Diagnosis of human herpesvirus 6B primary infection by polymerase chain reaction in young children with exanthematic disease

INTRODUCTION: Exanthem subitum is a classical rash disease of early childhood caused by human herpesvirus 6B (HHV-6B). However, the rash is frequently misdiagnosed as that of either measles or rubella. METHODS: In this study, a nested multiplex polymerase chain reaction (PCR) was used to diagnose HHV-6B primary infection, differentiate it from infections caused by HHV-6A and compare it to antibody avidity tests. The samples were separated into case group and control group according to the results of the indirect immunofluorescence assay (IFA) technique. RESULTS: From the saliva samples analyzed, HHV-6A DNA was detected in 3.2% of the case group and in 2.6% of the control group. Regarding HHV-6B, PCR detected viral DNA in 4.8% of the case group and in 1.3% of the control group. Among the serum samples studied, a frequency of 1.7% was determined for HHV-6A in the case group and 1.2% in the control group. PCR did not detect HHV-6B DNA in serum samples. The sensitivity and specificity of the PCR technique ranged from 0% to 4.8% and 97.5% to 100%, respectively, compared to IFA. CONCLUSIONS: The PCR technique was not suitable for diagnosing primary infection by HHV-6B in children with exanthematic disease and should not substitute the IFA.

Comparison among three polymerase chain reaction assays on detection of DNA from Leishmania in biological samples from patients with american cutaneous leishmaniasis

INTRODUCTION: The study analyzed positivity of polymerase chain reaction (PCR) on detection of DNA from Leishmania in patients' samples. METHODS: Extracted DNA was submitted to L150/L152, 13Y/13Z, and seminested PCR (snPCR). RESULTS: Results were evidenced by bands of approximately 120, 720, and 670 bp for L150/L152, 13Y/13Z, and snPCR, respectively. L150/L152, 13Y/13Z, and snPCR positivity indexes were 76.9, 56.4, and 9.2 (p>0.05), respectively, for suspected and 93.7, 68.7, and 84.4 (p<0.05), respectively, for confirmed. CONCLUSIONS: Preliminary results showed that these assays, mainly L150/L152 and snPCR, can detect Leishmania DNA and carry potential on laboratory diagnosis of leishmaniasis.

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Introduction Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. Methods We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

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.

Modifications de la coagulation sanguine dans la fièvre jaune

L'étude des modifications de la coagulation sanguine à l'aide de la technique BRAZIL-VELLARD, réalisée chez 25 malades atteints de formes diverses, mortelles, graves, et bénignes, entre le lléme et le XIIIéme jour de maladie, et chez 12 convalescents, a permis d'arriver aux conclusions suivantes: 1º. Les variations du pouvoir coagulant du serum sont très irrégulières et peu différentes des celles observées chez de sujets normaux. 2º. La coagulabilité du plasma, dès le IIème jour de la maladie, présente une diminution marquée, constante chez tous les malades, atteignant son chiffre le plus bas entre le VIIème et le IXème jour et revenant progressivement á la normale pendant la convalescence. 3º. Cette diminution de la coagulabilité est due principalement á l'apparition de grandes quantités d'antithrombines dans la circulation; la diminution du fibrinogène observée dans quelques cas, est toujours peu accusée. 4º. La diminution de la coagulabilité du plasma, qui n'a jusqu'ici été observée avec cette intensité que dans la fièvre jaune, peut être d'un certain secours pour le diagnostic précoce de cette affection; elle n'a jamais été vérifiée chez des malades atteints d'autres affections fébriles. 5º. Au point de vue du pronostic, la diminution précoce et très accentuée de la coagulabilité est un symptôme grave, indiquant une lésion profonde de la cellule hépatique. 6º. Dans la fièvre jaune expérimentale du Macacus rhesus, les altérations de la coagulation sanguine sont de même nature, mais paraissent plus tardives et moins accusées que chez l'homme.

Identification of species related to Anopheles (Nyssorhynchus) albitarsis by random amplified polymorphic DNA-Polymerase chain reaction (Diptera: Culicidae)

Species-specific Random Amplified Polymorphic DNA-Polymerase chain Reaction (RAPD-PCR) markers were used to identify four species related to Anopheles (Nyssorhynchus) albitarsis Lynch-Arribàlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wilkerson et al. 1995), which included sites in Paraguay and Argentina, these four species were designated "A", "B", "C" and "D". It was hypothesized that species A is An. (Nys.) albitarsis, species B is undescribed, species C is An. (Nys) marajoara Galvão and Damasceno and species D is An. (Nys.) deaneorum Rosa-Freitas. Species D, previously characterized by RAPD-PCR from a small sample from northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys.) deaneorum, Guajará-Mirim, state of Rondônia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, state of Rondônia, Brazil. Species A and C have yet to be encountered at the same locality. The RAPD markers for species C were found to be conserved over 4,620 km; from Iguape, state of São Paulo, Brazil to rio Socuavo, state of Zulia, Venezuela. RAPD-PCR was determined to be an effective means for the identification of unknown species within this species complex.