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Abstract. The leishmaniases are protozoan, zoonotic diseases transmitted to human and other mammal hosts by the bite of phlebotomine sandflies. Bolivia has the highest incidence of cutaneous leishmaniasis (CL) in Latin America (LA), with 33 cases per 100,000 population reported in 2006. CL is endemic in seven of the country's nine administrative departments. Visceral leishmaniasis (VL) is comparatively rare and is restricted to one single focus. Most CL cases are caused by Leishmania ( Viannia ) braziliensis (85% cases); VL is caused by L. (L.) infantum. Seven sandfly species are incriminated as vectors and Leishmania infections have been detected in several non-human mammal hosts. Transmission is associated with forest-related activities, but recently, cases of autochthonous, urban transmission were reported. Because most cases are caused by L. (V.) braziliensis, Bolivia reports the greatest ratio (i.e., up to 20% of all cases) of mucosal leishmaniasis to localized CL cases in LA. Per national guidelines, both CL and VL cases are microscopically diagnosed and treated with pentavalent antimony.

Species identification is highly relevant for improved prognosis and adequate treatment of American tegumentary leishmaniasis (ATL). PCR-based methods are available for this purpose but should be simplified to improve accessibility. As a first step in this process, this paper describes a simplified protocol for collection of clinical samples. Using samples from 44 Bolivian patients with confirmed ATL, we demonstrated that hsp70 PCR-RFLP on skin scrapings collected with a tooth pick allowed identification of the parasite species with a sensitivity of 95% and specificity of 100%. Our method should greatly facilitate individual patient management and epidemiological surveillance of ATL.

Eleven of 27 decameric primers were found to be suitable for random amplification of polymorphic DNA (RAPD) from triatomine bugs on the basis that they produced discrete profiles and distinguished among Panstrongylus megistus (Burmeister), Rhodnius prolixus Stål, and Triatoma infestans (Klug). The legs, or single leg segments, of individual bugs were used as the source of DNA so that the taxonomic value of the bug was conserved. Within the scope of the specimens studied, RAPD profiles allowed assignment to species even when bugs were kept dry for up to 12 mo. Profiles for individuals within a species were not identical. RAPD profiles, with the specimens tested, distinguished among species of 3 pairs considered to be morphologically similar and closely related, namely, Rhodnius ecuadorensis Lent & León and Rhodnius pictipes Stål; Rhodnius nasutus Stål, and Rhodnius neglectus Lent; Rhodnius prolixus Stål and Rhodnius robustus Larrousse. RAPD data conformed with the perceived affinities among these species. RAPD polymorphisms were seen with T. infestans from 3 different localities, but none of the polymorphisms was confined to 1 source. RAPD provided a molecular basis to reassess taxonomic relationships within the Triatomine subfamily. The accurate distinction of triatomine species and of intraspecific bug populations may contribute to elimination of vector-borne Chagas disease from the Americas.

Human-made and environmental changes constitute a major risk factor for the (re-)emergence and spread of leishmaniasis; surveillance of the transmission cycle is essential in this context. This study integrated entomological and molecular parasitological techniques to document the transmission pattern of a peridomestic focus of Leishmania in the Isiboro Secure area of Bolivia. First the spatial distribution and relative density of phlebotomine sand flies, genus Lutzomyia, were established. Lutzomyia shawi was the predominant species in domestic and peridomestic environments (90% from all collections). Second, direct application of the hsp70 PCR to sand fly extracts detected Leishmania infections in Lu. shawi only, and gave an estimated infection rate of 0.21 to 0.38%. The cleavage of the hsp70 amplicon with restriction enzymes (hsp70 PCR-RFLP) allowed identification of Le. (V.) braziliensis and Le. (V.) guyanensis in Lu. shawi captured in the same village. These two parasite species were also found in humans from the study region, supporting the co-existence of two transmission cycles involving the same sand fly species. This study demonstrated the use of PCR-RFLP in the identification of Leishmania in sand fly pools which could lead to the development of methods for screening large sand fly populations in Latin America.

Genetic variation within triatomine bugs was investigated by amplification of a 400 bp portion of the mitochondrial 16S ribosomal RNA gene by polymerase chain reaction (PCR), using evolutionarily conserved primers, from a selection of species representative of the genera Rhodnius, Triatoma and Panstrongylus. Amplification products were subsequently screened for sequence variation using single strand conformational polymorphism analysis (SSCP) and also subjected to direct sequencing. Single strand conformational polymorphism analysis could detect variation within and between genera; intraspecific variation was also detected and SSCP profiles appear to be useful for identification purposes at the inter-and intraspecific levels. A 290 bp multiple alignment of 15 sequences obtained from nine species was generated, phylogenetic inference subsequently used three methods; a distance estimate, maximum parsimony and maximum likelihood. This 16S region exhibited considerable variation which ranged from intergeneric to intraspecific levels. Phylogenies from these three methods of inference were in broad agreement. Triatoma and Panstrongylus were more closely related to each other than either was to Rhodnius, in keeping with the current taxonomic appraisal.

Data on the distribution and abundance of Lutzomyia spp. (Diptera: Psychodidae) in Bolivia is scarce. Sand flies from an area of Leishmania (Viannia) braziliensis endemicity in the Isiboro-Secure National Park in the Department of Cochabamba were captured and identified to species. In total, 945 sand flies (789 females and 156 males) belonging to 15 species were collected from the four collection points in two study villages in 2007. With 549 (58.1%) specimens, Lutzomyia shawi was the most abundant species, followed by Lutzomyia (Trichophoromyia) sp. (22.2%), Lutzomyia llanosmartinsi (8.3%), Lutzomyia antunesi (4.3%), and Lutzomyia olmeca (2.1%). Abundance and species composition varied between rainy and dry seasons, with 99.3% of all sand flies being collected outdoors. Because of species abundance and confirmed Leishmania infection in previous entomological collections, we believe Lu. shawi is the vector of L. (Viannia) braziliensis in Isiboro-Secure National Park.

et al. Variation of the hepatic C virus 5′ non-coding region: implications for secondary structure, virus detection and typing. J Gen Virol. 1995;76:174976: -61. DOI: 10.109976: /0022-131776: -76-7-1749 Leishmaniasis in Chaparé, BoliviaTo the Editor: In Bolivia, most cases of leishmaniasis are caused by Leishmania (Viannia) braziliensis (1). The parasite is transmitted zoonotically by several sandfl y species and, when transmitted to humans, may cause cutaneous leishmaniasis (CL), and potentially, mucosal leishmaniasis (ML) (2).Data on the prevalence and effects of CL in Bolivia have been scarce, even though anecdotal and offi cial reports indicate a dramatic increase in the number of human CL cases in Bolivia in the past decade (1,3). Also, although CL was originally a sylvatic disease in Bolivia, some evidence indicates that the transmission cycle has adapted to the peridomestic habitat. However, this evidence is largely based on individual case reports. No information is available on parasite species, vectors, and reservoirs in such a peridomestic transmission cycle.A preliminary study to guide future research focus and assist in immediate leishmaniasis prevention and control policy decision making is underway in Isiboro-Secure National Park, Chaparé, Bolivia. Our objectives were to collect data on the prevalence of leishmaniasis in that area and evidence for peridomestic Leishmania transmission.A survey was carried out during April-July 2007 in 2 communities in Isiboro-Secure National Park, San Gabriel (16°40′31′′S and 65°37′38′′W) and San Julian (16°41′59′′S and 65°38′10′′W). These 2 communities were selected because of local knowledge of disease in the community, their moderate degree of urbanization (i.e., ≈50% of the communities' houses are clustered around the main access road), and the accessibility of the sites to the fi eld team. In this area, CL is transmitted from April through October. We surveyed 133 and 52 households in San Gabriel and San Julian, which represented 86% and 80% of the total households of the respective communities; 21 and 13 households, respectively, were visited but did not participate because the owners refused or were not present. Of the 965 persons surveyed, 488 (50.6%) were male and 476 (49.3%) were female; 9 (0.9%) had active CL lesions and 62 (6.4%) had CL scars. One person had ML, and 3 had evidence of past ML; all ML patients were male. Of those with CL lesions, all had 1 lesion only. The mean lesion size was 2.3 cm (range 1.5-3 cm), and the mean lesion duration (to survey date) was 5.6 months (range 1-11 months). The clinical CL lesions were parasitologically confi rmed by microscopy (n = 4) or PCR (n = 8). Parasite culture was performed on patient isolates (n = 6), and L. (V.) braziliensis was identifi ed and characterized as the etiologic agent of these CL cases.Active lesion and scar prevalence were associated with male sex Whether the surveyed population is representative of the total population living in the study area is debatable. However on the basis of curre...