The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy smears. By hybridizing the PCR product to new kDNA * Corresponding author.

An entomological survey was undertaken from January 1991 to February 1992 in El Ingenio, Miranda State, Venezuela, an endemic area of cutaneous leishmaniasis: prevalence of 10.7 cases per 100,000 inhabitants. A total of 4863 female sandflies (Phlebotominae) of fourteen species were collected in Shannon traps, then dissected and examined for leishmanial infections. Lutzomyia ovallesi (85.4%) and Lu. gomezi (11.2%) were the predominant anthropophilic species of sandfly. Fifty-one (1.19%) Lu.ovallesi and two (0.47%) Lu.gomezi had natural infection with Leishmania promastigotes. Identification of the parasites was done by using the polymerase chain reaction (PCR) and DNA hybridization. Two isolates from Lu.gomezi and forty-nine from Lu.ovallesi were typed as Leishmania braziliensis and three of the latter reacted with Le.mexicana also. This is the first report of Lu.gomezi with parasites typed as Le.braziliensis. We concluded that Lu.ovallesi is the primary vector of cutaneous leishmaniasis in the north-central area of Venezuela and Lu.gomezi should be regarded as an additional vector.

Oil-in-water emulsions have gained consideration as vaccine adjuvants in recent years due to their ability to elicit a differentiated immunogenic response compared to traditional aluminum salt adjuvants. Squalene, a cholesterol precursor, is a natural product with immunostimulatory properties, making it an ideal candidate for such oil-in-water emulsions. Particle size is a key parameter of these emulsions and its relationship to stability and adjuvanticity has not been extensively studied. This study evaluates the effect of particle size on the stability and immunogenicity of squalene emulsions. We investigated the effect of formulation parameters such as surfactant concentration on particle size, resulting in particles with average diameter of 80 nm, 100 nm, 150 nm, 200 nm, or 250 nm. Emulsions were exposed to shear and temperature stresses, and stability parameters such as pH, osmolarity, size, and in-depth visual appearance were monitored over time. In addition, adjuvanticity of different particle size was assessed in a mouse model using Respiratory Syncytial Virus Fusion protein (RSV-F) as a model antigen. Temperature dependent phase separation appeared to be the most common route of degradation occurring in the higher particle sizes emulsions. The emulsions below 150 nm size maintained stability at either 5 C or 25 C, and the 80 nm diameter ones showed no measurable changes in size even after one month at 40 C. In vivo studies using the emulsions as an adjuvant with RSV F antigen revealed that superior immunogenicity could be achieved with the 80 nm particle size emulsion.

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