Guido Valverde, Viterman Alí, Pamela Durán, Luis Castedo, José Luis Paz, Eddy Martínez

Objective: This study was designed to search for ancient parasites in abdominal content and coprolites from Bolivian mummies. Materials: Twelve mummified individuals from the  Andean highlands, housed at  the  National Museum of Archaeology (MUNARQ) in La Paz, Bolivia. Methods: Microscopic analysis of  rehydrated samples (coprolites and abdominal content), following Lutz's spontaneous sedimentation technique. Results: Eggs of Enterobius vermicularis were identified in coprolites from one mummy, and capillariid eggs in the organic abdominal content from another individual. Conclusions: This is the first evidence of ancient intestinal parasites in Bolivian mummies. Significance: This pioneering study focused on the search of ancient intestinal parasites in human remains of the Bolivian Andes and contributes to greater knowledge of paleoparasitology in South America. Limitations: All mummies in the MUNARQ belonged to the Andean Bolivian highlands (post-Tiwanaku era or Late Intermediate Period), although the exact provenance of the material and the associated contexts are not well recorded. Suggestions for further research: Considering the great number of well-known archaeological sites and other un-explored sites in Bolivia, in addition to large collections in museums, further paleopathological and paleopar-asitological molecular studies in mummies and skeletons are called for.  

V. Ali, E. Martinez, P. Durán, M.A. Seláez, M. Barragan, P. Nogales, A. Peña y Lillo, M. Castañares, Y. Claros, P. Deplazes, C.A. Alvarez Rojas

Depickère, S., Durán, P., López, R., Martínez, E., & Chávez, T.

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is transmitted to humans by blood-sucking bugs (Triatominae).  Eratyrus mucronatus is a well-dispersed triatomine species, found sometimes in dwellings. In the Apolo region of Bolivia, people continue to complain of bites by this bug despite five years of chemical control. For the first time, the domiciliation process of . E. mucronatus was evaluated in a zone treated by insecticide, and it was compared with a previous situation 14 years ago. Both an entomological and a serological study were carried out: 152 dwellings were visited and 491 blood samples were analyzed with Stat-Pack and ELISA tests. In 34 dwellings (23% of the total visited dwellings), 160 triatomines of all stages were collected belonging to two species, E. mucronatus (98% of the capture) and . Panstrongylus geniculatus. A domiciliation of . E. mucronatus is demonstrated, particularly in the peridomicile. Bugs were captured more frequently in bedrooms in the intradomicile, and in curahuas (boundary walls in rammed mud) in the peridomicile. A . P. geniculatus adult was found infected with . T. cruzi. No evidence of a local vectorial transmission was found. In particular, the limited effect of the insecticide on the presence of . E. mucronatus and the role of the curahuas as a source of intrusion/domiciliation process are discussed.

Buitrago, R., Cupolillo, E., Bastrenta, B., Le Pont, F., Martinez, E., Barnabé, C., & Brenière, S. F.

Human leishmaniasis is highly endemic in Bolivia and shows a growing incidence. This report reveals the genetic variability of 35 isolates mainly belonging to Leishmania braziliensis and Leishmania amazonensis species. Among them, 31 were from human patients with different clinical presentations, 3 strains from Lutzomya nuneztovari anglesi (the proven vector of L. amazonensis) and 1 strain of a mammal (Conepatus chinga). The isolates were analyzed by isoenzyme electrophoresis (MLEE) and PCR-RFLP of ITS rRNA genes, a genetic marker highly polymorphic and better adapted to sub-structuring of populations. MLEE and RFLP-ITS were in agreement to discriminate the species, 12 belong to L. (V.) braziliensis, 21 to L. (L.) amazonensis, 1 to Leishmania (V.) lainsoni and 1 to Leishmania (L.) chagasi. Among L. (V.) braziliensis the RFLP-ITS only highlights variability. Ten isolates from either cutaneous or mucocutaneous clinical forms, were grouped together (bootstrap value of 99.8%) apart from two others, one from a mammal (C. chinga), the other from a patient with a cutaneous form. Among L. (L.) amazonensis both markers detect variability but no significant sub-division was identified including isolates from different clinical forms. Moreover, the high frequency of several isolates from cutaneous forms occurred during an outbreak, with putative hybrid character (multiloci heterozygous patterns depicted by MLEE) could be linked to better fitness of these parasites. However, in the absence of observation of hypothetical parents, their hybrid status remains a question.

Ježek, J., Le Pont, F., Martinez, E., & Mollinedo, S.

A study of Psychodinae, tribes Paramormiini, Psychodini and Pericomini, from Bolivia is completed, and three new species are described: Eurygarka freyrei sp. nov., Tinearia boliviensis sp. nov. and Alepia santacruz sp. nov. Differential diagnoses are included and important diagnostic characters illustrated. All known species of the genera Eurygarka Quate, 1959, Tinearia Schellenberg, 1803 and Alepia Enderlein, 1937 are listed. Comments on the classification of all three genera are given, and the conception of Neotropical Setomimini is discussed. The species Eurygarka aliciae (Ibáñez-Bernal & Cáceres, 2005) comb. nov., from Peru, is transferred from Philosepedon Eaton, 1904; seven species are transferred from Psychoda Latreille, 1796 to Tinearia – Tinearia alia (Quate, 1962) comb. nov. (Philippines, Borneo and New Guinea), T. capitipenis (Ibáñez-Bernal, 1992) comb. nov. (Mexico), T. efflatouni (Tonnoir, 1922) comb. nov. (Egypt), T. formosiensis (Tokunaga, 1957) comb. nov. (Taiwan, Borneo), T. platilobata (Tokunaga, 1957) comb. nov. (Taiwan, Philippines, Borneo, Jamaica, Trinidad), T. subquadrilobata (Tokunaga, 1957) comb. nov. (Taiwan), and T. vagabunda (Quate, 1962) comb. nov. (Sri Lanka, Borneo). A new subtribal synonymy is proposed: Threticina Vaillant, 1991 syn. nov. Trichopsychodina Ježek, 1985.

Martínez, E.

Martinez, E.

Martínez, E., Achocalla, A., Avila, J. C., Matías, A., Mollinedo, R., Escobar, J. P., … Melgar, G.

Se realizó un estudio de la eficacia de la cloroquina en el tratamiento de la malaria no complicada por Plasmodium vivax, en el sitio centinela de Riberalta, Beni, localizado en la región amazónica de Bolivia. Fue utilizada la metodología estandarizada de OPS/OMS para la evaluación in vivo de la eficacia de drogas antimaláricas. Fueron incluidos en el estudio 63 pacientes con malaria debida a P. vivax, todos recibieron tratamiento estrictamente supervisado a base de cloroquina 25 mg base por Kg, dividido en 3 días (primer día 10mg/Kg, segundo y tercer días 7,5mg/Kg). Se realizó el seguimiento de los pacientes durante 28 días (evaluación clínica y parasitológica) y se tomaron muestras de sangre para medir la concentración de cloroquina y desetilcloroquina y para genotipificación de los parásitos. De los 63 pacientes incluidos en el estudio 57 tuvieron un seguimiento completo. Mediante microscopía se detectó parasitemia recurrente tardía en 9 pacientes (15,5%), ocho en el día 28 y uno en el día 21, este último fue excluido del estudio por alcoholismo. Los pacientes recibieron un segundo tratamiento en base a cloroquina (misma dosis), en todos los casos desapareció la parasitemia, pero un paciente presentó una nueva recurrencia el día 28, el mismo que recibió terapia combinada en base a mefloquina (12mg/Kg día por 2 días) y artesunato (4mg/Kg día por 3 días) con cura clínica y parasitológica. En 4 casos se confirmó la parasitemia recurrente mediante PCR, que además identificó que las cepas recurrentes eran las mismas que las del día 0 para todos estos pacientes. Basados en estos resultados, el Ministerio de Salud y Deportes de Bolivia, mantiene el uso de la cloroquina más primaquina para el tratamiento de las infecciones por P. vivax. Sin embargo, se incluyó un segundo tratamiento con el mismo esquema y en caso de parasitemia recurrente o persistente después del segundo tratamiento, considerando la posibilidad de cloroquino- resistencia, fue incluida en la política de medicamentos de Bolivia, como segunda línea, la terapia combinada basada en mefloquina y artesunato, complementada con primaquina. Son necesarios estudios futuros para definir las razones de la parasitemia recurrente por P. vivax en la Amazonia boliviana.

Martinez, E, Yucra W O, Castro V, Figueroa R V, De La Cruz L, Tellez C, Avila JC, Mollinedo R, Escobar JP, Duran P.

Se realizó una evaluación in vivo de la eficacia de la cloroquina para el tratamiento de la malaria por Plasmodium vivax, en el sitio centinela de Palmar Chico, Municipio de Yacuiba, Provincia Gran Chaco, Departamento de Tarija al Sur de Bolivia. Fueron incluidos en el estudio 61 pacientes entre 5 y 59 años de edad que presentaban monoinfección por P. vivax. Todos los pacientes recibieron una dosis total de cloroquina de 25 mg/Kg en 3 días (10mg/Kg el primer día; 7,5 mg/Kg el segundo y tercer días). De acuerdo al protocolo estandarizado de la OPS/OMS, todos los pacientes fueron seguidos por 28 días (controles clínicos y parasitológicos). Se completó el seguimiento de 60 pacientes, ninguno de los pacientes tuvo recurrencias de la parasitemia o presentó manifestaciones clínicas después del tercer día de tratamiento. Este estudio mostró 100% de sensibilidad de P. vivax a la cloroquina, lo que justifica su permanencia en la política de medicamentos antimaláricos como la droga más adecuada para el tratamiento de las infecciones por P. vivax en el Sur de Bolivia.

Martinez, E., Yucra, W. O., Castro, V., Figueroa, R. V, De la Cruz, L., Tellez, C., … Duran, P.

Se realizó una evaluación in vivo de la eficacia de la cloroquina para el tratamiento de la malaria por Plasmodium vivax, en el sitio centinela de Palmar Chico, Municipio de Yacuiba, Provincia Gran Chaco, Departamento de Tarija al Sur de Bolivia. Fueron incluidos en el estudio 61 pacientes entre 5 y 59 años de edad que presentaban monoinfección por P. vivax. Todos los pacientes recibieron una dosis total de cloroquina de 25 mg/Kg en 3 días (10mg/Kg el primer día; 7,5 mg/Kg el segundo y tercer días). De acuerdo al protocolo estandarizado de la OPS/OMS, todos los pacientes fueron seguidos por 28 días (controles clínicos y parasitológicos). Se completó el seguimiento de 60 pacientes, ninguno de los pacientes tuvo recurrencias de la parasitemia o presentó manifestaciones clínicas después del tercer día de tratamiento. Este estudio mostró 100% de sensibilidad de P. vivax a la cloroquina, lo que justifica su permanencia en la política de medicamentos antimaláricos como la droga más adecuada para el tratamiento de las infecciones por P. vivax en el Sur de Bolivia.

Martinez, E., Chavez, T., Sossa, D., Aranda, R., Vargas, B., & Vidaurre, P.

Presentamos la descripción de Triatoma boliviana sp. n. en base a caracteres morfológicos externos de 3 machos y 3 hembras siguiendo las claves de Lent y Wygodzinsky (1979) y Carcavallo y col. (1997). La nueva especie es muy similar a Triatoma nigromaculata y fue capturada en valles subandinos de la Provincia Muñecas, Departamento de La Paz, Bolivia

ABSTRACT. We present the description of Triatoma boliviana sp. n. based on morphological external characters of 3 males and 3 females following the keys of Lent and Wygodzinsky (1979) and Carcavallo et al. (1997). The new species are very similar to Triatoma nigromaculata and was captured in Sub Andean valleys from Muñecas Province, Department of La Paz, Bolivia.

Dujardin, J. P., Le pont, F., & Maritnez, E.

We describe a multivariate metric comparison of three sandfly species showing strong differences in size, which questions the geographical distribution of one of them. Two species are represented by a single population (L. robusta and L. guilvardae) and one by two populations (L. serrana). All of them belong to the series serrana (Diptera, Psychodidae, Phlebotominae). The morphometric data confirm that L. guilvardae is a distinct species. However, they suggest that L. robusta and L. serrana in Ecuador are the same taxon, and that it is distinguishable from the population of L. serrana in Bolivia. A multilocus enzyme electrophoresis analysis comparing L. serrana in Bolivia and L. robusta in Ecuador adds further evidence that these two populations are distinct species. Thus, our data seem to indicate that, in Ecuador, the population previously identified as L. serrana is actually the same species as the allopatric population previously identified as L. robusta. Accepting L. serrana in Ecuador as small-sized L. robusta, the resulting geographic distribution of this latter becomes in closer agreement with ecology and epidemiology.

Le Pont, F., Matias, A., Martinez, E., & Dujardin, J. P.

Bolivian Phlebotomines: description of four new species of Lutzomyia (Diptera, Psychodidae). Four new species of phlebotomine sandflies from the genus Lutzomyia an: described and illustrated from specimens caught in light-traps. in humid of different leishmaniasis foci. Lutzomyia huacalquensis n. sp. belongs to the species group oswadoi; L dereuri n. sp. belongs to the same species group, but is caught in allopatric situation. L pradobarrientosi n. sp. belongs to the species-group aragaoi. L. caceresi n. sp. belongs to the Helcocyrtomyia subgenus and is the unique anthropophilic species. The last two species were only described from the male.

Matias, A., De La Riva, J., Martinez, E., Torrez, M., & Dujardin, J. P.

We report a systematic collection of Triatominae inside houses and in the peridomestic environment of Alto Beni, department of La Paz, Bolivia. This area is free of Triatoma infestans and although we detected previously seropositivity for Trypanosoma cruzi, the Alto Beni region is not officially considered as endemic for Chagas disease. From 11 houses of five localities, we collected adults, nymphs and eggs of a Rhodnius species, which was confirmed by morphological and morphometric analysis as Rhodnius stali. This little-known species has long been confused with R. pictipes, and was originally described from museum specimens labelled as R. pictipes. Our data show that R. stali is able to establish colonies in domestic and peridomestic habitats in Bolivia, and it is probably the vector responsible for Chagas disease seropositivity observed in the indigenous population of Alto Beni.

Martinez, E., Mollinedo, S., Torrez, M., Muñoz, M., Bañuls, A. L., & Le Pont, F.

We present the first report of a co-infection by Leishmania amazonensis and L. infantum/L. chagasi isolated in 1993 from a patient with diffuse cutaneous leishmaniasis (DCL), living in the sub-Andean region of Bolivia. This is the third reported case of DCL in Bolivia, but the first one with isoenzymatic identification of the aetiological agents involved and the first one giving evidence for a mixed infection by 2 Leishmania parasites in the same lesion.

Martinez, E., Le Pont, F., Mollinedo, S., & Cupolillo, E.

We present the first known case of cutaneous leishmaniasis due to Leishmania (Viannia) lainsoni detected in Bolivia. The parasite was isolated from a young girl living in the subtropical region of Carrasco (900-1000 m above sea level, Caranavi Province, Department of La Paz, Bolivia). The parasite identification was confirmed by multilocus enzyme electrophoresis.

De la Riva, J., Matias, A., Torrez, M., Martínez, E., & Dujardin, J. P.

This is the first report of adult and nymphs (20 nymphs of all stages and 4 adults) of Microtriatoma trinidadensis (Lent 1951) (Hemiptera: Reduviidae: Triatominae) collected in peridomestic environment, in the department of La Paz, Bolivia. These specimens were associated to Rhodnius stali Lent, Jurberg & Galvão 1993. The exceptional finding of M. trinidadensis in peridomestic environment, illustrates the general tendency of triatominae to adapt to human dwellings and dependences.

Campbell-Lendrum, D., Dujardin, J. P., Martinez, E., Feliciangeli, M. D., Perez, J. E., Passerat De Silans, L. N. M., & Desjeux, P.

Predictions that deforestation would reduce American cutaneous leishmaniasis incidence have proved incorrect. Presentations at a recent international workshop, instead, demonstrated frequent domestication of transmission throughout Latin America. While posing new threats, this process also increases the effectiveness of vector control in and around houses. New approaches for sand fly control and effective targeting of resources are reviewed.

Dujardin, J., Le Pont, F., & Martinez, E.

Elucidating the evolution of Phlebotominae is important not only to revise their taxonomy, but also to help understand the origin of the genus Leishmania and its relationship with humans. Our study is a phenetic portrayal of this history based on the genetic relationships among some New Word and Old Word taxa. We used both multilocus enzyme electrophoresis and morphometry on 24 male specimens of the Old Word genus Phlebotomus (with three of its subgenera: Phlebotomus, Spelaeophlebotomus and Australophlebotomus), and on 67 male specimens of the three New World genera, Warileya, Brumptomyia and Lutzomyia, (with three subgenera of Lutzomyia: Lutzomyia, Oligodontomyia and Psychodopygus). Phenetic trees derived from both techniques were similar, but disclosed relationships that disagree with the present classification of sand flies. The need for a true evolutionary approach is stressed.

Telleria, J., Bosseno, M., Tarifa, T., Buitrago, R., Martinez, E., Torrez, M., … Brenière, S.

Dujardin, J. P., Le Pont, F., & Martinez, E.

Morphological variation among geographic populations of the New World sand fly Lutzomyia quinquefer (Diptera, Phlebotominae) was analyzed and patterns detected that are probably associated with species emergence. This was achieved by examining the relationships of size and shape components of morphological attributes, and their correlation with geographic parameters. Quantitative and qualitative morphological characters are described, showing in both sexes differences among local populations from four Departments of Bolivia. Four arguments are then developed to reject the hypothesis of environment as the unique source of morphological variation: (1) the persistence of differences after removing the allometric consequences of size variation, (2) the association of local metric properties with meristic and qualitative attributes, rather than with altitude, (3) the positive and significant correlation between metric and geographic distances, and (4) the absence of a significant correlation between altitude and general-size of the insects.

Martinez, E., Le Pont, F., Torrez, M., Telleria, J., Vargas, F., Dujardin, J. C., & Dujardin, J. P.

Recently, a new Leishmania amazonensis focus was described in a sub-Andean region (1,450-2,100 meters above sea level) of Bolivia. In this area, three anthropophilic sandfly species were identified: Lutzomyia nuneztovari anglesi Le Pont & Desjeux, 1984, which represented 86-99% of the captures, Lu. galatiae Le Pont et al., 1998, and Lu. shannoni Dyar 1929. Only Lu. nuneztovari anglesi was found naturally infected by flagellates (16 of 1,715 females). Three Leishmania stocks were isolated and analyzed by isoenzyme electrophoresis at 11 loci. No significant isoenzymatic differences were demonstrated between them and 7 stocks isolated from patients from the same area, and previously characterized as L. amazonensis. Moreover, in a simplified protocol, the experimental infection of Lu. nuneztovari anglesi by L. amazonensis was successful in 92% of the surviving specimens. These data are discussed in relation to the Killick-Kendrick criteria. These results strongly suggest that Lu. nuneztovari anglesi is the vector of L. amazonensis at Cajuata, Inquisivi, La Paz, Bolivia.

E. Martınez, F. Le Pont, M. Torrez, J. Tellerı, F. Vargas ,M. Muñoz, S. De Doncker, J.C. Dujardin, J.P. Dujardin.

We detected a new outbreak focus with high incidence of cutaneous leishmaniasis in the Sub Andean region of La Paz. This area was never considered previously as an endemic zone of leishmaniasis. Leishmania stocks from human lesions were isolated: three stocks were explored by pulse field gradient electrophoresis, showing evidence for their affiliation to theL. mexicana complex. Eight stocks were submitted to isoenzyme electrophoresis and compared with five reference strains: L. amazonensis, L. braziliensis, L. chagasi, L. mexicana and L. pifanoi. Close genetic proximity was evidenced between newly isolated parasites and the reference stock of L. amazonensis, whereas high divergence was observed between them and either the L. pifanoi, L. mexicana, L. braziliensis and L. chagasi reference strains.

Le Pont F, Martinez E, Torrez Espejo JM, Dujardin JP.

Le Pont, F., Martinez, E., Torrez-Espejo, J. M., & Dujardin, J. P.

Bolivian Phlebotomines: description of five new species of Lutzomyia from subandean area (Diptera, Psychodidae). Five new species of phlebotomine sandflies from the genus Lutzomyia are described fiom specimens caught in light-traps in the subandean region of the foothills of La Paz. Lutzomyia galatiae sp. n. belongs to the Helcocyrtomyia subgenus, peruensis series; it is an anthropophilic species with endophagic behaviour, caught only in the leishmaniasis focus of Cajuata. L. kuscheli sp. n. belongs to the Pintomyia sub- genus. L. lereyi sp. n. and L. guilvardae sp. n. belong respectively to the Psathyromyia and Pifanomyia subgenera and are described only eom the males; females of these taxons may be cryptic with L. shannoni and L. serrana respectively. We describe the two sexes of L. duncanae sp. n. belonging to the Pressatia subgenus.