Development and comparative evaluation of two antigen detection tests for Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL) can be fatal without timely diagnosis and treatment. Treatment efficacies vary due to drug resistance, drug toxicity and co-morbidities. It is important to monitor treatment responsiveness to confirm cure and curtail relapse. Currently, microscopy of spleen, bone marrow or lymph node biopsies is the only definitive method to evaluate cure. A less invasive test for treatment success is a high priority for VL management. METHODS: In this study, we describe the development of a capture ELISA based on detecting Leishmania donovani antigens in urine samples and comparison with the Leishmania Antigen ELISA, also developed for the same purpose. Both were developed as prototype kits and tested on patient urine samples from Sudan, Ethiopia, Bangladesh and Brazil, along with appropriate control samples from endemic and non-endemic regions. Sensitivity and specificity were assessed based on accurate detection of patients compared to control samples. One-Way ANOVA was used to assess the discrimination capacity of the tests and Cohen's kappa was used to assess their correlation. RESULTS: The Leishmania Antigen Detect ELISA demonstrated >90% sensitivity on VL patient samples from Sudan, Bangladesh and Ethiopia and 88% on samples from Brazil. The Leishmania Antigen ELISA was comparable in performance except for lower sensitivity on Sudanese samples. Both were highly specific. To confirm utility in monitoring treatment, urine samples were collected from VL patients at days 0, 30 and 180 post-treatment. For the Leishmania Antigen Detect ELISA, positivity was high at day 0 at 95%, falling to 21% at day 30. At day 180, all samples were negative, corresponding well with clinical cure. A similar trend was also seen for the Leishmania Antigen ELISA albeit; with lower positivity of 91% at Day 0 and more patients, remaining positive at Days 30 and 180. DISCUSSION: The Leishmania Antigen Detect and the Leishmania Antigen ELISAs are standardized, user- friendly, quantitative and direct tests to detect Leishmania during acute VL as well as to monitor parasite clearance during treatment. They are a clear improvement over existing options. CONCLUSION: The ELISAs provide a non-invasive method to detect parasite antigens during acute infection and monitor its clearance upon cure, filling an unmet need in VL management. Further refinement of the tests with more samples from endemic regions will define their utility in monitoring treatment.

Immunochemotherapy of persistent post-kala-azar dermal leishmaniasis: a novel approach to treatment.

Post-kala-azar dermal leishmaniasis (PKDL) is a recognized dermatosis that follows successful treatment of visceral leishmaniasis in the Sudan. This randomized and double-blind study aimed to assess safety, immunogenicity and curative potentials of a novel immunochemotherapy regimen in patients with persistent PKDL. Following informed consent, 30 patients were randomized to receive alum-precipitated autoclaved Leishmania major (Alum/ALM) vaccine+Bacille Calmette-Guérin (BCG) and sodium stibogluconate (SSG) or vaccine diluent and SSG. The SSG+Alum/ALM+BCG proved safe with minimal local adverse events. In the SSG+vaccine group, 87% of the patients were cured by day 60 compared with 53% in the SSG alone group (SSG+vaccine efficacy=71%, 95% CI for risk ratio 0.7-1.16). On day 90 of follow-up there were two relapses in the SSG alone arm and none in the SSG+vaccine arm. Pre-treatment cytokines showed high IFN-gamma or high IFN-gamma/IL-10 levels and leishmanin skin test (LST) non-reactivity, while healing/clinical improvement were associated with LST reactivity and low IFN-gamma levels in both study groups (P=0.004). In conclusion, SSG+Alum/ALM+BCG is safe and immunogenic with significant healing potentials in persistent PKDL lesions. Immunochemotherapy probably augmented IFN-gamma production, which induced healing. Leishmanin skin reactivity is a good surrogate marker of cure in persistent PKDL lesions.

Efficacious, effective, and embedded interventions: implementation research in infectious disease control.

BACKGROUND: Research in infectious disease control is heavily skewed towards high end technology; development of new drugs, vaccines and clinical interventions. Oft ignored, is the evidence to inform the best strategies that ensure the embedding of interventions into health systems and amongst populations. In this paper we undertake an analysis of the challenge in the development of research for the sustainable implementation of disease control interventions. RESULTS: We highlight the fundamental differences between the research paradigms associated with the development of technologies and interventions for disease control on the one hand and the research paradigms required for enhancing the sustainable uptake of those very same interventions within the communities on the other. We provide a definition for implementation research in an attempt to underscore its critical role and explore the multidisciplinary science needed to address the challenges in disease control. CONCLUSION: The greatest value for money in health research lies in the sustainable and effective implementation of already proven, efficacious solutions. The development of implementation research that can help provide some solutions on how this can be achieved is sorely needed.

Consultation meeting on the development of therapeutic vaccines for post kala azar dermal leishmaniasis.

BACKGROUND: Post kala azar dermal leishmaniasis (PKDL) is a disease that appears after treatment of visceral leishmaniasis (VL). The highest incidence of PKDL in the world is in Sudan. Many patients heal spontaneously within 6 months but those who don't are difficult to treat, often requiring months of daily injections. These patients harbour parasite in their skin and are believed to be a source of infection and possibly epidemics. Present treatment modalities of PKDL are inadequate and impractical due to cost, duration of treatment required and side effects. New approach for treatment of PKDL is required. A joint meeting of the UNICEF/UNDP/World Bank/WHO Special Programme for research and training in Tropical Disease (TDR) and the Infectious Disease Research Institute (IDRI) Seattle, USA was held to review the progress of therapeutic vaccines and plan the development of treatment modalities for PKDL. METHODS: The history of leishmaniasis vaccine development for prophylaxis and therapy was reviewed. Other than previous infection - simulated by inoculation of live Leishmania as a vaccine (leishmanization), none of the preparations of killed parasite with or without adjuvants have shown significant prophylactic efficacy. Killed L. major absorbed with alum and mixed with BCG remains to be tested as a prophylactic vaccine. RESULTS: Killed parasite preparations i.e. L. mexicana mixed with BCG and L. amazonensis (combined with low dose of antimonial), have shown efficacy in immunotherapy and immuno-chemotherapy, respectively. In addition combined full antimonial plus alum-absorbed autoclaved L. major vaccine has been shown to significantly improve therapy of refractory PKDL patients. These are all crude preparations of parasites and are difficult to define and standardize. However, there is now a new, second generation vaccine, Leish-111f + MPL-SE, composed of a recombinant protein comprising three leishmanial antigens and a defined adjuvant in clinical development. CONCLUSION AND RECOMMENDATIONS: Immuno-chemotherapy has the potential of becoming a practical and affordable treatment modality for PKDL and other forms of leishmaniasis. The encouraging results with alum-autoclaved L. major + antimonial should be pursued. However, before further trials, availability of the vaccine and its production under Good Manufacturing Product, hence quality control must be assured. Following satisfactory safety profile of Leish-111f+MPL-SE, clinical trials using this vaccine initially with antimonials should be initiated. Similarly immunotherapy of VL should be considered with the view to controlling development of PKDL. Some immunological studies are required prior to initiation of immunotherapy in VL patients.

Diagnostic accuracy of rK28-based immunochromatographic rapid diagnostic tests for visceral leishmaniasis: a prospective clinical cohort study in Sudan.

BACKGROUND: Rapid diagnostic tests (RDTs) for visceral leishmaniasis (VL) based on rK39 antigen showed suboptimal sensitivity in East Africa. A prospective clinical cohort study in Sudan was designed to validate a novel rK28-based RDT for Leishmania donovani VL. METHODS: Patients (n=285) suspected of VL by residency, fever for ≥2 weeks, splenomegaly with no prior reported VL, and negative for malaria were consecutively enrolled at three Sudanese sites in 2012-2013 and informed consent obtained. Two human readers, who were blinded to the clinical status and other RDT results, evaluated patient whole blood (WB) and serum on the rK28 RDT. Based on Leishmania parasite detection in lymph node or bone marrow aspirates (Giemsa-stained smears or culture in Novy-MacNeal-Nicolle medium), patients were categorized as VL cases (n=200) or VL controls (n=85). RESULTS: The rK28 RDT had high specificity using either WB (100% [85/85]) or serum (97.6% [83/85]) and exhibited greater sensitivity (WB, 92.5% [185/200]; serum, 94.5% [189/200]) than a direct agglutination test performed with the same sera (92.9% [79/85] and 83.5% [167/200] for specificity and sensitivity, respectively). Two blinded readers scored a given WB or serum sample the same on the rK28 RDT 99.6% and 100% of the time, respectively. A reader scored each individual donor's paired WB and serum rK28 RDT results the same 97.2% of the time. CONCLUSIONS: An inexpensive rK28 RDT that performs robustly with WB or serum will be valuable for diagnosing cases of VL in East Africa.

Proinflammatory cytokines in open versus laparoscopic cholecystectomy.

OBJECTIVE: Laparoscopic cholecystectomy, a minimal access surgery, is fast replacing open cholecystectomy and is being associated with less trauma. The objective of this study was to compare the proinflammatory cytokine levels in both laparoscopic cholecystectomy and open cholecystectomy. METHODS: This study was carried out at Aseer Central Hospital, Aseer region, Abha Private Hospital and the College of Medicine and Medical Sciences, King Khalid University, Abha, Kingdom of Saudi Arabia, during the time period October 1998 through to November 2000. Sixty-one patients were included in the study, 27 of them had laparoscopic cholecystectomy and 34 had open cholecystectomy. Cytokines [Interleukin-6 Interleukin-1b, Tumor necrosis factor -a and Interleukin- 8] were measured in blood samples collected from the patients before, at and 24 hours post surgery, using commercially available kits. RESULTS: Interleukin-6 levels were significantly increased at 24 hours post surgery in the open cholecystectomy group of patients compared to the laparoscopic cholecystectomy group (P<0.04). No differences were found in the other cytokines levels (Interleukin-1b, tumor necrosis factor -a and Interleukin-8) between the open cholecystectomy and laparoscopic cholecystectomy groups. CONCLUSION: Laparoscopic cholecystectomy, a minimal access surgery, is associated with lower levels of the proinflammatory interleukin-6 cytokine compared to open cholecystectomy.

Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

BACKGROUND: Infection with Leishmania parasites causes mainly cutaneous lesions at the site of the sand fly bite. Inflammatory metastatic forms have been reported with Leishmania species such as L. braziliensis, guyanensis and aethiopica. Little is known about the factors underlying such exacerbated clinical presentations. Leishmania RNA virus (LRV) is mainly found within South American Leishmania braziliensis and guyanensis. In a mouse model of L. guyanensis infection, its presence is responsible for an hyper-inflammatory response driven by the recognition of the viral dsRNA genome by the host Toll-like Receptor 3 leading to an exacerbation of the disease. In one instance, LRV was reported outside of South America, namely in the L. major ASKH strain from Turkmenistan, suggesting that LRV appeared before the divergence of Leishmania subgenera. LRV presence inside Leishmania parasites could be one of the factors implicated in disease severity, providing rationale for LRV screening in L. aethiopica. METHODOLOGY/PRINCIPAL FINDINGS: A new LRV member was identified in four L. aethiopica strains (LRV-Lae). Three LRV-Lae genomes were sequenced and compared to L. guyanensis LRV1 and L. major LRV2. LRV-Lae more closely resembled LRV2. Despite their similar genomic organization, a notable difference was observed in the region where the capsid protein and viral polymerase open reading frames overlap, with a unique -1 situation in LRV-Lae. In vitro infection of murine macrophages showed that LRV-Lae induced a TLR3-dependent inflammatory response as previously observed for LRV1. CONCLUSIONS/SIGNIFICANCE: In this study, we report the presence of an immunogenic dsRNA virus in L. aethiopica human isolates. This is the first observation of LRV in Africa, and together with the unique description of LRV2 in Turkmenistan, it confirmed that LRV was present before the divergence of the L. (Leishmania) and (Viannia) subgenera. The potential implication of LRV-Lae on disease severity due to L. aethiopica infections is discussed.

Comparison of point-of-care tests for the rapid diagnosis of visceral leishmaniasis in East African patients.

The development of rK39-based rapid diagnostic tests (RDTs) has greatly aided the diagnosis of visceral leishmaniasis, especially in the Indian subcontinent and Brazil, by offering high sensitivity and specificity. However, these tests have been less sensitive and less specific in sub-Saharan Africa. To improve upon the performance of rK39 in Africa, we engineered the fusion molecule rK28, which retained some of the rK39 repeats and combined them with repeat sequences from two additional Leishmania genes. This polyprotein was used in the development of several prototype RDTs by different commercial manufacturers with the goal of assessing relative performance in inexpensive formats. Here, we report field studies showing that the rK28 antigen could be readily adapted to a variety of RDT formats to achieve high sensitivity, generally > 90%, and adequate specificity to aid in the diagnosis of human visceral leishmaniasis in East Africa, Asia, and South America.

Pharmacovigilance methods in public health programmes: the example of miltefosine and visceral leishmaniasis.

Pharmacovigilance is concerned with the assessment of benefit and harm. Disease burden, status of healthcare delivery through government centres and practitioners, existing pharmacovigilance programmes, relevant pre-marketing studies and the likely effectiveness and risks of drugs must be considered for planning pharmacovigilance activity. The risk of a drug may be known, unknown, potential or specific to the context of the programme. The potential benefits of a public health programme aimed at reducing or eliminating a specific condition will depend on the health burden due to that condition, which is a function of the seriousness of the condition and its frequency, as well as the likely efficacy of the programme in reaching its goals. The present article has outlined an approach to pharmacovigilance for such a donor-funded programme, using pharmacovigilance in leishmaniasis as an example.

Ambisome plus miltefosine for Indian patients with kala-azar.

The combination of one intravenous administration of 5mg/kg Ambisome and oral administration of miltefosine, 2.5mg/kg/day for 14 days, was evaluated in 135 Indian patients with kala-azar. The Intent-to-Treat cure rate at 6 months was 124 of the 135 enrolled patients (91.9%: 95% CI = 86-96%), and the per protocol cure rate was 124 of 127 evaluable patients (97.6%: 95% CI = 93-100%). Side effects could be attributed to each drug separately: fevers, rigors and back pain due to Ambisome; gastrointestinal side effects due to miltefosine. This combination is attractive for reasons of efficacy, tolerance, and feasibility of administration, although the gastrointestinal side effects of miltefosine require medical vigilance. Clinical Trials.gov identification number: NCT00371995.

Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?

Visceral leishmaniasis (VL) is a systemic protozoan disease that is transmitted by phlebotomine sandflies. Poor and neglected populations in East Africa and the Indian sub-continent are particularly affected. Early and accurate diagnosis and treatment remain key components of VL control. In addition to improved diagnostic tests, accurate and simple tests are needed to identify treatment failures. Miltefosine, paromomycin and liposomal amphotericin B are gradually replacing pentavalent antimonials and conventional amphotericin B as the preferred treatments in some regions, but in other areas these drugs are still being evaluated in both mono- and combination therapies. New diagnostic tools and new treatment strategies will only have an impact if they are made widely available to patients.

Maternal serum interleukin-6, interleukin-8, tumor necrosis factor-alpha and interferon-gamma in preterm labor.

BACKGROUND: To find out whether preterm labor is associated with raised maternal serum concentrations of interleukin (IL)-6, IL-8, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) and whether the measurement of these cytokines can be used to detect early intrauterine infection in preterm labor. METHODS: Cross-sectional study: 77 women in preterm labor, 47 controls of healthy preterm women not in labor and 19 women in term labor. The serum cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). The newborns of women who were in labor were followed up for evidence of infection. Differences between groups were tested using analysis of variance, Student's t-test and chi2-test. RESULTS: There was no significant difference in the concentration of all the cytokines measured between the different groups. No statistical difference was found in the concentration of the cytokines between women in preterm labor with ruptured membranes and those with intact membranes. There was also no difference found in the concentration of cytokines between women whose newborns had positive bacterial culture and those with negative culture. There was a positive correlation between the concentrations of IL-6, IL-8 and TNF-alpha. CONCLUSION: Serum levels of interleukin-6, interleukin-8 and tumor necrosis factor-alpha were not increased in preterm labor compared to normal control women. There is doubt regarding the usefulness of maternal serum measurement of these cytokines for the detection of early fetal infection in preterm labor, but this needs further evaluation.