More than 10 million people are thought to be infected with Trypanosoma cruzi, primarily in the Americas. The clinical manifestations of Chagas' disease (CD) are variable, but most subjects remain asymptomatic for decades. Only 15 to 30% eventually develop terminal complications. All current diagnostic tests have limitations. New approaches are needed for blood bank screening as well as for improved diagnosis and prognosis. Sera from subjects with asymptomatic CD (n ‫؍‬ 131) were compared to those from uninfected controls (n ‫؍‬ 164) and subjects with other parasitic diseases (n ‫؍‬ 140), using protein array mass spectrometry. To identify biomarkers associated with CD, sera were fractionated by anion-exchange chromatography and bound to two commercial ProteinChip array chemistries: WCX2 and IMAC3. Multiple candidate biomarkers were found in CD sera (3 to 75.4 kDa). Algorithms employing 3 to 5 of these biomarkers achieved up to 100% sensitivity and 98% specificity for CD. The biomarkers most useful for diagnosis were identified and validated. These included MIP1 alpha, C3a anaphylatoxin, and unusually truncated forms of fibronectin, apolipoprotein A1 (ApoA1), and C3. An antipeptide antiserum against the 28.9-kDa C terminus of the fibronectin fragment achieved good specificity (90%) for CD in a Western blot format. We identified full-length ApoA1 (28.1 kDa), the major structural and functional protein component of high-density lipoprotein (HDL), as an important negative biomarker for CD, and relatively little full-length ApoA1 was detected in CD sera. This work provides proof of principle that both platform-dependent (i.e., mass spectrometry-based) and platform-independent (i.e., Western blot) tests can be generated using high-throughput mass profiling.

SUMMARYThe interruption of vectorial transmission of Chagas disease in Venezuela is attributed to the combined effects of ongoing entomoepidemiological surveillance, ongoing house spraying with residual insecticides and the concurrent building and modification of rural houses in endemic areas during almost five decades. The original endemic areas which totaled 750,000 km², have been reduced to 365,000 km². During 1958During -1968, initial entomological evaluations carried out showed that the house infestation index ranged between 60-80%, the house infection index at 8-11% and a house density index of 30-50 triatomine bugs per house. By 1990-98, these indexes were further reduced to 1.6-4.0%, 0.01-0.6% and 3-4 bugs per house respectively. The overall rural population seroprevalence has declined from 44.5% (95% C.I.: 43.4-45.3%) to 9.2% (95% C.I.: 9.0-9.4%) for successive grouped periods from 1958 to 1998. The annual blood donor prevalence is firmly established below 1%. The population at risk of infection has been estimated to be less than four million. Given that prevalence rates are stable and appropriate for public health programmes, consideration has been given to potential biases that may distort results such as: a) geographical differences in illness or longevity of patients; b) variations in levels of ascertainment; c) variations in diagnostic criteria; and d) variations in population structure, mainly due to appreciable population migration. The endemic areas with continuous transmission are now mainly confined to piedmonts, as well as patchy foci in higher mountainous ranges, where the exclusive vector is Rhodnius prolixus. There is also an unstable area, of which landscapes are made up of grasslands with scattered broad-leaved evergreen trees and costal plains, where transmission is very low and occasional outbreaks are reported.

There is currently no "gold standard" test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease (n ‫؍‬ 195), healthy controls (n ‫؍‬ 400), and patients with other parasitic diseases (n ‫؍‬ 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain Trypanosoma cruzi trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with Leishmania-infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA IA proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA IA proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with Leishmania. These data suggest the need for field validation studies of TESA-and TESA IA -based assays in regions where Chagas' disease is endemic.

Abstract. We stratified the risk of malaria transmission (Plasmodium vivax) in 35 villages along a coastal range in northeastern Venezuela (51 km 2 ) where the main vector is the mosquito Anopheles aquasalis. After 20 years without local malaria transmission, reinfection of the entire area occurred from May to December 1985 by local (continuous) and jump (discontinuous) dispersal. Epidemiologic, environmental, and vector variables were investigated with the aid of a Geographic Information System. Risk factors for malaria transmission were human population density, proximity to pre-adult mosquito habitats (Ͻ 500 m), and the number of pre-adult habitats nearby. Most inhabitants, immature mosquito habitats, and malaria cases were located at low elevations and on gentle slopes. High prevalence of malaria during the dry seasons was associated with the presence of permanent bodies of water containing An. aquasalis. Occurrence of a La Niña event in 1988 (wet and cool phase of the El Niño Southern Oscillation) triggered malaria transmission to unusually high levels, consolidating infection in the area, and rendering traditional control efforts useless. We recommend tracking malaria persistence per village and associated risk factors as methods to reduce the cost of malaria control programs.A malaria focus is a circumscribed locality situated in a current or former malarious area that includes the epidemiologic factors necessary for malaria transmission. These include a human community, at least 1 source of infection, a vector population, and the appropriate environmental conditions.

SUMMARY. Many serological tests have been developed for the diagnosis of ChagasÕ disease, but few have been subjected to a rigorous field evaluation. We have recently described several novel enzyme immunoassays (EIAs) based on fixed-whole organisms or trypomastigote excretory-secretory antigens (TESA) from different Trypanosoma cruzi strains (Tulahuen or Brazil). This study evaluated the most promising of these novel assays (e.g. fixed-epimastigotes, fixedtrypomastigotes, TESA Brazil and TESA Tulahuen antigens) in a field study of Venezuelan blood bank specimens. The assays were tested in an operatorblinded fashion using 2038 blood bank samples obtained from low and high T. cruzi prevalence regions of Venezuela (n ¼ 1050 and n ¼ 988 from Bolivar and Portuguesa states, respectively). Based on National Laboratory for Chagas Immunodiagnosis (NLCI)Ôgold standardÕ results, all novel EIAs were superior to the commercial kit currently used in Venezuela, achieving 100% sensitivity and >99% specificity at optimal cut-off values. The novel assays identified seven false-negative samples compared with the routine screening performed by the Venezuelan blood bank although two samples were also misclassified as positive. Minor differences in the performance of the four novel assays were observed at lower arbitrary cutoff values. This study confirms the potential utility of both the fixed-organism and the TESA-based assays in the diagnosis of T. cruzi infection.