Protective immunogenicity of the paraflagellar rod protein 2 of Leishmania mexicana.

Paraflagellar rod proteins (PFR) are a potent immunogen against experimental Trypanosoma cruzi infection. PFR are highly conserved among kinetoplastid parasites. We therefore evaluated the immunogenicity of the Leishmania mexicana pfr-2 gene and protein product in the hamster model of American cutaneous leishmaniasis. Immunization with pfr-2 DNA-induced specific antibody, confirming immunogenicity. Subsequent challenge with 10,000 and 500 stationary phase L. mexicana promastigotes respectively, resulted in delayed appearance of lesions, and significant reduction in lesions post infection in male hamsters, yet exacerbated lesions in female hamsters. Immunization with recombinant PFR-2 protein (rPFR-2) prevented lesion development in female hamsters challenged with L. panamensis, but was ineffective against L. mexicana. Nevertheless, prime boost immunization of female hamsters with rPFR and pfr-2 DNA significantly reduced lesion size following challenge with 500 L. mexicana promastigotes, supporting the relevance of PFR-2 as a potential vaccine constituent.

Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential diagnosis of pleural tuberculosis.

STUDY OBJECTIVES: Pleural tuberculosis (TB) is a diagnostic challenge because of its nonspecific clinical presentation and paucibacillary nature. The inefficiency of conventional laboratory methods and the reliance on pleural biopsy have motivated the evaluation of alternative diagnostic strategies. We have evaluated polymerase chain reaction (PCR) directed to the IS6110 sequence of Mycobacterium tuberculosis, the determination of adenosine deaminase (ADA) activity, and measurement of interferon (IFN)-gamma levels in pleural fluid in the diagnosis of pleural TB. PATIENTS: ADA activity, IFN-gamma levels, and PCR were evaluated in 140 cases of pleural effusion, 42 with confirmed pleural TB, 19 with probable pleural TB, 70 with a nontuberculous etiology, and 9 having an undetermined etiology. RESULTS: ADA activity, IFN-gamma levels, and PCR were 88%, 85.7%, and 73.8% sensitive, respectively, and 85.7%, 97.1%, and 90% specific, respectively, for pleural TB that had been confirmed by either culture or pleural biopsy specimens. The combination of PCR, IFN-gamma measurement, and ADA activity determination allowed the selective increase of sensitivity and specificity for probable and confirmed cases compared to individual methods. Positive and negative predictive values for these individual or combined methods were maintained over a wide range of prevalence of pleural TB in the patient population presenting with pleural effusions. Fever and younger age were associated with tuberculous pleural effusion (p < 0. 0001), while blood in sputum and older age were associated with malignant etiology (p < 0.008). CONCLUSIONS: These clinical variables together with the use of ADA activity determination, PCR, and measurement of IFN-gamma levels provide the basis for the rapid and efficient diagnosis of pleural TB in different clinical settings.

Clonal diversity in the expression and stability of the metastatic capability of Leishmania guyanensis in the golden hamster.

Metastatic disease is a major concern of dermal leishmaniasis caused by Leishmania of the Viannia subgenus. The golden hamster provides an experimental model of systemic dissemination and cutaneous metastasis of Leishmania Viannia. We have exploited this model to examine the expression of parasite virulence in cloned populations derived from a strain of L. guyanensis previously shown to be highly metastatic in the hamster. Metastatic capacity manifested as dissemination throughout the lymphoid organs; cachexia and secondary cutaneous lesions were found to differ among clones, yielding a spectrum of virulence. The metastatic phenotype of clonal populations was stable over 5 sequential passages in hamsters. In addition, the low or high propensity to disseminate and produce cutaneous metastatic lesions was reproduced. Capacity to disseminate from the inoculation site was conserved following subcloning of metastatic clones that had been passaged in culture for several generations; clinical manifestations, cachexia, and cutaneous metastatic lesions were variably expressed. Dissemination of parasites and cachexia were significantly related (P = 0.004). Overall, cachexia was an earlier manifestation of dissemination than cutaneous metastases (P < 0.001). The reproducible expression of virulence phenotypes by discrete populations of Leishmania in the golden hamster provides an experimental model for clinically relevant expression of virulence in human leishmaniasis.

Distinct innate and acquired immune responses to Leishmania in putative susceptible and resistant human populations endemically exposed to L. (Viannia) panamensis infection.

Mechanisms of constitutive and acquired susceptibility/resistance to Leishmania Viannia panamensis (L. (V ) p.) were investigated in endemically exposed human populations presenting either recurrent disease (putative susceptible) or subclinical infection (clinically resistant). Cutaneous delayed type hypersensitivity response to leishmanin was significantly lower among individuals experiencing recurrent leishmaniasis than among those whose skin test converted without developing the disease. Monocyte derived macrophages from individuals with recurrent disease were more permissive in vitro to the entry of parasites than macrophages from subclinically infected individuals. In vitro proliferation of CD4 and CD8 T lymphocytes in response to intracellular amastigotes was significantly lower among individuals with a history of recurrent disease compared with subclinically infected individuals. Linear regression analyses revealed a strong direct relationship between the production of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 by peripheral blood mononuclear cells (PBMC) from resistant (subclinically infected) individuals and no correlation in the production of these cytokines by PBMC from individuals who experienced recurrent disease. The results provide evidence of differences in the innate and acquired responses to Leishmania according to the outcome of the natural infection. These findings support the feasibility of identifying the immunological bases of innate and acquired resistance through studies in naturally exposed human populations.

Intraspecific heterogeneity in the mini-exon gene localization of Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis from Colombia.

Intraspecific heterogeneity was demonstrated in the mini-exon gene localization from Leishmania (Viannia) panamensis and L. (Viannia) guyanensis. Different karyotypes were detected in human isolates circulating in endemic areas of Colombia. The presence of mini-exon gene sequences on chromosomes of different sizes, ranging from 370 to 800 kb in L. (V.) panamensis and from 500 to 800 kb in L. (V.) guyanensis, was observed and was neither strain nor species specific. In some cases, hybridization with 2 chromosomes in the same strain was observed. The variability of chromosomal localization of mini-exon gene sequences of these 2 species highlights the genetic variability of the Viannia subgenus and the potential utility of the mini-exon gene as a molecular epidemiologic marker.

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.

Sensitivity of Leishmania viannia panamensis to pentavalent antimony is correlated with the formation of cleavable DNA-protein complexes.

The emergence of Leishmania less sensitive to pentavalent antimonial agents (SbVs), the report of inhibition of purified topoisomerase I of Leishmania donovani by sodium stibogluconate (Pentostam), and the uncertain mechanism of action of antimonial drugs prompted an evaluation of SbVs in the stabilization of cleavable complexes in promastigotes of Leishmania (Viannia). The effect of camptothecin, an inhibitor of topoisomerase, and additive-free meglumine antimoniate (Glucantime) on the stabilization of cleavable DNA-protein complexes associated with the inhibition of topoisomerase was assessed in the human promonocytic cell line U-937, promastigotes of L. (Viannia) panamensis selected for SbV resistance in vitro, and the corresponding wild-type strain. The stabilization of cleavable complexes and the 50% effective dose (ED50) of SbVs for parasites isolated from patients with relapses were also evaluated. The median ED50 for the wild-type strain was 16. 7 microg of SbV/ml, while that of the line selected for resistance was 209.5 microg of SbV/ml. Treatment with both meglumine antimoniate and sodium stibogluconate (20 to 200 microg of SbV/ml) stabilized DNA-protein complexes in the wild-type strain but not the resistant line. The ED50s of the SbVs for Leishmania strains from patients with relapses was comparable to those for the line selected for in vitro resistance, and DNA-protein complexes were not stabilized by exposure to meglumine antimoniate. Cleavable complexes were observed in all Leishmania strains treated with camptothecin. Camptothecin stabilized cleavable complexes in U-937 cells; SbVs did not. The selective effect of the SbVs on the stabilization of DNA-protein complexes in Leishmania and the loss of this effect in naturally resistant or experimentally derived SbV-resistant Leishmania suggest that topoisomerase may be a target of antimonial drugs.

Epidemiologic, genetic, and clinical associations among phenotypically distinct populations of Leishmania (Viannia) in Colombia.

Phenotypic characterization of 511 strains of Leishmania, subgenus Viannia, isolated from Colombian patients was conducted based on electrophoretic polymorphisms of 13 isoenzymes. Ninety-one Colombian strains of L. braziliensis were the most heterogeneous, constituting seven zymodemes while 397 L. panamensis and 22 L. guyanensis strains yielded five and three zymodemes, respectively. Phosphogluconate dehydrogenase, nucleoside hydrolase, and superoxide dismutase were the most polymorphic enzymes in this collection of strains, and together with glucose-6-phosphate dehydrogenase, allowed the discrimination of the three aforementioned species. Hierarchical cluster analysis of the zymodemes using Jaccard's coefficient of similarities revealed two clusters, one constituted by L. braziliensis zymodemes, and another by three subgroups consisting of zymodemes of L. panamensis closely related to the species reference strain, another consisting of L. guyanensis zymodemes, and a third group distinguished by new electromorphs of proline iminopeptidase and aspartate aminotransferase that reacted with the L. panamensis-specific monoclonal antibody B-11. Multiple zymodemes of L. panamensis and L. guyanensis were found to be sympatrically transmitted in foci along the Pacific coast. Leishmania braziliensis variants were ubiquitous throughout the territory of Colombia; L. panamensis was prevalent in the western region and L. guyanensis was prevalent in the Orinoco and Amazon river basins in the eastern half of the country. Distinct zymodemes of L. panamensis predominated in the northern and southern regions of the Pacific coast. Nine zymodemes of all three species were isolated from mucosal lesions. Zymodeme 1.1 of L. braziliensis had the highest frequency of mucosal involvement (10% of the cases), and disease caused by this zymodeme had the longest mean time of evolution (31 months; P = 0.002). In addition to being useful in describing epidemiologic relationships, the intraspecific heterogeneity of strains of the Viannia subgenus within and among foci can be used to understand such fundamental questions as the pathogenicity of different populations of parasites, and the induction of cross-protection against related parasites.

Frequency of the Asn-108 and Thr-108 point mutations in the dihydrofolate reductase gene in Plasmodium falciparum from southwest Colombia.

Several point mutations in the dihydrofolate reductase (DHFR) gene of Plasmodium falciparum have been correlated with in vitro anti-folate drug resistance of laboratory and field isolates. Furthermore, two different point mutations that generate amino acid substitutions at the same position of the enzyme have been observed in all the isolates studied to date. These point mutations change a serine (Ser-108) in the wild type to an asparagine (Asn-108 mutation) or to a threonine (Thr-108 mutation). Using the polymerase chain reaction (PCR), it is possible to identify isolates that present these mutations. We used a mutation-specific PCR to screen 71 samples from several geographic locations of Colombia for the Asn-108 mutation (pyrimethamine resistance). In this initial screening 53 of 71 yielded amplification product with the DHFR mutation-specific primers. We further analyzed the 18 samples that did not amplify using a mutation-specific nested PCR. Of those 18 samples, seven amplified with primers specific for the Thr-108 mutation (proguanil resistance), one with the wild type (Ser-108), and 10 did not amplify. Of these 10 samples, three were identified as P. falciparum using a species-specific diagnostic nested PCR base on sequences from the small ribosomal RNA subunit gene. Overall, 51.6% of the samples amplified for the Asn-108 mutation, 10.9% for the Thr-108 mutation, 35.9% with the wild type specific primer, and 4.8% did not amplify with any of the DHFR primers. We observed variability in the frequency of the mutation between the different geographic location. The frequency of the Asn-108 and Thr-108 mutations in the state of Narifio was 25% each, while in Valle del Cauca the frequencies were 59% and 11%, respectively. These results contrast with observations in Brazil in which the Asn-108 mutation was found in 90% of the blood samples screened.

Permissiveness of human monocytes and monocyte-derived macrophages to infection by promastigotes of Leishmania (Viannia) panamensis.

During natural infections, Leishmania is in contact with a variety of mononuclear phagocytic cells in different tissues, including resident macrophages and monocytes mobilized to the site of infection from the bone marrow and blood circulation. Because the functional capabilities of fully differentiated macrophages and blood monocytes differ, the outcome of infection by Leishmania may depend upon the stage of differentiation of the host cells. To address this question, we evaluated Leishmania panamensis infection of (1) the human promonocytic/histiocytic cell line U-937 before and after induction of differentiation by phorbol myristate acetate; (2) fresh human peripheral blood monocytes; and (3) macrophages derived from monocytes by differentiation in vitro. Based on the percentage of cells infected and the number of parasites per cell, macrophages derived from monocytes or by induction of differentiation of U-937 cells were significantly more permissive to infection by stationary-phase L. (Viannia) panamensis promastigotes than monocytes. Increasing time and maturation in culture prior to exposure to infective promastigotes was associated with the increased permissiveness of differentiated macrophages to infection (P<0.05). The percentage of cells infected and number of amastigotes per cell increased with time postinfection for both monocytes and macrophages but remained significantly greater for macrophages. The increased expression of CD68, CD16, and lysozyme, and decreased expression of peroxidase by macrophages cultured for 5 days in vitro compared with fresh monocytes, whether adherent or in suspension, supported the distinct maturation status of these cells.

Carbohydrate and LPG expression in Leishmania viannia subgenus.

Glycosylated molecules expressed on the cell surface of Leishmania promastigotes contribute to the outcome of contact between the parasite and its invertebrate and vertebrate hosts. The expression of several such molecules is growth phase dependent. Information on the expression of carbohydrates by Leishmania of the Viannia subgenus (braziliensis complex), a widespread cause of morbidity in the Americas, is fragmentary. We have examined the relationship between growth phase and the expression of glycosylated surface structures in WHO reference strains of 3 species of the Viannia subgenus, i.e., L. panamensis, L. guyanensis, and L. braziliensis. Agglutination with lectins and the monoclonal antibody specific for the repeat unit of L. donovani lipophosphoglycan, CA7AE, distinguished logarithmic and stationary-phase promastigotes of all 3 species. Flow cytometry revealed increased heterogeneity and disparity in the expression of the repeat unit epitope in stationary-as compared to logarithmic-phase promastigotes. Biochemical analyses showed the LPG repeat unit of all 3 species reference strains to be constituted by mannose and galactose with little or no substitution and, hence, to be similar to the LPG of L. donovani. Initial quantitative analyses of L. braziliensis LPG indicated a 10-fold lower quantity of LPG in this species than L. donovani and an increase in the size of LPG in the stationary phase. These findings provide bases for isolating and biologically characterizing phenotypically distinct populations of promastigotes and for identifying molecular determinants of the host parasite-relationship among Leishmania Viannia.

In situ characterization of the human host response to Leishmania panamensis.

Both host and parasite determinants influence the outcome of Leishmania infections. Human host responses in cutaneous leishmaniasis of limited duration caused by a single species of the Viannia (V) subgenus were studied in skin biopsies obtained from lesions caused by Leishmania (V) panamensis in 31 male patients from the Colombian Pacific Coast. Dermal infiltrates and histopathologic changes were characterized using monoclonal antibodies and an indirect immunoperoxidase method. Dermal distribution of T-cell subpopulations and B-lymphocytes was nonrandom: CD4+ and CD8+ T cells were most frequent in the upper dermis, and B cells were most abundant in the lower dermis. Parasites, macrophages, and neutrophils were localized predominantly in the middermis. Multiple regression analyses to establish associations between lesion type (ulcer, nodule, or papule), immune response data (Montenegro skin test, indirect fluorescence antibody test titers, lymphocyte blastogenesis), and particular cell populations demonstrated statistically significant correlations between CD4+ lymphocytes and macrophages (p < 0.05). CD8+ lymphocytes were associated with plasma cells (p < 0.001), as was the presence of amastigotes (p < 0.05). These associations and the in situ divergence of CD4 and CD8 ratios suggest that prognostic indicators for disease evolution could be identified by prospective analysis of cellular relationships and response to therapy.

The inflammatory response promotes cutaneous metastasis in hamsters infected with Leishmania (Viannia) panamensis.

The influence of nonspecific and immunologically elicited inflammatory responses on the development of metastatic lesions was examined in the hamster model of Leishmania (Viannia) panamensis infection. Delayed type hypersensitivity (DTH) responses were induced using the contact sensitizing agent DNFB (2, 4-dinitro-1-fluorobenzene) and infection with L. panamensis followed by intradermal application of leishmanin. Nonspecific inflammatory response was achieved by the surgical excision of toes. The inductive and eliciting procedures were performed on the ears and fore and hind paws of the right side of experimental groups of hamsters that were inoculated in the snout with a highly metastatic strain of L. panamensis (MHOM/COL/84/1099). Skin metastases were detected by physical evaluation at 15-day intervals over a period of 7-8 mo. Suspected metastases were parasitologically confirmed by culture of tissue fluid aspirated from the lesion. The frequency of metastatic lesions was greater in hamsters subjected to inflammatory stimuli (14/38) than control animals (6/33; P = 0.035). Likewise, the frequency of metastases at the site of induction and elicitation of inflammation (18/22 lesions) in the experimental groups was greater than that observed at the same site in control animals (5/11 lesions; P = 0.017). These findings support a causal relationship between inflammatory response and the development of lesions in this model of secondary disease caused by L. panamensis.

Infectivity of Trypanosoma rangeli in a promonocytic mammalian cell line.

The existence of an intracellular stage of Trypanosoma rangeli in the vertebrate host was evaluated by experimental infection of the U937 histiocytic cell line with the San Agustín strain and the Ub66-5b clone. The identity of the parasites at the beginning and end of the experiments was confirmed through biological behavior in the vector and mammal hosts, isoenzymes, polymerase chain reaction (PCR), and monoclonal antibodies. Infectivity to U937 cells of T. rangeli obtained from culture and salivary glands was evaluated under different experimental conditions. These included 34 C vs. 37 C, opsonized vs. nonopsonized parasites, and 2, 4, 6, 24, 48, and 72 hr of cell-parasite contact. Trypanosoma rangeli adopted a characteristic nondividing amastigote-like form within U937 cells, which was different in size (P = 0.001) from Trypanosoma cruzi amastigotes. Culture forms of T. rangeli were more infective than parasites from salivary glands (P = 0.049) but were less infective than T. cruzi (P = 0.0001). Variations in temperature (34-37 C) and complement opsonization did not affect infectivity. Viability of intracellular forms was confirmed by feeding Rhodnius prolixus with T. rangeli-infected cells. Resistance of T. rangeli to the intracellular milieu could be an important mechanism in producing chronic infections in mammals and in the infection of triatomines.

Genotypic polymorphisms in experimental metastatic dermal leishmaniasis.

Molecular karyotype and kDNA restriction analyses were utilized to examine the genetic heterogeneity and plasticity of the Leishmania (Viannia) guyanensis strain WHI/BR/78/M5313, composed of metastatic and non-metastatic populations. Cloning revealed that the strain was constituted by multiple closely related populations that were distinguishable by restriction fragment polymorphisms in kDNA. Size polymorphisms in molecular karyotype were not detected. Passage of clones in hamsters and recovery of parasites from cutaneous metastatic lesions yielded evidence of further genetic heterogeneity among some of the progeny populations. Overall, six kDNA minicircle restriction patterns or schizodemes were observed among clones, subclones and progeny. Although the possibility that population heterogeneity was not resolved by cloning cannot be ruled out, subcloning and kDNA restriction analysis to determine whether the putative clones consisted of homogeneous populations showed the schizodeme of subclones of 3 out of 4 clones to be identical to the clone of origin, while a subclone of the fourth had a co-efficient of similarity of 0.95. Metastasis did not segregate with a particular schizodeme: all six restriction profiles were represented among populations isolated from metastatic lesions and some clones with the same restriction profile did not produce metastatic lesions. The strain from which the clones, subclones and progeny were derived had a kDNA restriction pattern identical to the most prevalent schizodeme (38%) among these subpopulations. This finding together with the reappearance of the repertoire of schizodemes found among clones in the populations recovered from metastatic lesions in hamsters inoculated with a single clone, suggest that sequence polymorphisms in kDNA can emerge during infection.

Epidemiology of cutaneous leishmaniasis in Colombia: a longitudinal study of the natural history, prevalence, and incidence of infection and clinical manifestations.

This prospective study measured the incidence of Leishmania infection, by Leishmanin skin test (LST) conversion, and leishmaniasis, by new acquisition of lesions, in a Leishmania braziliensis endemic area of Colombia, during 7243 person-years. The incidence rate of infection and leishmaniasis varied greatly by village, ranging from 2.8 to 23.0/100 person-years and 0.0 to 20.4/1000 person-years, respectively. Adult males experienced greater rates of both infection and leishmaniasis. Most primary infections (91%) were subclinical initially. Typical scars were predictive of subsequent leishmaniases both for persons initially LST-reactive (risk ratio = 11.3, P = .003) and for those initially nonreactive (risk ratio = 3.2, P = .02). Only one-third of the diagnosed leishmaniasis cases (24/77) were due to newly acquired infections in naive hosts. The relative contribution of existing lesions, recurrences, and new infections to the burden of disease should be considered in the planning of leishmaniasis control programs.

Epidemiology of cutaneous leishmaniasis in Colombia: environmental and behavioral risk factors for infection, clinical manifestations, and pathogenicity.

Through a longitudinal, active surveillance for Leishmania (Viannia) braziliensis and Leishmania (Viannia) panamensis infection and lesions on the Pacific Coast of Colombia, risk factors for infection (leishmanin skin test conversion), leishmanial lesions, and pathogenicity were examined. Risk factor information was obtained prior to and independently of case ascertainment. Similar factors were associated with acquisition of infection and of leishmaniasis, including male sex, age > 10 years, and farming occupation. The behaviors of entering the forest after sunset, hunting, and lumbering were most strongly associated with Leishmania infection independently of age, sex, and farming occupation. Environmental conditions associated with infection, including tall trees near the home, home located > 15 m from the nearest neighbor, and floor and roof made of open material, were less strong predictors of risk. Pathogenicity, the risk of lesion given a new infection, was reduced in those > 30 years of age and those entering the forest frequently.