Infectivity of the subspecies of the Leishmania braziliensis complex in vivo and in vitro.

The infectivity of Leishmania braziliensis ssp. in relation to their growth kinetics in Senekjie's medium was determined using the human macrophage cell line U937 and inbred hamsters. In both systems, infectivity was shown to be distinctive for each subspecies. While L. b. panamensis promastigotes from 6-day-old cultures (early stationary phase) were more infective than parasites from any other culture day, L. b. guyanensis and L. b. braziliensis reached maximum infectivity on days 8-10 and day 10 (late stationary phase of growth), respectively. Although maximum infectivity occurred during stationary growth, strict growth phase dependency was not observed. The populations of parasites on these culture days were composed mostly of small, highly motile promastigotes with flagella 2-3 times the length of their cell bodies. These promastigotes resembled the infective forms transmitted by the sand fly vector. A distinct pathological picture characterized the disease caused by the different WHO reference strains for these subspecies in hamsters: L. b. guyanensis developed the most severe lesions, while moderate and inconspicuous lesions were observed when L. b. panamensis and L. b. braziliensis, respectively, constituted the inocula.

Sensitivity to Glucantime of Leishmania viannia isolated from patients prior to treatment.

In vitro sensitivity to pentavalent antimony (SbV) as meglumine antimoniate (Glucantime) of Leishmania of the Viannia subgenus isolated prior to treatment from patients with uncomplicated cutaneous leishmaniasis was evaluated for intracellular amastigotes in the U-937 human monocytic cell line and log phase promastigotes. The 50% effective dose (ED50) of pharmaceutical and additive-free formulations of Glucantime were determined based on the kinetics of the response of Leishmania Viannia to SbV in vitro. ED50 to SbV was inversely related to time of exposure to drug. The potency of the additive-free formulation of Glucantime was significantly greater than that of the pharmaceutical formulation, irrespective of the parasite form. In vitro sensitivity to SbV ranged from < 5.3 micrograms/ml to > 170.0 micrograms/ml. Under the conditions used, 11 (39%) of 28 strains were sensitive to clinically achievable serum concentrations of SbV. No correlation was observed between the total amount of SbV required for healing of lesions and the in vitro response to the pharmaceutical formulation of Glucantime. In contrast, a significant correlation (P = 0.001) was observed between clinical response and the in vitro sensitivity of promastigotes to the additive-free formulation of Glucantime. The greater potency of the additive-free formulation of Glucantime, the correlation of in vitro sensitivity of promastigotes to this formulation and the clinical response to treatment, and the effect of time of exposure to SbV demonstrate the importance of assay conditions on the outcome and interpretation of in vitro evaluation of drug sensitivity.

Metastatic capability of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis in golden hamsters.

The pattern and kinetics of internal dissemination and frequency of cutaneous metastatic lesions resulting from experimental infection of golden hamsters with Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis were examined. Nineteen strains were evaluated: 16 L. (V.) panamensis isolated from patients and 3 L. (V.) guyanensis, 2 isolated from human cases and 1 WHO reference strain originating from a sandfly vector. Lymphatic dissemination occurred within 3 mo and was observed for 16 of 16 (100%) of L. (V.) panamensis and 3 of 3 (100%) of L. (V.) guyanensis. Parasites were cultured infrequently from liver and spleen: 3 of 125 (2%) L. (V.) panamensis and 1 of 22 (5%) L. (V.) guyanensis. Decreased frequency of isolation from the inoculation site and draining lymph nodes over time was accompanied by increased frequency of isolation from distant lymph nodes. Dilution of triturated tissue samples resulted in an increased efficiency of parasite culture. Both primary lesions and secondary cutaneous metastatic lesions were more severe in hamsters infected with L. (V.) guyanensis than with L. (V.) panamensis. Cutaneous metastatic lesions were produced more frequently by L. (V.) guyanensis, 24 of 46 hamsters (52%), than by L. (V.) panamensis, 28 of 252 hamsters (11%). Individual Leishmania strains displayed distinctive propensities to produce cutaneous metastases, manifested as a reproducible phenotype. Metastatic pathogenicity was independent of the inoculum dose, supporting the dissociation of infectivity and pathogenicity.

Human monocyte infection by Leishmania (Viannia) panamensis. Role of complement receptors and correlation of susceptibility in vitro with clinical phenotype.

Peripheral blood monocytes (PBMs) from healthy individuals who had experienced distinctive clinical outcomes after natural infection with Leishmania (Viannia) were evaluated in vitro with respect to susceptibility to infection by stationary phase promastigotes of L. (V). panamensis. Concomitantly, the role of complement receptors (CR) CR1 and CR3 in the attachment and entry of L. (V). panamensis into human monocytes was analyzed using mAbs to CR1 (CD35) and CR3 (CD11b) to inhibit competitively these early events in the host-parasite interaction. Cell adherence to fibronectin was examined to determine how modulation of CR activity affected the attachment and uptake of this parasite species. The human monocyte cell line U-937 was also evaluated and found to provide a reproducible control for L. (V). panamensis infection in vitro. Opsonization with fresh AB+ serum markedly enhanced uptake by both PBMs and U-937 cells, and the fluid phase blocking of CR1 and CR3 resulted in partial inhibition of attachment and/or internalization. Uptake rather than attachment was abrogated by antireceptor antibodies in PBMs from previously infected individuals, whereas attachment was diminished in PBMs from unexposed controls. Adherence of PBMs to fibronectin resulted in decreased infection. PBMs from persons who had experienced chronic disease 5 to 8.4 yr before these studies were significantly more susceptible to in vitro infection by L. (V). panamensis than PBMs from asymptomatically infected or control individuals based on the percentage of cells infected, the number of parasites per cell, and viability of intracellular parasites at 48 h postinfection. Neither blocking of CR nor modulation by fibronectin altered the pattern of susceptibility of PBMs from the different clinical groups. These findings provide evidence for the participation of CR in the infection of human monocytes by L. (V). panamensis and demonstrate a correlation between clinical phenotype and in vitro infection of PBMs cultured in the presence of autologous plasma before experimental infection.