Blood Parasite Load as an Early Marker to Predict Treatment Response in Visceral Leishmaniasis in Eastern Africa

Background: To expedite the development of new oral treatment regimens for visceral leishmaniasis (VL), there is a need for early markers to evaluate treatment response and predict long-term outcomes. Methods: Data from 3 clinical trials were combined in this study, in which Eastern African VL patients received various antileishmanial therapies. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative polymerase chain reaction (qPCR) before, during, and up to 6 months after treatment. The predictive performance of pharmacodynamic parameters for clinical relapse was evaluated using receiver-operating characteristic curves. Clinical trial simulations were performed to determine the power associated with the use of blood parasite load as a surrogate endpoint to predict clinical outcome at 6 months. Results: The absolute parasite density on day 56 after start of treatment was found to be a highly sensitive predictor of relapse within 6 months of follow-up at a cutoff of 20 parasites/mL (area under the curve 0.92, specificity 0.91, sensitivity 0.89). Blood parasite loads correlated well with tissue parasite loads (ρ = 0.80) and with microscopy gradings of bone marrow and spleen aspirate smears. Clinical trial simulations indicated a > 80% power to detect a difference in cure rate between treatment regimens if this difference was high (> 50%) and when minimally 30 patients were included per regimen. Conclusions: Blood Leishmania parasite load determined by qPCR is a promising early biomarker to predict relapse in VL patients. Once optimized, it might be useful in dose finding studies of new chemical entities.This work was supported by the European Union Seventh Framework Programme Africoleish (grant number 305178); the World Health Organization—Special Programme for Research and Training in Tropical Diseases (WHO-TDR); the French Development Agency, France (grant number CZZ2062); UK aid, UK; the Federal Ministry of Education and Research through KfW, Germany; the Medicor Foundation, Liechtenstein; Médecins Sans Frontières, International; the Swiss Agency for Development and Cooperation (SDC), Switzerland (grant number 81017718); the Dutch Ministry of Foreign Affairs (DGIS), the Netherlands (grant number PDP15CH21); the French Ministry for Europe and Foreign Affairs (MEAE), France; The Rockefeller Foundation, USA; BBVA Foundation, Spain; the European Union—AfriKADIA project of the Second European and Developing Countries Clinical Trials Partnership Programme (EDCTP2) (grant number RIA2016S1635); and ZonMw/Dutch Research Council (NWO) Veni grant (project number 91617140 to T. P. C. D.).S

Dynamics of Parasite Clearance in Cutaneous Leishmaniasis Patients Treated with Miltefosine

Parasite loads were quantified in repeated skin biopsies from lesions of 2 patients with Old-World cutaneous leishmaniasis (CL) caused by Leishmania major and L. infantum during and after treatment with miltefosine. Miltefosine induced a rapid therapeutic effect on both infections with an initial decline of parasites of ∼1 log/week for the L. major infection. These observations illustrate the usability of quantifying parasite loads in skin lesions as a pharmacodynamic measure and quantitative descriptor of drug effect for CL supporting clinical assessment

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Diagnosis plays a central role in the control of human African trypanosomiasis (HAT) whose mainstay in disease control is chemotherapy. However, accurate diagnosis is hampered by the absence of sensitive techniques for parasite detection. Without concentrating the blood, detection thresholds can be as high as 10,000 trypanosomes per milliliter of blood. The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) are promising molecular diagnostics that generally yield high sensitivity and could improve case detection. Recently, these two tests were coupled to oligochromatography (OC) for simplified and standardized detection of amplified products, eliminating the need for electrophoresis. In this study, we evaluated the diagnostic accuracy of these two novel tests on blood specimens from HAT patients and healthy endemic controls from D.R. Congo and Uganda. Both tests exhibited good sensitivity and specificity compared to the current diagnostic tests and may be valuable tools for sensitive and specific parasite detection in clinical specimens. These standardized molecular test formats open avenues for improved case detection, particularly in epidemiological studies and in disease diagnosis at reference centres

Diagnostic Accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for Diagnosis of Leishmaniasis in Sudan

The leishmaniases are a group of vector-borne diseases caused by protozoan parasites of the genus Leishmania. The parasites are transmitted by phlebotomine sand flies and can cause, depending on the infecting species, three clinical manifestations of leishmaniasis: visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) including the mucocutaneous form. VL, PKDL as well as CL are endemic in several parts of Sudan, and VL especially represents a major health problem in this country. Molecular tests such as the polymerase chain reaction (PCR) or nucleic acid sequence based assay (NASBA) are powerful techniques for accurate detection of the parasite in clinical specimens, but broad use is hampered by their complexity and lack of standardisation. Recently, the Leishmania OligoC-TesT and NASBA-Oligochromatography were developed as simplified and standardised PCR and NASBA formats. In this study, both tests were phase II evaluated for diagnosis of VL, PKDL and CL in Sudan

An ab initio and AIM investigation into the hydration of 2-thioxanthine

<p>Abstract</p> <p>Background</p> <p>Hydration is a universal phenomenon in nature. The interactions between biomolecules and water of hydration play a pivotal role in molecular biology. 2-Thioxanthine (2TX), a thio-modified nucleic acid base, is of significant interest as a DNA inhibitor yet its interactions with hydration water have not been investigated either computationally or experimentally. Here in, we reported an <it>ab initio </it>study of the hydration of 2TX, revealing water can form seven hydrated complexes.</p> <p>Results</p> <p>Hydrogen-bond (H-bond) interactions in 1:1 complexes of 2TX with water are studied at the MP2/6-311G(d, p) and B3LYP/6-311G(d, p) levels. Seven 2TX^...H₂O hydrogen bonded complexes have been theoretically identified and reported for the first time. The proton affinities (PAs) of the O, S, and N atoms and deprotonantion enthalpies (DPEs) of different N-H bonds in 2TX are calculated, factors surrounding why the seven complexes have different hydrogen bond energies are discussed. The theoretical infrared and NMR spectra of hydrated 2TX complexes are reported to probe the characteristics of the proposed H-bonds. An improper blue-shifting H-bond with a shortened C-H bond was found in one case. NBO and AIM analysis were carried out to explain the formation of improper blue-shifting H-bonds, and the H-bonding characteristics are discussed.</p> <p>Conclusion</p> <p>2TX can interact with water by five different H-bonding regimes, N-H^...O, O-H^...N, O-H^...O, O-H^...S and C-H^...O, all of which are medium strength hydrogen bonds. The most stable H-bond complex has a closed structure with two hydrogen bonds (N(7)-H^...O and O-H^...O), whereas the least stable one has an open structure with one H-bond. The interaction energies of the studied complexes are correlated to the PA and DPE involved in H-bond formation. After formation of H-bonds, the calculated IR and NMR spectra of the 2TX-water complexes change greatly, which serves to identify the hydration of 2TX.</p

Safety and efficacy of single dose versus multiple doses of AmBisome for treatment of visceral leishmaniasis in eastern Africa: a randomised trial.

BACKGROUND: Anti-leishmanial drug regimens that include a single dose AmBisome could be suitable for eastern African patients with symptomatic visceral leishmaniasis (VL) but the appropriate single dose is unknown. METHODOLOGY: A multi-centre, open-label, non-inferiority, randomized controlled trial with an adaptive design, was conducted to compare the efficacy and safety of a single dose and multiple doses of AmBisome for the treatment of VL in eastern Africa. The primary efficacy endpoint was definitive cure (DC) at 6 months. Symptomatic patients with parasitologically-confirmed, non-severe VL, received a single dose of AmBisome 7.5 mg/kg body weight or multiple doses, 7 times 3 mg/kg on days 1-5, 14, and 21. If interim analyses, evaluated 30 days after the start of treatment following 40 or 80 patients, showed the single dose gave significantly poorer parasite clearance than multiple doses at the 5% significance level, the single dose was increased by 2·5 mg/kg. In a sub-set of patients, parasite clearance was measured by quantitative reverse transcriptase (qRT) PCR. PRINCIPAL FINDINGS: The trial was terminated after the third interim analysis because of low efficacy of both regimens. Based on the intention-to-treat population, DC was 85% (95%CI 73-93%), 40% (95%CI 19-64%), and 58% (95%CI 41-73%) in patients treated with multiple doses (n = 63), and single doses of 7·5 (n = 21) or 10 mg/kg (n = 40), respectively. qRT-PCR suggested superior parasite clearance with multiple doses as early as day 3. Safety data accorded with the drug label. CONCLUSIONS: The tested AmBisome regimens would not be suitable for VL treatment across eastern Africa. An optimal single dose regimen was not identified. TRIALS REGISTRATION: www.clinicaltrials.govNCT00832208