Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate.

In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.

In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses.

The parasitic protozoan Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a Leishmania species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to in vitro susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.

Factors associated with relapse and hospital death in patients coinfected with visceral leishmaniasis and HIV: a longitudinal study.

OBJECTIVE: Visceral leishmaniasis (VL) is an endemic parasitic disease in Latin America, and its clinical picture is aggravated in coinfections with the human immunodeficiency virus (HIV). The objective of this study was to investigate clinical factors and laboratory variables associated with VL relapse and death in VL/HIV coinfected patients. METHODS: A prospective longitudinal study was conducted from January 2013 to July 2020 among 169 patients coinfected with VL and HIV. The outcomes investigated were the occurrence of VL relapse and death. Chi-square test, Mann-Whitney test and logistic regression models were used for statistical analysis. RESULTS: The occurrence rates were 41.4% for VL relapse and 11.2% for death. Splenomegaly and adenomegaly were associated with the increased risk of VL relapse. Patients with VL relapse had higher levels of urea (p = .005) and creatinine (p < .001). Patients who died had lower red blood cell counts (p = .012), hemoglobin (p = .017) and platelets (p < .001). The adjusted model showed that antiretroviral therapy for more than 6 months was associated with a decrease in VL relapse, and adenomegaly was associated with an increase in VL relapse. In addition, edema, dehydration, poor general health status, and paleness were associated with an increase in hospital death. CONCLUSION: The findings suggest that adenomegaly, antiretroviral therapy, and renal abnormalities can be associated with VL relapse, while hematological abnormalities, and clinical manifestations like paleness, and edema can be associated with an increased odds of hospital death. TRIAL REGISTRATION NUMBER: The study was submitted to the Ethics and Research Committee of the Federal University of Maranhão (Protocol: 409.351).

Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis.

In the Americas, Brazil is responsible by 90% of the cases registered of visceral leishmaniasis (VL), and Leishmania infantum is the most common parasite species responsible by disease in Brazilian dogs and humans. A precise diagnosis may allow to a faster and more effective treatment against the disease, which increases the possibility of cure, as well as to induce less toxic effects, due to a lower time exposition for the chemotherapeutics. In a previous study, two L. infantum mimotopes, B10 and C01 clones, were recognized by antibodies in VL dogs sera by a phage display technology, and were well-successfully evaluated as vaccine candidates against visceral and tegumentary leishmaniasis. In the present work, the diagnostic efficacy of these clones, as well as of their exogenous peptides (B10: LSFPFPG and C01: FTSFSPY), was evaluated to diagnose canine and human VL. ELISA assays were performed with the four antigens, and results showed that both clones, as well as their synthetic peptides; showed high sensitivity and specificity values to identify VL samples, presenting an excellent performance to serologically diagnose VL-developing humans and dogs. On the other hand, a wild-type phage, a random non-specific clone and a L. infantum antigenic preparation were used as controls, and showed worst sensitivity and specificity results. In conclusion, besides their biological action as vaccine, B10 and C01 phages and their synthetic peptides could be considered as new markers for the serodiagnosis of canine and human VL.

Phage-fused epitopes from Leishmania infantum used as immunogenic vaccines confer partial protection against Leishmania amazonensis infection.

Two mimotopes of Leishmania infantum identified by phage display were evaluated as vaccine candidates in BALB/c mice against Leishmania amazonensis infection. The epitope-based immunogens, namely B10 and C01, presented as phage-fused peptides; were used without association of a Th1 adjuvant, and they were administered isolated or in combination into animals. Both clones showed a specific production of interferon-gamma (IFN-γ), interleukin-12 (IL-12) and granulocyte/macrophage colony-stimulating factor (GM-CSF) after in vitro spleen cells stimulation, and they were able to induce a partial protection against infection. Significant reductions of parasite load in the infected footpads, liver, spleen, bone marrow and paws' draining lymph nodes were observed in the immunized mice, in comparison with the control groups (saline, saponin, wild-type and non-relevant clones). Protection was associated with an IL-12-dependent production of IFN-γ, mediated mainly by CD8(+) T cells, against parasite proteins. Protected mice also presented low levels of IL-4 and IL-10, as well as increased levels of parasite-specific IgG2a antibodies. The association of both clones resulted in an improved protection in relation to their individual use. More importantly, the absence of adjuvant did not diminish the cross-protective efficacy against Leishmania spp. infection. This study describes for the first time two epitope-based immunogens selected by phage display technology against L. infantum infected dogs sera, which induced a partial protection in BALB/c mice infected with L. amazonensis.

Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic Leishmania infantum antigens with potential serodiagnosis applications.

Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n = 31) compared to those from vaccinated dogs (n = 21), experimentally infected dogs with cross-reactive parasites (n = 23), and healthy controls (n = 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.