cent and negative predictive value = 98.2 per cent.The ELISA test was found to be of great use as a diagnostic tool for the establishment of amoebic etiology in patients with clinical supposition of ALA. The test could also be used for seroepidemiological surveys of the prevalence of invasive amoebiasis in a given population, since it allows the processing of a greater number of samples at a lower cost tahn other serological tests.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 ¼g of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Conjugation by Mosquito Pathogenic Strains of Bacillus sphaericus

A mosquito pathogenic strain of Bacillus sphaericus carried out the conjugal transfer of plasmid pAMß1 to other strains of its own and two other serotypes. However, it was unable to conjugate with mosquito pathogens from three other serotypes, with B. sphaericus of other DNA homology groups or with three other species of Bacillus. Conjugation frequency was highest with a strain having an altered surface layer (S layer). Conjugal transfer of pAMß1 was not detected in mosquito larval cadavers. B. sphaericus 2362 was unable to mobilize pUB110 for transfer to strains that had served as recipients of pAMß1. These observations suggest that it is unlikely that genetically engineered B. sphaericus carrying a recombinant plasmid could pass that plasmid to other bacteria

Cellulose acetate as solid phase in ELISA for plague

Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 µg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.

IgG Anti-IgE Autoantibodies in Visceral Leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 ± 0.43) than sera from healthy individuals (OD = 1.35 ± 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 ± 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

The ue of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.

Basic biology of Pneumocystis carinii: a mini review

Basic aspects of cell biology of Pneumocystis carinii are reviewed with major emphasis on its life cycle and the structural organization of the trophozoites and cyst forms. Initially considered as a protozoan it is now established that Pneumocystis belongs to the Fungi Kingdom. Its life cycle includes two basic forms: (a) trophozoites, which are haploid cells that divide by binary fission and may conjugate with each other forming an early procyst and (b) cysts where division takes place through a meiotic process with the formation of eight nuclei followed by cytoplasmic delimitation and formation of intracystic bodies which are subsequently released and transformed into trophozoites. Basic aspects of the structure of the two developmental stages of P. carinii are reviewed.

Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

A dot enzyme-linked immunosorbent assay (dot-ELISA) was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 µg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silicoana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and SmBAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

Evaluation of Haemophilus influenzaetype b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzaetype b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.