Extending the spectrum of antimalarial treatment: Artemisinin combinations for the treatment of rare Plasmodium species infections and the development of dyes as antimalarials

Malaria, an infectious disease caused by a parasite of the genus Plasmodium, was responsible for about 198 Million cases and 584 000 deaths in 2013, mainly in Sub-Saharan Africa. The first line treatment for uncomplicated Plasmodium falciparum infections are artemisinin combination therapies (ACTs). Although the efficacy of ACTs against P. falciparum has been well documented, there is only anecdotal evidence of efficacy of ACTs against other human Plasmodium species because chloroquine is still recommended and commonly used. However, in highly endemic regions non-falciparum species can occur together with P. falciparum and, moreover, exact microscopic species identification and detection of residual parasites is usually not possible in those settings. Availability of broadly acting drugs would ease clinical management of suspected non-falciparum and mixed infections. This thesis presents data on the efficacy of artemether-lumefantrine (AL), a widely used ACT, against uncomplicated non-falciparum or mixed-species malaria in Lambaréné, Gabon and assesses the performance of microscopy in an African routine laboratory setting. Forty patients presenting with uncomplicated malaria caused by P. malariae, P. ovale or a mixed infection (including P. falciparum) were treated with a three days AL regimen and were followed up to 28 days after start of the treatment. All evaluable patients presented an adequate clinical and parasitological response. All adverse events were mild or moderate and resolved by the end of the follow-up. The parasitological cure rate was 100% and the drug was well tolerated. These findings support the use of AL for the treatment of uncomplicated malaria in Gabon, even when non-falciparum or mixed infections are suspected. Microscopic identification of Plasmodium species other than P. falciparum did not perform well. From 39 samples microscopically determined as non-falciparum or mixed infection, only 19 were confirmed by PCR. This might be due to the very low parasitaemia found in most cases and underlines the need for the development of broadly acting antimalarials. The second part of this thesis reports on the in vitro activity of fluorescent dyes against cultured malaria parasites. The emergence of resistance against most of the drugs in current use urges the development of new antimalarial compounds. Since x several antimicrobials were derived from molecular modification of synthetic dyes, which often have a broad spectrum of activity, screening of dyes may represent an interesting strategy to identify novel antimalarial lead structures. The original motivation to screen fluorescent dyes in Plasmodium were in vivo labeling experiments. Here, a remarkably high antiparasitic activity was noted for some compounds. Hence, the activity of fourteen fluorescent dyes was systematically investigated by in vitro growth assays using the two laboratory P. falciparum strains 3D7 and Dd2. Five had an activity comparable to the control drug chloroquine and were not cytotoxic against human HeLa cells (Rhodamine B, MitoTracker Red, and DiOC6, Hoechst33342, SYTO 9). Rhodamine B and DiOC6 were further tested in a mouse model but DiOC6 was toxic and Rhodamine B, although less toxic, did not lead to a notable reduction of parasitaemia. Despite of this, this pilot experiment shows that pre-screening for staining properties may be an interesting way to build highly efficient pathogen-specific compound libraries. There is a need for broadly acting antimalarial drugs. This requirement includes multi-stage activity as well as activity against different parasite species. AL showed high in vivo activity against different Plasmodium species, but does not act against pre-erythrocytic stages (sporozoites and infected hepatocytes) and reduces gametocytes only modestly. Dyes are a group of chemically heterogeneous compounds that may extent the spectrum of activity. In fact, the dye methylene blue was shown to meet many of the requirements. Investigation of larger dye libraries may result in highly interesting novel antimalarial compounds and may lead to the development of novel, broadly acting drugs

Prospective evaluation of artemether-lumefantrine for the treatment of non-falciparum and mixed-species malaria in Gabon

Background: The recommendation of artemisinin combination therapy (ACT) as first-line treatment for uncomplicated falciparum malaria is supported by a plethora of high quality clinical trials. However, their recommendation for the treatment of mixed-species malaria and the large-scale use for the treatment of non-falciparum malaria in endemic regions is based on anecdotal rather than systematic clinical evidence. Methods: This study prospectively observed the efficacy of artemether-lumefantrine for the treatment of uncomplicated non-falciparum or mixed-species malaria in two routine district hospitals in the Central African country of Gabon. Results: Forty patients suffering from uncomplicated Plasmodium malariae, Plasmodium ovale or mixed-species malaria (including Plasmodium falciparum) presenting at the hospital received artemether-lumefantrine treatment and were followed up. All evaluable patients (n = 38) showed an adequate clinical and parasitological response on Day 28 after oral treatment with artemether-lumefantrine (95% confidence interval: 0.91,1). All adverse events were of mild to moderate intensity and completely resolved by the end of study. Conclusions: This first systematic assessment of artemether-lumefantrine treatment for P. malariae, P. ovale and mixed-species malaria demonstrated a high cure rate of 100% and a favourable tolerability profile, and thus lends support to the practice of treating non-falciparum or mixed-species malaria, or all cases of malaria without definite species differentiation, with artemether-lumefantrine in Gabon. Trial Registration: ClinicalTrials.gov Identifier: NCT0072577

Species and genotype diversity of Plasmodium in malaria patients from Gabon analysed by next generation sequencing

Background Six Plasmodium species are known to naturally infect humans. Mixed species infections occur regularly but morphological discrimination by microscopy is difficult and multiplicity of infection (MOI) can only be evaluated by molecular methods. This study investigated the complexity of Plasmodium infections in patients treated for microscopically detected non- falciparum or mixed species malaria in Gabon. Methods Ultra-deep sequencing of nucleus (18S rRNA), mitochondrion, and apicoplast encoded genes was used to evaluate Plasmodium species diversity and MOI in 46 symptomatic Gabonese patients with microscopically diagnosed non-falciparum or mixed species malaria. Results Deep sequencing revealed a large complexity of coinfections in patients with uncomplicated malaria, both on species and genotype levels. Mixed infections involved up to four parasite species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale curtisi, and P. ovale wallikeri). Multiple genotypes from each species were determined from the asexual 18S rRNA gene. 17 of 46 samples (37%) harboured multiple genotypes of at least one Plasmodium species. The number of genotypes per sample (MOI) was highest in P. malariae (n = 4), followed by P. ovale curtisi (n = 3), P. ovale wallikeri (n = 3), and P. falciparum (n = 2). The highest combined genotype complexity in samples that contained mixed-species infections was seven. Conclusions Ultra- deep sequencing showed an unexpected breadth of Plasmodium species and within species diversity in clinical samples. MOI of P. ovale curtisi, P. ovale wallikeri and P. malariae infections were higher than anticipated and contribute significantly to the burden of malaria in Gabon

Direct venous inoculation of Plasmodium falciparum sporozoites for controlled human malaria infection: a dose-finding trial in two centres

BACKGROUND: Controlled human malaria infection (CHMI) accelerates development of anti-malarial interventions. So far, CHMI is done by exposure of volunteers to bites of five mosquitoes carrying Plasmodium falciparum sporozoites (PfSPZ), a technique available in only a few centres worldwide. Mosquito-mediated CHMI is logistically complex, exact PfSPZ dosage is impossible and live mosquito-based interventions are not suitable for further clinical development. METHODS: An open-labelled, randomized, dose-finding study in 18-45 year old, healthy, malaria-naive volunteers was performed to assess if intravenous (IV) injection of 50 to 3,200 aseptic, purified, cryopreserved PfSPZ is safe and achieves infection kinetics comparable to published data of mosquito-mediated CHMI. An independent study site verified the fully infectious dose using direct venous inoculation of PfSPZ. Parasite kinetics were assessed by thick blood smear microscopy and quantitative real time PCR. RESULTS: IV inoculation with 50, 200, 800, or 3,200 PfSPZ led to parasitaemia in 1/3, 1/3, 7/9, and 9/9 volunteers, respectively. The geometric mean pre-patent period (GMPPP) was 11.2 days (range 10.5-12.5) in the 3,200 PfSPZ IV group. Subsequently, six volunteers received 3,200 PfSPZ by direct venous inoculation at an independent investigational site. All six developed parasitaemia (GMPPP: 11.4 days, range: 10.4-12.3). Inoculation of PfSPZ was safe. Infection rate and pre-patent period depended on dose, and injection of 3,200 PfSPZ led to a GMPPP similar to CHMI with five PfSPZ-infected mosquitoes. The infectious dose of PfSPZ predicted dosage of radiation-attenuated PfSPZ required for successful vaccination. CONCLUSIONS: IV inoculation of PfSPZ is safe, well tolerated and highly reproducible. It shall further accelerate development of anti-malarial interventions through standardization and facilitation of CHMI. Beyond this, rational dose selection for whole PfSPZ-based immunization and complex study designs are now possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT01624961 and NCT01771848

In Vitro Activity of Fluorescent Dyes against Asexual Blood Stages of Plasmodium falciparum

Many successful antimicrobial drugs originate from synthetic dyes. This paper reports the in vitro activity of 14 fluorescent dyes against Plasmodium falciparum. Five of these dyes (Hoechst 33342, MitoRed, DiOC(6), SYTO 9, and rhodamine B) show activity at a low nanomolar concentration against two P. falciparum strains in the histidine-rich protein 2 drug sensitivity assay, while toxicity in HeLa cells is low. These dyes may be a starting point for developing new drugs against P. falciparum

Limit of blank and limit of detection of Plasmodium falciparum thick blood smear microscopy in a routine setting in Central Africa

BACKGROUND: Proper malaria diagnosis depends on the detection of asexual forms of Plasmodium spp. in the blood. Thick blood smear microscopy is the accepted gold standard of malaria diagnosis and is widely implemented. Surprisingly, diagnostic performance of this method is not well investigated and many clinicians in African routine settings base treatment decisions independent of microscopy results. This leads to overtreatment and poor management of other febrile diseases. Implementation of quality control programmes is recommended, but requires sustained funding, external logistic support and constant training and supervision of the staff. This study describes an easily applicable method to assess the performance of thick blood smear microscopy by determining the limit of blank and limit of detection. These two values are representative of the diagnostic quality and allow the correct discrimination between positive and negative samples. METHODS: Standard-conform methodology was applied and adapted to determine the limit of blank and the limit of detection of two thick blood smear microscopy methods (WHO and Lambaréné method) in a research centre in Lambaréné, Gabon. Duplicates of negative and low parasitaemia thick blood smears were read by several microscopists. The mean and standard deviation of the results were used to calculate the limit of blank and subsequently the limit of detection. RESULTS: The limit of blank was 0 parasites/μL for both methods. The limit of detection was 62 and 88 parasites/μL for the Lambaréné and WHO method, respectively. CONCLUSION: With a simple, back-of-the-envelope calculation, the performance of two malaria microscopy methods can be measured. These results are specific for each diagnostic unit and cannot be generalized but implementation of a system to control microscopy performance can improve confidence in parasitological results and thereby strengthen malaria control

Species and genotype diversity of Plasmodium in malaria patients from Gabon analysed by next generation sequencing

Abstract Background Six Plasmodium species are known to naturally infect humans. Mixed species infections occur regularly but morphological discrimination by microscopy is difficult and multiplicity of infection (MOI) can only be evaluated by molecular methods. This study investigated the complexity of Plasmodium infections in patients treated for microscopically detected non-falciparum or mixed species malaria in Gabon. Methods Ultra-deep sequencing of nucleus (18S rRNA), mitochondrion, and apicoplast encoded genes was used to evaluate Plasmodium species diversity and MOI in 46 symptomatic Gabonese patients with microscopically diagnosed non-falciparum or mixed species malaria. Results Deep sequencing revealed a large complexity of coinfections in patients with uncomplicated malaria, both on species and genotype levels. Mixed infections involved up to four parasite species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale curtisi, and P. ovale wallikeri). Multiple genotypes from each species were determined from the asexual 18S rRNA gene. 17 of 46 samples (37%) harboured multiple genotypes of at least one Plasmodium species. The number of genotypes per sample (MOI) was highest in P. malariae (n = 4), followed by P. ovale curtisi (n = 3), P. ovale wallikeri (n = 3), and P. falciparum (n = 2). The highest combined genotype complexity in samples that contained mixed-species infections was seven. Conclusions Ultra-deep sequencing showed an unexpected breadth of Plasmodium species and within species diversity in clinical samples. MOI of P. ovale curtisi, P. ovale wallikeri and P. malariae infections were higher than anticipated and contribute significantly to the burden of malaria in Gabon

Broad-spectrum antimalarial activity of peptido sulfonyl fluorides, a new class of proteasome inhibitors

Despite declining numbers of cases and deaths, malaria remains a major public health problem in many parts of the world. Today, case management relies heavily on a single class of antimalarial compounds: artemisinins. Hence, development of resistance against artemisinins may destroy current malaria control strategies. Beyond malaria control are elimination and eradication programs that will require drugs with good activity against acute infection but also with preventive and transmission-blocking properties. Consequently, new antimalarials are needed not only to ensure malaria control but also for elimination and eradication efforts. In this study, we introduce peptido sulfonyl fluorides (PSF) as a new class of compounds with antiplasmodial activity. We show that PSF target the plasmodial proteasome and act on all asexual stages of the intraerythrocytic cycle and on gametocytes. PSF showed activities at concentrations as low as 20 nM against multidrug-resistant and chloroquine-sensitive Plasmodium falciparum laboratory strains and clinical isolates from Gabon. Structural requirements for activity were identified, and cytotoxicity in human HeLa or HEK 293 cells was low. The lead PSF PW28 suppressed growth of Plasmodium berghei in vivo but showed signs of toxicity in mice. Considering their modular structure and broad spectrum of activity against different stages of the plasmodial life cycle, proteasome inhibitors based on PSF have a great potential for further development as preclinical candidate compounds with improved species-specific activity and less toxicity