Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis

<p>Abstract</p> <p>Background</p> <p>Molecular methods to detect <it>Leishmania </it>parasites are considered specific and sensitive, but often not applied in endemic areas of developing countries due to technical complexity. In the present study isothermal, nucleic acid sequence based amplification (NASBA) was coupled to oligochromatography (OC) to develop a simplified detection method for the diagnosis of leishmaniasis. NASBA-OC, detecting <it>Leishmania </it>RNA, was evaluated using clinical samples from visceral leishmaniasis patients from East Africa (n = 30) and cutaneous leishmaniasis from South America (n = 70) and appropriate control samples.</p> <p>Results</p> <p>Analytical sensitivity was 10 parasites/ml of spiked blood, and 1 parasite/ml of culture. Diagnostic sensitivity of NASBA-OC was 93.3% (95% CI: 76.5%-98.8%) and specificity was 100% (95% CI: 91.1%-100%) on blood samples, while sensitivity and specificity on skin biopsy samples was 98.6% (95% CI: 91.2%-99.9%) and 100% (95% CI: 46.3%-100%), respectively.</p> <p>Conclusion</p> <p>The NASBA-OC format brings implementation of molecular diagnosis of leishmaniasis in resource poor countries one step closer.</p

Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

BACKGROUND: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (<20 parasites/μl) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at <100 parasites/μl. METHODS: This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA) assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. RESULTS: The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood). The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. CONCLUSION: Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood) and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus making it an effective tool for diagnostic purposes and useful for epidemiological and drug studies

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

Efficacy and Safety of AmBisome in Combination with Sodium Stibogluconate or Miltefosine and Miltefosine Monotherapy for African Visceral Leishmaniasis: Phase II Randomized Trial.

BACKGROUND: SSG&PM over 17 days is recommended as first line treatment for visceral leishmaniasis in eastern Africa, but is painful and requires hospitalization. Combination regimens including AmBisome and miltefosine are safe and effective in India, but there are no published data from trials of combination therapies including these drugs from Africa. METHODS: A phase II open-label, non-comparative randomized trial was conducted in Sudan and Kenya to evaluate the efficacy and safety of three treatment regimens: 10 mg/kg single dose AmBisome plus 10 days of SSG (20 mg/kg/day), 10 mg/kg single dose AmBisome plus 10 days of miltefosine (2.5mg/kg/day) and miltefosine alone (2.5 mg/kg/day for 28 days). The primary endpoint was initial parasitological cure at Day 28, and secondary endpoints included definitive cure at Day 210, and pharmacokinetic (miltefosine) and pharmacodynamic assessments. RESULTS: In sequential analyses with 49-51 patients per arm, initial cure was 85% (95% CI: 73-92) in all arms. At D210, definitive cure was 87% (95% CI: 77-97) for AmBisome + SSG, 77% (95% CI 64-90) for AmBisome + miltefosine and 72% (95% CI 60-85) for miltefosine alone, with lower efficacy in younger patients, who weigh less. Miltefosine pharmacokinetic data indicated under-exposure in children compared to adults. CONCLUSION: No major safety concerns were identified, but point estimates of definitive cure were less than 90% for each regimen so none will be evaluated in Phase III trials in their current form. Allometric dosing of miltefosine in children needs to be evaluated. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov, number NCT01067443

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

Global Virtual Time Approximation with Distributed Termination Detection Algorithms

It is shown that distributed termination detection algorithms can be transformed into efficient algorithms to approximate the so-called Global Virtual Time (GVT) of a distributed monotonic computation. Typical instances of such computations are optimistic distributed simulations based on the timewarp principle. The transformation is exemplified for two termination detection algorithms, namely an algorithm by Dijkstra et al. and a new scheme based on the principle of &quot;sticky flags&quot;. The general idea of the transformation is that many termination detection algorithms (viz., one for each possible GVT value) run in parallel. Each algorithm determines a specific lower bound The work of H. Mehl is supported by the German National Science Foundation (Deutsche Forschungsgemeinschaft) under grant SPP-322671. y The work of A.A. Schoone and G. Tel is supported by the ESPRIT II Basic Research Actions Program of the EC under contract no. 3075 (project ALCOM). on the current GVT value. In a ..