Pilotfallstudie zur Bewertung der Ausgleichszulage in benachteiligten Gebieten im Landkreis Freyung-Grafenau

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Auswirkungen der Luxemburger Beschlüsse auf ländliche Räume, Agrarumweltmaßnahmen und die Ausgleichszulage: Studie im Auftrag des BMVEL

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Misclassification of Plasmodium infections by conventional microscopy and the impact of remedial training on the proficiency of laboratory technicians in species identification.

BACKGROUND: Malaria diagnosis is largely dependent on the demonstration of parasites in stained blood films by conventional microscopy. Accurate identification of the infecting Plasmodium species relies on detailed examination of parasite morphological characteristics, such as size, shape, pigment granules, besides the size and shape of the parasitized red blood cells and presence of cell inclusions. This work explores misclassifications of four Plasmodium species by conventional microscopy relative to the proficiency of microscopists and morphological characteristics of the parasites on Giemsa-stained blood films. CASE DESCRIPTION: Ten-day malaria microscopy remedial courses on parasite detection, species identification and parasite counting were conducted for public health and research laboratory personnel. Proficiency in species identification was assessed at the start (pre) and the end (post) of each course using known blood films of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale and Plasmodium vivax infections with densities ranging from 1,000 to 30,000 parasites/μL. Outcomes were categorized as false negative, positive without speciation, P. falciparum, P. malariae, P. ovale, P. vivax and mixed infections. DISCUSSION AND EVALUATION: Reported findings are based on 1,878 P. falciparum, 483 P. malariae, 581 P. ovale and 438 P. vivax cumulative results collated from 2008 to 2010 remedial courses. Pre-training false negative and positive misclassifications without speciation were significantly lower on P. falciparum infections compared to non-falciparum infections (p < 0.0001). Post-training misclassifications decreased significantly compared to pre- training misclassifications which in turn led to significant improvements in the identification of the four species. However, P. falciparum infections were highly misclassified as mixed infections, P. ovale misclassified as P. vivax and P. vivax similarly misclassified as P. ovale (p < 0.05). CONCLUSION: These findings suggest that the misclassification of malaria species could be a common occurrence especially where non-falciparum infections are involved due to lack of requisite skills in microscopic diagnosis and variations in morphological characteristics within and between Plasmodium species. Remedial training might improve reliability of conventional light microscopy with respect to differentiation of Plasmodium infections

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

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine

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

Lessons learnt from the first controlled human malaria infection study conducted in Nairobi, Kenya

BACKGROUND: Controlled human malaria infection (CHMI) studies, in which healthy volunteers are infected with Plasmodium falciparum to assess the efficacy of novel malaria vaccines and drugs, have become a vital tool to accelerate vaccine and drug development. CHMI studies provide a cost-effective and expeditious way to circumvent the use of large-scale field efficacy studies to deselect intervention candidates. However, to date few modern CHMI studies have been performed in malaria-endemic countries. METHODS: An open-label, randomized pilot CHMI study was conducted using aseptic, purified, cryopreserved, infectious P. falciparum sporozoites (SPZ) (Sanaria® PfSPZ Challenge) administered intramuscularly (IM) to healthy Kenyan adults (n = 28) with varying degrees of prior exposure to P. falciparum. The purpose of the study was to establish the PfSPZ Challenge CHMI model in a Kenyan setting with the aim of increasing the international capacity for efficacy testing of malaria vaccines and drugs, and allowing earlier assessment of efficacy in a population for which interventions are being developed. This was part of the EDCTP-funded capacity development of the CHMI platform in Africa. DISCUSSION: This paper discusses in detail lessons learnt from conducting the first CHMI study in Kenya. Issues pertinent to the African setting, including community sensitization, consent and recruitment are considered. Detailed reasoning regarding the study design (for example, dose and route of administration of PfSPZ Challenge, criteria for grouping volunteers according to prior exposure to malaria and duration of follow-up post CHMI) are given and changes other centres may want to consider for future studies are suggested. CONCLUSIONS: Performing CHMI studies in an African setting presents unique but surmountable challenges and offers great opportunity for acceleration of malaria vaccine and drug development. The reflections in this paper aim to aid other centres and partners intending to use the CHMI model in Africa. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-015-0671-x) contains supplementary material, which is available to authorized users

Longitudinal study on Plasmodium falciparum gametocyte carriage following artemether-lumefantrine administration in a cohort of children aged 12–47 months living in Western Kenya, a high transmission area

BACKGROUND: The effects that artemether-lumefantrine (AL) has on gametocyte dynamics in the short-term have recently been described. However there is limited long-term longitudinal data on the effect of AL on gametocyte dynamics in asymptomatic children. METHODS: An epidemiological study was conducted in Kombewa, Western Kenya, in which 270 asymptomatic children aged between 12 and 47 months were enrolled. The subjects were randomized to receive either a course of AL or placebo at enrolment. Active follow-up was conducted for one year. RESULTS: The gametocyte prevalence and density dynamics throughout the study period mirrored that of the asexual forms. The proportion of initially parasitaemic subjects becoming gametocytaemic was significantly lower in the AL arm for the first 12 weeks following randomization. The geometric mean gametocyte density was lower in the AL arm for 2 weeks following randomization. None of the variables of interest had a statistically significant effect on the duration of gametocytaemia. There is no effect seen in subjects who are not parasitaemic at the time of drug administration. CONCLUSIONS: The treatment of asymptomatic parasitaemic subjects with AL results in a significant reduction in the proportion of subjects who become gametocytaemic for at least 12 weeks