Genoma mitocondrial de Simulium spp. e Onchocerca volvulus na Amazônia brasileira / Mitochondrial genome of Simulium spp. and Onchocerca volvulus in the Brazilian Amazon

As espécies de simulídeos são vetores de filárias, como as do gênero Onchocerca e Mansonella, que são os agentes etiológicos da oncocercose e mansonellose, respectivamente. Essas duas filárias ocorrem na região Amazônica brasileira e são transmitidas pelas seguintes espécies de vetores: Simulium incrustatum, S. limbatum, S. oyapockense, S. exiguum, S. guianense, e S. roraimense. As espécies de Simulium tem sido designada com base em caracteres morfológicos, os quais, em alguns casos, não são bem discriminativos. Recentemente, o gene mitocondrial Citocromo c-oxidase 1 (CO1) e a região nuclear Internal Transcribed Spacer (ITS) tem sido utilizados para descriminar espécies e definir populações dentro deste gênero. Entretanto, existe um grande gap acerca da informação genética de Simulium, o qual é considerado a linha de base para estudos ecológicos e populacionais. Considerando este cenário, nosso objetivo foi aplicar a metagenômica para recuperar genomas mitocondriais de amostras brasileiras S. incrustatum e S. oyapockense do foco de oncocercose e também a informação genética a respeito de seus microbiomas. O DNA total de dez simulídeos, morfologicamente identificados como S. incrustatum (3) e S. oyapockense (7) foram sequenciados randomicamente na plataforma Illumina HiSeq 2500. Nós recuperamos dez genomas mitocondriais com cobertura média de 15,591 bp e conteúdo médio de GC de 22,94 %, apresentando o mesmo conteúdo gênico e em sintenia. Baseado nestes mitogenomas, no gene mitocondrial CO1, e também na região nuclear (ITS), realizamos análises filogenéticas que mostraram a presença de três espécies conhecidas dentre as amostras: S. incrustatum, S. oyapockense e S. guianense, e também um grupo de amostras pertencentes à Simulium spp. Nós também recuperamos um genoma mitocondrial de Onchocerca volvulus da amostra aqui identificada como S. guianense.

Análises taxonômicas do microbioma dos simulídeos revelaram Proteobacteria e Ascomycota como os filos mais abundantes. A análise funcional revelou que a família de enzimas das Transcriptases Reversas são as mais abundantes. Portanto, nós contribuímos com informação genética original preenchendo parte do viés a respeito das espécies de Simulium associadas ao foco brasileiro de oncocercose. (AU)

Arthropod-Borne Onchocerciasis: is it deserved to be neglected?

Onchocerciasis a filarial parasitic nematode, also known as river blindness and Robles disease, is a neglected tropical disease infecting more than 18 million people mainly in subSaharan of Africa, the Middle East, South and Central America and many other countries. Disease infectivity initiates from Onchocerca volvulus [Filarioidea: Onchocercidae] transmitted by the blackfly, Simulium sp. which introduces the infective stage larva with its saliva into the skin. Within human body, adult females [macrofilaria] produce thousands of larvae [microfilariae] which migrate in skin and eye. Infection results in severe visual impairment or blindness for about 2 million, as being the world's second-leading cause of blindness after trachoma, as well as skin onchocercomata

Outstanding insecurities concerning the use of an Ov16-based ELISA in the Amazonia onchocerciasis focus

In a recent issue of Memórias do Instituto Oswaldo Cruz, published in Rio de Janeiro in February 2014 (109: 87-92), Adami et al. have published a survey reporting Mansonella parasite prevalence in the Amazon Region. This report makes a useful contribution to the existing knowledge of filarial parasite distribution within the Amazon area, parasite prevalence rates in relation to age and occupation and provides observations on the possible clinical impact of Mansonella ozzardi. Their publication also provides an account of what appears to be a novel ELISA that has recently been used in the Simuliidae and Onchocerciasis Laboratory of the Oswaldo Cruz Institute, Rio de Janeiro, Brazil. We are concerned that the publication of this ELISA may have created an excessively positive impression of the effectiveness of the onchocerciasis recrudescence serological surveillance tools that are presently available for use in the Amazonia onchocerciasis focus. In this letter we have, thus, sought to highlight some of the limitations of this ELISA and suggest how continuing insecurities concerning the detection of antibodies to Onchocerca volvulus within the Amazonia onchocerciasis focus might be minimised.

New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region

Mansonella ozzardi infections are common in the riverside communities along the Solimões, Negro and Purus Rivers in the state of Amazonas (AM). However, little is known about the presence of this parasite in communities located in regions bordering AM and the state of Acre. The prevalence rate of M. ozzardi infections was determined in blood samples from volunteers according to the Knott method. A total of 355 volunteers from six riverine communities were enrolled in the study and 65 (18.3%) were found to be infected with M. ozzardi. As expected, most of the infections (25%) occurred in individuals involved in agriculture, cattle rearing and fishing and an age/sex group analysis revealed that the prevalence increased beginning in the 40-50-years-of-age group and reached 33% in both sexes in individuals over 50 years of age. Based on the described symptomatology, articular pain and headache were found to be significantly higher among infected individuals (56 and 65% prevalence, respectively, p < 0.05). Sera from volunteers were subjected to ELISA using a cocktail of recombinant proteins from Onchocerca volvulus to evaluate the specificity of the test in an endemic M. ozzardi region. No cross-reactions between M. ozzardi-infected individuals and recombinant O. volvulus proteins were detected, thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric.

Transmission de l'onchocercose par Simulium squamosum (Diptera : Simuliidae) dans le foyer urbain de Kinsuka a Kinshasa/Republique democratique du Congo

Contexte. Les etudes realisees dans le foyer de Kinsuka entre 1985 et 2001 ont montre une tendance a la reemergence de la transmission de l'onchocercose dans ce foyer. Le but de la presente etude exploratoire etait de determiner le niveau actuel de la transmission de cette parasitose; en vue d'apprehender des facteurs explicatifs du niveau d'endemicite constate. Methodes. Les simulies ont ete capturees dans deux points de capture (P1 et P2); pour une periode de 12 mois (mars 2008-fevrier 2009); par le procede classique de capture sur appats humains. Dans l'ensemble; 82;1des simulies capturees ont ete dissequees pour la determination de la parite; et toutes les simulies pares ont ete dissequees pour denombrer les larves d'O. volvulus. Les autres especes de simulies ont ete inventoriees; et les supports larvaires ont ete prospectes. Resultats. Au total; 12282 simulies ont ete capturees dont 5018 au S1; et 7264 au S2. Le cycle d'agressivite journaliere a presente une courbe concave avec deux pics; dont un mineur entre 8 et 9 heures; et un majeur entre 17h et 18h. Dans l'ensemble de simulies dissequees; 42;4etaient pares. Seules 25 simulies pares etaient infectees par les larves d'O. volvulus; dont 14 au S1; et 11 au S2. Une seule simulie etait infectante. Les taux annuels de piqures (TAP) etaient de 564453; et 78111 piqures par personne par an respectivement. Le potentiel de transmission est pratiquement nul : 0 au S1; et 28 larves infectantes par personne par an au S2. Conclusion. Le taux d'agressivite est environ 12 fois plus eleve que celui observe il y a 27 ans; dans le meme site. Le taux observe est en partie du a l'influence des activites realisees par la nouvelle entreprise d'exploitation de moellons dans la carriere de Kinsuka. Ces activites ont provoque entre autres; le retrecissement du lit du fleuve pendant la saison seche; qui entraine une augmentation du niveau du fleuve; creant une grande disponibilite des supports larvaires. La transmission de l'onchocercose est presque nulle. Des etudes ulterieures permettront d'en identifier les determinants

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardiand Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region.

Determination of aspartic protease gene dosage in the Onchocerca volvulus genome

Aspartic proteases are a relatively small group of enzymes which express in various nematodes including Onchocerca volvulus. An estimation of the gene copy number corresponding to the OV7A clone, which contains a cDNA insert encoding approximately two-thirds of the entire coding sequence of aspartic protease of O. volvulus, was made by slot blot analysis in a closely related species O. gibsoni genome. Nylon membrane was loaded with serial dilutions of genomic DNA alongside the OV7A plasmid DNA before hybridizing the membrane to that [32]P-labeled cDNA insert. To prepare the initial probe, OV7A cDNA insert was amplified using gene-specific primers. By comparing the signal intensity of slot blot hybridization of known amounts of genomic DNA and plasmid DNA containing the cDNA insert under similar conditions, the abundance of sequence homologues to the [32]P-labeled cDNA insert in the genome was calculated. For confirmation, southern blot analysis was performed by digesting genomic DNA with a panel of different restriction enzymes. Hybridizing patterns of the same probe revealed a single band except when predicted internal restriction sites were affected. It was confirmed that Onchocerca contains a single copy of the gene corresponding to this cDNA insert per haploid genome

Clinical manifestations of mesoendemic onchocerciasis in an area with multiple filarial species

The Imo River Basin, Nigeria is endemic for onchocerciasis, bancroftian filariasis, loaiasis and mansonellosis. This study was aimed at determining the clinical manifestations of onchocerciasis in the region. A cross- sectional study was carried out in 2006 in Umuowaibu I and Ndiorji communities in Okigwe Local Government Area of Imo State, Nigeria. Consenting individuals were examined for various gradations of skin manifestations, subcutaneous nodules, and visual impairments by qualified medical doctors. Five categories of skin manifestations were observed, namely permanent itching, onchodermatitis, atrophy of ski, leopard skin, and sowda. A total of 1024 individuals were examined. The prevalence of the skin manifestations were permanent itching [2.5%], onchodermatitis [3.9%], atrophy of the skin [5.8%], leopard skin [22.1%], and sowda [0.1%]. The prevalence of subcutaneous nodules was 25.3%, but 88.9% among the oldest age group, and mostly found in lower half of body. The majority of cases of visual acuity problems [6.8% overall prevalence] were in the oldest age groups. Among those who were >/= 20 years old, the prevalence of visual acuity problems was significantly higher in females than in males [chi[2] -test; P<0.05]. Only two of the examined persons were observed to be blind. Clinical manifestations of onchocerciasis are perhaps more intense in the area probably because of presence of endemic infections of other filarial species

Molecular cloning of adenylate kinase from the human filarial parasite Onchocerca volvulus

Adenylate kinases [ADK] are ubiquitous enzymes that contribute to the homeostasis of adenine nucleotides in living cells. In this study, the cloning of a cDNA encoding an adenylate kinase from the filaria Onchocerca volvulus has been described. Using PCR technique, a 281 bp cDNA fragment encoding part of an adenylate kinase was isolated from an O. volvulus cDNA library. Use of this fragment as a probe allowed the isolation of a larger cDNA clone through the searching the GenBank expressed sequence tag database. The full-length cDNA encodes 236 amino acid residues with a predicted molecular mass of 26.177 kDa. The deduced amino acid sequence exhibited 80% identity to the homologous adenylate kinase identified from Caenorhabditis elegans. Domain analysis of the resulting protein sequence was found to contain "adenylate kinase signature" motif which is highly conserved in all known ADKs. Multiple alignments showed that the N-terminal is well conserved, whereas the C-terminal is the most variable region

Histochemical Characterization of Rain-Forest Strain of Onchocerca Volvulus Microfilariae Isolated in Akamkpa; Cross River State; Nigeria

The histochemical characterization of rain-forest strain of Onchocerca volvulus isolated in Akamkpa of Cross River State; Nigeria was studied. In a preliminary survey of 350 persons from eight villages; 75(21.4) were found to be positive for the parasite. Males (23.6) were more infected than the females but there was no statistically significant difference in the presence of infection by gender (p=0.506). Staining pattern of the parasite using Lead nitrate method for the demonstration of acid phosphatase activity revealed that of the six patterns; four corresponded to the enzyme types already described by some authors in West African rain-forest; and Sudan. One corresponded to the type found in Guinea savannas while the remaining did not correspond to any of the strains described elsewhere. The multiple enzyme staining patterns of Onchocerca volvulus microfilariae in the study area is indicative of polymorphic strain than that of West African strains. This variation in strain might contribute to the degree of pathogenicity and clinical manifestation of onchocerciasis in this endemic zone

Reacción en cadena de polimerasa (PCR) para detectar formas infectivas de Onchocerca volvulus en Simulium metallicum s.l. (Diptera: Simuliidae), en áreas endémicas de Venezuela / Polymerase chain reaction (PCR) for detecting infective forms of Onchocerca volvulus in Simulium metallicum s.l. (Diptera: Simuliidae) in Venezuelan endemic areas

Presentamos los resultados obtenidos después de un estudio parasitológico realizado en tres áreas endémicas de oncocercosis localizadas en los focos nor-central y nor-oriental de Venezuela. Durante aproximadamente 1 año, se capturaron hembras de simúlidos posándose sobre atractante humano en las márgenes de los ríos y las proximidades de las viviendas donde habían sido detectados casos de oncocercosis. Se capturaron un total de 95.251 moscas paras; de los cuales solo 0,05% correspondió a Simulium exiguum s.l. y el resto a Simulium metallicum s.l. Todos fueron sometidos a ruptura mecánica para separar cabezas y cuerpo. Las cabezas fueron separadas en grupos de 50 para la extracción de ADN genómico. El ADN fue utilizado para evaluar la infectividad natural por Onchocerca volvulus, mediante la Reacción en Cadena de la Polimerasa con oligonucleotidos especie-específicos. La prevalencia de moscas infectadas fue calculada mediante el programa Poolsreen ó prueba de grupo. Los resultados revelan larvas infectivas de O. volvulus en La Cuesta (Foco nor- oriental, estado Anzoátegui) con una prevalencia de 9/10.000; seguido por Santa Rosa (Foco nor-central, Estado Carabobo) con 5/10.000, el menor índice de prevalencia fue encontrado en La Carapa (Foco nor-oriental, Estado Monagas) con 3/10.000 de positividad, calculado con un intervalo de confianza de 95%. Estos resultados demuestran la utilidad de la técnica para estudiosentomológicos a gran escala y la detección especifica de larvas infectivas de O. volvulus causante de la oncocercosis en la población humana.

Population dynamics of onchocerca volvulus microfilariae in human host after six years of drug control.

BACKGROUND & OBJECTIVES: Mass administration of ivermectin drug was carried out annually between 1995 and 2001 in three villages that were endemic for onchocerciasis in the Lower Cross River Basin, Nigeria. The aim of this study was to evaluate the population dynamics (dispersion patterns, distribution, prevalence and intensity) of Onchocerca volvulus microfilariae in their human host after six years of ivermectin treatment. METHODS: A total of 1014 subjects from three rural areas in Etung Local Government Area of Cross River State, Nigeria were screened for skin microfilariae using standard parasitological method of diagnosis. RESULTS: Ivermectin drug intervention had significantly reduced the prevalence of skin microfilariae (PMF) from 69.3% pre-control to 39.3% and community microfilarial load (CMFL) from 7.11 to 2.31 microfilariae per skin snip. Males (45%) were significantly (p <0.05) more infected than females (34%). Both microfilarial prevalence and intensity increased with age. Pearson correlation test between intensity and age was not significant (r = 0.37; p >0.05). The correlation between age-dependent parasite prevalence and mean abundance was also not significant (r = 0.42; p >0.05). The degree of dispersion as measured by variance to mean ratio (VMR), coefficient of variation (CV) and exponent 'K' of the negative binomial model of distribution showed that the parasite aggregated, clumped and overdispersed in their human host. The relative index of potential infection of each age group showed that adults between the age of 21 and 50 yr accounted for 52.7% of microfilariae positive cases. INTERPRETATION & CONCLUSION: Aggregated and overdispersion of O. volvulus observed in this study showed that active transmission could still be going on, because the tendency of the vector, Simulium damnosum ingesting more microfilariae was high due to the aggregated and overdispersed nature of the parasite with its host.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

Detection of Onchocerca volvulus (Nematoda: Onchocercidae) infection in vectors from Amazonian Brazil following mass Mectizan™ distribution

Detection of Onchocerca volvulus in Simulium populations is of primary importance in the assessment of the effectiveness of onchocerciasis control programs. In Brazil, the main focus of onchocerciasis is in the Amazon region, in a Yanomami reserve. The main onchocerciasis control strategy in Brazil is the semi-annually mass distribution of the microfilaricide ivermectin. In accordance with the control strategy for the disease, polymerase chain reaction (PCR) was applied in pools of simuliids from the area to detect the helminth infection in the vectors, as recommended by the Onchocerciasis Elimination Program for the Americas and the World Health Organization. Systematic sampling was performed monthly from September 1998 to October 1999, and a total of 4942 blackflies were collected from two sites (2576 from Balawaú and 2366 from Toototobi). The molecular methodology was found to be highly sensitive and specific for the detection of infected and/or infective blackflies in pools of 50 blackflies. The results from the material collected under field conditions showed that after the sixth cycle of distribution of ivermectin, the prevalence of infected blackflies with O. volvulus had decreased from 8.6 to 0.3 percent in Balawaú and from 4 to 0.1 percent in Toototobi.

Onchocerciasis: a review of a filarial disease of significant importance for dermatologists and ophthalmolodists

Onchocerciasis is a common, chronic, multisystemic disease caused by the nematode Onchocerca volvulus. The disease characteristically includes dermatologic, lymphatic, ophthalmologic, and systemic manifestations. It is transmitted to humans by a bite from the intermediate host, the black fly [Simulium damnosum]. It is endemic in Western and Central Africa and approximately 95% of all infected people live in these regions. Onchocerciasis has been associated with a high incidence of detrimental effects on socioeconomic development and public health in endemic areas. We, here in Asia, hardly see any case of this disease but due to globalization, frequent international traveling and deployment of military troops from Asian countries [as a part of UN forces] in endemic African countries. We should expect such diseases in our region also and should have a high index of suspicion. Here is a review of this troublesome filarial disease with emphasis of its clinical spectrum, so that dermatologists, ophthalmologis ts and physicians in our region would be able to keep this disease in mind as a differential diagnosis of some unusual case of dermatitis, blindness or arthritis etc. Patients described in this article are author`s own collection during one year stay in Sierra Leone [an endemic country of West Africa for the disease]