Human Endemic Areas Research Articles

DNA multigene sequencing of topotypic specimens of the fascioliasis vector Lymnaea diaphana and phylogenetic analysis of the genus Pectinidens (Gastropoda)

Freshwater lymnaeid snails are crucial in defining transmission and epidemiology of fascioliasis. In South America, human endemic areas are related to high altitudes in Andean regions. The species Lymnaea diaphana has, however, been involved in low altitude areas of Chile, Argentina and Peru where human infection also occurs. Complete nuclear ribosomal DNA 18S, internal transcribed spacer (ITS)-2 and ITS-1 and fragments of mitochondrial DNA 16S and cytochrome c oxidase (cox)1 genes of L. diaphana specimens from its type locality offered 1,848, 495, 520, 424 and 672 bp long sequences. Comparisons with New and Old World Galba/Fossaria, Palaearctic stagnicolines, Nearctic stagnicolines, Old World Radix and Pseudosuccinea allowed to conclude that (i) L. diaphana shows sequences very different from all other lymnaeids, (ii) each marker allows its differentiation, except cox1 amino acid sequence, and (iii) L. diaphana is not a fossarine lymnaeid, but rather an archaic relict form derived from the oldest North American stagnicoline ancestors. Phylogeny and large genetic distances support the genus Pectinidens as the first stagnicoline representative in the southern hemisphere, including colonization of extreme world regions, as most southern Patagonia, long time ago. The phylogenetic link of L. diaphana with the stagnicoline group may give light to the aforementioned peculiar low altitude epidemiological scenario of fascioliasis.

Fasciola hepatica phenotypic characterization in Andean human endemic areas: Valley versus altiplanic patterns analysed in liver flukes from sheep from Cajamarca and Mantaro, Peru

Fascioliasis is a zoonotic parasitic disease caused by Fasciola hepatica and Fasciola gigantica. Of both species, F. hepatica is the only one described in the Americas, mainly transmitted by lymnaeid snail vectors of the Galba/Fossaria group. Human fascioliasis endemic areas are mainly located in high altitude areas of Andean countries. Given the necessity to characterize F. hepatica populations involved, the phenotypic features of fasciolid adults infecting sheep present in human fascioliasis endemic areas were analysed in the Cajamarca Valley and Mantaro Valley (valley transmission patterns) and the northern Bolivian Altiplano (altiplanic transmission pattern). A computer image analysis system (CIAS) was applied on the basis of standardized measurements. The aforementioned highland populations were compared to standard lowland natural and experimental populations of European origin. Liver fluke size was studied by multivariate analyses. Two phenotypic patterns could be distinguished in F. hepatica adult size: the valley pattern (Cajamarca and Mantaro, Peru) and the altiplanic pattern (northern Altiplano, Bolivia). Results showed that the Andean valley population and European standard populations presented a phenotypic homogeneity. The Altiplano population showed a large size range with a pronouncedly lower minimum size indicating that uterus gravidity is reached at a smaller size than in valley populations. The results of this study demonstrate that there is no apparent relationship between the shape of fasciolid adults with regard to altitudinal difference or geographical origin and that allometry-free shape appears as a more stable trait than size in fasciolid species. Results are analysed in terms of intensity/crowding effect aspects and permanent/seasonal transmission characteristics.

A new baseline for fascioliasis in Venezuela: lymnaeid vectors ascertained by DNA sequencing and analysis of their relationships with human and animal infection.

BackgroundHuman and animal fascioliasis poses serious public health problems in South America. In Venezuela, livestock infection represents an important veterinary problem whereas there appear to be few human cases reported, most of which are passively detected in health centres. However, results of recent surveys suggest that the situation may be underestimated in particular areas. To obtain a baseline for future fascioliasis assessment, studies were undertaken by means of rDNA ITS-2 and ITS-1 and mtDNA cox1 sequencing to clarify the specific status of Venezuelan lymnaeids, their geographical distribution and fascioliasis transmission capacity, by comparison with other American countries and other continents.ResultsResults obtained completely change the lymnaeid scenario known so far. The relatively rich lymnaeid fauna of Venezuela has been proven to include (i) Lymnaea meridensis and L. neotropica as the only native members, (ii) L. cubensis and Pseudosuccinea columella introduced from the Caribbean area, and (iii) Galba truncatula and L. schirazensis introduced from the Old World. The absence of representatives of the stagnicoline and Radix groups is remarkable. Four species are fascioliasis vectors: G. truncatula, L. cubensis and L. neotropica, which have the capacity to give rise to human endemic areas, and P. columella, which is a source of animal infection and is responsible for the spread of disease. Vector capacity in the apparently highland endemic L. meridensis is to be confimed, although may be expected given its phylogenetic relationships. Similarly as elsewhere, the non-transmitting L. schirazensis has been confused with L. cubensis, also with G. truncatula and possibly with L. neotropica.ConclusionsThe new scenario leads to the re-opening of many disease aspects. In Venezuela, altitude appears to be the main factor influencing fascioliasis distribution. Human infection shows an altitude pattern similar to other Andean countries, although a differing highland/lowland impact on animal infection does not appear evident. The overlap of G. truncatula, L. cubensis and probably also L. neotropica in temperate and cold zones suggests a higher risk for human infection in mid and high altitude areas. A lymnaeid species mapping by means of DNA markers becomes a priority to determine human and animal fascioliasis distribution in Venezuela, owing to the importance of lymnaeid vectors in defining transmission and epidemiological patterns.

Characterisation of fascioliasis lymnaeid intermediate hosts from Chile by DNA sequencing, with emphasis on Lymnaea viator and Galba truncatula

In South America, Fasciola hepatica infection poses serious health problems in both humans and livestock. In Chile, the medical impact appears yearly stable and mainly concentrated in central regions, where the veterinary problem is highlighted by higher animal prevalences. Studies were undertaken by rDNA ITS-2 and ITS-1 and mtDNA cox1 sequencing to clarify the specific status of the lymnaeids, their geographical distribution and fascioliasis transmission capacity in Chile, by comparison with other American countries and continents. Results change the lymnaeid scenario known so far. The lymnaeid fauna of mainland Chile shows to be poor, including only two authochthonous species, Lymnaea viator and Pectinidens diaphana, and a third introduced species of Palaearctic origin Galba truncatula. Both Lymnaea lebruni and Lymnaea patagonica proved to be synonyms of P. diaphana. G. truncatula appears to have always been confused with L. viator and seems distributed from Región VI to Región IX, overlapping with human endemic areas. DNA sequencing results suggest that the absence of correlation between remote sensing data and disease prevalences could be due to transmission capacity differences between L. viator and G. truncatula. Results furnish a new baseline on which to undertake future appropriate studies on transmission, epidemiology and control.

Fluke egg characteristics for the diagnosis of human and animal fascioliasis by Fasciola hepatica and F. gigantica

In trematodiases, shape and size of the fluke eggs shed with faeces are crucial diagnostic features because of their typically reduced intraspecific variability. In fascioliasis, the usual diagnosis during the biliary stage of infection is based on the classification of eggs found in stools, duodenal contents or bile. The aim of the present study is to validate the identification of Fasciola species based on the shape and size of eggs shed by humans, characterizing their morphometric traits using a computer image analysis system (CIAS). The influence of both the geographical location and of the host (human and livestock) has been analysed. Coprological studies were carried out in fascioliasis human endemic areas, where only F. hepatica is present (the northern Bolivian Altiplano and the Cajamarca valley in Peru), and where F. hepatica and F. gigantica coexist (the Kutaisi region of Georgia, the Nile Delta in Egypt, and the Quy Nhon province in Vietnam). Classically, it is considered that at the abopercular end of the shell of Fasciola eggs there is often a roughened or irregular area. Nevertheless, results show that the frequency of the presence of this feature in F. hepatica is population-dependent, and therefore is not a pathognomonic criterion in diagnosis. The study reveals that eggs shed by humans show morphological traits different from eggs shed by animals. In humans, F. hepatica eggs are bigger and F. gigantica eggs are smaller than reported to date from livestock, and their measurements overlap when compared. The material analysed in this study shows that the size of eggs shed by humans from Georgia and Egypt corresponds to the F. hepatica morph, while the size of eggs shed by humans from Vietnam corresponds to the F. gigantica morph. Measurements of F. hepatica and F. gigantica eggs originating from humans and animals from sympatric areas overlap, and, therefore, they do not allow differential diagnosis when within this overlapping range. In this sense, the new results should aid clinicians since the application of the classic egg size range in human samples may lead to erroneous conclusions. Fasciolid egg size in human stool samples ought to be corrected in books and monographs related to medical parasitology and/or tropical medicine as well as in guides for clinicians and parasitic disease diagnosis analysts.

First phenotypic description of Fasciola hepatica/ Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta, Egypt

Fasciola gigantica is the main fasciolid species in Africa; however, F. hepatica and F. gigantica overlap in some countries. Egypt deserves mentioning because of the emerging situation of human fascioliasis in the Nile Delta area. The morphometric characteristics of fasciolid adults infecting the main livestock species present in the Nile Delta human endemic area are analyzed through a computer image analysis system (CIAS) on the basis of standardized measurements known to be useful for the differentiation of both fasciolid species. This is the first time that such a study is performed in an African country and, therefore, the results are compared to (i) F. hepatica (European Mediterranean area) and F. gigantica (Burkina Faso) standard populations, i.e. geographical areas where both species do not co-exist, and (ii) F. hepatica and F. gigantica populations from geographical areas where both species do co-exist, including the presence of intermediate forms (Iran). Results indicate the presence of F. hepatica, F. gigantica and intermediate forms ( Fasciola sp.) in Egypt for the first time, and demonstrate the usefulness of CIAS for the phenotypic characterization of liver fluke adults from a concrete fascioliasis endemic area. Body roundness, body length over body width, and distance between the ventral sucker and the posterior end of the body provide useful tools for studying inter- and intraspecific morphological diversity in Fasciola adults. The application of these markers to specimens from geographical areas where F. hepatica and F. gigantica co-exist, such as in Egypt and Iran, suggest a strong population-level variation in Fasciola adult morphology.

366 POSTER Mechanism of the initiation of DNA methylation de novo by small RNA

Khon Kaen, a northeastern province of Thailand, has been considered as one of the human opisthorchiasis endemic areas with continuing high prevalence. Unsuccessful eradication of the disease is probably from the culture of eating raw and undercooked fish of local residence and the parasitic persistency in animal reservoir hosts, such as cats and dogs. In cooperation with the other human opisthorchiasis control programs in an endemic area of 29 villages in Ban Haet, Ban Phai, Chonnabot and Muncha Khiri Districts, Khon Kaen, this study investigated the prevalence of Opisthorchis viverrini infection using a formalin-ether sedimentation method as the gold standard, and hematology and blood chemistry of the reservoir hosts in this endemic area. The results showed that cats had much higher prevalence (76 of 214, 35.51%) than dogs (3 of 821, 0.37%). Hematology between the infected and uninfected cats was not different. Complete blood count and biochemistry reflected some altered hepatic functions. However, only severely infected cats showed apparent clinical signs, including lethargy, diarrhea, ocular and nasal discharges. Moreover, the ultrasonogram of infected cats with very high egg per gram (> 1500 EPG) showed apparent thickening of the gall bladder wall with hyperechoicity of hepatic parenchyma. This study suggests that cat is the most important animal reservoir of human opisthorchiasis, especially in this endemic area. It is also interesting that villages with infection are mostly located in the vicinity of Chi River and two large water reservoirs (Lawa and Nong Kongkaew Lakes), but people without infection were away from Chi River, on the south of Kudkhow Lake. Further investigation on this particular geofactor is essential for effective opisthorchiasis control programs.

EVALUATION OF FAS2-ELISA FOR THE SEROLOGICAL DETECTION OF FASCIOLA HEPATICA INFECTION IN HUMANS

The performance of Fas2-ELISA for the diagnosis of Fasciola hepatica infection in children living in areas of high endemicity for fascioliasis in the Peruvian Andes is analyzed. Fas2-ELISA is based on the detection of circulating IgG antibodies elicited in infected individuals against a F. hepatica antigen termed Fas2. The study was conducted in three Andean localities, Huertas-Julcan in Junin, Asillo in Puno, and Cajamarca, with a total population of 634 children in an age range 1 to 16 years old. Child fascioliasis prevalence was 21.1% in Huertas-Julcan, 25.4% in Asillo, and 24% in Cajamarca, estimated by coprological inspection. The seroprevalence of F. hepatica infection, determined by Fas2-ELISA, was 27.8% in Huertas-Julcan, 44.6% in Asillo, and 29.1% in Cajamarca. The overall sensitivity of Fas2-ELISA was 92.4%, the specificity 83.6%, and the negative predictive value 97.2%. No association between OD(450) Fas2-ELISA and infection intensity measured by egg counting was observed. Results show that Fas2-ELISA is a highly sensitive immunodiagnostic test for the detection of F. hepatica infection in children living in human fascioliasis endemic areas.

Lymnaea cousinni (Gastropoda: Lymnaeidae) as transmitter of fascioliasis

Dear Editor,In the article entitled “First report of Lymnaea cou-sini Jousseaume, 1887 naturally infected with Fasciolahepatica (Linnaeus, 1758) (Trematoda: Digenea) inMachachi, Ecuador” recently published by AngelVillavicencio A and Mauricio Carvalho de Vasconcellosin Memorias do Instituto Oswaldo Cruz (vol. 100, is-sue 7, pages 735-737, November 2005), it is stated thattheir finding in Ecuador represents the first report ofspecimens of this lymnaeid species naturally infectedby the liver fluke. However, it is well known from longago that this species acts as intermediate host of fascio-liasis in Andean countries. In the first half of the lastcentury, Brumpt et al. (1939-1940) already demonstratedthat L. bogotensis Pilsbry, 1935, a synonym of L. cousiniproposed and established by Hubendick (1951) and al-ways accepted (see Pointier et al. 2004), even also rec-ognized by Villavicencio and Carvalho de Vasconcellos(2005) in their paper here in question, is the intermedi-ate host of Fasciola hepatica in the surroundings of SantaFe de Bogota, Colombia.Materials studied by Villavicencio and Carvalho deVasconcellos (2005) were from Machachi, in the Andeanregion of Ecuador. These authors further note that fas-cioliasis prevalence in humans in the Andean region ofEcuador ranges from 24 to 53% and add the referenceof the Servicio Ecuatoriano de Sanidad Animal (SESA2003) concerning this information. However, these veryhigh human prevalences do not fit with results obtainedin surveys carried out up to the present in Andean com-munities of Ecuador, in which human prevalences alwaysappear to be low or very low: 6% by serology (Trueba etal. 2000); 0.5% by coprology (Gozalbo et al. 2004).Authors additionaly note that, according to WHO (1995),almost 200,000 people in Ecuador are infected, but itshall be clarified that these WHO data were only esti-mations available at the beginning of the 90s decade.Villavicencio and Carvalho de Vasconcellos (2005)emphasize the high prevalence of infection of 31.43%by F. hepatica they detected in L. cousini snails as thehighest value ever reported for lymnaeid snails naturallyinfected with this parasite. However, although this preva-lence is evidently high when compared to currentprevalences by fasciolids in lymnaeid snails, which areusually less than 5%, F. hepatica prevalences in snailssimilar and even higher than that reported by Villavicencioand Carvalho de Vasconcellos (2005) have been foundelsewhere. In fascioliasis human endemic areas of An-dean countries, for instance, a confirmed 31.6% preva-lence by F. hepatica in Galba truncatula was detectedin the center of the Northern Bolivian Altiplano villageof Tambillo (Bargues et al. 1995), that is, even in a hu-man settlemen instead of in a field place inhabited bylivestock.The fasciolid prevalence reported by Villavicencioand Carvalho de Vasconcellos (2005) was found in 70 L.cousini snail specimens collected in a 4.5-m

Remote sensing and climate data as a key for understanding fasciolosis transmission in the Andes: review and update of an ongoing interdisciplinary project

Fasciolosis caused by Fasciola hepatica in various South American countries located on the slopes of the Andes has been recognized as an important public health problem. However, the importance of this zoonotic hepatic parasite was neglected until the last decade. Countries such as Peru and Bolivia are considered to be hyperendemic areas for human and animal fasciolosis, and other countries such as Chile, Ecuador, Colombia and Venezuela are also affected. At the beginning of the 1990s a multidisciplinary project was launched with the aim to shed light on the problems related to this parasitic disease in the Northern Bolivian Altiplano. A few years later, a geographic information system (GIS) was incorporated into this multidisciplinary project analysing the epidemiology of human and animal fasciolosis in this South American Andean region. Various GIS projects were developed in some Andean regions using climatic data, climatic forecast indices and remote sensing data. Step by step, all these GIS projects concerning the forecast of the fasciolosis transmission risk in the Andean mountain range were revised and in some cases updated taking into account new data. The first of these projects was developed on a regional scale for the central Chilean regions and the proposed model was validated on a local scale in the Northern Bolivian Altiplano. This validated mixed model, based on both fasciolosis climatic forecast indices and normalized difference vegetation index values from Advanced Very High Resolution Radiometer satellite sensor, was extrapolated to other human and/or animal endemic areas of Peru and Ecuador. The resulting fasciolosis risk maps make it possible to show the known human endemic areas of, mainly, the Peruvian Altiplano, Cajamarca and Mantaro Peruvian valleys, and some valleys of the Ecuadorian Cotopaxi province. Nevertheless, more climate and remote sensing data, as well as more accurate epidemiological reports, have to be incorporated into these GIS projects, which should be considered the key in understanding fasciolosis transmission in the Andes.

Epidemiology of fascioliasis in human endemic areas

Considered a secondary zoonotic disease until the mid-1990s, human fascioliasis is at present emerging or re-emerging in many countries, including increases of prevalence and intensity and geographical expansion. Research in recent years has justified the inclusion of fascioliasis in the list of important human parasitic diseases. At present, fascioliasis is a vector-borne disease presenting the widest known latitudinal, longitudinal and altitudinal distribution. Fasciola hepatica has succeeded in expanding from its European original geographical area to colonize five continents, despite theoretical restrictions related to its biology and in turn dependent upon environmental and human activities. Among the different epidemiological situations, human hypo- to hyperendemic areas, including epidemics, are noteworthy. A global analysis of the distribution of human cases shows that the expected correlation between animal and human fascioliasis only appears at a basic level. Areas presenting very high human prevalences and intensities, especially in children and females, have been recently described. In hypo- to hyperendemic areas of Central and South America, Europe, Africa and Asia, human fascioliasis presents a range of epidemiological characteristics related to a wide diversity of environments. Thus far well-known epidemiological patterns of fascioliasis may not always explain the transmission characteristics in any given area and control measures should consider the results of ecoepidemiological studies undertaken in the zones concerned.

Human and animal fascioliasis in Mazandaran province, northern Iran.

The fascioliasis situation in humans and livestock of Iranian Mazandaran is analysed for the first time. Coprological studies showed 7.3% and 25.4% global prevalences in sheep and cattle, respectively. Studies in slaughterhouses indicate that sheep and cattle may be the main reservoir species, buffaloes may play local roles in the transmission, and goats and horses probably only participate sporadically. Morphometric studies by computer image analysis showed that forms intermediate between Fasciola hepatica and F. gigantica appear in addition to both species. A total of 107 infected humans were diagnosed during the 1999-2002 period, which suggests that fascioliasis may be widespread. The absence of differences in human fascioliasis among gender and age groups differs from other human endemic areas. Both human and animal infections show marked differences between western and eastern Mazandaran. Traditions in herbal condiments for human consumption, methods of animal husbandry and annual rainfall may explain the higher prevalences in western Mazandaran.

Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America.

Fascioliasis due to the digenean species Fasciola hepatica has recently proved to be an important public health problem, with human cases reported in countries of the five continents, including severe symptoms and pathology, with singular epidemiological characteristics, and presenting human endemic areas ranging from hypo- to hyperendemic. One of the singular epidemiological characteristics of human fascioliasis is the link of the hyperendemic areas to very high altitude regions, at least in South America. The Northern Bolivian Altiplano, located at very high altitude (3800-4100 m), presents the highest prevalences and intensities of human fascioliasis known. Sequences of the internal transcribed spacers ITS-1 and ITS-2 of the nuclear ribosomal DNA of Altiplanic Fasciola hepatica and the intermediate snail host Lymnaea truncatula suggest that both were recently introduced from Europe. Studies were undertaken to understand how the liver fluke and its lymnaeid snail host adapted to the extreme environmental conditions of the high altitude and succeeded in giving rise to high infection rates. In experimental infections of Altiplanic lymnaeids carried out with liver fluke isolates from Altiplanic sheep and cattle, the following aspects were studied: miracidium development inside the egg, infectivity of miracidia, prepatent period, shedding period, chronobiology of cercarial emergence, number of cercariae shed by individual snails, survival of molluscs at the beginning of the shedding process, survival of infected snails after the end of the shedding period and longevity of shedding and non-shedding snails. When comparing the development characteristics of European F. hepatica and L. truncatula, a longer cercarial shedding period and a higher cercarial production were observed, both aspects related to a greater survival capacity of the infected lymnaeid snails from the Altiplano. These differences would appear to favour transmission and may be interpreted as strategies associated with adaptation to high altitude conditions.

CD30-Positive anaplastic large cell lymphoma in a human T-cell lymphotropic virus-I endemic area

Clinicopathological and cytogenetic studies were performed on specimens from 43 patients with CD30-positive anaplastic large cell lymphoma (ALCL). All patients were born and lived in a human T-cell lymphotropic virus (HTLV)-I endemic area. Twenty-one patients (48.8%) had serum anti-HTLV-I antibody suggesting the presence of adult T-cell leukemia/lymphoma (ATL). Seventeen of them showed a clonal integration of complete HTLV-I proviral DNA by the Southern blot hybridization method in materials obtained by biopsy. Their ages ranged from 37 to 81 years (median, 67.0), and they frequently presented with lymphadenopathy (82.4%) and extranodal tumors with (76.5%) as well as with rare leukemic changes (5.9%). Immunohistologically the lymphoma cells in 15 ATL patients showed a T-cell phenotype. In only one patient (5.9%) was there an expression of epithelial membrane antigen (EMA) in most of the lymphoma cells. Cytogenetically aberration of chromosome band 5q35 was not found in seven patients with HTLV-I proviral DNA. The overall median length of survival was 11.9 months for the patients with HTLV-I proviral DNA, indicating a worse prognosis compared with that of the age-matched patients with negative anti-HTLV-I antibody ( P < .01). The specimens of the patients with HTLV-I proviral DNA had unique clinicopathological and cytogenetic features. These findings suggested that T-cell ALCL with HTLV-I proviral DNA should be considered to represent the lymphoma type of ATL.

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene.

Schistosoma mansoni and Sch. haematobium Found as a Natural Double Infection in the Nile Rat, Arvicanthis n. niloticus, from a Human Endemic Area in Egypt

Schistosoma mansoni and Sch. haematobium Found as a Natural Double Infection in the Nile Rat, Arvicanthis n. niloticus, from a Human Endemic Area in Egypt