Sample-ready multiplex qPCR assay for detection of malaria

BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. METHODS: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The C(T) values and the standard deviations (SD) were used in the analysis of the assay performance. RESULTS: The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the “wet” assay. CONCLUSION: The MMSR assay has the same robust performance characteristics as the “wet” assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget

Sustainable development of a GCP-compliant clinical trials platform in Africa: the Malaria Clinical Trials Alliance perspective

BACKGROUND: The Malaria Clinical Trials Alliance (MCTA), a programme of INDEPTH network of demographic surveillance centres, was launched in 2006 with two broad objectives: to facilitate the timely development of a network of centres in Africa with the capacity to conduct clinical trials of malaria vaccines and drugs under conditions of good clinical practice (GCP); and to support, strengthen and mentor the centres in the network to facilitate their progression towards self-sustaining clinical research centres. CASE DESCRIPTION: Sixteen research centres in 10 African malaria-endemic countries were selected that were already working with the Malaria Vaccine Initiative (MVI) or the Medicines for Malaria Venture (MMV). All centres were visited to assess their requirements for research capacity development through infrastructure strengthening and training. Support provided by MCTA included: laboratory and facility refurbishment; workshops on GCP, malaria diagnosis, strategic management and media training; and training to support staff to undertake accreditation examinations of the Association of Clinical Research Professionals (ACRP). Short attachments to other network centres were also supported to facilitate sharing practices within the Alliance. MCTA also played a key role in the creation of the African Media & Malaria Research Network (AMMREN), which aims to promote interaction between researchers and the media for appropriate publicity and media reporting of research and developments on malaria, including drug and vaccine trials. CONCLUSION: In three years, MCTA strengthened 13 centres to perform GCP-compliant drug and vaccine trials, including 11 centres that form the backbone of a large phase III malaria vaccine trial. MCTA activities have demonstrated that centres can be brought up to GCP compliance on this time scale, but the costs are substantial and there is a need for further support of other centres to meet the growing demand for clinical trial capacity. The MCTA experience also indicates that capacity development in clinical trials is best carried out in the context of preparation for specific trials. In this regard MCTA centres involved in the phase III malaria vaccine trial were, on average, more successful at consolidating the training and infrastructure support than those centres focussing only on drug trials

Implementation of a structured paediatric admission record for district hospitals in Kenya – results of a pilot study

BACKGROUND: The structured admission form is an apparently simple measure to improve data quality. Poor motivation, lack of supervision, lack of resources and other factors are conceivably major barriers to their successful use in a Kenyan public hospital setting. Here we have examined the feasibility and acceptability of a structured paediatric admission record (PAR) for district hospitals as a means of improving documentation of illness. METHODS: The PAR was primarily based on symptoms and signs included in the Integrated Management of Childhood Illness (IMCI) diagnostic algorithms. It was introduced with a three-hour training session, repeated subsequently for those absent, aiming for complete coverage of admitting clinical staff. Data from consecutive records before (n = 163) and from a 60% random sample of dates after intervention (n = 705) were then collected to evaluate record quality. The post-intervention period was further divided into four 2-month blocks by open, feedback meetings for hospital staff on the uptake and completeness of the PAR. RESULTS: The frequency of use of the PAR increased from 50% in the first 2 months to 84% in the final 2 months, although there was significant variation in use among clinicians. The quality of documentation also improved considerably over time. For example documentation of skin turgor in cases of diarrhoea improved from 2% pre-intervention to 83% in the final 2 months of observation. Even in the area of preventive care documentation of immunization status improved from 1% of children before intervention to 21% in the final 2 months. CONCLUSION: The PAR was well accepted by most clinicians and greatly improved documentation of features recommended by IMCI for identifying and classifying severity of common diseases. The PAR could provide a useful platform for implementing standard referral care treatment guidelines

Experience and Challenges from Clinical Trials with Malaria Vaccines in Africa.

Malaria vaccines are considered amongst the most important modalities for potential elimination of malaria disease and transmission. Research and development in this field has been an area of intense effort by many groups over the last few decades. Despite this, there is currently no licensed malaria vaccine. Researchers, clinical trialists and vaccine developers have been working on many approached to make malaria vaccine available.African research institutions have developed and demonstrated a great capacity to undertake clinical trials in accordance to the International Conference on Harmonization-Good Clinical Practice (ICH-GCP) standards in the last decade; particularly in the field of malaria vaccines and anti-malarial drugs. This capacity is a result of networking among African scientists in collaboration with other partners; this has traversed both clinical trials and malaria control programmes as part of the Global Malaria Action Plan (GMAP). GMAP outlined and support global strategies toward the elimination and eradication of malaria in many areas, translating in reduction in public health burden, especially for African children. In the sub-Saharan region the capacity to undertake more clinical trials remains small in comparison to the actual need.However, sustainability of the already developed capacity is essential and crucial for the evaluation of different interventions and diagnostic tools/strategies for other diseases like TB, HIV, neglected tropical diseases and non-communicable diseases. There is urgent need for innovative mechanisms for the sustainability and expansion of the capacity in clinical trials in sub-Saharan Africa as the catalyst for health improvement and maintained

Performance of a fully‐automated system on a WHO malaria microscopy evaluation slide set

Background: Manual microscopy remains a widely-used tool for malaria diagnosis and clinical studies, but it has inconsistent quality in the field due to variability in training and field practices. Automated diagnostic systems based on machine learning hold promise to improve quality and reproducibility of field microscopy. The World Health Organization (WHO) has designed a 55-slide set (WHO 55) for their External Competence Assessment of Malaria Microscopists (ECAMM) programme, which can also serve as a valuable benchmark for automated systems. The performance of a fully-automated malaria diagnostic system, EasyScan GO, on a WHO 55 slide set was evaluated. Methods: The WHO 55 slide set is designed to evaluate microscopist competence in three areas of malaria diagnosis using Giemsa-stained blood films, focused on crucial field needs: malaria parasite detection, malaria parasite species identification (ID), and malaria parasite quantitation. The EasyScan GO is a fully-automated system that combines scanning of Giemsa-stained blood films with assessment algorithms to deliver malaria diagnoses. This system was tested on a WHO 55 slide set. Results: The EasyScan GO achieved 94.3 % detection accuracy, 82.9 % species ID accuracy, and 50 % quantitation accuracy, corresponding to WHO microscopy competence Levels 1, 2, and 1, respectively. This is, to our knowledge, the best performance of a fully-automated system on a WHO 55 set. Conclusions: EasyScan GO’s expert ratings in detection and quantitation on the WHO 55 slide set point towards its potential value in drug efficacy use-cases, as well as in some case management situations with less stringent species ID needs. Improved runtime may enable use in general case management settings

Fully-automated patient-level malaria assessment on field-prepared thin blood film microscopy images, including Supplementary Information

Malaria is a life-threatening disease affecting millions. Microscopy-based assessment of thin blood films is a standard method to (i) determine malaria species and (ii) quantitate high-parasitemia infections. Full automation of malaria microscopy by machine learning (ML) is a challenging task because field-prepared slides vary widely in quality and presentation, and artifacts often heavily outnumber relatively rare parasites. In this work, we describe a complete, fully-automated framework for thin film malaria analysis that applies ML methods, including convolutional neural nets (CNNs), trained on a large and diverse dataset of field-prepared thin blood films. Quantitation and species identification results are close to sufficiently accurate for the concrete needs of drug resistance monitoring and clinical use-cases on field-prepared samples. We focus our methods and our performance metrics on the field use-case requirements. We discuss key issues and important metrics for the application of ML methods to malaria microscopy.Comment: 16 pages, 13 figure

A randomized, open-label, comparative efficacy trial of artemether-lumefantrine suspension versus artemether-lumefantrine tablets for treatment of uncomplicated Plasmodium falciparum malaria in children in western Kenya

<p>Abstract</p> <p>Background</p> <p>Artemether/lumefantrine (AL) has been adopted as the treatment of choice for uncomplicated malaria in Kenya and other countries in the region. Six-dose artemether/lumefantrine tablets are highly effective and safe for the treatment of infants and children weighing between five and 25 kg with uncomplicated <it>Plasmodium falciparum </it>malaria. However, oral paediatric formulations are urgently needed, as the tablets are difficult to administer to young children, who cannot swallow whole tablets or tolerate the bitter taste of the crushed tablets.</p> <p>Methods</p> <p>A randomized, controlled, open-label trial was conducted comparing day 28 PCR corrected cure-rates in 245 children aged 6–59 months, treated over three days with either six-dose of artemether/lumefantrine tablets (Coartem^®) or three-dose of artemether/lumefantrine suspension (Co-artesiane^®) for uncomplicated falciparum malaria in western Kenya. The children were followed-up with clinical, parasitological and haematological evaluations over 28 days.</p> <p>Results</p> <p>Ninety three percent (124/133) and 90% (121/134) children in the AL tablets and AL suspension arms respectively completed followed up. A per protocol analysis revealed a PCR-corrected parasitological cure rate of 96.0% at Day 28 in the AL tablets group and 93.4% in the AL suspension group, p = 0.40. Both drugs effectively cleared gametocytes and were well tolerated, with no difference in the overall incidence of adverse events.</p> <p>Conclusion</p> <p>The once daily three-dose of artemether-lumefantrine suspension (Co-artesiane^®) was not superior to six-dose artemether-lumefantrine tablets (Coartem^®) for the treatment of uncomplicated malaria in children below five years of age in western Kenya.</p> <p>Trial registration</p> <p>ClinicalTrials.gov NCT00529867</p

Community screening and treatment of asymptomatic carriers of Plasmodium falciparum with artemether-lumefantrine to reduce malaria disease burden: a modelling and simulation analysis

<p>Abstract</p> <p>Background</p> <p>Asymptomatic carriers of <it>Plasmodium falciparum </it>serve as a reservoir of parasites for malaria transmission. Identification and treatment of asymptomatic carriers within a region may reduce the parasite reservoir and influence malaria transmission in that area.</p> <p>Methods</p> <p>Using computer simulation, this analysis explored the impact of community screening campaigns (CSC) followed by systematic treatment of <it>P. falciparum </it>asymptomatic carriers (AC) with artemether-lumefantrine (AL) on disease transmission. The model created by Okell <it>et al </it>(originally designed to explore the impact of the introduction of treatment with artemisinin-based combination therapy on malaria endemicity) was modified to represent CSC and treatment of AC with AL, with the addition of malaria vector seasonality. The age grouping, relative distribution of age in a region, and degree of heterogeneity in disease transmission were maintained. The number and frequency of CSC and their relative timing were explored in terms of their effect on malaria incidence. A sensitivity analysis was conducted to determine the factors with the greatest impact on the model predictions.</p> <p>Results</p> <p>The simulation showed that the intervention that had the largest effect was performed in an area with high endemicity (entomological inoculation rate, EIR > 200); however, the rate of infection returned to its normal level in the subsequent year, unless the intervention was repeated. In areas with low disease burden (EIR < 10), the reduction was sustained for over three years after a single intervention. Three CSC scheduled in close succession (monthly intervals) at the start of the dry season had the greatest impact on the success of the intervention.</p> <p>Conclusions</p> <p>Community screening and treatment of asymptomatic carriers with AL may reduce malaria transmission significantly. The initial level of disease intensity has the greatest impact on the potential magnitude and duration of malaria reduction. When combined with other interventions (e.g. long-lasting insecticide-treated nets, rapid diagnostic tests, prompt diagnosis and treatment, and, where appropriate, indoor residual spraying) the effect of this intervention can be sustained for many years, and it could become a tool to accelerate the reduction in transmission intensity to pre-elimination levels. Repeated interventions at least every other year may help to prolong the effect. The use of an effective diagnostic tool and a highly effective ACT, such as AL, is also vital. The modelling supports the evaluation of this approach in a prospective clinical trial to reduce the pool of infective vectors for malaria transmission in an area with marked seasonality.</p