Pentoxifylline in the Treatment of Cutaneous Leishmaniasis: A Randomized Clinical Trial in Colombia.

Addition of the immunomodulator pentoxifylline (PTX) to antimonial treatment of mucosal leishmaniasis has shown increased efficacy. This randomized, double-blind, placebo-controlled trial evaluated whether addition of pentoxifylline to meglumine antimoniate (MA) treatment improves therapeutic response in cutaneous leishmaniasis (CL) patients. Seventy-three patients aged 18−65 years, having multiple lesions or a single lesion ≥ 3 cm were randomized to receive: intramuscular MA (20 mg/kg/day × 20 days) plus oral PTX 400 mg thrice daily (intervention arm, n = 36) or MA plus placebo (control arm, n = 37), between 2012 and 2015. Inflammatory gene expression was evaluated by RT-qPCR in peripheral blood mononuclear cells from trial patients, before and after treatment. Intention-to-treat failure rate was 35% for intervention vs. 25% for control (OR: 0.61, 95% CI: 0.21−1.71). Per-protocol failure rate was 32% for PTX, and 24% for placebo (OR: 0.50, 95% CI: 0.13−1.97). No differences in frequency or severity of adverse events were found (PTX = 142 vs. placebo = 140). Expression of inflammatory mediators was unaltered by addition of PTX to MA. However, therapeutic failure was associated with significant overexpression of il1ß and ptgs2 (p < 0.05), irrespective of study group. No clinical benefit of addition of PTX to standard treatment was detected in early mild to moderate CL caused by Leishmania (V.) panamensis.

Parasitological Confirmation and Analysis of Leishmania Diversity in Asymptomatic and Subclinical Infection following Resolution of Cutaneous Leishmaniasis.

BACKGROUND: The contribution of individuals with subclinical infection to the transmission and endemicity of cutaneous leishmaniasis (CL) is unknown. Immunological evidence of exposure to Leishmania in residents of endemic areas has been the basis for defining the human population with asymptomatic infection. However, parasitological confirmation of subclinical infection is lacking. METHODS: We investigated the presence and viability of Leishmania in blood and non-invasive mucosal tissue samples from individuals with immunological evidence of subclinical infection in endemic areas for CL caused by Leishmania (Viannia) in Colombia. Detection of Leishmania kDNA was conducted by PCR-Southern Blot, and parasite viability was confirmed by amplification of parasite 7SLRNA gene transcripts. A molecular tool for genetic diversity analysis of parasite populations causing persistent subclinical infection based on PCR amplification and sequence analysis of an 82bp region between kDNA conserved blocks 1 and 2 was developed. PRINCIPAL FINDINGS: Persistent Leishmania infection was demonstrated in 40% (46 of 114) of leishmanin skin test (LST) positive individuals without active disease; parasite viability was established in 59% of these (27 of 46; 24% of total). Parasite burden quantified from circulating blood monocytes, nasal, conjunctival or tonsil mucosal swab samples was comparable, and ranged between 0.2 to 22 parasites per reaction. kDNA sequences were obtained from samples from 2 individuals with asymptomatic infection and from 26 with history of CL, allowing genetic distance analysis that revealed diversity among sequences and clustering within the L. (Viannia) subgenus. CONCLUSIONS: Our results provide parasitological confirmation of persistent infection among residents of endemic areas of L. (Viannia) transmission who have experienced asymptomatic infection or recovered from CL, revealing a reservoir of infection that potentially contributes to the endemicity and transmission of disease. kDNA genotyping establishes proof-of-principle of the feasibility of genetic diversity analysis in previously inaccessible and unexplored parasite populations in subclinically infected individuals.

Miltefosine and antimonial drug susceptibility of Leishmania Viannia species and populations in regions of high transmission in Colombia.

BACKGROUND: Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance. METHODOLOGY/PRINCIPAL FINDINGS: In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (Sb(V)); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to Sb(V) was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980-1989 and 2000-2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to Sb(V). Resistance to HePC and Sb(V) occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb(V). Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb(V) were discerned among L. V. panamensis strains isolated during 1980-1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2. CONCLUSIONS/SIGNIFICANCE: Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.

Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.

Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 µmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 µmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 µmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/ß-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = -0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis.

Validez y confiabilidad de una prueba tamiz del lenguaje para preescolares en edades de 2 a 3 años / Validity and reliability of a language screening test for preschoolers from 2 to 3 years of age

El objetivo del trabajo fue determinar la validez y confiabilidad de la prueba tamiz del lenguaje en preescolares de 2 a 3 años del municipio de Juan De Acosta del Departamento del Atlántico. Fue un estudio descriptivo, cuantitativo, realizado a treinta preescolares del plan de atención "Educación y Desarrollo Integral para cada Colombiano". Se evaluó la validez del instrumento por parte de los jueces expertos, en relación a cada uno de los aspectos del lenguaje que contiene la prueba. La confiabilidad del instrumento, fue hallada mediante el estadístico ALPHA DE CRONBACH, aplicado al total de la muestra. Se encontró que el 66,7% están de acuerdo con que el aspecto pragmático es válido y el 33.3% que este aspecto es pertinente, claro y completo. Referente a los aspectos semántico y morfosintáctico en un 8.43, 8.46 y8.75 respectivamente. El aspecto fonético-fonológico de la prueba obtuvo una confiabilidad de 4.49, lo que determina que dicho aspecto presentó una baja consistencia interna. Se concluyó que los índices obtenidos en la validación del instrumento generados por el coeficiente de correlación, demuestran que la prueba tamiz de lenguaje diseñada para preescolares de 2 a 3 años es válida, pertinente, clara y completa.

The purpose was determinate the validity and reliability of the screening test of language in pres-choolers 2 to 3 years in Juan de Acosta town located in the department of Atlántico. A descriptive and quantitative approach to the validity and reliability of a test about language assessment was done to thirty (30) preschool from the ages 2 to 3. Based on the " Education was determined and for each Colombian Integral development " plan, applied Juan de Acosta in the department of the Atlantic. These aspects (validity and reliability) of the test made to the children from Juan de Acosta were endorsed by expert judges that determined, and validated each of the aspects of language related to the contains in the test. The values show that 66.7 % of the experts agreed that the pragmatic aspect is valid adding that is relevant, and the other 33.3 % ensure that is clear and complete. Regarding to the semantic aspect, the results show that 100 % of the judges agree that this aspect is valid 66.7 % affirm is relevant, clear and complete, while 33.3% somehow agreed with this judgments. In the morphosyntactic aspect, the results show that 11% of judges ensure this aspect is valid, relevant, clear and complete. As for the phonetic-phonological aspect, the results gave a 100% validation from the judges in its validation and relevance, 66.7% agreed that is clear and complete while 33.3 % somewhat agree with this. The reliability of the instrument, which was found by Cronbach's alpha, after it was applied to a sample of 30 preschool students established that this test is reliable in the pragmatic, semantic and morphosyntactic aspects a 8.43, 8.46 and 8.75 respectively . The phonetic - phonological aspect of the test obtained a reliability of 4.49, which determines that this aspect has low internal consistency

Human macrophage response to L. (Viannia) panamensis: microarray evidence for an early inflammatory response.

BACKGROUND: Previous findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response. CONCLUSIONS: Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48-96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

Novel approach to in vitro drug susceptibility assessment of clinical strains of Leishmania spp.

Resistance to antimonial drugs has been documented in Leishmania isolates transmitted in South America, Europe, and Asia. The frequency and distribution of resistance to these and other antileishmanial drugs are unknown. Technical constraints have limited the assessment of drug susceptibility of clinical strains of Leishmania. Susceptibility of experimentally selected lines and 130 clinical strains of Leishmania panamensis, L. braziliensis, and L. guyanensis to meglumine antimoniate and miltefosine was determined on the basis of parasite burden and percentage of infected U-937 human macrophages. Reductions of infection at single predefined concentrations of meglumine antimoniate and miltefosine and 50% effective doses (ED(50)s) were measured and correlated. The effects of 34°C and 37°C incubation temperatures and different parasite-to-host cell ratios on drug susceptibility were evaluated at 5, 10, and 20 parasites/cell. Reduction of the intracellular burden of Leishmania amastigotes in U-937 cells exposed to the predefined concentrations of meglumine antimoniate or miltefosine discriminated sensitive and experimentally derived resistant Leishmania populations and was significantly correlated with ED(50) values of clinical strains (for meglumine antimoniate, ρ = -0.926 and P < 0.001; for miltefosine, ρ = -0.906 and P < 0.001). Incubation at 37°C significantly inhibited parasite growth compared to that at 34°C in the absence of antileishmanial drugs and resulted in a significantly lower ED(50) in the presence of drugs. Susceptibility assessment was not altered by the parasite-to-cell ratio over the range evaluated. In conclusion, measurement of the reduction of parasite burden at a single predetermined drug concentration under standardized conditions provides an efficient and reliable strategy for susceptibility evaluation and monitoring of clinical strains of Leishmania.

Murine model of chronic L. (Viannia) panamensis infection: role of IL-13 in disease.

Leishmania (Viannia) organisms are the most prevalent etiologic agents of human cutaneous leishmaniasis in the Americas. Nevertheless, our knowledge of the immunological mechanisms exploited by L. (Viannia) organisms remains limited and the mechanisms underlying disease are not well understood. Here, we report the development of a BALB/c mouse model of L. (V.) panamensis infection that is able to reproduce chronic disease, with persistent infection and clinically evident lesions for over 1 year. The immune response of the mouse resembles that found for L. (V.) panamensis-infected patients with chronic and recurrent lesions, presenting a mixed Th1/Th2 response with the presence of TNF-α, IFN-γ, IL-10 and IL-13. Using immunodeficient mice, the critical role for IL-13 and/or IL-4Rα in determining susceptibility to chronic infection was evident. With the induction of healing in the immunodeficient mice, increases in IFN-γ and IL-17 were found, concomitant with parasite control and elimination. Specifically, increases in CD4(+) (but not CD8(+)) T cells producing IFN-γ were observed. These results suggest that IL-13 represents an important target for disease control of L. (V.) panamensis infection. This murine model should be useful to further understand the pathology associated with chronic disease and to develop methods for the treatment and prevention of leishmaniasis caused by L. (Viannia) parasites.

T-bet, GATA-3, and Foxp3 expression and Th1/Th2 cytokine production in the clinical outcome of human infection with Leishmania (Viannia) species.

BACKGROUND: T cell differentiation determines susceptibility and resistance to experimental cutaneous leishmaniasis, yet mixed T1/Th2 responses characterize the clinical spectrum of human infection with Leishmania (Viannia) species. MATERIALS AND METHODS: To discern the interrelationship of T cell differentiation and outcome of human infection, we examined factors that regulate T cell differentiation and Th1/Th2 cytokine responses in asymptomatic infection, active and historical chronic and recurrent cutaneous leishmaniasis. T-bet, GATA-3, Foxp3, and cytokine gene expression were quantified by real-time polymerase chain reaction and correlated with interleukin 2, interferon gamma, tumor necrosis factor alpha, interleukin 4, interleukin 13, and interleukin 10 secretion during in vitro response to live Leishmania panamensis. RESULTS: Higher GATA-3 expression than T-bet expression occurred throughout the 15 days of coculture with promastigotes; however, neither transcription nor secretion of interleukin 4 was detected. A sustained inverse correlation between GATA-3 expression and secretion of proinflammatory cytokines interferon gamma and tumor necrosis factor alpha was observed in asymptomatic infection. In contrast, higher T-bet expression and a higher ratio of T-bet to GATA-3 characterized active recurrent disease. Down-regulation of T-bet and GATA-3 expression and increased interleukin 2 secretion, compared with control subjects, was directly correlated with Foxp3 expression and interleukin 13 secretion in chronic disease. CONCLUSIONS: Regulation of the inflammatory response rather than biased Th1/Th2 response distinguished asymptomatic and recalcitrant outcomes of infection with Leishmnania viannia species.

Antimony resistance and trypanothione in experimentally selected and clinical strains of Leishmania panamensis.

The participation of trypanothione in clinical and experimental antimony (Sb) resistance in Leishmania panamensis was examined by using specific inhibitors. Buthionine sulfoximine (BSO) significantly reversed the resistance to trivalent Sb (Sb(III)) of promastigotes of experimentally derived Sb-resistant lines, supporting the participation of a trypanothione-mediated mechanism of resistance. In contrast, promastigotes of strains isolated at the time of clinical treatment failure and resistant to pentavalent Sb (Sb(V)) as intracellular amastigotes were not cross resistant to Sb(III), and BSO had little or no effect on susceptibility. Difluoromethylornithine did not alter the Sb(III) susceptibilities of experimentally selected lines or clinical strains. The mechanisms of acquired resistance emerging in clinical settings may differ from those selected by in vitro exposure to Sb.

Treponema pallidum elicits innate and adaptive cellular immune responses in skin and blood during secondary syphilis: a flow-cytometric analysis.

BACKGROUND: Syphilis is caused by the spirochetal pathogen Treponema pallidum. The local and systemic cellular immune responses elicited by the bacterium have not been well studied in humans. METHODS: We used multiparameter flow cytometry to characterize leukocyte immunophenotypes in skin and peripheral blood from 23 patients with secondary syphilis and 5 healthy control subjects recruited in Cali, Colombia. Dermal leukocytes were obtained from fluid aspirated from epidermal suction blisters raised over secondary syphilis skin lesions. RESULTS: Compared with peripheral blood (PB), blister fluids (BFs) were enriched for CD4(+) and CD8(+) T cells, activated monocytes/macrophages, and CD11c(+) monocytoid and CD11c(-) plasmacytoid dendritic cells (mDCs and pDCs, respectively). Nearly all mDCs in BFs expressed the human immunodeficiency virus (HIV) coreceptors CCR5 and DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and high levels of human leukocyte antigen (HLA)-DR. Dermal pDCs expressed both HIV coreceptors without increases in HLA-DR intensity. Compared with normal blood, circulating mDCs in patients with syphilis expressed higher levels of both CCR5 and DC-SIGN, whereas circulating pDCs in patients expressed only higher levels of DC-SIGN. Most dermal T cells were CCR5(+) and displayed a memory (CD27(+)/CD45RO(+)) or memory/effector (CD27(-)/CD45RO(+)) immunophenotype. A corresponding shift toward memory and memory/effector immunophenotype was clearly discernible among circulating CD4(+) T cells. Compared with PB from control subjects, a larger percentage of CD4(+) T cells in PB from patients with syphilis expressed the activation markers CD69 and CD38. CONCLUSIONS: During secondary syphilis, T. pallidum simultaneously elicits local and systemic innate and adaptive immune responses that may set the stage for the bidirectional transmission of HIV.

Comparative immunohistological analysis of the Montenegro skin test reaction in asymptomatic infection and in acute and chronic cutaneous leishmaniasis / Análisis inmunohistopatológico comparativo de la reacción a la prueba cutánea de Montenegro en infección asintomática y en leishmaniasis cutánea aguda y crónica

Introducción. La reacción a la prueba cutánea de Montenegro (a leishmanina) y la lesión de leishmaniasis comparten características fundamentales de la reacción de hipersensibilidad de tipo retardado. Objetivos. Determinar si la respuesta cutánea a leishmanina se aproxima y podría modelar la respuesta inflamatoria e inmune temprana a la infección por Leishmania. Materiales y métodos. Este estudio comparó la respuesta inflamatoria de biopsias de lesiones agudas (tiempo de evolución £1 mes) y lesiones crónicas (tiempo de evolución ³6 meses) con su respectiva reacción a la prueba cutánea de Montenegro, y con la reacción a la prueba de Montenegro de individuos infectados asintomáticos. Resultados. La proporción de linfocitos T CD4+ y CD8+, células fagocíticas mononucleares y granulocitos fue similar entre lesiones agudas y sus reacciones a la prueba cutánea a leishmanina. En contraste, los linfocitos CD4+ (32,6 por ciento) representaron una proporción significativamente mas baja, y los linfocitos B (20 por ciento) y macrófagos (27 por ciento) una proporción significativamente más alta del infiltrado celular en lesiones crónicas que en sus correspondientes reacciones a la prueba cutánea de Montenegro (CD4+: 43,7 por ciento, linfocitos B: 0.9 por ciento; macrófagos: 17,5 por ciento). Se encontró una asociación positiva entre linfocitos T CD8+ y macrófagos ( P=0,038) en la reacción a la prueba cutánea de Montenegro de los individuos con infección asintomática, mientras que los linfocitos T CD8+ y CD4+ estuvieron asociados positivamente en las reacciones a la prueba cutánea de Montenegro de pacientes crónicos ( P=0,002). Adicionalmente, la proporción de linfocitos B en lesiones crónicas (20 por ciento) fue más alta que en lesiones agudas (5,3 por ciento) ( P=0,002). Conclusión. La reacción cutánea de Montenegro permitió diferenciar la respuesta celular inmune entre infección asintomática y enfermedad crónica, y simula la respuesta temprana a la infección.

Introduction. The Montenegro skin test reaction and leishmaniasis lesions share fundamental characteristics of a delayed type hypersensitivity reaction. Objectives. To determine whether the Montenegro skin test reaction (response to leishmanin) might approximate and thereby provide insight into the early inflammatory and immune response to Leishmania infection. Materials and methods. We compared the inflammatory response in biopsies of acute (evolution time £one month), and chronic lesions (evolution time ³6 months) with the Montenegro skin test reaction in the corresponding patients, and with the Montenegro skin test of asymptomatically infected volunteers. Results. The proportion of CD4+ and CD8+ T lymphocytes, mononuclear phagocytes and granulocytes were similar in acute lesions and in their corresponding Montenegro skin test reactions. In contrast, CD4+ lymphocytes (32.6%) represented a significantly lower, and B cells (20%) and macrophages (27%) a significantly higher proportion of the cellular infiltrate in chronic lesions as compared to reactions in the corresponding skin test site (CD4+: 43.7%, B cells: 0.9%; macrophages: 17.5%). CD8+ T lymphocytes and macrophages were positively associated ( P=0.038) in the Montenegro skin test of asymptomatically infected individuals whereas CD8+ and CD4+ T cells were positively associated in the Montenegro skin test of chronic patients ( P=0.002). Notably, B cells were markedly more frequent in chronic lesions (20%) than in acute lesions (5.3%) ( P=0.002). Conclusion. The Montenegro skin test distinguished the cellular immune response to Leishmania in asymptomatic infection and chronic disease and may provide a surrogate of the early response to infection.

Evidence for leishmania (viannia) parasites in the skin and blood of patients before and after treatment.

BACKGROUND: American cutaneous leishmaniasis is considered to be a zoonotic disease transmitted by sand flies that feed on infected sylvatic mammals. However, the "domestication" of transmission and the increase in treatment failure with antimonial drugs have raised the suspicion of anthroponotic transmission of American cutaneous leishmaniasis. METHODS: The objective of the present study was to explore the potential of humans as a source of infection for sand flies. Biological (xenodiagnosis and culture) and molecular (polymerase chain reaction/Southern blot) detection methods were used to evaluate peripheral-blood monocytes and tissue fluids from sites accessible to sand flies from 59 adult patients with parasitologically confirmed American cutaneous leishmaniasis. RESULTS: Overall, 44.1% of patients (26/59) presented biological and/or molecular evidence of Leishmania parasites in normal skin, peripheral-blood monocytes, lesion scars, or lesion border (by xenodiagnosis) before (18/59 [30.5%]) or after (10/27 [37.0%]) treatment. Leishmania parasites were cultured from the unaffected skin of 2 (3.6%) of 55 patients, and xenodiagnosis gave positive results for 5 (8.8%) of 57 patients before treatment. CONCLUSIONS: The presence of Leishmania parasites in the unaffected skin and peripheral-blood monocytes of a high proportion of patients even after treatment and the acquisition of infection by sand flies support the plausibility of anthroponotic transmission of American cutaneous leishmaniasis.

Resistance to antimony and treatment failure in human Leishmania (Viannia) infection.

BACKGROUND: Failure of antimonial therapy has been increasingly reported in anthroponotic visceral leishmaniasis and in cutaneous disease. The role of drug resistance in treatment failure has been difficult to ascertain because therapeutic response is multifactorial, and the efficacy of antimonial drugs depends on an effective immune response. In this study, we sought to determine whether standard treatment selects for resistant organisms and whether drug resistance contributes to treatment failure. METHODS: We evaluated the susceptibility to antimony of 19 strains isolated before treatment with meglumine antimoniate and 21 strains isolated at treatment failure from 20 patients. The 50% effective dose (ED50) of antimony in the form of additive-free meglumine antimoniate was determined for intracellular amastigotes in human promonocytic U-937 cells. RESULTS: Before treatment, 16% of strains (3/19) showed primary resistance (ED50 of >128 microg Sb/mL), whereas 84% (16/19) were susceptible (ED50 of <20 microg Sb/mL). However, 88% of susceptible strains (14/16) had ED90 values of >128 microg Sb/mL. At treatment failure, 40% of strains (8/20) were resistant. Secondary resistance was documented in 4 patients. CONCLUSIONS: Primary and secondary resistance to antimony can contribute to treatment failure in American cutaneous leishmaniasis. Selection for resistance to antimony occurs during standard treatment with antimonial drugs, and primary resistance to antimony supports the plausibility of anthroponotic transmission.

Pathogenicity and protective immunogenicity of cysteine proteinase-deficient mutants of Leishmania mexicana in non-murine models.

This study demonstrates that deletion of cysteine proteinase (CP) genes diminishes pathogenicity of Leishmania mexicana in non-murine experimental host models while preserving immunogenicity. Both cpb and cpa/cpb-deficient lines induced delayed disease onset, smaller lesions and lower parasite burden in hamsters. cpa/cpb-deficient L. mexicana grew more slowly as promastigotes and presented lower infectivity and growth in human mononuclear phagocytic host cells. Protection against homologous challenge comparable to that induced by infection with the virulent wild-type (WT) L. mexicana strain was achieved in the highly susceptible hamster model by immunization with 1000 cpb-deficient promastigotes. CP-deficient L. mexicana elicited significantly lower levels of Th2-associated cytokines IL-10 and TGF-beta than the WT in the primary lesion of hamsters. These findings support the feasibility of using genetically attenuated live Leishmania to achieve protective immunity.

Comparative immunohistological analysis of the Montenegro skin test reaction in asymptomatic infection and in acute and chronic cutaneous leishmaniasis.

INTRODUCTION: The Montenegro skin test reaction and leishmaniasis lesions share fundamental characteristics of a delayed type hypersensitivity reaction. OBJECTIVES: To determine whether the Montenegro skin test reaction (response to leishmanin) might approximate and thereby provide insight into the early inflammatory and immune response to Leishmania infection. MATERIALS AND METHODS: We compared the inflammatory response in biopsies of acute (evolution time < or = one month), and chronic lesions (evolution time > or = 6 months) with the Montenegro skin test reaction in the corresponding patients, and with the Montenegro skin test of asymptomatically infected volunteers. RESULTS: The proportion of CD4+ and CD8+ T lymphocytes, mononuclear phagocytes and granulocytes were similar in acute lesions and in their corresponding Montenegro skin test reactions. In contrast, CD4+ lymphocytes (32.6%) represented a significantly lower, and B cells (20%) and macrophages (27%) a significantly higher proportion of the cellular infiltrate in chronic lesions as compared to reactions in the corresponding skin test site (CD4+: 43.7%, B cells: 0.9%; macrophages: 17.5%). CD8+ T lymphocytes and macrophages were positively associated (P = 0.038) in the Montenegro skin test of asymptomatically infected individuals whereas CD8+ and CD4+ T cells were positively associated in the Montenegro skin test of chronic patients (P = 0.002). Notably, B cells were markedly more frequent in chronic lesions (20%) than in acute lesions (5.3%) (P = 0.002). CONCLUSION: The Montenegro skin test distinguished the cellular immune response to Leishmania in asymptomatic infection and chronic disease and may provide a surrogate of the early response to infection.

Heterogeneity, geographic distribution, and pathogenicity of serodemes of Leishmania viannia in Colombia.

Leishmania Viannia strains from 1,092 patients who acquired dermal leishmaniasis in endemic regions of Colombia were analyzed for expression of species and subgenus specific epitopes. Eight monoclonal antibodies prepared against membranes of the major species of the Viannia subgenus and previously shown to distinguish these species, recognized low molecular mass (< 45kD) membrane components. Thirteen widely but non-uniformly distributed serodemes were identified: one unique to L. panamensis, four unique to L. braziliensis and eight that were common to L. braziliensis and L. guyanensis. Thirty-seven percent of Colombian L. braziliensis strains concomitantly typed by isoenzymes were null, i.e., not recognized by the corresponding species-specific B-16 or B-18 antibodies. No Colombian L. guyanensis strains were recognized by the antibody specific for this species (B-19). In contrast, L. panamensis-specific B-4 and B11 antibodies recognized > 98% of the L. panamensis strains. Null strains of L. braziliensis and L. panamensis were more frequently isolated from mucosal leishmaniasis than strains that expressed species specific epitopes, suggesting that these strains may be more pathogenic.

Evaluación del factor de necrosis tumoral en tuberculosis

El factor de necrosis tumoral (TNF) es una citoquina que juega un papel muy importante en la patogénesis de la tuberculosis. Como un producto de monocitos-macrófagos y de linfocitos, puede ejercer efectos benéficos y perjudiciales como resultado de la infección con M. tuberculosis. En este estudio se implementaron y optimizaron las condiciones de un bioensayo con células L-929 para cuantificar TNF en un grupo de 14 pacientes tuberculosos y 15 controles sanos. Todos los pacientes fueron diagnosticados por baciloscopia y las muestras obtenidas antes de iniciar el tratamiento. En ambos grupos se realizó la prueba de tuberculina por el método de Mantoux, se obtuvieron células mononucleares de sangre periférica para efectuar ensayos de linfoproliferación y purificación de céluls adherentes. En los sobrenadantes obtenidos de los cultivos celulares se determinaron los niveles de TNF. Los experimento dosis-respuesta al PPD indicaron que 10 ug/ml era la dosis óptima a usar. Al comparar la liberación de TNF por células adherentes (monocitos) y células mononucleares completas (monocitos y linfocitos) encontramos que ambas poblaciones celulares producen TNF espontáneamente y en respuesta al PPD, con mayor liberación por parte de los monocitos. Con respecto a la producción de TNF y la respuesta proliferativa al PPD, se encontró correlación estadísticamente significativa entre estas dos variables (p=0,001). Nuestros hallazgos concuerdan con el modelo de respuesta Th1, en el cual se libera INF-gamma como respuesta inicial a la infección por la micobacteria, induciendo la posterior liberación de TNF con el propósito de controlar la infección.