Iván Darío Vélez

Leishmaniasis Worldwide and Global Estimates of Its Incidence

As part of a World Health Organization-led effort to update the empirical evidence base for the leishmaniases, national experts provided leishmaniasis case data for the last 5 years and information regarding treatment and control in their respective countries and a comprehensive literature review was conducted covering publications on leishmaniasis in 98 countries and three territories (see ‘Leishmaniasis Country Profiles Text S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S18, S19, S20, S21, S22, S23, S24, S25, S26, S27, S28, S29, S30, S31, S32, S33, S34, S35, S36, S37, S38, S39, S40, S41, S42, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S65, S66, S67, S68, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, S100, S101’). Additional information was collated during meetings conducted at WHO regional level between 2007 and 2011. Two questionnaires regarding epidemiology and drug access were completed by experts and national program managers. Visceral and cutaneous leishmaniasis incidence ranges were estimated by country and epidemiological region based on reported incidence, underreporting rates if available, and the judgment of national and international experts. Based on these estimates, approximately 0.2 to 0.4 cases and 0.7 to 1.2 million VL and CL cases, respectively, occur each year. More than 90% of global VL cases occur in six countries: India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil. Cutaneous leishmaniasis is more widely distributed, with about one-third of cases occurring in each of three epidemiological regions, the Americas, the Mediterranean basin, and western Asia from the Middle East to Central Asia. The ten countries with the highest estimated case counts, Afghanistan, Algeria, Colombia, Brazil, Iran, Syria, Ethiopia, North Sudan, Costa Rica and Peru, together account for 70 to 75% of global estimated CL incidence. Mortality data were extremely sparse and generally represent hospital-based deaths only. Using an overall case-fatality rate of 10%, we reach a tentative estimate of 20,000 to 40,000 leishmaniasis deaths per year. Although the information is very poor in a number of countries, this is the first in-depth exercise to better estimate the real impact of leishmaniasis. These data should help to define control strategies and reinforce leishmaniasis advocacy.

Autosomal, mtDNA, and Y-Chromosome Diversity in Amerinds: Pre- and Post-Columbian Patterns of Gene Flow in South America

To evaluate sex-specific differences in gene flow between Native American populations from South America and between those populations and recent immigrants to the New World, we examined the genetic diversity at uni- and biparental genetic markers of five Native American populations from Colombia and in published surveys from native South Americans. The Colombian populations were typed for five polymorphisms in mtDNA, five restriction sites in the beta-globin gene cluster, the DQA1 gene, and nine autosomal microsatellites. Elsewhere, we published results for seven Y-chromosome microsatellites in the same populations. Autosomal polymorphisms showed a mean G(ST) of 6.8%, in agreement with extensive classical marker studies of South American populations. MtDNA and Y-chromosome markers resulted in G(ST) values of 0.18 and 0.165, respectively. When only Y chromosomes of confirmed Amerind origin were used in the calculations (as defined by the presence of allele T at locus DYS199), G(ST) increased to 0.22. G(ST) values calculated from published data for other South American natives were 0.3 and 0.29 for mtDNA and Amerind Y chromosomes, respectively. The concordance of these estimates does not support an important difference in migration rates between the sexes throughout the history of South Amerinds. Admixture analysis of the Colombian populations suggests an asymmetric pattern of mating involving mostly immigrant men and native women.

The wMel strain of Wolbachia Reduces Transmission of Zika virus by Aedes aegypti.

Zika virus (ZIKV) is causing an explosive outbreak of febrile disease in the Americas. There are no effective antiviral therapies or licensed vaccines for this virus, and mosquito control strategies have not been adequate to contain the virus. A promising candidate for arbovirus control and prevention relies on the introduction of the intracellular bacterium Wolbachia into Aedes aegypti mosquitoes. This primarily has been proposed as a tool to control dengue virus (DENV) transmission; however, evidence suggests Wolbachia infections confer protection for Ae. aegypti against other arboviruses. At present, it is unknown whether or not ZIKV can infect, disseminate, and be transmitted by Wolbachia-infected Ae. aegypti. Using Ae. aegypti infected with the wMel strain of Wolbachia that are being released in Medellin, Colombia, we report that these mosquitoes have reduced vector competence for ZIKV. These results support the use of Wolbachia biocontrol as a multivalent strategy against Ae. aegypti-transmitted viruses.

Inefficacy of Allopurinol as Monotherapy for Colombian Cutaneous Leishmaniasis: A Randomized, Controlled Trial

New World cutaneous leishmaniasis classically presents as an ulcerating papule or nodule. The ulcer then reepithelializes during a period of a few to many months. Infection frequently spreads to draining lymph nodes; less frequently, it spreads to the mucous membranes of the nose and mouth. The disease is treated to relieve the discomfort of a weeping ulcer and to prevent mucosal disease. Treatment currently requires large daily doses of pentavalent antimony (20 mg/kg of body weight per day) by injection for 20 days [1]. This regimen creates considerable morbidity: Severe arthralgias or myalgias occur in at least 50% of patients, gastrointestinal discomfort develops in 30% of patients, hepatocellular enzyme levels are elevated in 30% of patients, diminution of T-wave height as seen on electrocardiography occurs in at least 10% of patients, and-rarely-thrombocytopenia and neutropenia occur [1]. Antimony treatment also causes chemical pancreatitis in almost all patients; gastrointestinal discomfort may be a symptom of clinical pancreatitis [2]. The treatment of cutaneous leishmaniasis, therefore, leads to morbidity similar to that of the disease itself. Such considerations have led to a 20-year search for an oral, well-tolerated therapeutic agent. The hypoxanthine analogue allopurinol, an inhibitor of uric acid formation in mammalian cells, was shown by LaFon and colleagues [3] to inhibit purine anabolism in Leishmania. In a randomized study done in Colombia, Martinez and Marr [4] found that a daily regimen of allopurinol (20 mg/kg per day) and antimony (20 mg/kg per day) for 15 days was much more effective than therapy with antimony alone (20 of 25 patients receiving allopurinol plus antimony [80%] recovered completely compared with 12 of 33 patients [36%] receiving antimony alone). However, the rate of cure for antimony alone in this study was surprisingly low [5]. Because of the controversy about the Colombian study and the lack of a well-controlled study on the efficacy of allopurinol in treating clinical leishmaniasis, we planned a randomized, blinded, controlled, large-scale clinical trial of the efficacy of allopurinol. To improve our ability to evaluate the efficacy of therapy, we chose the same endemic area (Colombia) and parasite (Leishmania panamensis) on which the major 1992 report focused. Methods Study Design In this randomized, controlled, partially double-blinded phase III study, we compared allopurinol with placebo and Glucantime (Rhone Poulenc, Paris, France). One hundred eighty-seven patients with cutaneous leishmaniasis were randomly assigned to one of three treatment groups. Patients in the first group received allopurinol, 300 mg (three 100-mg tablets) four times daily for 28 days, so that the dosage given was approximately 5 mg/kg four times daily or 20 mg/kg daily for 28 days. Patients in the second group received placebo, three tablets four times daily for 28 days. Patients in the third group received Glucantime, 20 mg of antimony/kg daily (no maximum daily dose) intramuscularly for 20 days. Patients in the allopurinol and placebo groups were assigned to treatment in a double-blinded manner. Study Sample, Inclusion Criteria, and Exclusion Criteria The study sample was composed of patients who were clinically suspected of having cutaneous leishmaniasis and who were from the following regions of Colombia: Arma, Dabeiba, Herveo, La Mesa, Marquetalia, Medellin, San Carlos, San Luis, Taraza, Valdivia, and Victoria. Patients were eligible for the study if they were 6 to 60 years of age, had cutaneous leishmaniasis as confirmed by the presence of parasites, had not received treatment for leishmaniasis with recognized agents during the previous 6 months, did not have lesions close to the eyes or on the mucosa, had body weight that was appropriate for height, and were amenable to prolonged follow-up. Exclusion criteria were the presence of concomitant diseases that required medical intervention, abnormalities in the complete blood count, abnormal glutamate oxaloacetate aminotransferase levels, abnormal creatinine levels, abnormal uric acid levels, and pregnancy. The ethical review committee of the Antioquia University School of Medicine and Hospital San Vicente de Paul, Medellin, Colombia, approved the study, and all patients gave written, informed consent. Parasitologic Diagnosis Leishmaniasis was diagnosed by visualizing amastigotes in lesion material or culturing promastigotes from lesion material. Two scrapings from one lesion on each patient (one scraping taken from the border of the lesion and one taken from the center) were examined with Giemsa stain for amastigotes; needle aspirates of the same lesion were added to culture medium to permit promastigotes to grow. Cultures were maintained at 26 C for 4 weeks before they were reported as negative. In each isolate that was cultured, species were identified by the use of monoclonal antibodies [6]. Conduct of Clinical Trial The first patient was entered into the study in April 1992; the last follow-up examination was completed in November 1995. Before the start of therapy, a complete history, a physical examination, and laboratory testing were done for each patient as described above. A photograph of each patients lesions was also taken. Patients were monitored every 10 days during the treatment phase of the study. Oral therapy was self-administered, and compliance was self-recorded. At each monitoring session, a 10-day supply of pills was dispensed and the patients compliance record was checked and verified by counting the number of pills. Glucantime was administered by medical support personnel. In addition, the occurrence and severity of anticipated adverse effects were recorded at each monitoring session. Lesions were examined before the start of therapy; at the end of therapy; and 1.5, 3, 6, 9, and 12 months after the end of therapy. Attendance at monitoring sessions and follow-up appointments was aided by telephone calls and home visits by study staff. At each evaluation, the induration (measured by using the ballpoint-pen technique) and the area of ulceration were both measured in two directions, which we designated R1 and R2. The areas of the indurated and ulcerated regions were calculated using the formula R1 x R2. Definition of Responses Response of lesions to therapy was determined clinically. Lesions treated with standard Glucantime regimens may increase in size or may not completely heal by the end of therapy; however, they generally heal by 1.5 months after therapy. We therefore used the response pattern of lesions treated with Glucantime as our standard in developing our definitions. For each lesion, we used the following definitions and dispositions: Complete clinical response: Complete reepithelialization of the ulcer and disappearance of all induration. Lesions that showed a complete clinical response were followed for as long as 12 months to verify lack of relapse. Clinical improvement: Fifty percent to 99% reepithelialization of the ulcer area and diminution of induration relative to the previous examination. Lesions that showed clinical improvement at the end of therapy or 1.5 months after the end of therapy were followed until either a complete clinical response or no clinical response was seen at subsequent follow-up sessions. No clinical response: Less than 50% enlargement or diminution of the ulcer area and of induration. If no clinical response was seen at the end of therapy, the lesion was monitored further. If no clinical response was seen at a later follow-up, therapy was considered to have failed. Failure to respond: 1) Greater than 50% enlargement of lesion size at the end of therapy or at subsequent follow-up or 2) no clinical response at an examination done 1.5 months or more after the end of therapy. Relapse: The reappearance of the lesion at the original site after a complete clinical response or the appearance of lesions involving the mucosa. A patient was considered cured if all of the patients lesions had a complete clinical response by the third month of therapy and no relapse had occurred by the 12-month follow-up appointment. Therapy was considered to have failed if any of the patients lesions did not respond to therapy or relapsed. Evaluation of Responses Patients, study investigators, and monitors were blinded to therapy with allopurinol compared with placebo. After the end of follow-up for the last patient, three independent, blinded evaluators determined efficacy and reached a consensus for each patient. The randomization code was then broken. Toxicity was determined by one evaluator before the code was broken. Results Patient Characteristics Five of the original 187 randomly assigned patients were excluded from the study: Two patients violated the study protocol, 1 had an uncertain parasitologic diagnosis, 1 had a clinical course that could not be interpreted, and 1 had co-infection with Sporothrix schenckii. The characteristics of the 182 patients studied are shown in Table 1. The groups were well matched for age, sex, number of lesions per patient, location and characteristics of lesions, and duration of the presence of lesions before treatment. Table 1. Patient Characteristics* Study Site Characteristics and Parasitology Patients from Arma and La Mesa had disease caused by L. braziliensis. Patients from the other nine regions had disease caused by L. panamensis. Ninety-seven percent to 100% of lesions were smear positive and 61% to 75% were culture positive for Leishmania organisms. Because our study was designed to investigate the efficacy of treatment for infection with L. panamensis, we stopped entering patients into the study at Arma and La Mesa as soon as the strain present at those sites was recognized. For the 182 analyzable patients, 153 patients (84%) had documented infection with L. panamensis or were from regions in which L. panamensis was endemic; 29 patients (16%) had infection

Safety and immunogenicity of a recombinant live attenuated tetravalent dengue vaccine (DENVax) in flavivirus-naive healthy adults in Colombia: a randomised, placebo-controlled, phase 1 study

BACKGROUND Dengue virus is the most serious mosquito-borne viral threat to public health and no vaccines or antiviral therapies are approved for dengue fever. The tetravalent DENVax vaccine contains a molecularly characterised live attenuated dengue serotype-2 virus (DENVax-2) and three recombinant vaccine viruses expressing the prM and E structural genes for serotypes 1, 3, and 4 in the DENVax-2 genetic backbone. We aimed to assess the safety and immunogenicity of tetravalent DENVax formulations. METHODS We undertook a randomised, double-blind, phase 1, dose-escalation trial between Oct 11, 2011, and Nov 9, 2011, in the Rionegro, Antioquia, Colombia. The first cohort of participants (aged 18-45 years) were randomly assigned centrally, via block randomisation, to receive a low-dose formulation of DENvax, or placebo, by either subcutaneous or intradermal administration. After a safety assessment, participants were randomly assigned to receive a high-dose DENVax formulation, or placebo, by subcutaneous or intradermal administration. Group assignment was not masked from study pharmacists, but allocation was concealed from participants, nurses, and investigators. Primary endpoints were frequency and severity of injection-site and systemic reactions within 28 days of each vaccination. Secondary endpoints were the immunogenicity of DENVax against all four dengue virus serotypes, and the viraemia due to each of the four vaccine components after immunisation. Analysis was by intention to treat for safety and per protocol for immunogenicity. Because of the small sample size, no detailed comparison of adverse event rates were warranted. The trial is registered with ClinicalTrials.gov, number NCT01224639. FINDINGS We randomly assigned 96 patients to one of the four study groups: 40 participants (42%) received low-dose vaccine and eight participants (8%) received placebo in the low-dose groups; 39 participants (41%) received high-dose vaccine, with nine (9%) participants assigned to receive placebo. Both formulations were well tolerated with mostly mild and transient local or systemic reactions. No clinically meaningful differences were recorded in the overall incidence of local and systemic adverse events between patients in the vaccine and placebo groups; 68 (86%) of 79 participants in the vaccine groups had solicited systemic adverse events compared with 13 (76%) of 17 of those in the placebo groups. By contrast, 67 participants (85%) in the vaccine group had local solicited reactions compared with five (29%) participants in the placebo group. Immunisation with either high-dose or low-dose DENVax formulations induced neutralising antibody responses to all four dengue virus serotypes; 30 days after the second dose, 47 (62%) of 76 participants given vaccine seroconverted to all four serotypes and 73 (96%) participants seroconverted to three or more dengue viruses. Infectious DENVax viruses were detected in only ten (25%) of 40 participants in the low-dose group and 13 (33%) of 39 participants in the high-dose group. INTERPRETATION Our findings emphasise the acceptable tolerability and immunogenicity of the tetravalent DENVax formulations in healthy, flavivirus-naive adults. Further clinical testing of DENVax in different age groups and in dengue-endemic areas is warranted. FUNDING Takeda Vaccines.

In Vitro and In Vivo Cytotoxicities and Antileishmanial Activities of Thymol and Hemisynthetic Derivatives

ABSTRACT The in vitro and in vivo antileishmanial and cytotoxic activities of thymol and structural derivatives in comparison to those of Glucantime were studied. The results showed here suggest that thymol and hemisynthetic derivatives have promising antileishmanial potential and could be considered as new lead structures in the search for novel antileishmanial drugs.

Phlebotomine sand flies (Diptera: Psychodidae) associated with the appearance of urban leishmaniasis in the city of Sincelejo, Colombia

Although once associated only with rural areas, the American leishmaniasis vectors now appear to be associated also with urban and suburban areas of the Neotropics. Following the appearance of the first autochthonous visceral and cutaneous leishmaniasis cases in the urban area of the city of Sincelejo, Colombia, a preliminary entomological survey of the sand fly species composition was performed using Shannon and CDC light traps. A total of 486 sand flies representing six Lutzomyia species were collected. L. evansi, L. panamensis and L. gomezi, known vectors of Leishmania spp. were the predominant sand fly species around dwellings. The finding of these species in relation to the appearance of the first cases of leishmaniasis in the city mentioned is discussed.

Efficacy of Miltefosine for the Treatment of American Cutaneous Leishmaniasis

Miltefosine is an oral agent used for cutaneous leishmaniasis treatment. An open-label, randomized, phase III clinical trial was carried out in the Colombian army population. Miltefosine, 50 mg capsule was taken orally three times per day for 28 days (N = 145) or meglumine antimoniate, 20 mg/kg body weight per day for 20 days by intramuscular injection (N = 143). The efficacy of miltefosine by protocol was 69.8% (85/122 patients) and 58.6% (85/145 patients) by intention to treat. For meglumine antimoniate, the efficacy by protocol was 85.1% (103/121 patients) and 72% (103/143 patients) by intention to treat. No association was found between drug efficacy and L. (V.) braziliensis or L. (V.) panamensis species of Leishmania responsible for infection. Adverse gastrointestinal events were associated with the use of miltefosine, the meglumine antimoniate treatment was associated with adverse effects on the skeletal musculature, fever, cephalea, and higher toxicity in kidney, liver, pancreas, and hematological system.

The wMel Strain of Wolbachia Reduces Transmission of Chikungunya Virus in Aedes aegypti

Background New approaches to preventing chikungunya virus (CHIKV) are needed because current methods are limited to controlling mosquito populations, and they have not prevented the invasion of this virus into new locales, nor have they been sufficient to control the virus upon arrival. A promising candidate for arbovirus control and prevention relies on the introduction of the intracellular bacterium Wolbachia into Aedes aegypti mosquitoes. This primarily has been proposed as a tool to control dengue virus (DENV) transmission; however, evidence suggests Wolbachia infections confer protection for Ae. aegypti against CHIKV. Although this approach holds much promise for limiting virus transmission, at present our understanding of the ability of CHIKV to infect, disseminate, and be transmitted by wMel-infected Ae. aegypti currently being used at Wolbachia release sites is limited. Methodology/Principal Findings Using Ae. aegypti infected with the wMel strain of Wolbachia that are being released in Medellin, Colombia, we report that these mosquitoes have reduced vector competence for CHIKV, even with extremely high viral titers in the bloodmeal. In addition, we examined the dynamics of CHIKV infection over the course of four to seven days post feeding. Wolbachia-infected mosquitoes remained non-infective over the duration of seven days, i.e., no infectious virus was detected in the saliva when exposed to bloodmeals of moderate viremia, but CHIKV-exposed, wild type mosquitoes did have viral loads in the saliva consistent with what has been reported elsewhere. Finally, the presence of wMel infection had no impact on the lifespan of mosquitoes as compared to wild type mosquitoes following CHIKV infection. Conclusions/Significance These results could have an impact on vector control strategies in areas where Ae. aegypti are transmitting both DENV and CHIKV; i.e., they argue for further exploration, both in the laboratory and the field, on the feasibility of expanding this technology beyond DENV.

Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs.

Development of new therapeutic approaches for leishmaniasis treatment requires new high throughput screening methodologies for the antileishmanial activity of the new compounds both in vitro and in vivo. Reporter genes as the GFP have become one of the most promissory and widely used tools for drug screening in several models, since it offers live imaging, high sensibility, specificity and flexibility; additionally, the use of GFP as a reporter gene in screening assays eliminates all the drawbacks presented in conventional assays and also those technical problems found using other reporter genes. The utility of the GFP as a reporter gene in drug screening assays with Leishmania parasites depends on the homogeneity and stability of the GFP transfected strains. Stable expression of the GFP in the Old World Leishmania species has been demonstrated using integration vectors; however, no reports exist yet about the success of this methodology in the New World species. Here we report the generation of New World Leishmania strains expressing the GFP protein from an integration vector, which replaces one copy of the 18S RNA in the chromosome with the GFP coding sequence by homologous recombination. We also prove that the expression of the integrated GFP is stable and homogeneous in the transfected parasites after months in culture without selective pressure or during its use in hamster infection assays. The fluorescent strains are useful for in vitro, ex vivo and in vivo drug screening assays since no considerable variations in virulence or infectivity where seen attributable to the genetic manipulation during both in vitro and in vivo infection experiments. The platform described here for drug testing assays based on the use of stable fluorescent Leishmania strains coupled to flow cytometry and fluorescent microscopy is more sensitive, more specific and faster than conventional assays used normally for the evaluation of compounds with potential antileishmanial activity.