The different impact of drug-resistant Leishmania on the transcription programs activated in neutrophils.

Drug resistance threatens the effective control of infections, including parasitic diseases such as leishmaniases. Neutrophils are essential players in antimicrobial control, but their role in drug-resistant infections is poorly understood. Here, we evaluated human neutrophil response to clinical parasite strains having distinct natural drug susceptibility. We found that Leishmania antimony drug resistance significantly altered the expression of neutrophil genes, some of them transcribed by specific neutrophil subsets. Infection with drug-resistant parasites increased the expression of detoxification pathways and reduced the production of cytokines. Among these, the chemokine CCL3 was predominantly impacted, which resulted in an impaired ability of neutrophils to attract myeloid cells. Moreover, decreased myeloid recruitment when CCL3 levels are reduced was confirmed by blocking CCL3 in a mouse model. Collectively, these findings reveal that the interplay between naturally drug-resistant parasites and neutrophils modulates the infected skin immune microenvironment, revealing a key role of neutrophils in drug resistance.

High prevalence of dengue, Zika, and chikungunya viruses in blood donors during a dengue outbreak and an endemic period in Colombia.

Objective: Arboviruses pose a challenge in ensuring the supply of pathogen-free blood components because they are not routinely screened in blood banks, and blood components from infected asymptomatic donors could be transfused. This study aimed to detect and characterize arboviral infections in Colombian blood donors. Methods: In a cross-sectional study, the prevalence of dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses and co-infections of blood donors were compared between an epidemic period (November 2019-February 2020, n = 462) and an endemic period (November 2021-August 2022, n = 1,119). Viral RNA from each donor serum was purified, and the viruses were detected using a previously standardized multiplex hemi-nested RT-PCR protocol. Subsequently, donors who tested positive were surveyed 15 days after the detection of the virus to identify clinical characteristics related to the arboviral infection. The prevalences of each virus were presented as percentages and compared between epidemic and endemic periods. Results: Significantly higher prevalences were found in the epidemic period compared with the endemic period for DENV (14.5 vs. 1.9%), ZIKV (7.8 vs. 0.3%), CHIKV (8 vs. 3.3%), and co-infections (4.3 vs. 0.2%). The survey response rate of positive donors in the two periods was 83/175 (47%). In total, 57% of the donors surveyed were asymptomatic. Symptomatic donors most frequently reported headache (31%), malaise (13%), arthralgia (10%), and fever/chills (8%). Conclusion: The prevalence observed in epidemic and endemic periods was higher than that reported in other studies in the Americas. The high proportion of asymptomatic cases found, in addition to the mild and nonspecific manifestations among the symptomatic, may limit the effectiveness of the donor selection criteria used to mitigate the risk of transfusion-transmitted arboviruses.

Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 µg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

Potency and Preclinical Evidence of Synergy of Oral Azole Drugs and Miltefosine in an Ex Vivo Model of Leishmania (Viannia) panamensis Infection.

Failure of treatment of cutaneous leishmaniasis with antimonial drugs and miltefosine is frequent. Use of oral combination therapy represents an attractive strategy to increase efficacy of treatment and reduce the risk of drug resistance. We evaluated the potency of posaconazole, itraconazole, voriconazole, and fluconazole and the potential synergy of those demonstrating the highest potency, in combination with miltefosine (HePC), against infection with Leishmania (Viannia) panamensis. Synergistic activity was determined by isobolograms and calculation of the fractional inhibitory concentration index (FICI), based on parasite quantification using an ex vivo model of human peripheral blood mononuclear cells (PBMCs) infected with a luciferase-transfected, antimony and miltefosine sensitive line of L. panamensis. The drug combination and concentrations that displayed synergy were then evaluated for antileishmanial effect in 10 clinical strains of L. panamensis by reverse transcription-quantitative (qRT-PCR) of Leishmania 7SLRNA. High potency was substantiated for posaconazole and itraconazole against sensitive as well as HePC- and antimony-resistant lines of L. panamensis, whereas fluconazole and voriconazole displayed low potency. HePC combined with posaconazole (Poz) demonstrated evidence of synergy at free drug concentrations achieved in plasma during treatment (2 µM HePC plus 4 µM Poz). FICI, based on 70% and 90% reduction of infection, was 0.5 for the sensitive line. The combination of 2 µM HePC plus 4 µM Poz effected a significantly greater reduction of infection by clinical strains of L. panamensis than individual drugs. Orally administrable miltefosine/posaconazole combinations demonstrated synergistic antileishmanial capacity ex vivo against L. panamensis, supporting their potential as a novel therapeutic strategy to improve efficacy and effectiveness of treatment.

Neutrophil Activation: Influence of Antimony Tolerant and Susceptible Clinical Strains of L. (V.) panamensis and Meglumine Antimoniate.

Emerging evidence indicates that innate host response contributes to the therapeutic effect of antimicrobial medications. Recent studies have shown that Leishmania parasites derived by in vitro selection for resistance to pentavalent antimony (SbV) as meglumine antimoniate (MA) modulate the activation of neutrophils. However, whether modulation of neutrophil activation extends to natural resistance to this antileishmanial drug has not been established. We have evaluated the influence of clinical strains of L. (V.) panamensis having intrinsic tolerance/resistance to SbV, on the inflammatory response of neutrophils during ex vivo exposure to MA. Accordingly, neutrophils obtained from healthy donors were infected with clinical strains that are sensitive (n = 10) or intrinsically tolerant/resistant to SbV (n = 10) and exposed to a concentration approximating the maximal plasma concentration (Cmax) of SbV (32 µg/ml). The activation profile of neutrophils was evaluated as the expression of the surface membrane markers CD66b, CD18, and CD62L by flow cytometry, measurement of reactive oxygen species (ROS) by luminometry, and NET formation using Picogreen to measure dsDNA release and quantification of NETs by confocal microscopy. These parameters of activation were analyzed in relation with parasite susceptibility to SbV and exposure to MA. Here, we show that clinical strains presenting intrinsic tolerance/resistance to SbV induced significantly lower ROS production compared to drug-sensitive clinical strains, both in the presence and in the absence of MA. Likewise, analyses of surface membrane activation markers revealed significantly higher expression of CD62L on cells infected with intrinsically SbV tolerant/resistant L. (V.) panamensis than cells infected with drug-sensitive strains. Expression of other activation markers (CD18 and CD66b) and NET formation were similar for neutrophils infected with SbV sensitive and tolerant clinical strains under the conditions evaluated. Exposure to MA broadly impacted the activation of neutrophils, diminishing NET formation and the expression of CD62L, while augmenting ROS production and CD66b expression, independently of the parasite susceptibility phenotype. These results demonstrated that activation of human neutrophils ex vivo is differentially modulated by infection with clinical strains of L. (V.) panamensis having intrinsic tolerance/resistance to SbV compared to sensitive strains, and by exposure to antimonial drug.

Resistance of Leishmania (Viannia) Panamensis to Meglumine Antimoniate or Miltefosine Modulates Neutrophil Effector Functions.

Leishmania (Viannia) panamensis (L. (V.) p.) is the main causative agent of cutaneous leishmaniasis in Colombia and is usually treated with either meglumine antimoniate (MA) or miltefosine (MIL). In recent years, there has been increasing evidence of the emergence of drug-resistance against these compounds. Neutrophils are known to play an important role in immunity against Leishmania. These cells are rapidly recruited upon infection and are also present in chronic lesions. However, their involvement in the outcome of infection with drug-resistant Leishmania has not been examined. In this study, human and murine neutrophils were infected in vitro with MA or MIL drug-resistant L. (V.) p. lines derived from a parental L. (V.) p. drug-susceptible strain. Neutrophil effector functions were assessed analyzing the production of reactive oxygen species (ROS), the formation of neutrophil extracellular trap (NET) and the expression of cell surface activation markers. Parasite killing by neutrophils was assessed using L. (V.) p. transfected with a luciferase reporter. We show here that MA and MIL-resistant L. (V.) p. lines elicited significantly increased NET formation and MA-resistant L. (V.) p. induced significantly increased ROS production in both murine and human neutrophils, compared to infections with the parental MIL and MA susceptible strain. Furthermore, neutrophils exposed to drug-resistant lines showed increased activation, as revealed by decreased expression of CD62L and increased expression of CD66b in human neutrophils yet presented higher survival within neutrophils than the drug-susceptible strain. These results provide evidence that parasite drug-susceptibility may influences neutrophil activation and function as well as parasite survival within neutrophils. Further investigaton of the inter-relationship of drug susceptibility and neutrophil effector function should contribute to better understanding of the factors involved in susceptibility to anti-Leishmania drugs.

Ex vivo host and parasite response to antileishmanial drugs and immunomodulators.

BACKGROUND: Therapeutic response in infectious disease involves host as well as microbial determinants. Because the immune and inflammatory response to Leishmania (Viannia) species defines the outcome of infection and efficacy of treatment, immunomodulation is considered a promising therapeutic strategy. However, since Leishmania infection and antileishmanial drugs can themselves modulate drug transport, metabolism and/or immune responses, immunotherapeutic approaches require integrated assessment of host and parasite responses. METHODOLOGY: To achieve an integrated assessment of current and innovative therapeutic strategies, we determined host and parasite responses to miltefosine and meglumine antimoniate alone and in combination with pentoxifylline or CpG 2006 in peripheral blood mononuclear cells (PBMCs) of cutaneous leishmaniasis patients. Parasite survival and secretion of TNF-α, IFN-γ, IL-10 and IL-13 were evaluated concomitantly in PBMCs infected with Luc-L. (V.) panamensis exposed to meglumine antimoniate (4, 8, 16, 32 and 64 µg SbV/mL) or miltefosine (2, 4, 8, 16 and 32 µM HePC). Concentrations of 4 µM of miltefosine and 8 µg SbV/mL were selected for evaluation in combination with immunomodulators based on the high but partial reduction of parasite burden by these antileishmanial concentrations without affecting cytokine secretion of infected PBMCs. Intracellular parasite survival was determined by luminometry and cytokine secretion measured by ELISA and multiplex assays. PRINCIPAL FINDINGS: Anti- and pro-inflammatory cytokines characteristic of L. (V.) panamensis infection were evaluable concomitantly with viability of Leishmania within monocyte-derived macrophages present in PBMC cultures. Both antileishmanial drugs reduced the parasite load of macrophages; miltefosine also suppressed IL-10 and IL-13 secretion in a dose dependent manner. Pentoxifylline did not affect parasite survival or alter antileishmanial effects of miltefosine or meglumine antimoniate. However, pentoxifylline diminished secretion of TNF-α, IFN-γ and IL-13, cytokines associated with the outcome of infection by species of the Viannia subgenus. Exposure to CpG diminished the leishmanicidal effect of meglumine antimoniate, but not miltefosine, and significantly reduced secretion of IL-10, alone and in combination with either antileishmanial drug. IL-13 increased in response to CpG plus miltefosine. CONCLUSIONS AND SIGNIFICANCE: Human PBMCs allow integrated ex vivo assessment of antileishmanial treatments, providing information on host and parasite determinants of therapeutic response that may be used to tailor therapeutic strategies to optimize clinical resolution.

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.

Phase I safety and immunogenicity trial of Plasmodium vivax CS derived long synthetic peptides adjuvanted with montanide ISA 720 or montanide ISA 51.

We assessed the safety, tolerability, and immunogenicity of a mixture of three synthetic peptides derived from the Plasmodium vivax circumsporozoite protein formulated in Montanide ISA 720 or Montanide ISA 51. Forty healthy malaria-naive volunteers were allocated to five experimental groups (A-E): four groups (A-D) were immunized intramuscularly with 50 and 100 µg/dose injections of a mixture of N, R, and C peptides formulated in the two different adjuvants at 0, 2, and 4 months and one group was administered placebo. Vaccines were immunogenic, safe, well tolerated, and no serious adverse events related to the vaccine occurred. Seroconversion occurred in > 90% of the vaccines and antibodies recognized the sporozoite protein on immunofluorescent antibody test. Vaccines in Montanide ISA 51 showed a higher sporozoite protein recognition and interferon production. Results encourage further testing of the vaccine protective efficacy.

Safety and elicitation of humoral and cellular responses in colombian malaria-naive volunteers by a Plasmodium vivax circumsporozoite protein-derived synthetic vaccine.

Substantial experimental evidence indicates that the Plasmodium circumsporozoite (CS) protein has great potential as a vaccine candidate. We tested the safety and immunogenicity of vaccines composed of P. vivax CS-derived synthetic peptides. Sixty-nine healthy, malaria-naive volunteers were randomized to receive three injections of placebo or synthetic proteins N, R, or C (10, 30, or 100 microg/dose) in a double-blinded fashion. Vaccines were well tolerated and no serious adverse events were observed. Peptides N and R elicited humoral responses at all doses; peptide C elicicted these responses only at doses of 30 and 100 microg. The N peptide at a dose of 100 microg elicited the greatest antibody response. Antibodies to the three peptides recognized P. vivax sporozoites in an immunofluorescent antibody test. Peripheral blood mononuclear cells from most immunized volunteers also produced interferon-gamma upon peptide in vitro stimulation. These vaccines appear safe, well tolerated, and immunogenic in malaria-naive volunteers. Further optimization and development of this vaccine is being attempted to conduct phase II clinical trials.