Fats & Fakes

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Fats & Fakes

Towards improved control of malaria

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Benjamin Jelle Visser

Fats & Fakes Towards improved control of malaria

Benjamin Jelle Visser

This thesis was prepared at the Department of Tropical Medicine and Travel Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. The printing of this thesis was ecologically compensated by planting trees in China, Brazil, Israel and The Netherlands. Layout:

Benjamin Jelle Visser

Front cover:

Ralf Kayser & Benjamin Jelle Visser

Printed by:

Ipskamp Printing, Enschede, the Netherlands

ISBN:

978-94-028-0511-6

CC BY 4.0 - 2017 B.J. Visser. This thesis is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. © Chapter 2 may not be reproduced, stored or transmitted in any form or by any means without the prior permission of the publisher of the included book chapter.

Fats & Fakes Towards improved control of malaria

ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op vrijdag 3 maart 2017, te 12:00 uur door Benjamin Jelle Visser geboren te Amsterdam

Promotiecommissie Promotor:

prof. dr. M.P. Grobusch

Universiteit van Amsterdam

Copromotor:

dr. M. van Vugt

Universiteit van Amsterdam

Overige leden:

dr. T. van Gool dr. P.F. Mens

Universiteit van Amsterdam Koninklijk Instituut voor de Tropen Universiteit van Amsterdam Universiteit van Amsterdam Universiteit van Amsterdam Universiteit Leiden Bernhard-Nocht-Institut Hamburg

prof. dr. M. Boele van Hensbroek prof. dr. F.G.J. Cobelens prof. dr. T.F. Rinke de Wit prof. dr. L.G. Visser prof. dr. J. May Faculteit der Geneeskunde

Contents Chapter 1.

General introduction and outline of this thesis

Part I:

Treatment of malaria

Chapter 2.

Discovery of the Malaria Parasites and their Vectors - A Short History 21 Discoveries in Modern Science: Exploration, Invention, Technology, 2015

Chapter 3.

Malaria: an update on current chemotherapy Expert Opinion on Pharmacotherapy 2014; (15):2219-54

35

Chapter 4.

Efficacy and safety of artemisinin combination therapy (ACT) for non-falciparum malaria: a systematic review Malaria Journal 2014; 13:463

85

Chapter 5.

The influence of pregnancy on the pharmacokinetic properties of artemisinin combination therapy (ACT): a systematic review Malaria Journal 2016; 15:99

115

Chapter 6.

Health workers' compliance to rapid diagnostic tests (RDTs) to guide malaria treatment: a systematic review and meta-analysis Malaria Journal 2016; 15:163

175

Part II:

The quality of anti-malarial drugs

Chapter 7.

Assessing the quality of anti-malarial drugs from Gabonese Pharmacies using the MiniLab®: a field study Malaria Journal 2015; 14:273

195

Chapter 8.

The diagnostic accuracy of the hand-held Raman spectrometer for the identification of anti-malarial drugs Malaria Journal 2016; 15:160

223

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Part III: Lipids during malaria infection Chapter 9.

Serum lipids and lipoproteins in malaria: a systematic review and meta-analysis Malaria Journal 2013; 12:442

Chapter 10. Serum lipids and lipoproteins during uncomplicated malaria: a cohort study in Lambaréné, Gabon American Journal of Tropical Medicine and Hygiene, in Press

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Part IV: Epilogue Chapter 11. Reporting of medical research costs. Improving transparency and reproducibility of medical research Methods of Information in Medicine 2014;53(4):329-31

293

Chapter 12. Summary and general discussion

299

Addendum Nederlandse samenvatting References Africa – the real view? Abbreviations Contribution authors PhD Portfolio Publications Dankwoord Curriculum vitae

313 325 362 365 369 373 375 379 381

“The map above shows the lithological properties of the surface geology of Africa. Lithology describes the mineral composition and structure of geological material which is based on rock formation (i.e. whether it is igneous, sedimentary, metamorphic) and mineralogy (e.g. carbonate, silicic, mafic). This map is a good proxy for soil parent material as it only reflects surface conditions and not the underlying bedrock. The preponderance of wind-blown sediments across Africa is striking as are the volcanic areas.” Adapted from: Soil Atlas of Africa, 2013. European Commission, Publications Office of the European Union, Luxembourg.1

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Chapter 1 General introduction and outline of this thesis

Benjamin J. Visser

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Chapter 1

Introduction Malaria is an ancient disease and the most important parasitic disease of human beings, leading to approximately 438 000 deaths per year and 214 million cases of malaria (World Health Organization (WHO) estimates December 2015).2 It is transmitted in 95 countries inhabited by roughly 3 billion people.2 More than 85% of malaria cases and 90% of malaria deaths occur in Africa, south of the Sahara (sub-Saharan Africa), mainly in children younger than 5 years.3, 4 In addition to its health toll, Africa alone is estimated to lose at least € 12 billion per year in direct losses (e.g. to illness, premature death, cost of treatment), and many times more than that in lost economic growth due to malaria. 2, 5 Malaria is transmitted via the bite by female Anopheles mosquitoes.6-9 This occurs mainly between dusk and dawn. Five species of the genus Plasmodium are known to cause human malaria infections.10-12 Most are caused by P. falciparum and P. vivax, but can also be caused by P. malariae and P. ovale (P. ovale curtisi and P. ovale wallikeri) and in some parts of South East Asia P. knowlesi.13 The majority of malaria deaths are due to infection with the protozoan parasite Plasmodium falciparum.

Epidemiology Malaria is prevalent in most of the tropical regions of the world with P. falciparum causing the largest burden of disease, followed by P. vivax. P. falciparum is more prevalent in subSaharan Africa and New Guinea.12 P. vivax is more prevalent in South-America and the Pacific. In Asia and Oceania, the prevalences are approximately the same.12 P. malaria is less prevalent and found in most endemic regions, particularly in sub-Saharan Africa. P. ovale is also less common, and is also found mainly in sub-Saharan Africa.11 The fifth species causing malaria in humans, P. knowlesi, discovered in 1927 in its natural host, the long-tailed macaque, is currently an important cause of zoonotic malaria in Borneo, Peninsular Malaysia and beyond.14-18 Malaria caused by Plasmodium vivax and P. malariae was also endemic in the Netherlands and transmitted by the mosquito Anopheles atroparvus, which still prevails in the Netherlands.19 This mosquito cannot carry P. falciparum parasites. The last endemic case in the Netherlands was reported in 1959 and in November 1970 the World Health Organization (WHO) declared The Netherlands “Malaria free”. Elimination took many years, involving effective and prompt malaria treatment, vector control (extensive spraying of DDT), education of the public (see Figure 1), improved housing and political commitment.20

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General introduction

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Figure 1. Poster used in the fight against malaria in the Netherlands, titled “destroy mosquitoes, no mosquitoes, no malaria”. Designer: D. Straus, printer: Van Geelen & Co, 1921, Utrecht.21

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Chapter 1

This history of the disease shows that the elimination of malaria is possible. Since the year 2000, large campaigns against malaria have resulted in unprecedented levels of intervention coverage across sub-Saharan Africa.11, 22-33 A study that linked a large database of malaria field surveys including intervention coverages investigated trends from 2000 to 2015 found that P. falciparum infection prevalence in sub-Saharan Africa halved in children (2-10 years old).34

Changing epidemiology Due to remarkable success in reducing the malaria burden, malaria epidemiology has changed and has become more complex. The changing epidemiology is caused by several factors, including effective vector control, strengthening of health systems, increased funding for malaria research and elimination, improved case detection, reporting and diagnosis (the widespread availability of rapid diagnostic tests). 11 Simultaneously, malariaeliminating countries became richer and their GDP (gross-domestic product) increased, which potentially formed a less favourable setting because of improved housing and urbanisation. In the past decades, the burden of malaria was probably also overestimated; many randomised controlled trials evaluating the efficacy and safety of antimalarials found lower prevalences of malaria than expected beforehand. This can be explained by the fact that “symptomatic diagnosis” remained the basis of the management and care of febrile patients in many malaria endemic regions. Since symptomatic diagnosis of malaria overestimates malaria prevalence, a parasitological confirmation before malaria treatment is pivotal. 35

Diagnosis of malaria There are many benefits of a reliable parasitological diagnosis for malaria for patients and health care providers. The two most routinely used methods of diagnosis are traditional light microscopy with thick and thin blood smears (still the most common method and the gold standard)36 and immunochromatographic rapid diagnostic tests (RDTs).36, 37 Both types of methods have advantages and disadvantages not discussed here. Important to consider is that each test has its own limitations regarding sensitivity and specificity, and quality assurance programs are vital. Essential is that antimalarial drug treatment should only be given to those who have confirmed positive parasitological diagnosis (microscopy or RDT); patients with negative malaria diagnostic test results should either be re-assessed for malaria at a later point in time, or should be assessed for other common causes of malaria-like symptoms (and treated appropriately according to the test result). However, in patients with suspected complicated malaria, absence or delay of parasitological diagnosis should not delay prompt treatment.

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General introduction

Compliance to Rapid Diagnostic Tests RDTs allow malaria endemic countries to provide healthcare workers access to adequate malaria diagnosis in rural areas, i.e. settings without regular access to expert light microscopy, by means of a (fairly) simple-to-use, point-of-care rapid test. The WHO recommends that every treated malaria patient has received parasitological diagnosis, and that the patient is treated according to their test result (so a positive test results means an antimalarial administrated, and negative results means assessment for other causes of fever).38 The appropriate use of RDTs in resource-poor settings enables basing treatment onto a confirmed diagnosis; enhances speeding up considering an alternative diagnosis, prevents the over-prescription of antimalarial drugs, reduces cost and avoids unnecessary exposure to adverse drug effects. It is known that antimalarial drugs are often overprescribed, in the sense that they are prescribed to patients with negative RDT results. In this thesis, the compliance to RDTs is further investigated and factors associated with compliance are discussed.

Effective treatments and medicine quality Prompt diagnosis followed by treatment is essential to prevent excess morbidity and mortality. Prompt treatment is most effective if initiated within 24-48 hours after the onset of symptoms suggestive of malaria.38 Only patients who have confirmed malaria should be treated, and adherence to the full regimen should be promoted. To prevent or delay global resistance (and to increase efficacy) all cases of malaria should be treated with at least two effective antimalarial drugs (combination therapy). The WHO currently recommends artemisinin-based combination therapy (ACT) for the treatment of uncomplicated P. falciparum malaria.10 By combining two active pharmaceutical compounds with different mechanisms of action, the highest efficacy is obtained. The WHO recommends currently five different antimalarial ACTs. Since there are almost no new antimalarial drugs are expected to enter phase II/III/IV trials for at least the coming years, their effectiveness must be guaranteed. Therefore, drug resistance patterns as well as drug efficacy is continuously monitored.34, 39, 40 P. vivax malaria is still treated with chloroquine as first-line treatment in most endemic countries, but resistance is increasing. Chloroquine resistance was present in 53% in a study involving approximately 22 000 patients from 179 study sites. 41 Therefore, P. vivax infections from areas with levels of resistance should be treated with an ACT as well, preferably one in which the partner drug has a long half-life. Severe or uncomplicated malaria should be treated with intravenous (or intramuscular) injectable artesunate. Prereferral treatment with rectal artesunate can be a life-saving option, before receiving the appropriate treatment.42 Since malaria can be fatal or cause severe illness if not treated, it is essential that the administered drugs are of good quality.43 Low concentrations of the active pharmaceutical ingredient and subsequent sub-therapeutic concentrations in the patient may

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increase the risk of drug resistance.44 Poor quality drugs can also lead to financial loss for patients and their relatives and healthcare systems and pharmaceutical companies producing the genuine product. The general public can lose confidence in a pharmaceutical brand, drugs, pharmacies, or healthcare providers. With almost no new antimalarial drugs in the pipeline, and reports of ACT resistance of P. falciparum, good quality anti-malarial drugs are a top-priority for malaria control. Unfortunately, poor-quality antimalarial drugs are a crucial health problem in certain developing countries, particularly in sub Saharan Africa and South-East Asia. The production, sale and distribution of falsified antimalarials is a huge market evaluated to several billions of dollars. Recent studies showed the alarming scale of poor-quality antimalarials in malaria-endemic countries.45 On the other hand, there are also major geographical gaps with no large-scale data for many malaria endemic regions, such as Central Africa.45 We conducted a randomized field survey in Gabon, Central Africa, to assess the quality of antimalarial drugs. No systematic data was available for Gabon, and the limited data of surrounding countries showed high prevalences of poor-quality antimalarial drugs.

The pathophysiology of malaria In the past decades we augmented our understanding of the pathogenesis of malaria. This involved studying different mechanisms including parasite invasion, the life cycle of malaria, the immunologic host defence and many other aspects. The clinical symptoms and laboratory abnormalities have also been studied extensively. The literature on lipid profile changes during malaria is relatively scarce. In this thesis, we studied lipid profile changes during malaria infection. This is not solely an academic quest, but may be of significant clinical relevance. A better understanding of the pathophysiological process that are responsible for the many manifestations of malaria may lead to the discovery of potential adjunctive therapies for (severe) malaria, ultimately reducing mortality and morbidity.

Lipid profile changes during malaria In the acute phase of malaria infection, lipid profile changes have been observed.46-48 Changes in serum lipid profile and lipid metabolism are due to a whole range of at least partially disease-specific mechanisms. Although the extent of lipid profile changes seems to be related to the parasitaemia of malaria in several studies, others found no association between the severity of malaria and the magnitude of lipid profile changes. Those transient lipid profile changes in the parasitaemic phase of the disease have been suggested by some researchers as a potential adjuvant diagnostic tool for malaria. Part III of this thesis is aims at improving our understanding of these lipid profile changes during malaria.

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General introduction

Study site – Gabon Patients described in this thesis were recruited in Lambaréné, the provincial capital of Moyen-Ogooué, in Gabon, Central Africa. Gabon, officially the Republic of Gabon, is a state on the west coast of Africa, located on the Equator. It is bordered by Equatorial Guinea and Cameroon in the North, Republic of the Congo in the East and South. The country is nearly 270,000 square kilometres, inhabited by 1,5-2 million people (estimated vary widely). The country is covered by vast tropical rainforest and is warm (average 26°C) with a high humidity. There is little infrastructure and the health services are limited. There are approximately 75 pharmacies open and functioning in Gabon. For the medicine quality survey described in this thesis, anti-malarial drugs were collected from more than 50% of Gabonese pharmacies. Malaria is highly endemic in Gabon. P. falciparum malaria is responsible for 94% of the cases, 6% are mixed infections or mono-infections with P. ovale or P. malariae. 49, 50 P. vivax is almost never seen in Gabon. Patients with fever and malaria like symptoms in this thesis were recruited in the Albert Schweitzer Hospital in Lambaréné. This hospital was found by Albert Schweitzer (14 January 1875 – 4 September 1965), who was a French-German theologian, organist, and physician. In 1981, the third generation of the hospital was build. Around 1990, a medical research unit was established. Renamed “Medical Research Unit” in 2001, the centre became administrative and financially independent from the hospital. The research focus has expended during the last decades to tuberculosis, soil-transmitted helminths and malaria. Large scale studies were conducted here, such as the RTS,S malaria vaccine trial. In 2011, the centre was renamed the to “Centre de Recherches Médicale de Lambaréné” in 2011 and received a new legal status as a nonprofit association in Gabon. The research lab is quite well equipped. However, the supportive care for critically ill patients, for example severe malaria is limited. Mechanical ventilation, blood pressure and heart rate monitoring and CT-scans are not (yet) available.

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Aim and Outline This thesis intended to contribute to the ultimate goal to control malaria by studying the epidemiology of poor-quality antimalarial drugs in sub-Saharan Africa and to contribute to the development of a novel, cheap and easy-to-use method to assess the quality of antimalarial drugs in the field. Furthermore, this thesis aimed to better understand lipid profile changes during malaria infection. This thesis is organised in three parts.

Part I: Treatment of malaria The first step in this thesis was to discover the history of malaria. This history of malaria is important because it helps us to understand our current state of knowledge of malaria. In many aspects, the multifaceted history of malaria (Chapter 2) illuminates the evolution of modern science and medicine over centuries, from myths and supernatural beliefs to evidence-based scientific insights. Chapter 3 and 4 are systematic reviews summarizing antimalarial drug treatment. Chapter 3 summarized the current treatment chemotherapy strategies for malaria and discussed novel developments as far as they underwent Phase III/IV clinical trials. ACTs are currently recommended as first-line treatment of uncomplicated falciparum malaria, whereas chloroquine is still commonly used for the treatment of non-falciparum species (P. vivax, P. ovale and P. malariae). Chapter 4 reviewed whether a more simplified, more uniform treatment approach across all malaria species is worthwhile to be considered and therefore investigated the efficacy and safety of ACT for non-falciparum malaria. A patient group with special risks are pregnant women. Pregnancy has been reported to alter the pharmacokinetic properties of anti-malarial drugs, including the different components of artemisinin-based combination therapy. Because it is difficult to draw strong conclusions based on individual pharmacokinetic studies, we summarized the evidence of the influence of pregnancy on the pharmacokinetic properties of ACTs (Chapter 5). The treatment of a patient with malaria can only start following establishment of the correct diagnosis. The WHO recommends malaria to be confirmed by light microscopy or a Rapid Diagnostic Tests (RDT). In Chapter 6, we investigated the health workers’ compliance to RDTs and investigated factors associated with compliance to results, thus health workers treating patients according to the RDT result.

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General introduction

Part II: The quality of anti-malarial drugs In Chapter 7 we investigated the prevalence of poor-quality drugs in Gabon, Central-Africa, using a rigorous standardized study design and following, where appropriate, the Medicine Quality Assessment Reporting Guidelines. In Chapter 8, we investigated a non-invasive (non-destructive) method as a screening and identification tool for antimalarial drugs. The study described the evaluation of a handheld Raman spectrometer, for testing anti-malarial drugs.

Part III: Lipids and coagulation during malaria infection Lipid profile changes have been reported during the course of malaria infection. In Chapter 9, we reviewed and meta-analysed previous conducted studies to identify serum lipid profile changes with respect to commonly used laboratory parameters such as (total) cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and triglycerides. The meta-analysis in Chapter 9 forms the basis of an observational clinical cohort study (Chapter 10), in which we prospectively measured lipid profile changes in patients with malaria versus patients who tested negative for malaria by microscopy.

Part IV: Epilogue In Chapter 11, we discussed our proposal with pros and cons of reporting the costs of medical research. We proposed to publish the cost of research in order to increase transparency, efficiency, quality and ultimately reproducibility of scientific studies.

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Part I: Treatment of malaria

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“Land cover map of Africa. This map was produced for the year 2000 using data collected by sensors on satellites.” Adapted from: Soil Atlas of Africa, 2013. European Commission, Publications Office of the European Union, Luxembourg.1

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Chapter 2 Discovery of the Malaria Parasites and their Vectors – A Short History

Benjamin J. Visser Martin P. Grobusch

Discoveries in Modern Science: Exploration, Invention, Technology, 1st ed. 2015, Chapter: Malaria Is Transmitted by Mosquitoes, Publisher: Macmillan Reference USA, Editors: James Trefil, Patricia Daniels, Donna McPhie, Craig Schiffries, pp.640-647

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Chapter 2

Abstract Malaria is an ancient disease continuing to pose an enormous health, social, and economic burden. It is caused by infection with protozoan parasites belonging to the genus Plasmodium transmitted via the bite of female Anopheles species mosquitoes. Of more than 100 different species infecting a wide range of animals from rodents and birds to mammals, five species of malaria parasites are known to infect humans: Plasmodium falciparum, P. vivax, P. ovale (now being recognized as consisting of two subspecies), P. malariae and P. knowlesi. P. falciparum is most likely to cause severe disease and, if not promptly treated, may lead to death. References to the disease occur in the Chinese canon of medicine, clay tablets from Mesopotamia, Egyptian papyri and Indian medical works. Descriptions of malaria from classic Greece and the Roman Empire are abundant. It was commonly believed that malaria was caused by marsh water and foul vapors emanating from swamps, hence the word mal’aria, from the Italian for “bad air”. For thousands of years, no effective treatment was available. This changed with the discovery of Artemisia annua (sweet wormwood) in China and the use of quinine from Peruvian bark as potent and effective drugs against malaria. The current understanding of the malaria parasites and their lifecycle starts in the end of the nineteenth century with the discovery of the malaria parasites in the blood of malaria patients by Alphonse Laveran in 1880. Subsequently, Ronald Ross discovered in 1897 that a bird malaria parasite was transmitted by mosquitoes. In 1898 Giovanni Grassi, Camillo Golgi, Ettore Marchiafava, Amico Bignami, Angelo Celli and Giuseppe Bastianelli confirmed that malaria in humans was also a mosquito-borne disease, in this case Anopheles species. Grassi and Filetti introduced the names of P. vivax and P. malariae in 1890. The causative agent of what was dubbed ‘malignant malaria’ was baptized P. falciparum by William Welch in 1897 and P. ovale by John Stephens in 1922. The discovery of a liver stage before malaria enters the bloodstream was made by Henry Shortt and Cyril Garnham in 1948. The existence of dormant stages, in P. vivax and P. ovale was shown in 1982 by Wojciech Krotoski. This chapter describes the key discoveries and provides a short overview of the multifaceted history of malaria.

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Introduction Malaria is an ancient disease causing an enormous health, social, and economic burden51, 52. It has been described for more than four millennia and is understood to have changed human populations and the course of human history53, 54. Malaria continues to sicken hundreds of millions of people, resulting in an estimated number of preventable deaths exceeding one million per year (1.2 million in 2010), most of them children under five years of age in subSaharan Africa3. In many aspects, the multifaceted history of malaria illuminates the evolution of modern science and medicine over the centuries, from myths and supernatural beliefs to evidence-based scientific insights55. Poverty and inequality, as much as geography and climate, made malaria only recently a ‘‘tropical’’ disease: even in the early twentieth century it afflicted temperate regions of North America stretching northward into Canada, western and northern Europe, and Central and East Asia (see Figure 1)56, 57. Elimination of the disease was achieved progressively from many of these areas, and following these successes, the World Health Organization (WHO) Global Malaria Eradication Program was launched in the late 1950s. There were many accomplishments; however, in the heartlands of malaria (especially sub-Saharan Africa) the disease remains a dangerous and resilient foe58. Climate change is predicted to have unexpected consequences on malaria distribution and incidence59. Changes in rainfall, and rising as well as fluctuating temperatures, influence the Anopheles mosquito vectors60. In addition, the malaria life cycle can be altered by temperature variations that influence parasite development within the mosquito (the extrinsic incubation development)61. Climate change, which may lead to altitudinal and latitudinal temperature increases, pose a particular risk for high-elevation areas and regions with a temperate climate59, 62. Climate change can result in reduced prevalence in some areas, while it may increase or be (re-)introduced in others60, 63.

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Figure 1. World distribution of malaria, Mid-19th century to 2010. Note: This composite map does not claim to be complete. It is intended to illustrate where malaria transmission existed over the years. Source: Adapted from: “Eliminating Malaria: Learning from the Past, Looking Ahead. “ P&I series report. Available at: http://www.rollbackmalaria.org/microsites/wmd2014/report9.html

Malaria in ancient history Malaria was known in China long before classical antiquity and the Christian era 64. The symptoms of malaria, or disease resembling malaria, was described in the Chinese medical classic Nei Ching (the Canon of Medicine) in 2700 B.C.E., edited by the legendary Yellow Emperor Huang Ti.65 It contains descriptions of different types of fevers and already differentiated tertian (every third day) from quartan (every fourth day) fevers with enlargements of the spleen. Clay tablets from Mesopotamia from 2000 B.C.E., and Egyptian papyri from 1570 B.C.E. describe malaria66. In India, the symptoms of malarial fever were described in the Charaka Samhita and the Susrata Samhita and other medical works67. Tertian and quartan fevers and a ‘‘splenic’’ belly were described and were considered as a most dreaded affliction, attributed to the anger of the god Shiva. Five types of mosquitoes were described and blamed for the transmission of this ‘‘king of diseases’’ 68. These ancient historical records must be regarded with caution as less than firm and controversial compared to sources from classical antiquity. Literature from Greek sources is extensive; malaria was widely endemic in Greece by the sixth century B.C.E., and it was responsible for the decline of many of the city-state populations. In about 550 B.C.E. the philosopher Empedocles of Agrigentum, who resolved a ‘‘plague’’ in Sicily by draining marshes, demonstrates how

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early the Greeks inductively associated malaria with swamps68. Hippocrates, in about 400 B.C.E., noted the most important symptoms, described different stages of a malaria attack, and noticed the seasonal character of the disease and the noxious consequences of wet springs and dry summers. The same familiarity with malaria is shown in the writings of Plato, Aristotle, and later Greek authors68. When malaria was introduced into Italy is unclear, yet the evidence that Rome and major parts of the Roman Empire were highly malarious are overwhelming. Several authors, such as M. Porcius Cato, Cicero, Marcus Terentius Varro, and others, described malaria and warned residents of marshy places68. Both the Greeks and Romans became more certain that there were some causal relations between intermittent fevers and swampy terrain. This, among other things, resulted in the use of drains to mitigate the detrimental effect of stagnant water. It was suggested that marsh water and marsh vapours were causal factors, hence the name mal’aria, from the Italian for ‘‘bad air.’’

Malaria treatments The rediscovery of artemisinin by Chinese scientists for malaria treatment was one of the greatest achievements in medicine in the twentieth century69. Archaeological findings suggest that Artemisia annua, also known as sweet wormwood, has been used in China for thousands of years to treat many illnesses, including malaria. During the second century B.C.E., the herbal remedy qinghaosu, obtained from the qinghao plant (Artemisia annua) was described as anti-fever medicine in the Fifty-Two Remedies, a medical treatise discovered in the Mawangdui Tomb (Changsha, China) in 197270, 71. In 340 B.C.E., the Artemisia annua plant was first described in the Handbook of Prescriptions for Emergency Treatments, by the alchemist Ge Hong of the East Yin Dynasty72. Yet for a long time this ancient drug had fallen out of common use. In 1967 the top secret Project 523 was established by the Chinese government during the Vietnam War, involving 500 scientists in about 60 laboratories and research institutes, to develop new antimalarials to aid NorthVietnamese troops73. During the war, malaria caused by chloroquine-resistant Plasmodium falciparum was a major health problem that urged malaria research efforts on both sides. Because the research was secret, publication in scientific journals was forbidden, and it is therefore hard to decide who exactly should be credited with the (re)discovery of artemisinin. In 1971 Chinese scientists led by Youyou Tu (1930– ), recognized its potential for treating malaria and isolated the active ingredient of qinghao, known as artemisinin73. Its rapid action, low toxicity, and potent effect against the most dangerous species, Plasmodium falciparum malaria, made it a favored subject for research. Between 1976 and 1978 the molecular structure of artemisinin was discovered, and new artemisinin derivatives were developed74. In 1979 artemisinin-based antimalarial drugs were successfully used in the combat zone in the Sino-Vietnamese War (the Third Indo-China War). Derivates of this compound are today the most potent and effective antimalarial drugs. Artemisinin

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combination therapies (ACTs) are recommended by the World Health Organization and are the first-line treatment in nearly all malaria-endemic countries.

Quinine from the New World The curative virtues of the Peruvian bark, also known as Jesuit’s bark, first became known to Europeans about 1630, following the arrival of Spanish Jesuit missionaries in the New World68, 75. The legendary story that the Countess of Chinchόn, wife of the Viceroy of Peru, was cured of her fever by using this bark, which she supposedly later brought to Spain, has been disputed68. It is more likely that this indigenous remedy was discovered by observing practices of indigenous Indian herbalists by the Jesuits. Nevertheless, the tree was given the generic name cinchona, after the Countess of Chinchόn. The acceptance of the cinchona bark as antimalarial was arduous and slow, and doubts regarding its efficacy lingered for a long time68. Many physicians hesitated to use the bark because of the many attributed toxicities. Yet the rising demand for an effective antimalarial prompted botanical expeditions to the New World by the British and the Dutch76. This was hampered when in the nineteenth century South American countries began outlawing the export of cinchona seeds. After a successful attempt to smuggle the famous Cinchona ledgeriana strain from Bolivia, the South American monopoly was destroyed. Plantations were set up in the British Empire in India and Ceylon (Sri Lanka) and by the Dutch in Java, the Dutch East Indies, to produce large quantities of the bark. This resulted in a situation in which for many years the Netherlands East India cinchona plantations have produced 97 percent of the total global production while British India has produced 2.5 percent and the rest of the world 0.5 percent. For a long time the Dutch held a monopoly on cinchona bark. The use of the cinchona bark enabled settlers and explorers to enter dangerous malarial areas and therefore to some extent facilitated imperial expansion by Western powers. The active antimalarial principle from the bark, quinine (from kina-kina, Quechua for ‘‘barks of barks’’), is now chemically synthesized and is, along with artemisinins, one of the most effective and potent drugs.

Discovery of Chloroquine and Mefloquine Chloroquine was discovered by Johann ‘‘Hans’’ Andersag (1902–1955) at the Bayer laboratories in Elberfeld, Germany77. In July 1934 Andersag chemically modified atabrine by replacement of a part with a quinoline ring77. The drug was initially named resochin. The drug was shelved for more than ten years and not used in practice because it was considered too toxic for practical use in humans (it was tested against blood-induced P. vivax malaria in four paretic patients in a psychiatric clinic)78. Finally, during World War II, US government-sponsored trials unequivocally recognized and established chloroquine as an effective and safe antimalarial drug. For many years it was the first-line antimalarial drug.

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However, as for many antimicrobial drugs, eventually (two decades later) drug-resistant strains developed77. In the second half of the twentieth century, another important antimalarial drug was also developed in a time of war: mefloquine was number 142,490 of a total of 250,000 antimalarial compounds screened during the US Army’s antimalarial drug discovery program 79. It was discovered shortly after the Vietnam War in the 1970s, and subsequently marketed worldwide.

Discovery of the life cycle of the malaria parasite in humans With the discovery of bacteria by the Dutch tradesman and scientist Antoni van Leeuwenhoek (1632–1723) in the Netherlands in 1676—along with the subsequent discoveries of the French chemist and microbiologist Louis Pasteur (1822–1895) and the German physician and microbiologist Robert Koch (1843–1910) in 1878–1879 that many diseases are caused by microbes (known as the germ theory)—the search for a bacterial origin of malaria intensified80. Many studies subsequently searched unsuccessfully for an infectious agent in marshland soil.

Alphonse Laveran Discovers the Malaria Parasite It was in this setting that Charles Louis Alphonse Laveran (1845–1922), a student of Pasteur and a French military doctor based in a hospital in Algeria, started searching for the causative agent80, 81 (Cox 2010). By means of the autopsy of malaria patients and examining lesions of affected organs and blood, he found that the only constant factor was the presence of granules of black pigment in the blood82, 83. He examined fresh unstained blood of hundreds of patients and observed ‘‘several different forms of erythrocytic organism including crescents, spherical motionless bodies with pigment, spherical moving bodies with pigment and bodies that extruded flagella-like structures all of which he thought were on the outside of the red cells’’80. He noticed these in all patients, but never in those without malaria. Furthermore, he noted that quinine removed these stages from the blood. On November 6, 1880, he observed a motile parasite in blood from a malaria patient. What he saw was the exflagellation of the parasite, later identified as the male gametocyte, a phase in the life cycle of the parasite that normally occurs in the midgut of the Anopheles mosquito. Laveran realized he had discovered the causative parasitic protozoan, which he named Oscillaria malariae80. His theory was not readily accepted by eminent microbiologists or malariologists of his time, but he did convince the Italian malariologists Camillo Golgi (1843–1926), Amico Bignami (1862–1929), and Ettore Marchiafava (1847–1935) that malaria was caused by a protozoan and not a bacterium. Later he also convinced leading microbiologists including Pasteur, Robert Koch, Charles Chamberland (1851–1908), and Pierre Roux (1853–1933)80. The discoveries of Laveran were particularly remarkable because he used

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only fresh blood (without any kind of staining) and a dry objective lens with a maximum magnification of x400. In the search for malaria parasites in animals, by accident, the methylene blue-eosin stain was discovered by the Russian physician Dimitri Leonidovich Romanowsky (1861–1921) in 189184. This would serve as the basis for blood staining methods developed later by others. Staining methods could not only be used for malaria, but also for other tropical diseases and is therefore a most significant breakthrough in the history of parasitology. For Laveran’s extensive work on protozoa, he was awarded the Nobel Prize in Physiology or Medicine in 190785.

Naming of the disease Camillo Golgi, an Italian neurophysiologist, had observed in 1885–1886 that there were at least two separate types of malaria: tertian (48-hour periodicity) and quartan (72-hour periodicity). The causing organisms were later called Plasmodium vivax and Plasmodium malariae. He also established that the two forms produced differing amounts of merozoites into the bloodstream upon maturity and that the fever coincided with the rupture and release of merozoites into the blood stream. Finally, he observed that the severity of symptoms correlated with the number of parasites in the blood80. The Italian malariologists Giovanni Battista Grassi (1854–1925) and Raimondo Feletti (1887–1927) first introduced the names of Plasmodium vivax and Plasmodium malariae in 189086. This was in contrast with the belief of Laveran that there was only one species, Oscillaria malariae. The malignant tertian malaria was named Plasmodium falciparum by an American physician and bacteriologist, William Henry Welch (1850–1934). In West Africa in 1918 the British physician John William Watson Stephens (1865–1946) discovered and named a fourth species that resembled Plasmodium vivax, which he described as Plasmodium ovale in 192287. Plasmodium knowlesi, a zoonotic malaria species, was first described by Robert Knowles and Biraj Mohan Das Gupta in 1932 in wild-caught primates. The first documented human infection with Plasmodium knowlesi was in 196588, and until 1971 only two cases had been described. Since 2004 several hundred cases of human infections have been reported, mainly from Malaysia and other parts of Southeast Asia89.

Ross’s discovery that mosquitoes transmit malaria parasites Despite Laveran’s flagellum discovery, the transmission conundrum of the malaria parasite was yet to be unravelled. The solution to the problem of transmission was due in very large part to the efforts of Ronald Ross (1857–1932), a British doctor working with avian malaria parasites in India68. However, enormous credit is due to the Scottish physician Sir Patrick Manson (1844–1922), who played a pivotal role in providing the scientific background that eventually led to Ross’s accomplishments90, 91.

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The fundamental principle of the transmission of malaria by mosquitoes came to be deemed possible only when Manson (considered by many as the father of tropical medicine) demonstrated in 1878 that the parasite for elephantiasis (Bancroft’s filaria) could infect a mosquito. Working with the hypothesis of Manson and Laveran that mosquitoes were involved in the transmission of malaria between humans, Ross made his most important discovery on August 20, 189780. While dissecting a culicine mosquito, he found the avian malaria parasite Plasmodium relictum in the stomach tissue of the mosquito. Using the microscope, Ross also identified the malaria parasite by its darkly pigmented cells (now known as haemozoin, the black malaria pigment, derived from discomposed haemoglobin) 92 . Using an experimental malaria model in birds, he demonstrated in 1898 that the parasite developed in mosquitoes and migrated to its salivary glands, allowing the infected mosquito to infect other birds during consecutive bites. He argued that human malaria parasites, analogous to the avian model, are also transmitted by mosquitoes 93. However, because of military duties (he was sent to India to investigate an epidemic of plague) he was not allowed to test his theory at that time. The incrimination of mosquitoes as vectors in human malaria was demonstrated shortly after the discoveries of Ross by the Italian scientists Giovanni Battista Grassi, Amico Bignami, Giuseppe Bastianelli, Angelo Celli, Camillo Golgi, and Ettore Marchiafava80, 94. They demonstrated the complete sporogonic cycle of Plasmodium falciparum, P. vivax, and P. malariae with experiments involving Anopheles claviger mosquitoes fed on malarial patients and subsequently transmitted the disease to healthy individuals via the bite of these mosquitoes. Later on they proved that only female Anopheles mosquitoes could transmit malaria. The mosquito-vector link was established beyond doubt68. Meanwhile, Ross had been posted to Sierra Leone where, in 1899, he tested and confirmed his hypothesis and the discoveries of his Italian colleagues. The discovery made by Ross that a vector taking a blood meal from an infected host, to transmit this parasite somewhat later to a healthy host, would be one of the most far-reaching scientific insights to date. It took other scientists quite some time to realize that other diseases such as onchocerciasis, filariasis, loiasis, African trypanosomiasis, leishmaniasis, and others were transmitted by the bite of infected flies and mosquitoes. In 1902 Ross received the Nobel Prize in Physiology or Medicine for his work on malaria. The complete life cycle as we understand it from the ground-breaking contributions described above is depicted in Figure 2 (next page).

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Figure 2. Courtesy of H. Visser-Berenschot, 2012. Adapted and modified from AJ Cann. Original image from: http://www.flickr.com/photos/ajc1/6174379207/ (CC BY-SA 2.0) Malaria in humans is caused by five different Plasmodium species (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and the zoonotic Plasmodium knowlesi), all of which share essentially similar life cycles (Figure 2). Infection begins when, via a bite and blood meal, sporozoites are entering the blood stream from the salivary glands of an infected female Anopheles mosquito (1) and travel to the liver where they invade hepatocytes (liver cells) (2). There they undergo asexual multiplication resulting in thousands of daughter merozoites (new parasites) released into the bloodstream (3). This process, “exoerythrocytic schizogony”, is asymptomatic. Some sporozoites develop into ‘hypnozoites’ (Plasmodium vivax and Plasmodium ovale only) which are dormant forms that may resume replication after a period of months or years to cause relapses of malaria. In the bloodstream, the sporozoites invade red blood cells where they initiate a second phase of asexual multiplication (4). This results in the production of new merozoites which invade other red blood cells (5). This stage of ”erythrocytic schizogony” (from Greek ‘schizein’ – to divide), is repeated continuously and is responsible for the disease, malaria. During the infection, the gametocytes, male (microgametocytes) and female (macrogametocytes) are produced (6) which are ingested during a subsequent mosquito blood meal. The parasites’ sexual multiplication in the female

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Anopheles mosquito’s stomach (midgut) is known as the sporogonic cycle. In the mosquito’s stomach cavity the microgametes penetrate the macrogametes, generating zygotes by fusion and meiosis (7). The zygotes become motile and enlarged (ookinetes) and this form penetrates the midgut wall (8) of the mosquito where it develops into oocysts. The oocysts grow, burst, and release numerous sporozoites into the coelomic cavity of the mosquito (9). The sporozoites then make their way to the mosquito's salivary glands awaiting the mosquito to start feeding on another human host.

Consequences for Malaria Control. The discovery of the role of Anopheles mosquitoes provided new and effective tools for the fight against malaria. Grassi conducted a classical experiment in a malarious area in Italy, protecting 112 volunteers from mosquito bites between dusk and dawn and demonstrating that only five of them developed clinical malaria compared with 415 mosquito-exposed individuals who all developed the disease95. Hence the first weapon of controlling the disease was demonstrated to be extremely effective. Subsequently, other choices of the vector control menu were developed, such as the use of chemical larvicides (use of oils), biological control (larvivorous fish, biological larvicides), environmental source reduction (draining swamps and other mosquito habitats), and personal protection (bed-nets, insect repellents)68.

Mechanism of action of disease The clinical manifestations of malaria vary with geography, epidemiology, immunity, and age. Understanding the complex pathogenesis of malaria requires exploring mechanisms for parasite invasion and host immune response96. The pathogenesis of Plasmodium falciparum is the most studied, since this species causes the most severe clinical disease, highest parasitemias, and has a high mortality rate. Other forms are rarely fatal. Plasmodium knowlesi malaria, endemic in localized areas in Southeast Asia, can also cause fulminant disease. As described above, the plasmodial life cycle consists of an exoerythrocytic (asymptomatic) stage and an erythrocytic (symptomatic) stage. The discovery of a liver stage before malaria enters the bloodstream was made by the British protozoologists Henry E. Shortt (1901–1994) and Percy Cyril Claude Garnham (1901–1994) in 1948 97. The existence of dormant stages in Plasmodium vivax was shown in 1982 by the Polish-American physician and scientist Wojciech A. Krotoski (1937– ) and his international team98. Plasmodium falciparum malaria differs from the so called benign malarias in several ways: it affects all red blood cells, digesting haemoglobin and making the cell wall less deformable, thus resulting in haemolysis and/or splenic clearance (anaemia). Furthermore, endothelial binding of Plasmodium falciparum–infected red blood cells leads to sequestration within small blood vessels, which results in blood flow obstruction, inflammation, and endothelial barrier breakdown. Microvascular disease due to cytoadherence (formation of sticky knobs

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on the red blood cell surface and rosetting) of infected erythrocytes to endothelial cells lining the blood vessels, permitting sequestration, is fairly well understood 99. Although not described here, research into cerebral malaria has provided some insights and many theories, but we still have limited understanding on the pathogenesis of cerebral malaria, anaemia in malaria, and placental malaria. Based on concepts of pathophysiology, several adjuvant treatments have been recommended for cerebral malaria; however, none of those have stood the test of time.

Renewed optimism The fascinating quest of malariology’s early heroes, unravelling the mysteries of the plasmodia causing malaria, has paved the way for generations of scientists helping us to better understand malaria in order to fight it most efficiently. We are currently witnessing renewed optimism that malaria control can be optimized to a degree coming close to its elimination from many areas, on a global scale. While the success depends on sustained scientific, economic, and political commitment, we may be able to succeed in pushing back malaria on an unprecedented scale, with a mixed approach to malaria control encompassing optimized prevention measures, better tools to diagnose the disease, novel drugs to treat it efficiently and keep the development of drug resistance at bay, and possibly even a vaccination.

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“Map shows organic carbon in the uppermost 100 cm of soil. Organic carbon held deeper than 100 cm (e.g. deep peats, estuaries, etc) are not counted in this exercise. Consequently, there is more soil carbon in reality than shown by this map.” Adapted from: Soil Atlas of Africa, 2013. European Commission, Publications Office of the European Union, Luxembourg.1

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Chapter 3 Malaria: an update on current chemotherapy Benjamin J. Visser Michèle van Vugt Martin P. Grobusch

Expert Opinion on Pharmacotherapy 2014 Oct;15(15):2219-54 Appendices and supplementary material are available online at: http://www.tandfonline.com/doi/full/10.1517/14656566.2014.944499

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Abstract Introduction Chemotherapy of malaria has become a rapidly changing field. Less than two decades ago, treatment regimens were increasingly bound to fail due to emerging drug resistance against 4-aminoquinolines and sulfa compounds. By now, artemisinin-based combination therapies (ACTs) constitute the standard of care for uncomplicated falciparum malaria and are increasingly also taken into consideration for the treatment of non-falciparum malaria.

Areas covered This narrative review provides an overview of the state-of-art antimalarial drug therapy, highlights the global portfolio of current Phase III/IV clinical trials and summarizes current developments.

Expert opinion Malaria chemotherapy remains a dynamic field, with novel drugs and drug combinations continuing to emerge in order to outpace the development of large-scale drug resistance against the currently most important drug class, the artemisinin derivatives. More randomized controlled studies are urgently needed especially for the treatment of malaria in first trimester pregnant women. ACTs should be used for the treatment of imported malaria more consequently. Gaining sufficient efficacy and safety information on ACT use for nonfalciparum species including Plasmodium ovale and P. malariae should be a research priority. Continuous investment into malaria drug development is a vital factor to combat artemisinin resistance and successfully improve malaria control toward the ultimate goal of elimination.

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Introduction Chemotherapy of malaria has become a considerably rapidly changing field. Less than two decades ago, established treatment regimens were increasingly bound to fail due to emerging drug resistance against 4-aminoquinolines and sulfa compounds. By now, artemisinin-based combination therapies (ACTs) constitute the standard of care for uncomplicated falciparum malaria and are increasingly also taken into consideration for the treatment of nonfalciparum malaria. Most importantly, injectable quinghaosu (artesunate) derivatives are now rapidly becoming the therapeutic backbone of severe falciparum malaria around the globe. Unfortunately, little progress has been made so far on developing marketable tissue schizonticides as possible alternatives to the 8-aminoquinoline primaquine. Furthermore, early evidence of resistance development against the artemisinins 100, 101 highlights the need for continuous investment into the development of alternative drug classes.

Objectives This review summarizes the current treatment strategies for malaria and discusses novel developments as far as they are currently undergoing Phase III/IV clinical trials. Preclinical developments and the utilization of antimalarials for malaria chemoprophylaxis in high-risk groups (pregnant women and infants) and for travelers are not in the focus of this review and have been covered recently elsewhere.102-110

Methods This is a narrative review. Methods of the search strategy and inclusion and exclusion criteria were specified in advance and documented in a protocol. Recommendations made by the Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) group were followed where appropriate.111 The electronic databases Medline/PubMed, Embase, Cochrane Central Register of Controlled Trials (The Cochrane Library), Biosis Previews and the African Index Medicus were searched in order to identify studies published up to June 2014. In addition, major trial registries were searched to identify ongoing or future trials. The search strategy consisted of free-text words and subject headings related to the treatment of malaria with synthetic drugs. Malaria search terms chosen by consulting a medical subheading (MeSH) thesaurus, and were supplemented with search terms used by Cochrane Database reviews of malaria. For the search, also the function ‘All Fields and Title/Abstract’ was utilized to identify recent, not yet indexed publications. Main search terms were ‘malaria (MeSH)’ and ‘therapeutics (MeSH)’. The search strategy was not limited by language. We did not search the gray literature. The search was restricted to the past 5 years to avoid redundant data and to select more recent evidence. However, related or cited papers of crucial trials and guidelines before this period have also been included. All abstracts were read by the first author, and key articles were indentified based on inclusion criteria and consensus among all authors. Bibliographies of relevant studies retrieved from the studies were checked for additional publications. Selection criteria for inclusion of

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retrieved studies were as follows; randomized controlled trials (RCTs), meta-analyses, clinical trials, clinical guidelines were included in this review. Case series, case reports and animal studies were excluded. Only trials in Phase III of development and onward were included. The software program EndNote X7.0.2. (Thomson Reuters) was used to manage, de-duplicate and screen the references for eligibility. We did not assess risk of bias in included studies nor did we investigate publication bias. Treatment of uncomplicated Plasmodium falciparum malaria The causative species, the severity of signs and symptoms as well as patient age, immunity status and other risk determining factors (acute or chronic conditions, pregnancy and/or immune impairment) direct the choice of the most appropriate therapy. In addition, drug therapy should be in conjunction with relevant treatment guidelines and subject to local availability of drugs. Much evidence from RCTs and metaanalyses is available on the treatment of uncomplicated P. falciparum malaria.112-118 To overcome the threat of drug resistance of P. falciparum, and to augment treatment efficacy, most malaria-endemic countries have endorsed the World Health Organization (WHO) recommendation and adopted ACTs as first-line therapy for uncomplicated falciparum malaria38, following establishment of a correct diagnosis of malaria by rapid diagnostic tests. The history of artesunates from ‘household remedy’ against malarial fevers on the Chinese peninsula of Hainan to the modern-day backbone class of antimalarials has been summarized.119 The artesunate derivate components in combination treatments are active against all stages of the asexual malaria parasites and lead to significantly shorter parasite clearance time than other antimalarials.120 Moreover, they exhibit some effect on gametocytes, thus reducing the risk of life cycle perpetuation in post-therapeutic patients, which is important when it comes to optimizing malaria control/pre-elimination efforts in malaria-endemic areas.121 The rationale of administering an ACT, usually over 3 days in total, is twofold; first, administering two or more blood schizontocidal drugs with different modes of action and targets is most often more effective compared to a single drug. In the event that resistance conferring polymorphisms pre-exist, or arise from de novo mutations during treatment to one of the drugs, the mutant and resistant parasite will be probably killed by the still effective other drug. Secondly, artemisinin derivates should be given in combination since they exhibit an extremely short half-life. Recrudescence may result if given as monotherapy for too short. Artemisinins do have a favorable adverse effects profile.122 Several artemisinin derivates are available -- with no regimen having been unequivocally demonstrated to be superior over the others -- including artesunate (watersoluble: for oral, rectal, intramuscular or parenteral use) and artemether (lipid-soluble: for oral, rectal or intramuscular use). These agents are converted to the active agent dihydroartemisinin (DHA), which itself can also be administered directly as in the DHApiperaquine combination. These drugs differ in their pharmacokinetic and dynamic properties such as stability, bioavailability, metabolism, absorption and excretion. Serious side effects of ACTs have not been reported in humans, although neurotoxicity has been reported in animal studies.123 ACTs are generally not recommended in the first trimester of pregnancy, on the ground of lack of safety data (see under ‘treatment of malaria in

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pregnancy’ for details). ACT options now recommended for treatment of uncomplicated P. falciparum malaria in any order are: artemether + lumefantrine, artesunate + amodiaquine, artesunate + mefloquine, artesunate + sulfadoxine-pyrimethamine and DHA + piperaquine (PIP). A wealth of clinical trials have been performed to evaluate the efficacy and safety of artemether-lumefantrine (AL).124 This combination is well tolerated and regularly yielded cure rates of > 95% for P. falciparum malaria in several trials.122-145 Several ongoing trials (Table 1) are currently assessing the efficacy of AL compared to the relatively new regimen of DHA + PIP (Phase III: NCT01939886; Phase IV NCT01704508, NCT01755559). Several studies have shown that artesunate in combination with mefloquine is 90 -- 100% effective.146-151 As well, artesunate in combination with atovaquone-proguanil is highly effective and well-tolerated, as shown among 1596 patients in Thailand with uncomplicated multidrug-resistant falciparum malaria.152 The recently developed and now marketed fixeddose ACT is DHA + PIP, with cure rates > 95%.139, 153-156 DHA + PIP is currently under investigation in seven ongoing trials (Phase III: NCT018 45701, NCT01838902; NCT01736319 and NCT01640587; Phase IV: NCT01878357, NCT01755559 and NCT01704 508). Studies evaluating the combination artesunate + sulphadoxinepyrimethamine for the treatment of uncomplicated malaria show variable efficacies. 157-159 Artesunate-pyronaridine versus other ACTs in adults and children with uncomplicated P. falciparum malaria performed well in six trials39, 160-164, with a polymerase chain reaction (PCR)-adjusted treatment failure rate at day 28 below the 5% standard set by the WHO. 165 However, further efficacy and safety studies are needed whether this combination is an option as first-line treatment.165 Recently, the combination artesunate-amodiaquine showed a significantly higher unadjusted adequate clinical and parasitological response compared to AL (58.4 vs 46.1%) at day 28.166 The efficacy of the combination of fosmidomycin and clindamycin has been investigated in several trials167-170 and has been considered as a promising antimalarial combination as alternative to artemisinins. However, results are conflicting and a recent trial conducted by Lanaspa et al.171 showed inadequate efficacy of a new formulation of fosmidomycin-clindamycin combination treatment. Therefore, development of this combination has stalled. However, one Phase II study is still recruiting patients for this combination treatment (NCT01361269). Fosmidomycin-piperaquine appears to be a potential combination of interest and is currently entering clinical testing (G Mombo-Ngoma, personal communication). A new, not yet marketed, fixed-dose combination of artemisinin-naphthoquine (‘Arco’) has been evaluated in Phase III trials.172182 Naphthoquine is a 4-aminoquinoline, synthetic blood schizonticide antimalarial drug with a long half-life (276h 172) and is administered orally as a singledose treatment. A study evaluating the safety and efficacy of artemisinin-naphthoquine versus DHA-piperaquine in adult patients with uncomplicated malaria found a PCR-corrected cure rate of 96.3% (95% CI: 93.6 -- 99.0%) in Arco compared to 97.3% (95% CI: 95.0 -- 99.6%) in DHP groups.172 The drug was well tolerated with no adverse reactions. Although a highly effective singledose treatment for malaria seems to be a breakthrough, concerns have been raised. There is a considerable chance that widespread single-dose use of naphthoquine in this particular

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combination could generate enough pressure on the malaria parasites resulting in the emergence of increasingly less susceptible mutants and eventually to different levels of parasite resistance.172, 173, 183 Other compounds currently under investigation in Phase I and II studies are discussed in appendix 1 (see page 53).

Article highlights.  Artemisinin-based combination therapies (ACTs) constitute the standard of care for uncomplicated falciparum malaria and are increasingly also taken into consideration for the treatment of non-falciparum malaria (Plasmodium vivax, P. ovale, P. malariae and P. knowlesi).  For severe malaria, intravenous (i.v.) artesunate is superior to quinine; however, i.v. administered quinine remains an option for the treatment of severe malaria particularly if artesunate is unavailable. In the future, should emergence of resistance arise on a large scale before other alternatives become available then i.v. administered quinine will also be an option.  Artemisinin combination therapy is highly effective for both chloroquine-resistant and chloroquine- sensitive strains of P. vivax malaria. There is also evidence on a smaller scale that it is effective and safe for other non-falciparum species.  Pregnant women are systematically excluded from clinical trials, resulting in lack of evidence on the safety and efficacy of certain antimalarial drugs. Based on the available clinical data, which show no serious adverse effects of ACTs, the authors advocate conducting controlled clinical trials, including pharmacokinetic studies, for the treatment of malaria with ACT in all trimesters of pregnancy.  For malaria in returning travellers, ACTs should be most consequently used for the treatment of uncomplicated imported falciparum malaria in view of its favourable adverse effect profile as well as the rapid schizontocidal action. This box summarizes key points contained in the article.

Treatment of severe malaria With the increasing availability of injectable artesunates in Good Manufacturing Practice (GMP) quality - while availability seems to remain an issue in and outside endemic areas184, 185 - there is widespread acceptance of the SEAQUAMAT186 and AQUAMAT187 multicentre trial results that subsequently led to a WHO policy change from intravenously (i.v.) administered quinine to i.v. artesunate (followed by an oral single drug or drug combination38 as first-line treatment of complicated malaria). Notwithstanding open detail questions, SEAQUAMAT186 in adult patients from India and across Southeast Asia and AQUAMAT187 in children across sub-Saharan Africa established the superiority of

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artesunate not alone with regard to statistically significant mortality reductions (34.7 -- 95% CI 18.5 -- 47.6%; p = 0.0002 in SEAQUAMAT; 22.5 -- 95% CI 8.1 -- 36.9%; p = 0.0022 in AQUAMAT) but also in terms of easier handling (e.g., no rate-controlled infusion, no continuous cardiac monitoring, no frequent plasma glucose monitoring required) and an overall favourable adverse events profile (with regard to neurological consequences of severe malaria, no significant differences between both drugs have been observed). That notwithstanding, i.v. administered quinine remains an option for the treatment of severe malaria particularly if artesunate availability in adequate quality is not warranted yet, or in future, should emergence of resistance arise on a large scale before other alternatives become available. However, while artesunate resistance is not a major issue in practice to date, duration of treatment, a disadvantageous adverse events profile with cinchonism, induction of hypoglycemia and pharmacokinetical properties requiring skilled administration from loading dose to dose adaptation in due course facilitated fairly swift acceptance of a shift from quinine to artesunate as backbone drug against severe falciparum malaria. 188 Intramuscular administration of an oily emulsion of artemether is feasible, 189, 190 but where possible, preference is given to i.v. administrable artesunate. The intramuscular use of oily artemether might increase risk of neurotoxicity, although the current regimen dosing duration appears to be safe.191 Although there are no major safety issues with parenteral artesunate, there are some concerns regarding risk of prolonged and/or late hemolysis after high-dose artesunate treatment. Over the past years, up to 25% of patients from several cohorts treated with i.v. artesunate for severe falciparum malaria from Africa (children/malaria-endemic area: Gabon) and Europe (mainly adults, imported malaria) developed in some cases profound delayed hemolytic anaemia 7 -- 31 days after treatment.192-196 Up to date, the pathophysiology, causality and dimension of the problem remain to be fully elucidated. Rectal administration of artesunate prior to referral to/arrival at an appropriately equipped health-care referral unit197 has been proven to be potentially lifesaving, and all practical problems notwithstanding, repeated rectal administration have been suggested to further improving pre-referral outcomes in cases of suspected malaria in settings where prompt adequate diagnosis and treatment may not be at hand. 42, 198 Table 2 depicts all currently ongoing trials on chemotherapy of severe falciparum malaria. While this review focuses on malaria chemotherapy, it ought to be mentioned that in both complicated and uncomplicated disease, there is no room for adjuvant therapies other than unspecific supportive methods such as appropriate rehydration, or administration of (also controversially discussed) antipyretic drugs.199 An RCT of levamisole hydrochloride (an anthelminthic drug that inhibits cytoadherence in vitro and reduces sequestration) as adjunctive therapy in severe falciparum malaria with high parasitemia showed no benefit in a recent trial [105].200 A critical discussion on the value of exchange transfusion201-203 (or erythrocytapheresis, which is not identical, and the preferred method in some more affluent settings204) is on full swing; while there is no evidence for outcome improvement across studies but a recognition of potential benefits in individuals who are critically ill, 205 including improvement of the rheological profile, the rapidity of parasite clearance as encountered

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with i.v. artesunates shifts the debate toward it being increasingly judged upon as contraindicated. For example, the recently revised German guidelines for the treatment of malaria206 go as far as considering exchange transfusion as contraindicated, whereas others such as the Dutch guidelines, for example, do not go that far.

Treatment of non-falciparum malaria Non-falciparum malaria refers to disease due to infection with Plasmodium spp. other than P. falciparum; namely P. vivax, P. ovale subspecies curtisi and wallikeri, P. malariae and P. knowlesi. Although the cause of nearly all of the deaths due to malaria is due to P. falciparum, non-falciparum malarias (P. vivax and P. knowlesi) also carry the risk of severe and life-threatening illness. Plasmodium knowlesi, a parasite of macaque monkeys in Southeast Asia, has been identified as the cause of uncomplicated as well as severe and fatal malaria in Southeast Asia.207, 208 Severe malaria in P. malariae and P. ovale is extremely rare. Of the non-falciparum species, P. vivax has the largest geographic distribution and burden of disease in terms of health, longevity and socioeconomic development, and accounts for 40% of malaria cases worldwide.209 The other two human malaria Plasmodium species P. malariae and P. ovale are normally less prevalent, but they are distributed widely across malaria-endemic areas.

Treatment (P. ovale, P. vivax and P. malariae) The treatment of non-falciparum malaria consists of treating the erythrocytic asexual forms that induce symptoms and, for infections with P. vivax and P. ovale, assuring eradication of liver hypnozoites to prevent relapse of infection. Chloroquine is highly effective against P. malariae, P. ovale and the majority of P. vivax infections. Chloroquine, a synthetic compound of the 4-aminoquinoline group, is a powerful schizonticide with antiinflammatory action and so helps to reduce the nonspecific symptoms of malaria. Hydroxychloroquine is a good second-line alternative to chloroquine.210 The combination artesunate-amodiaquine, combined with primaquine, is also very effective for blood-stage parasite clearance of uncomplicated P. vivax malaria.211, 212 In most malaria guidelines, chloroquine is still the drug of choice for the treatment of blood forms of all non-falciparum species. Nevertheless, since the discovery of chloroquine-resistant P. vivax (CRPV) in the early 1990s, reports of CPRV are increasing and of a particular problem in the regions of Papua New Guinea, the Solomon Islands and Indonesia. Sporadically, CRPV has also been reported from Burma (Myanmar), India, Vietnam, Turkey, and Central and South America.213 The variability among P. vivax strains emphasizes that healthcare practitioners are required to consider geographical factors when initiating drug therapy for P. vivax infection. A recurrence of asexual parasitemia < 30 days after starting drug treatment suggests CRPV; recurrence after 30 days suggests primaquine-resistant P. vivax. Currently, three alternative drugs are recommend by the U.S. Centers for Disease Control and Prevention (CDC) for CRPV; quinine sulfate plus either doxycycline or tetracycline;

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atovaquone-proguanil; and mefloquine. All three drugs are recommended equivalently and are succeeded by primaquine, the only licensed hypnozoicidal drug that is able to reliably prevent relapses and achieve radical cure. Interestingly, for the therapeutic efficacy of primaquine (an 8-aminoquinoline) to eradicate hypnozoites it is shown that a 4aminoquinoline (e.g., chloroquine or quinine) is needed. Data from > 50 years ago showed that primaquine may exert its beneficial effect when combined with a 4-aminoquinoline drug such as chloroquine.214 Administration of a regimen of primaquine concurrently with quinine or chloroquine showed significantly higher cure rates for P. vivax malaria compared to primaquine alone.214 The potential for synergistic effects has never been evaluated for primaquine with mefloquine, doxycycline or atovaquone/proguanil.215 Primaquine is contraindicated in pregnant women and children, as discussed before. Because primaquine is never a critical or urgent treatment, patients should be screened (for glucose 6-phrosphate dehydrogenase [G6PD] deficiency) beforehand -- if this is available in a low-resources setting -- so that the regimen and dosage can be adjusted for those with G6PD deficiency. 216 A recent trial dose-ranging RCT evaluated a single-dose primaquine for clearance of P. falciparum gametocytes in children with uncomplicated malaria.217 It was shown that a lower dose (0.4 mg/kg primaquine) had similar gametocytocidal efficacy compared to the reference (0.75 mg/kg). However, these findings are not directly important for the individual patient, but rather on community level with regard to gametocyte transmission back to the vector.

ACTs for the treatment of non-falciparum malaria ACT is highly effective for both chloroquine-resistant and chloroquine-sensitive strains of P. vivax malaria. Currently, the WHO recommends, for areas with CRPV, artemisininbased therapies, particularly with those partner drugs that have long half-lives. AL reaches comparable cure rates to chloroquine in the treatment of P. vivax in areas with sensitive strains of P. vivax for chloroquine.218-220 Evidence indicates excellent cure rates of DHA + PIP. An RCT in Indonesia compared DHA + PIP with artesunate-amodiaquine where both groups were also given primaquine to clear hypnozoites. DHA + PP reduced the number of relapses by day 42 compared to Artesunate (AS) + Amodiaquine (AQ) (84 participants; relative risk [RR] 0.16, 95% CI 0.05 -- 0.49).212 DHA was more effective and better tolerated than AQ against P. vivax infections. Also, it was noted that PIP decreased the rate of recurrence of P. vivax infection, and reduced the risk of P. vivax gametocyte carriage and anaemia.212 Even though these data encompass patients with P. falciparum at inclusion, and even though recurrence of P. falciparum was also lower with DHA + PP, it is likely that this prophylactic property is related to the longer half-life of DHA + PP. Another trial in Indonesia showed DHA + PP (compared to AL) also showed that the number of relapses by day 42 was reduced (126 participants; RR 0.16, 95% CI 0.07 -- 0.38). More recent trials confirmed these findings (Table 3): An RCT comparing DHA + PP with the standard treatment chloroquine in Thailand demonstrated fever and parasite clearance times to be significantly slower in the chloroquine (CQ) than in the DHA + PP group.221 The cumulative

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risk of recurrence with P. vivax at 9 weeks was considerably higher in the chloroquine group compared to the artemisinin derivate group (Table 3) [126]. A randomized comparison of DHA + PP and AS + AQ, both combined with primaquine in Indonesia in 2013, showed comparable efficacy for blood-stage parasite clearance of uncomplicated P. vivax malaria. Of note, DHA + PP was better tolerated.211 The efficacy of 2- versus 3-day regimens of DHA-piperaquine for uncomplicated malaria (mainly P. vivax) was evaluated in Cambodia in an open-label RCT. Cure rate at day 42 was for the 2-day regimen 85% (95% CI 69 -94%) and for the 3-day regimen 90% (95% CI 75 -- 97%).222 So far, there are no trials registered comparing DHA + PP and artesunate + mefloquine in P. vivax mono-infection. Currently, a comparative study is conducted with DHA + PP with standard malaria treatment (AS + sulfadoxine-pyrimethamine [SP] and CQ) in Afghanistan (NCT00682578). A randomized cluster trial of mass screening and selective treatment using DHA-piperaquine plus primaquine (DHP + PQ) is currently conducted in Indonesia, evaluating an intervention arm with an interval of 6 weeks; 3 months and a control arm without mass screening and treatment. Another highly effective ACT for the treatment of non-falciparum malaria is AL, which has been investigated in several randomized and prospective clinical trials. 50, 218, 223, 224 A new combination (artemisinin-naphthoquine ‘ANQ,’ 3-day regimen), which is not yet marketed, was investigated and compared to chloroquine-primaquine (8-day regimen) in an open-label randomized and non-inferiority design trial in China.225 By day 42, no significant difference was found in the cure rates; 98.4%; 95% CI 94.4 -- 99.8% for the artemisininnaphthoquine versus 96.1%; 95% CI 91.1 -- 98.7% for chloroquine-primaquine. Side effects of this combination were found to be more mild compared to CQ-PQ. This trial demonstrated this new combination ANQ to be an effective blood schizonticide for P. vivax infections and is possibly an alternative for people not willing or able to take primaquine .225 For the radical treatment of P. vivax and P. ovale, according to the WHO, at least a 14-day course of primaquine is required.38 The best combinations for the treatment of P. vivax are those containing primaquine when given in antihypnozoite doses.211, 226 Less evidence is available on the treatment of the Plasmodium species P. ovale and P. malariae. It is no understatement that these two species are neglected in malaria research and intervention trials. In the past few years, only one nonrandomized prospective cohort study performed in Gabon investigated the therapeutic efficacy and safety of AL for these two species. 50 Day 28, overall cure rates were 100% (95% CI: 91 -- 100%) for both species. A limitation of this study, however, is the small number of participants (which is a limit in most settings, and a reason why there is less evidence from clinical trials than for other species) and its non-comparative study design. No ongoing trial (Table 4) is currently evaluating the efficacy and safety for the treatment of these two species, which account for a sizable cause of malaria, especially in sub-Saharan Africa.

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Malaria: an update on current chemotherapy

Treatment of P. knowlesi malaria Plasmodium knowlesi is a zoonotic malaria species originating from Sarawak, Malaysian Borneo. It can cause severe malaria with high morbidity and mortality. Effective treatment is available. Plasmodium knowlesi is highly sensitive to artemisinins and thus ACT combination treatments, variably and moderately sensitive to chloroquine and less sensitive to mefloquine. Therefore, it is recommended that treatment of P. knowlesi malaria is similar to uncomplicated P. falciparum malaria. Further studies investigating the effectiveness of ACTs for P. knowlesi malaria need to be undertaken. Recently, an extensive and comprehensive review and in vitro sensitivity of P. knowlesi was undertaken using a WHO schizont maturation assay.227 A Phase III trial that will be conducted in the near future is examining whether fixed combination of AL is superior to chloroquine in order to define the optimal treatment for both uncomplicated P. knowlesi infection in both adults and children in this region (NCT02001012).

Treatment of malaria in pregnancy Pregnant women are at increased risk of acquiring malaria and are susceptible to more severe disease. The treatment of malaria in pregnant women poses particular challenges, as the theoretical risks of teratogenicity of antimalarial drugs need to be weighed against the risk of undertreatment.228 In addition, safety and efficacy data from clinical trials are limited. Knowledge about adequate drug levels in pregnant women is scarce. More pharmacodynamic and pharmacokinetic data are needed to be able to adjust dosages according to body weight and not according to age groups, which allow a large deviation in exact therapeutic drug levels. For pregnant women, there is a need to adapt pharmacokinetic models and safety data need to be collected in a systemic way. Commonly, the newer the antimalarial drug, the more effective it is (to a certain extent due to the lack of time for drug resistance to emerge). However, less information will be at hand on safety and efficacy in pregnancy, in particular the first trimester, in the early years of usage of a drug/drug combination, as data will only accumulate on inadvertent use particularly in early, on time point of treatment initiation unrecognized pregnancy. Therefore, physicians should base their management on the clinical state of the pregnant patient, geographical data, resistance patterns, national guidelines, experience (of colleagues) and published data concerning safety of the drug in pregnancy. The safety of the mother should always prevail over that of the unborn child. Treatment involves antimalarial drugs and supportive measures preferably after parasitological confirmation by expert microscopy or, in the majority of settings in endemic areas, following a rapid diagnostic immunochromatographic antigen detection test. This will reduce the unnecessary exposure to antimalarials of both the mother and the unborn child. Prevention of malaria during pregnancy involves chemoprophylaxis ‘Intermittent Preventive Treatment in pregnancy (IPTp)’229, 230 and preventing mosquito bites, for example, with insecticide-treated bednets, are discussed elsewhere. 231

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First trimester Clinical trials that assess the safety and efficacy of new antimalarial drugs typically exclude pregnant women in the first trimester (gestational age < 14 weeks) of pregnancy. Therefore, evidence is scarce and is based on observational rather than interventional studies (Table 5). Current guidelines consider chloroquine, quinine, clindamycin and proguanil as safe in the first trimester.38 A drug safety database analysis of 2506 cases of mefloquine exposure during pregnancy or in the pre- and peri-conception period showed that the birth defect prevalence and foetal loss in maternal, prospectively monitored cases were comparable to background rates.232 A retrospective evaluation reviewing the effects of mefloquine treatment on pregnant women with suspected hyperreactive malarial splenomegaly showed significant smaller spleens and decreased anaemia and malaria antibody titres without negative consequences on the treated women or their newborns.233 Although data from animal studies234-236 suggest that artemisinin drugs are teratogenic in the first trimester of pregnancy, human data are reassuring; a recent systematic review assessing the safety and efficacy of AL against uncomplicated P. falciparum malaria during pregnancy237 shows no evidence of increased risks in 212 first trimester exposures. Animal studies have demonstrated toxic effects to the unborn foetus due to the depletion of primitive red blood cells at therapeutic doses of artemisinin derivates, and there is also information available that reticulocyte counts are decreased in individuals after the intake of artemisinins.238 Treatment of malaria in humans in the first trimester with artemisinin drugs was fairly safe. 239-244 A retrospective population-based study that included antenatal records of 17,613 women showed no difference in adverse effects and risk of miscarriage between artemisinin derivates (n = 44) and other drugs .240 Only one study performed in The Gambia in 2001 was a clinical trial that randomized participants to a single dose of the combination artesunate plus sulfadoxine-pyrimethamine or sulfadoxine-pyrimethamine plus placebo during a mass drug administration. There were no differences for pregnant women (first trimester) exposed to artesunate (n = 77) in the proportion of abortions, stillbirths or infant deaths compared to that of other pregnant women.243 A prospective cohort study conducted in Zambia evaluated the safety of AL in women (n = 106) during their first trimester of pregnancy. No particular risks were identified in terms of perinatal morbidity, malformations or developmental impairment in women exposed to AL.244 This study confirmed findings of an earlier observational study with 62 women exposed to artemisinin derivates.242 In Tanzania, 319 pregnant women using antimalarials in their first trimester were described in an observational study.245 Most of them (53.9%) used AL. Quinine showed an increased risk of stillbirth and premature birth as opposed to AL. No significant difference between congenital anomalies was found between the antimalarial drugs (AL, Q, SP, AQ). Globally, 555 first trimester artemisinin derivate exposures are documented in clinical trials with known pregnancy outcomes (Table 5). Many more have been reported in population-based surveys.246 However, the question is whether this number is too small or sufficient enough to draw any significant conclusions on the efficacy and safety of ACTs in first trimester pregnancy malaria treatment. Currently, the WHO recommends quinine plus clindamycin to

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Malaria: an update on current chemotherapy

be given for 7 days for uncomplicated P. falciparum malaria (given adequate safety data and low cost) or artesunate plus clindamycin for 7 days if this treatment fails. However, this latter recommendation is pragmatic and based on modest evidence; no randomized clinical trial has compared the efficacy of artesunate in pregnant women in their first trimester of pregnancy. Several trials did show high cure rates of artesunate alone or in combination with other antimalarials in the second and third trimester of pregnancy in Tanzania,247 Thailand239, 248 and Malawi (Table 5).249 No trials have compared the efficacy of artesunate + clindamycin in first trimester pregnant women. The WHO only recommends ACTs in the first trimester if it is the only treatment available, or if treatment with quinine plus clindamycin fails or uncertainty of compliance with a 7-day treatment exists. This, however, is in sharp contrast with daily practice: a population-based survey on self-reported antimalarial drugs showed that almost half of the malaria episodes in the first trimester are treated with ACTs, without obvious disadvantages compared to other antimalarial drugs ,246 a survey that is probably a good representation of clinical practice in sub-Saharan Africa. Hitherto, human data suggest that ACTs are safe in the first trimester, so the use of ACTs for the treatment of first trimester malaria in RCTs appears to be justifiable, as obtaining high-quality data from a controlled trial appears to be superior than collecting evidence from retrospective analysis of possibly insufficiently documented anecdotal evidence. So far, women in their first trimester of pregnancy remain excluded. Primaquine is contraindicated in pregnancy as it can cause a hemolytic anemia in persons with G6PD deficiency, and with the G6PD status of the unborn child naturally remaining unknown. Consequently, pregnant women should receive a treatment of chloroquine (if chloroquine sensitive) or another drug as described above, and then continue once weekly with chloroquine until after delivery, when primaquine can be given without danger for the neonate. The maintenance treatment of CRPV (from Papua New Guinea and Indonesia) remains unclear, but repetitive mefloquine or quinine can be considered. A recent systematic review showed no increased risk for the unborn child due to mefloquine use during pregnancy .250 Whether primaquine can be safely administered during lactation is currently under investigation (NCT 01780753) but is at present advised to be avoided during breastfeeding, along with tetracycline and doxycycline (Table 6).

Second and third trimester Much more evidence from observational as well as interventional trials is available on the use of artemisinin combination treatment in the second and third trimester of pregnancy. A recent review of the safety and efficacy of AL against uncomplicated P. falciparum malaria during pregnancy from studies conducted in 1989 -- 2011237 showed no evidence of increased risks (890 second/third trimester exposures), supporting the WHO recommendation to treat uncomplicated falciparum malaria with ACT known to be effective in the region in second and third trimester pregnancy. Also, treatment with artesunate plus clindamycin to be given for 7 days, or quinine plus clindamycin also for 7 days is possible. 38 For severe P. falciparum malaria, i.v. administration of artesunate to the mother is the

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preferable treatment. The poor tolerability and longer duration of treatment with quinine augments the risk of poor compliance, and therefore the risk of treatment failure and the development of drug resistance. Several artemisinin derivates, alone or in combination with other antimalarials, are evaluated as efficacious and safe in second and third trimester pregnancy. AL has been shown to be efficacious in pregnant women with uncomplicated P. falciparum malaria in Thailand ,248, 251 Uganda.252 Only one study253 compared the efficacy of AL with quinine, although quinine was previously the first-line WHO recommended treatment for malaria in pregnant women and is still the first-line drug for malaria in the first trimester of pregnancy. An open-label RCT performed in Uganda showed a day 28 cure rate of AL of 100%, where it was compared to chlorproguanil-dapsone (cure rate also 100%). Parasite and fever clearance time were comparable, and the treatment was well tolerated. However, these results are in contrast to findings from Thailand where the day 42 cure rate for AL was only 82% for the intention to treat population. This significant risk of recurrence of infection was most probable because of low plasma concentrations during pregnancy of both artemether and lumefantrine at day 7.248 As for other antimalarial drugs, plasma concentrations of artemether and its metabolite DHA, and lumefantrine, are lowered in pregnant women.251, 254-256 This raises the question of whether the standard adult dose should be modified for pregnant women. A pharmacokinetics study in 103 pregnant women with uncomplicated P. falciparum malaria treated with AL suggested that in order to maintain optimal lumefantrine concentrations the duration of AL in pregnant women should be prolonged to 5 days. Further studies are needed to collect pharmacokinetic data in pregnant women after an extended regimen or dose adjustment to investigate whether an adjusted course is warranted. A comparison of two regimens of AL (3 vs 5 days) is currently under investigation in the Democratic Republic of Congo (NCT01916954). In a pharmacovigilance study with 978 exposures to AL and follow-up until delivery, no specific safety concerns related to AL for uncomplicated falciparum malaria were described. 257 However, there were slightly more obstetric complications in the treatment group (compared to a matched, nonexposed control group); this could have been caused by the treatment itself or more probably have been caused by the malaria episode itself. Further assessment of possible obstetric complications is required. A population-based survey on self-reported antimalarial drugs showed that AL was the most widely used drug in the treatment of malaria in the second or third trimester of pregnancy (any use of AL 43.3%; 207/478 episodes). AL is currently being investigated in Thailand and several other African countries (Phase III: NCT01054248, NCT01916954, NCT01717885, NCT008 52423 and PACTR2010020001862624) (Table 6). Another drug that demonstrated to be highly effective is amodiaquine. A pharmacokinetic study conducted in Thailand reduced the risk of recurrent P. falciparum infections from 22.2 to 7.4% at day 35 in 27 women.258 This study also implied that no dose adjustments are required in pregnancy. This study supports previous research that there were no clinically relevant differences in the pharmacokinetics of amodiaquine and desethylamodiaquine between pregnant and postpartum women. 259 Amodiaquine was shown to be highly effective combined with sulfadoxine-pyrimethamine

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or artesunate in an RCT in Tanzania.247. By day 28, parasitological failure rates were 1% in the sulfadoxine-pyrimethamine-amodiaquine group and 9% in the amodiaquine-artesunate group. In a large RCT in Ghana, amodiaquine alone (n = 225) had a 3% PCR-corrected parasitological failure rate by day 28, compared to 0 in combination with sulfadoxinepyrimethamine.260 Amodiaquine is relatively safe and well tolerated; however, some side effects such as dizziness and nausea have been reported. Amodiaquine is currently under investigation in a clinical Phase III trial in Ghana (NCT01231113), where it is combined with artesunate and compared with DHA plus PIP. Two other Phase III trials are currently underway evaluating amodiaquine in pregnant women in Africa (PACTR20100 20001862624 and NCT00852423). Safety and efficacy data on quinine are widely available and not discussed in detail here. A randomized trial performed > 10 years ago, compared artesunate versus quinine plus clindamycin for the treatment of P. falciparum malaria, reported no difference in efficacy with 100% of the women in each treatment regimen cured.261 Efficacy data from a more recent randomized trial on the Thai-Burmese border showed that 63.4% (95% CI: 46.9 -- 77.4%) of pregnant women (second and third trimester) with uncomplicated P. falciparum malaria who received a 7-day course of quinine monotherapy were cured, based on PCR-corrected parasite clearance at day 63 of follow-up or delivery .262 This was in contrast with considerably higher proportion of cure rates in the arm with 3 days of artesunate-atovaquone-proguanil (94.9%; 95% CI: 81.37, -99.11%). The low cure rates of 63.4% of quinine262 may be explained by a combination of resistance of P. falciparum and by the pharmacokinetic properties of quinine during pregnancy. Furthermore, quinine is not well tolerated and often causes symptoms of cinchonism, and it can cause severe hypoglycemia with high insulin levels. Extensive clinical experience of prophylactic use of mefloquine in the first trimester of pregnancy showed no increased risks or teratogenic effects.250 Experience with a high dose as treatment is limited to three studies (Table 5): A high cure rate has been reported in Sudan (recrudescence or reinfection in 2.5%; 1/40),263 Thailand (mefloquine in combination with artesunate a cure rate of 98.2% by day 63)264 and Nigeria (in combination with artemether cure rate of 100% by day 14 and day 28).265 The treatment is generally well tolerated, and only minor adverse effects were reported. As for the use of mefloquine in nonpregnant individuals, safety concerns have been raised regarding the occurrence of neuropsychiatric disorders as adverse effects. A literature review suggested that females are at greater risk to develop neurotoxicity and it was advised to not combine mefloquine with other potentially neurotoxic agents such as the artemisinin antimalarials.266 That notwithstanding, a pharmacokinetic Phase II/III study investigating the combination mefloquine with artesunate in Burkina Faso is underway (NCT00701961). DHA-PIP was highly effective in women with uncomplicated P. falciparum malaria in Thailand.267, 268 However, all published studies evaluating DHA plus PIP are pharmacokinetic studies, and the first RCTs from both African and Asia are conducted at the time of writing (Phase III: NCT012 31113 and NCT00852423). In conclusion, the current recommended treatment for malaria in the second or third trimester of pregnancy is a locally effective ACT (AL, artesunate-amodiaquine, artesunate-mefloquine, artesunate-

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sulfadoxine-pyrimethamine), but do not (yet) include DHA plus PIP due to the lack of data. For the women who breastfeed, standard antimalarial treatment for adults (including ACTs) is recommended, except for dapsone, primaquine and tetracyclines.38

Treatment of malaria in children

Malaria is, on a global scale, a paediatric disease.12 Very much different from many other diseases, almost all clinical drug development trials have been performed in children in endemic areas, with treatment outcomes being extrapolated from those trials to inform treatment strategies for adults in malaria-endemic areas, as well as for children and adults exporting malaria to non-endemic, affluent countries. That notwithstanding, the most appropriate choice of combination therapy needs to take age and age-specific pharmacokinetic and -dynamic factors, body weight and specific paediatric risk factors (e.g., among others, the problems of administering tetracyclines to younger children) into account. AL is the ACT most commonly used for the treatment of uncomplicated malaria in children. AL has been demonstrated to be safe when compared with other antimalarials such as quinine, sulphadoxine-pyrimethamine and chloroquine.269 Several combinations have been investigated. Firstly, AL has been compared with dihydroartemisinin-piperaquine in 11 studies,138, 154, 270-279 involving 5958 children. No drug-related deaths were identified, and the risk of serious adverse events for AL was not significantly different for DHA + PP .269 Other trials compared AL with artesunate-amodiaquine (13 studies, 6018 children) [39,183,186-196],137, 277, 280-290 with chlorproguanil-dapsone- artesunate (three studies, 3366 children),277, 282, 291 with artesunate-mefloquine (two studies, 476 children)292, 293 and with artesunate-azithromycin (one study, 261 children).294 Regarding the safety and tolerability of AL, the authors of a recent systematic review269 demonstrate cough as the most common adverse event in children treated with AL. Other frequently reported adverse effects are gastrointestinal symptoms such as vomiting, abdominal pain and diarrhoea. Headache and anaemia were also described as common adverse events.

Treatment of malaria as an imported condition Whereas most cases of malaria remain to be paediatric in endemic countries, most imported cases are in adults -- yet our treatment strategies are everywhere based on data predominantly obtained from clinical trials conducted in young children in Africa. There is a wealth of national guidelines in place in the various countries where malaria is regularly encountered as an imported condition. Whereas those vary in some detail, atovaquone-proguanil, mefloquine and ACTs, with AL dominating and DHA-PIP now entering the Northern, affluent markets, are regularly featuring in various order of appearance with regard to preference.206, 295-297 In Europe, atovaquone-proguanil ranges high in many nonendemic countries among the preferred therapies for uncomplicated falciparum malaria,298 despite the fact that the slow action inherent to this drug combination, with comparably long parasite and fever clearance times, regularly leads to misperceptions about possible resistance, and

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to prolonged disease episodes compared to ACTs. Special recommendations for the treatment of children/pregnant women apply throughout all guidelines. A recently introduced black box warning regarding mefloquine use for the therapy of uncomplicated falciparum malaria299 will reduce its use as treatment for uncomplicated malaria further. However, for some indications (high-risk groups, such as long-term travelers, VFR travelers and families with small children), there is currently no replacement for mefloquine available or in the pipeline.300 In our view, ACTs should most consequently be used for the treatment of uncomplicated imported falciparum malaria in view of its favourable adverse events profile as well as the rapid schizontocidal action. There is also an increasing debate on whether to continue with non-ACTs (chloroquine in the first place, mostly followed by primaquine administration in non-G6PDdeficient individuals) for non-falciparum species (except for P. knowlesi) therapy as far as susceptibility is assumed. In some non-endemic countries, first shifts away from chloroquine for vivax and ovale malaria treatment toward ACTs on basis of good tolerance and swift clinical improvement due to quick parasite and fever clearance times can be observed,206 mainly based on data from malaria-endemic countries and based on expert opinion, as controlled trials being tedious to carry out at least in non-endemic countries. With the prospects of increased availability of GMP conform artesunates in non-malaria-endemic countries improving, there is an increasing shift toward adopting i.v. artesunate in place of i.v. quinine as chemotherapeutic backbone for the treatment of severe falciparum malaria. While controlled trials on the scale of the trials in Asia and Africa are not possible due to small patient numbers,301 there is evidence from small case series194 as well as growing expert opinion in favour of parenteral artesunate use.302 Due to space constraints, it is not possible to elaborate in detail on differences in all the factors that may influence clinical presentation and clinical course of malaria in patients in endemic versus those encountered in non-endemic areas, and possible (maybe only subtle) consequences for the choice of antimalarial treatment; a subject that would warrant a paper on its own.

Conclusion Malaria chemotherapy remains a dynamic field, with novel drugs and drug combinations continue to emerge in order to outpace the development of large-scale drug resistance against the currently most important drug class, the artesunates. Continuous investment into malaria drug development is a vital contribution to combat artemisinin resistance and successfully improve malaria control toward the ultimate elimination goal.

Expert opinion Knowledge about adequate drug levels in children and pregnant women is scarce. More pharmacodynamic and pharmacokinetic data are needed to be able to adjust dosages according to body weight and not according to age groups, which allow a large deviation in exact therapeutic drug levels, especially among children. For pregnant women, there is a

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need to adapt pharmacokinetic models and safety data need to be collected in a systemic way. We see a need to discuss openly, in view of the complexity of the ethical aspects of the issue, whether women should be excluded per se from these RCTs, as long as sufficient observational safety data for the drug under investigation are available. Researchers may argue it is unfeasible and unlikely they will intentionally expose pregnant women to potential teratogenic drugs; the only alternative to RCTs are sensitive pharmacovigilance systems for the monitoring of outcomes of unintentional first trimester exposures, but these need to be developed first and have many (practical) limitations. Another argument not to include consenting pregnant women in their first trimester in clinical trials is that in view of the widespread use of AL for the treatment of female adults of child-bearing age, a substantial number of women will be unintentionally exposed to an artemisinin derivate early in pregnancy. In areas with high transmission, people might receive as many as three treatments of artemisinin derivates every year, there is a 17% chance of the unborn child to be exposed during the putative sensitive period from week 3 to 9 weeks after conception. This may affect 8.5 million unborn children each year.303 An important note is that, due to such an early susceptible period in pregnancy, foetal deaths due to ACTs could be easily overlooked, because women may not yet know they are pregnant. Data suggest that a large proportion of women have malaria at the time of their first antenatal care visit, 304 another reason that highlights the importance of further studies into the safety and efficacy of ACTs for its potential use in the first trimester. Furthermore, and despite constraints in case numbers in individual sites, it is somewhat surprising that almost none of the past and ongoing clinical trials investigate ACTs for non-falciparum species, particularly P. vivax that causes the same complications as P. falciparum malaria, although less frequent and less severe. Treatment of non-falciparum malaria in pregnant women is nearly the same as for non-pregnant adults. Following treatment of infection due to P. ovale or P. vivax, non-pregnant patients, if not returning to an endemic region, are treated with primaquine to prevent relapse by eradicating hypnozoite forms that may remain dormant in the liver. For future trials, it is important to recognize that if primaquine is co-administered with a blood schizontocidal agent, the total effect is a sum of the synergistic efficacy of the schizontocidal drug and primaquine. Furthermore, more consequent use of ACTs for the treatment of important malaria in order to capitalize outside endemic areas on reduced parasite and fever clearance times resulting in patients improving in the shortest possible period of time after diagnosis and treatment initiation should become a priority. In the future, one important research area will be to further explore avenues toward identifying drug combinations that may further reduce the duration of treatment and allow reduction on the total number of doses administered; with single-dose regimens being the sought-after ‘magic bullet’ (which from today’s perspective may remain unsuitable for routine use for quite a while).

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Appendix 1 Phase I and II compounds currently under investigation for uncomplicated falciparum malaria. In 2013, researchers reported the discovery of an antimalarial drug candidate in a new class of compounds known as spiroindolones305. The spirotetrahydro-beta-carbolines, or spiroindolones, have the potential to clear the blood stages of P. vivax and P. falciparum at a low concentration and are compatible with once-daily oral dosing. These drugs work by rapidly inhibiting protein synthesis in P. falciparum. KAE609, an optimized spiroindolone, shows efficacy in a rodent malaria model and has advanced to the initial phase of testing in humans and is currently undergoing proof-of-concept testing (NCT01860989). Another promising new class of antimalarial drugs, the imidazolopiperazines, exhibit in vitro potency against blood, liver and gametocyte stages of Plasmodium.306 This class has demonstrated in vivo efficacy in a Plasmodium berghei mouse infection model. Having successfully completed Phase I studies one compound from the series, KAF156 is currently in Phase IIa clinical trials (NCT01753323). A new hope for a single-dose cure of uncomplicated falciparum malaria is offered by the synthetic ozonide drug candidate OZ439,307 which has successfully completed preclinical studies and clinical trials in human volunteers (Phase I). It demonstrated a higher potency and longer plasma exposure compared to the artemisinins, which might imply higher efficacy at lower doses. It is a fast-acting agent against all asexual erythrocytic P. falciparum stages with half maximal inhibitory concentration values comparable to those for artemisinin derivates. In contrast with synthetic peroxides and artemisinin derivates, OZ439 demonstrated a complete cure rate in a Plasmodium berghei malaria model with a single oral dose; furthermore, it exhibits prophylactic activities better than the chemoprophylactic agent mefloquine. A phase IIa study is currently conducted to evaluate the efficacy, stability of OZ439 and the effects on the recrudescence of falciparum malaria (NCT01713621). AQ-13,308 which is a close structural analog of chloroquine, shows potency against P. falciparum infected red blood cells, and also against those that are resistant for chloroquine. Phase I clinical trials have shown pharmacokinetic properties similar to chloroquine, and a phase II (proof of concept) study is currently conducted (NCT01614964).

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AL vs. AS+MQ

NCT01939886 (2013) Tanzania

DHA+PP + PQ in different dosages) AL + PQ (in different dosages)

The optimal timing of primaquine to prevent malaria transmission after NCT01906788 (2013) artemisinin-combination therapy Tanzania

AL vs. AS+AQ vs. DHA+PP

Primaquine's gametocytocidal efficacy in malaria asymptomatic carriers NCT01838902 (2013) (PRINOGAM) The Gambia

Effect of oral activated charcoal on parasite clearance rates in response to NCT01955382 (2013) intravenous artesunate in Malian children with uncomplicated P. falciparum Malaria Artemisinin-based antimalarial combinations and clinical response in Cameroon NCT01845701 (2013) Cameroon

DHA+PP Mass drug administration (MDA) – several time intervals Oral activated charcoal

AL (3 days) vs. AL (5 days)

NCT02020330 (2013) Myanmar

Surveillance and treatment with dihydroartemisinin-piperaquine plus primaquine NCT01878357 (2013) Indonesia

An open-label randomized controlled trial to evaluate the effectiveness and safety of a 3 day versus 5 day course of artemether-lumefantrine for the treatment of uncomplicated falciparum Malaria in Myanmar Treatment efficacy and malaria TRANSmission after Artemisinin Combination Therapy 2 (TRANSACT2)

Registration ID (year) Antimalarial(s) tested Country, status as of March 2014 Study to determine the efficacy of artesunate-mefloquine combination therapy for NCT02052323 (2014) AS + MQ vs. the treatment of uncomplicated P. falciparum Malaria in Thailand Thailand

Name of study

Table 1. Ongoing trials on malaria treatment for uncomplicated P. falciparum malaria (date of last search: June 2014).

KAE609 DHA+PP dosages)

A study to assess efficacy, safety of KAE609 in adult patients with acute malaria NCT01860989 (2013) mono-infection (Phase II) Thailand NCT01743820 (2012) Mali, Thailand NCT01736319 (2012) Cambodia NCT01728961 (2012) Malawi, Uganda

Phase2a primaquine dose escalation study

Artemisinin-resistant malaria in Cambodia

Pharmacology of antimalarial therapy with or without antiretroviral therapy

AL vs. DHA+PP

Improving anti-malarial treatment options in Guinea-Bissau - Part A

NCT01704508 (2012) Guinea-Bissau

DHA+PP vs. AS+MQ

(different

Comparing mefloquine-artesunate and dihydroartemisinin-piperaquine in malaria NCT01640587 (2012) treatment (MMA) Thailand

PQ

AL vs. DHA+PP vs. AS+AQ

+

Efficacy of three ACTs for the treatment of P. falciparum malaria in Maradi Niger NCT01755559 (2012) Niger

AL

DHA+PP

KAE609

Registration ID (year) Antimalarial(s) tested Country, status as of March 2014 NCT01930331 (2013) ARCO vs. DHA + PP Tanzania

A study to find the minimum inhibitory concentration of KAE609 in adult male NCT01836458 (2013) patients with P. falciparum mono-infection (Phase II) Thailand

Safety, tolerability, pharmacokinetics and efficacy of ARCO

Name of study

3

NCT01350856 (2011) Multiple countries

Tracking resistance to artemisinin (TRAC)

AS (two different regimens)

AQ-13 vs. AL

AP vs. AL AL AS+MQ

Evaluation of the Riamet® versus Malarone® in the treatment of uncomplicated NCT01150344 (2010) malaria (MalaRia) France

Surveillance of effectiveness/safety of artemether-lumefantrine in patients with NCT01228344 (2010) malaria United States

Effectiveness of the association artesunate and mefloquine in the treatment of NCT01144702 (2010) malaria by P. falciparum

Evaluation of fosmidomycin and clindamycin in the treatment of acute NCT01361269 (2011) (status Fosmidomycin and clindamycin uncomplicated P. falciparum malaria unknown, probably stopped) Gabon, Mozambique Impact of artemisinin-based combination therapy and quinine on treatment failure NCT01374581 (2011) AL vs. AS+AQ vs. Q + and resistance in uncomplicated malaria (QuinAct) Congo, Uganda clindamycine

NCT01614964 (2012) Mali

Studies of a candidate aminoquinoline antimalarial (AQ-13)

Registration ID (year) Antimalarial(s) tested Country, status as of March 2014 Efficacy, safety and pharmacokinetics of artemether-lumefantrine dispersible NCT01619878 (2012) Benin, AL dispersible tablet tablet in the treatment of malaria in infants < 5 kg Burkina Faso, Togo, Nigeria, Congo OZ439 PhIIa study in P. falciparum: extended observation (Phase II) NCT01713621 (2012) OZ439 Thailand

Name of study

NCT01023399 (2009) Ivory Coast

AS+AQ

Malaria Treatment With Injectable ArteSunate (MATIAS)

Intravenous Artesunate and Malaria (IVAS)

Name of study

NCT01828333 (2013) AS (completed) Democratic Republic of Congo

Registration ID (year) Antimalarial(s) tested Country, status as of March 2014 NCT01805232 (2013) AS vs. Q Sudan

Table 2. Ongoing trials on severe malaria treatment registered online in clinical trial registries (last search June 2014)

Drugs abbreviations: A=artemether, AL= artemether- lumefantrine, AP = atovaquone-proguanil, ARCO = artemisinin/naphthoquine, AQ = amodiaquine, AS = artesunate, AZ = azithromycine, CD = chlorproguanil-dapsone, CQ = chloroquine, DHA = dihydroartemisinin, MQ = mefloquine, PQ = piperaquine, Q = quinine, QC = quinine + clindamycine SP = sulfadoxine-pyrimethamine, PP = piperaquine.

Artesunate plus amodiaquine in malaria in Cote d'Ivoire

Registration ID (year) Antimalarial(s) tested Country, status as of March 2014 Efficacy, safety and tolerability of dihydroartemisinin-piperaquine for NCT01231113 (2010) DHA+PP uncomplicated malaria in pregnancy in Ghana (DHAPPQ/MIP) Ghana

Name of study

3

SAR97276A

AS

AS vs. AS + Azithromycin

Artemether sublingual spray vs. Q

Antimalarial(s) tested

Drugs: A=artemether, AL= artemether- lumefantrine, AP= atovaquone-proguanil, AQ=amodiaquine, AS=artesunate, AZ=Azithromycine, CD=chlorproguanil-dapsone, CQ=chloroquine, DHA=dihydroartemisinin, MQ=mefloquine, PQ=piperaquine, Q=quinine, QC= quinine + clindamycine SP=sulfadoxine-pyrimethamine

Registration ID (year) Country, status as of March 2014 Superiority of ArTiMist Versus Quinine in Children With Severe Malaria NCT01258049 (2012) (completed) Burkina Faso, Ghana, Rwanda Azithromycin Combination Therapy for the Treatment of Severe Malaria NCT01374126 (2011) (completed) Bangladesh Pharmacokinetics and Pharmacodynamics of Intravenous Artesunate for Severe NCT01122134 (2010) (status Malaria Treatment unknown) Uganda Study of SAR97276A in the Treatment of Uncomplicated and Severe Malaria in NCT00739206 (2008) Adults and Children. (suspended) Benin, Burkina Faso, Gabon

Name of study

et Thailand

China

Senn et al.223 Papua 2013 New Guinea

Pasaribu al.211 2013

2013

Liu et al.225

Number patients

Longitudinal prospective effectiveness study

N=594 vivax)

Open label N= 331 vivax) randomized controlled trial

Open label N= 251 vivax) randomized controlled trial

Source (first Countr Study author, year of y (study design publication) site), Timeframe

Measure of Findings (primary) outcome

(P. AL

42 By day 42, AS + PQ: 0/167 recurrent infection vs. 1/164 (0.6%; 95% CI, 0.01%-3.4%) for DHA + PP. Minor adverse events were more frequent with AQ + PP.

Day 7, 8 & Clinical treatment failure rates by 7, 28, and 42 days 28 clinical were 0.2%, 2.2%, and 12.0% treatment failure rates

(P. DHA+PP+P Day Q vs. efficacy AS+AQ+PQ

(P. AN vs. Day 42 cure By day 42, the number of patients free of recurrence was CQ+PQ rate 125 (98.4%; 95% Confidence interval, 94.4-99.8%) for ANQ arm and 123 (96.1%; 95%CI, 91.1-98.7%) for CQPQ, and non-significant (P = 0.4496). Compared with CQ-PQ, the side effect of ANQ was mild.

of Antimalaria l(s) tested

Table 3. Clinical studies 2008-2013 on treatment of non-falciparum malaria* (P. vivax, P. ovale & P. malariae)

3

Randomized, open-label controlled trial.

Hwang et al.218 2013

Prospective cohort study

Abdallah al.224 2012

et Sudan

Prospective study

Barber et al.305 Malaysi 2013 a

(P. AL vs. CQ Day efficacy

N=38 (P. vivax) AL

Day 28 cure Day 28, the cure rate was 100% and 88.4% for the per rate protocol analysis and for the intention to treat analysis, respectively.

Median parasite clearance time (PCT) for P. vivax was 2 days. 19 (44%) P. vivax patients smear negative by day 1.

28 Day 28 cure rates were 75.7% (95% (CI) 66.8-82.5) for AL and 90.8% (95% CI 83.6-94.9) for CQ. Day 28 cure rates were genotype adjusted to 91.1% (95% CI 84.195.1) for AL and to 97.2% (91.6-99.1) for CQ.

Relapse in 32 of 41 (78%) only AS (2.71 attacks/personyear), 7 of 36 (19%) PQ+Q (0.23 a/p-y), and 2 of 36 (6%) DHA+PP+PQ (0.06 a/p-y). The efficacy of PQ against relapse was 92% (95% CI 81% - 96%) for Q + PrQ and 98% (95% CI = 91% to 99%) for DHA+PP+PQ

Measure of Findings (primary) outcome

(P. PQ+Q vs. Therapeutic PQ+ efficacy DHA+PP (follow up 12 months) Relapse control: only AS

of Antimalaria l(s) tested

N=19 (P. vivax) CQ+PQ or NR ACT (AS)

N=242 vivax)

N=116 vivax)

Randomized, open-label, relapsecontrolled trial

Sutanto et al.226 Indonesi 2013 a

Ethiopia

Number patients

Source (first Countr Study author, year of y (study design publication) site), Timeframe

Phyo et al.221 Thailand 2011

2012

Randomized controlled trial

N=500 vivax)

parasitologic al response

Day 28 Day 28 overall cure rate was 100% (95% CI: 91-100%) for all species. adequate clinical and

Measure of Findings (primary) outcome

(P. DHA+PP vs. Day 63 risk Day 28, recurrent infections in 18 of 207 CQ vs. 5 of CQ of 230 DHA+PP (RR 4.0; 95% CI, 1.51-10.58; P = 0.0046). recurrence Risk of recurrence day 63: 79.1% (95% CI, 73.5%– 84.8%) in CQ vs. 54.9% (95% CI, 48.2%–61.6%) in DHA+PP ([HR, 2.27; 95% CI, 1.8–2.9; P 0.001). Both drugs well tolerated.

N=7 (P. m or P. o)

AL

of Antimalaria l(s) tested

N= 32 mixed infections (P. ovale or P. malariae with P. f.

N=38 (total)

Prospective cohort study

Mombo-ngoma et al.50

Gabon

Number patients

Source (first Countr Study author, year of y (study design publication) site), Timeframe

3

Prospective nonrandomized trial

Open-label randomized noninferiority trial

et Ethiopia

Yohannes al.219 2011

Awab et al.306 Afghani 2010 stan

Number patients

N=536 vivax)

N=132 vivax)

Randomized, N= 456 vivax) noninferiority trial (double dummy design)

et Cambod ia, Thailand , India & Indonesi a

Poravuth al.163 2011

Source (first Countr Study author, year of y (study design publication) site), Timeframe

Day 56, ↑ recurrent infections in the CQ arm (8.9%, 95% CI 6.0-13.1%) than the DHA+PP arm (2.8%, 95% CI 1.4-5.8%), a difference in cumulative recurrence rate of 6.1% (2-sided 90%CI +2.6 to +9.7%). Day 28 cure rate was 100% in both groups.

Day 28 Day 28 cumulative incidence treatment failure of 7.5% treatment (95% CI 2.9-18.9%) for CQ and 19% (95% CI 11failure 31.6%) for AL. CQ resistance was confirmed in 3 of 5 CQ treatment failures cases. The effectiveness of AL was ↓ than CQ; however, the findings were not conclusive, because the AL evening doses were not supervised.

(P. DHA+PP vs. Day 56 CQ overall cumulative parasitologic al failure rate

(P. AL vs. CQ

Day 14 CR: 99.5%, (217/218; 95%CI 97.5-100) AS+ P vs. 100% (209/209; 95%CI 98.3, 100) CQ. P= noninferior to CQ: treatment difference -0.5% (95%CI -2.6, 1.4) AS+P CR> non-inferior to CQ for D21, 28, 35 and 42. PCT: shorter for AS+P (median 23.0 h) vs. CQ (32.0 h; p8g/dL. Pregnant women in second or third trimester of pregnancy with recrudescent multi-drug resistant uncomplicated P. falciparum malaria after 7-day quinine treatment and Ht>25%.

Pregnant women in the second and third trimester of pregnancy with recrudescent uncomplicated multi-drug resistant P. falciparum malaria after 7-day quinine treatment.

Population

Number of women

ArtesunateAP (4/20/8 mg/kg p.o. q.d. for 3 days) + 300 mL chocolate

AP (1000/400 mg p.o. q.d. for 3 days) 24 pregnant women

26 pregnant women

fat) AL (80/480 13 pregnant mg p.o. women b.i.d. for 3 days) + 250 ml chocolate milk (7g fat)

Drug (dose)

Noncompart mental & compartmen tal analysis

Compartme ntal analysis

Noncompart mental & compartmen tal analysis

Pharmacok inetic analytic methodolog y

Cmax, Tmax, CL/F, V/F, AUC0-∞, AUC48-∞.

Cmax, Tmax, AUC0-∞, PG-CG ratio.

Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC024, AUC 60-84, AUC/dose.

Pharmacok inetic variables

Remarks

Country (time period)

Thailand (not reported)

Thailand (Sept 1986June 1988)

Thailand (not reported)

Author (year)

McGready (2003-2)

Na Bangchang (1994)

Wangboons kul (1993)

Clinical trial

Clinical trial

Clinical trial

Type of study

Pregnant women in first (n=2) and third (n=7) trimester of pregnancy with P. falciparum parasitaemia & non-pregnant women matched for age with P. falciparum parasitaemia. Pregnant women in third trimester of pregnancy without P. falciparum malaria & same women post partum (>2 months) without

Healthy pregnant women with an EGA>35wks & same women post partum (>2 months).

Population

Proguanil (200 mg p.o. once)

Mefloquine (15 mg/kg)

milk (8% fat) Proguanil (200 mg p.o. once)

Drug (dose)

Pharmacok inetic analytic methodolog y

Noncompart mental analysis

Compartme ntal analysis

Compartme ntal analysis

Number of women

45 pregnant women 45 postpartum women

9 pregnant women 8 non-pregnant women

10 pregnant women 4 postpartum women 9 male

5

Cmax, Tmax, CL/F, t1/2, AUC.

Total dose, Cmax (plasma and urine), 6h concentratio n (plasma and urine). Total dose, Cmax, Tmax, CL/F, V/F, t1/2.

Pharmacok inetic variables

Remarks

Thailand (not reported)

Nosten (1990)

Clinical trial

Type of study

Drug (dose)

P. falciparum malaria & healthy adult male volunteers without P. falciparum malaria*. Pregnant women in third Group 1: trimester of pregnancy. Mefloquine (250 mg per week)

Population

20 pregnant women

patients*

Number of women

Compartme ntal analysis

Pharmacok inetic analytic methodolog y

Cmax, Tmax, CL/F, t1/2, AUC.

Pharmacok inetic variables

Remarks

Group 2: Mefloquine (125 mg per week) q.d. = once a day; b.i.d. = twice a day; t.i.d. = three times a day; p.o. = per os (oral); i.v. (intravenous); AL = artemether-lumefantrine; DHA-PPQ = dihydroartemisinin-piperaquine; SP = Sulfadoxine-Pyrimethamine; AP = atovaquone-proguanil; PG = proguanil; CG = cycloguanil; Cmax = maximum concentration after administration; Tmax = time to maximum concentration after administration; CL/F = oral clearance; V/F = apparent volume of distribution; t1/2 = half-life; AUC = area under the curve (exposure); Hb = haemoglobin; Ht = haematocrit; wks = weeks; * Data for male subjects were included from a previous study for comparison.

Country (time period)

Author (year)

Uganda (March 2008 – Sept 2008)

Thailan d (April 2004-

McGready (2006-2)

Countr y (time period) Uganda (Oct 2006 – May 2009)

Tarning (2012-2)

Tarning (2013)

Artemether Author (year)

Pregnant women in the second and third trimester of pregnancy with recrudescent

Pregnant women in second and third trimester of pregnancy (EGA>13wks) with uncomplicated P. falciparum malaria.

Pregnant women with uncomplicated P. falciparum infection with an EGA>13wks & non-pregnant women matched for history of fever, temp. > 37.5°C, smoking status and level of parasitaemia.

Population

Table 2. Primary study outcomes per compound

AL (80/480 mg p.o. b.i.d. for 3 days) + 200 mL milk tea AL (80/480 mg p.o.

AL (80/480 mg p.o. b.i.d. for 3 days) + 200 mL milk tea

Drug (dose)

13 pregnant women

21 pregnant women

21 pregnant women

Number of women

No significant differences in the pharmacokinetic parameters of artemether and DHA between the second and third trimester. Comparison with data from literature

No statistically significant differences in pharmacokinetic properties between second and third trimester.

Estimated exposure to artemether and DHA was similar to that previously reported in pregnant Thai patients and lower than reported in adult non-pregnant Thai patients.

Result

5

Country (time period)

Thailand (April 2008 – March 2009)

Kloprogge (2015)

Clinical trial

Type of study

b.i.d. for 3 days) + 250 ml chocolate milk (7g fat)

Pregnant women in second and third trimester of pregnancy (Ht>25%) with uncomplicated P. falciparum malaria & the same women post partum (3 months) without malaria.

Population

uncomplicated multi-drug resistant P. falciparum malaria after 7-day quinine treatment.

Author (year)

Artesunate

Aug 2004)

20 pregnant women Group 1: 10 women Group 2: 10 women 14 postpartum women

Group 1 Artesunate (4 mg/kg) i.v. q.d. on day 0; artesunate (4mg/kg) p.o q.d. on day 1-6 Group 2

Number of women

Drug (dose)

Pharmacok inetic analytic methodolog y Compartme ntal analysis

AUC0-12, Cmax, Tmax, t1/2, CL/F, Vd/F.

Pharmacok inetic variables

Based on the same clinical study as McGready (2012).

Remarks

showed a lower AUC and Cmax of artemether compared to male Thai patients and of DHA compared to nonpregnant patients.

Burkina Faso (Sept 2008-Jan 2009)

Thailand (April 2008 – March 2009)

Valea (2014)

McGready (2012)

Clinical trial

Clinical trial

Pregnant women in second and third trimester of pregnancy (Ht>25%) with uncomplicated P. falciparum malaria & the same women post partum (3 months)

Pregnant women in second and third trimester of pregnancy with uncomplicated P. falciparum monoinfection & matched non-pregnant women with P. falciparum infection.

Noncompart mental analysis

Noncompart mental analysis

24 pregnant women 23 nonpregnant women

20 pregnant women Group 1: 10 women Group 2: 10 women

Artesunate (4 mg/kg) p.o. q.d. on day 0; artesunate (4 mg/kg) i.v. q.d. on day 1, artesunate (4 mg/kg) p.o. q.d. on day 2-6) Mefloquine + Artesunate (8/3.6 mg/kg q.d. for 3 days)

Group 1 Artesunate (4 mg/kg) i.v. q.d. on day 0; artesunate

5

Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2, AUC0-last, AUC0-∞, AUC∞/dose. Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2, AUC0-∞,

Based on the same clinical study as Kloprogge (2015).

Morris (2011)

DRC (May 2007-Nov 2008)

Clinical trial

Pregnant women in second (22-26wks) and third (3236wks) trimester of pregnancy with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL; Ht>30%) & same women post partum (3 months) with (n=2) or without (n=24) P. falciparum parasitaemia & non-pregnant

without malaria.

(4mg/kg) p.o q.d. on day 1-6 Group 2 Artesunate (4 mg/kg) p.o. q.d. on day 0; artesunate (4 mg/kg) i.v. q.d. on day 1, artesunate (4 mg/kg) p.o. q.d. on day 2-6) Artesunate (200 mg) p.o. q.d. on day 0 + SP (1725 mg) p.o. q.d. on day 1 26 pregnant women 26 postpartum women 25 nonpregnant women

14 postpartum women

Compartme ntal analysis

T1/2, CL/F, V/F.

AUC0∞/dose.

Based on the same clinical study as Onyamboko (2011).

DRC (May 2007-Nov 2008)

Thailand (Oct 2000July 2001)

Onyamboko (2011)

McGready (2006-1)

Clinical trial

Clinical trial

female volunteers with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL). Pregnant women in second (22-26wks) and third (3236wks) trimester of pregnancy with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL; Ht>30%) & same women post partum (3 months) with (n=2) or without (n=24) P. falciparum parasitaemia & non-pregnant female volunteers with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL). Pregnant women in second or third trimester of pregnancy with recrudescent uncomplicated P. falciparum malaria after 7-day quinine treatment and Ht>25%. ArtesunateAP (4/20/8 mg/kg p.o. q.d. for 3 days) + 200 mL chocolate

Artesunate (200 mg) p.o. q.d. on day 0 + SP (1725 mg) p.o. q.d. on day 1

24

26 pregnant women 26 postpartum women 25 nonpregnant women

5

Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC0∞.

Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC4872.

Noncompart mental analysis

Noncompart mental & compartmen tal analysis

Based on the same clinical study as Morris (2011).

Clinical trial

Papua New Guinee (…)

Burkina Faso (Sept 2008-Jan 2009)

Benjamin (2015)

Valea (2014)

Clinical trial

Type of study

Dihydroartemisinin Author Country (year) (time period)

Pregnant women in second and third trimester of pregnancy with uncomplicated P. falciparum monoinfection & matched

Pregnant women in second and third trimester of pregnancy (EGA>14wks) and age-matched nonpregnant women with uncomplicated with malaria infection.

Population

Number of women

32 pregnant women 33 nonpregnant women

24 pregnant women 23 nonpregnant women

Drug (dose)

Group 1 DHA-PPQ (7/58 mg/kg p.o. q.d. for 3 days) Group 2 PPQ (1280mg p.o. q.d. for 3 days) + SP (25mg/kg once) Mefloquine + Artesunate (8/3.6 mg/kg q.d. for 3 days)

milk (8% fat)

Noncompart mental analysis

Pharmacok inetic analytic methodolog y Compartme ntal analysis

Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2,

MTT, NN, CL/F, Vc/F, Q/F, Vp/F, t1/2, AUC0∞.

Pharmacok inetic variables

Remarks

McGready (2012)

Thailand (April 2008 – March 2009)

Clinical trial

Pregnant women in second and third trimester of pregnancy (Ht>25%) with uncomplicated P. falciparum malaria & the same women post partum (3 months) without malaria.

non-pregnant women with P. falciparum infection.

Group 2 Artesunate (4 mg/kg) p.o. q.d. on day 0; artesunate (4 mg/kg) i.v. q.d. on day 1, artesunate (4 mg/kg) p.o. q.d. on day 2-6)

Group 1 Artesunate (4 mg/kg) i.v. q.d. on day 0; artesunate (4mg/kg) p.o q.d. on day 1-6 20 pregnant women Group 1: 10 women Group 2: 10 women 14 postpartum women

5

Noncompart mental analysis

AUC0-last, AUC0-∞, AUC∞/dose. Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2, AUC0-∞, AUC0∞/dose.

Based on the same clinical study as Kloprogge (2015).

Clinical trial

Clinical trial

Uganda (March 2008 – Sept 2008)

DRC (May 2007-Nov 2008)

Tarning (2012-2)

Morris (2011)

Clinical trial

Thailand (June 2008Dec 2008)

Tarning (2012-1)

Pregnant women in second (22-26wks) and third (3236wks) trimester of pregnancy with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL; Ht>30%) & same women post partum (3 months) with (n=2) or without (n=24) P. falciparum parasitaemia & non-pregnant

Pregnant women in second and third trimester of pregnancy (EGA>13wks) with uncomplicated P. falciparum malaria.

Pregnant women in second and third trimester of pregnancy (Ht>25%) with uncomplicated P. falciparum malaria & matched nonpregnant women with P. falciparum malaria.

Artesunate (200 mg) p.o. q.d. on day 0 + SP (1725 mg) p.o. q.d. on day 1

AL (80/480 mg p.o. b.i.d. for 3 days) + 200 mL milk tea

DHA-PPQ (6.4/51.2 mg/kg p.o. q.d. for 3 days)

26 pregnant women 26 postpartum women 25 nonpregnant women

21 pregnant women

24 pregnant women 24 nonpregnant women

Compartme ntal analysis

Compartme ntal analysis

Compartme ntal analysis

T1/2, CL/F, V/F.

Cmax, Tmax, CL/F, V/F, t1/2, AUC024, AUC092, day 7 and 28 concentratio n. Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC60last, AUC/dose.

Based on the same clinical study as Onyamboko (2011).

Nested in larger efficacy study by Piola (2010).

Based on the same clinical study as Rijken (20112).

DRC (May 2007-Nov 2008)

Thailand (June 2008Dec 2008)

Onyamboko (2011)

Rijken (2011-2)

Clinical trial

Clinical trial

female volunteers with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL). Pregnant women in second (22-26wks) and third (3236wks) trimester of pregnancy with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL; Ht>30%) & same women post partum (3 months) with (n=2) or without (n=24) P. falciparum parasitaemia & non-pregnant female volunteers with asymptomatic P. falciparum parasitaemia (200-300,000 p/µL). Pregnant women in second and third trimester of pregnancy (Ht25%.

Artesunate- 24 AP (4/20/8 mg/kg p.o. q.d. for 3 days) + 200 mL chocolate milk (8% fat) AL (80/480 13 pregnant mg p.o. women b.i.d. for 3 days) + 250 ml chocolate milk (7g fat)

Noncompart mental & compartmen tal analysis

Noncompart mental & compartmen tal analysis

Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC024, AUC 60-84, AUC/dose.

∞/dose, AUC0-24, AUC24-48, AUC48-72, AUC72-∞, day 7, 14 and 28 concentratio n. Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC4872.

Type of study

Clinical trial

Lumefantrine Author Country (year) (time period)

Uganda (March 2008 – Sept 2008)

Uganda (Oct Clinical 2006 – May trial 2009)

Kloprogge (2013)

Tarning (2013)

Pregnant women with uncomplicated P. falciparum infection with an EGA>13wks & nonpregnant women matched for

Pregnant women in second and third trimester of pregnancy (EGA>13wks) with uncomplicated P. falciparum malaria.

Population

AL (80/480 mg p.o. b.i.d. for 3 days) + 200 mL milk tea

AL (80/480 mg p.o. b.i.d. for 3 days) + 200 mL milk tea

Drug (dose)

Lumefantrine:

AL: 21 pregnant women

115 pregnant women 26 venous samples 89 capillary samples 17 nonpregnant women (all venous samples)

Number of women

5

Noncompart mental analysis

Pharmacok inetic analytic methodolog y Compartme ntal analysis

Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2,

AUC0-∞, Cmax, T1/2, day 7 concentratio n.

Pharmacok inetic variables

Results for artemether and dihydroartemi sinin are

Nested in larger efficacy / safety study by Piola (2010).

Remarks

Thailand (not reported)

Thailand (April 2004-

Tarning (2009)

McGready (2008)

Clinical trial

Clinical trial

Uganda (Oct Clinical 2006-May trial 2009)

Piola (2010)

Pregnant women in second or third trimester of

Pregnant women in second or third trimester of pregnancy with uncomplicated symptomatic P. falciparum malaria.

Pregnant women with uncomplicated P. falciparum infection (13wks and Hb > 7g/dL.

history of fever, temp. > 37.5°C, smoking status and level of parasitaemia.

AL (80/480 mg p.o. b.i.d. for 3 days) + 200250 ml chocolate milk (6-7g fat) AL (80/480 mg p.o.

AL (80/480 mg p.o. b.i.d. for 3 days) + 200 mL milk

OR Quinine (10 mg/kg p.o. t.i.d. for 7 days)

85 pregnant women

103 pregnant women

Quinine: 21 pregnant women 97 pregnant women

26 pregnant women 17 nonpregnant women

Noncompart mental

Compartme ntal analysis

Noncompart mental analysis

Day 7 concentratio

Total dose, CL/F, V/F, day 7 concentratio n.

AUC0-last, AUC0-∞, AUC∞/dose, AUC72-last, AUC72-∞, day 7 concentratio n. Day 7 concentratio n.

Based on the same clinical

reported by Tarning (2012-2). Nested in larger efficacy / safety study by Piola (2010). Based on the same clinical study as Tarning (2012-2) and Tarning (2013). Nested in larger efficacy / safety study by McGready (2008).

Clinical trial

Thailand (Oct 2007May 2008)

Tarning (2012-3)

Clinical trial

Type of study

Thailand (April 2004Aug 2004)

Amodiaquine Author Country (year) (time period)

McGready (2006-2)

Aug 2006)

Pregnant women in second and third trimesters of pregnancy with acute P. vivax monoinfection & same women post partum (84-173 days) with (n=7) or without

Population

Pregnant women in the second and third trimester of pregnancy with recrudescent uncomplicated multi-drug resistant P. falciparum malaria after 7-day quinine treatment.

pregnancy with acute uncomplicated P. falciparum malaria.

Pharmacok inetic analytic methodolog y Compartme ntal analysis

Number of women

Amodiaquin e (10 mg/kg p.o. q.d. for 3 days)

27 pregnant women 19 postpartum women

Noncompart mental & compartmen tal analysis

AL (80/480 13 pregnant mg p.o. women b.i.d. for 3 days) + 250 ml chocolate milk (7g fat)

Drug (dose)

analysis

b.i.d. for 3 days) + 250 ml chocolate milk (7g fat)

5

Cmax, Tmax, t1/2, AUClast.

Pharmacok inetic variables

Total dose, Cmax, Tmax, CL/F, V/F, t1/2, AUC024, AUC 60-84, AUC/dose.

n.

Based on the same clinical study as Rijken (20111).

Remarks

study as McGready (2006-2) and Tarning (2009).

Valea (2014)

Mefloquine Author (year)

Rijken (2011-1)

Burkina Faso (Sept 2008-Jan 2009)

Country (time period)

Thailand (Oct 2007May 2008)

Clinical trial

Type of study

Clinical trial

Pregnant women in second and third trimester of pregnancy with uncomplicated P. falciparum monoinfection & matched

Population

(n=12) P. vivax malaria. Pregnant women in second and third trimesters of pregnancy with acute P. vivax monoinfection & same women post partum (84-173 days) with (n=7) or without (n=12) P. vivax malaria.

Mefloquine + Artesunate (8/3.6 mg/kg q.d. for 3 days)

Drug (dose)

Amodiaquin e (10 mg/kg p.o. q.d. for 3 days)

24 pregnant women 23 nonpregnant women

Number of women

24 pregnant women 18 postpartum women

Pharmacok inetic analytic methodolog y Noncompart mental analysis

Noncompart mental analysis

Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2,

Pharmacok inetic variables

Total dose, Cmax, Cmax/dose, Tmax, CL/F, V/F, t1/2, AUC0-last, AUC0-∞, AUC0∞/dose, day 7 concentratio n.

Remarks

Based on the same clinical study as Tarning (2012-3)

Thailand (not reported)

Nosten (1990)

Clinical trial

Clinical trial

Sulfadoxine-pyrimethamine Author Country Type of (year) (time study period)

Thailand (Sept 1986June 1988)

Na Bangchang (1994)

Population

Pregnant women in first (n=2) and third (n=7) trimester of pregnancy with P. falciparum parasitaemia & non-pregnant women matched for age with P. falciparum parasitaemia. Pregnant women in third trimester of pregnancy.

non-pregnant women with P. falciparum infection.

Compartme ntal analysis

Pharmacok inetic analytic

20 pregnant women

Number of women

Group 1: Mefloquine (250 mg per week)

Drug (dose)

Group 2: Mefloquine (125 mg per week)

Compartme ntal analysis

9 pregnant women 8 non-pregnant women

Mefloquine (15 mg/kg)

5

Pharmacok inetic variables

Cmax, Tmax, CL/F, t1/2, AUC.

AUC0-last, AUC0-∞, AUC∞/dose. Total dose, Cmax, Tmax, CL/F, V/F, t1/2.

Remarks

Mali, Mozambiqu e, Sudan & Zambia (not reported)

Papua New Guinee (Feb 2006-July 2006)

Nyunt (2010)

Karunajeew a (2009)

Clinical trial

Clinical trial

Pregnant women with an EGA 15-36wks without P. falciparum parasitaemia (Hb>8g/dL) & same women post partum (6-43wks) without P. falciparum parasitaemia and with Hb>8g/dL (Mali & Zambia) / postpartum women (> 6 months) without P. falciparum parasitaemia and with Hb>8g/dL (Mozambique & Sudan) & matched non-pregnant women with acute uncomplicated falciparum malaria (Mozambique). Pregnant women in second or third trimester of pregnancy without severe malaria (n=17: P. falciparum / vivax / malariae parasitaemia; n=13: no parasitaemia) & matched SP (1500/75 mg p.o. once) + Chloroquine (1350 mg p.o. q.d. for 3 days)

SP (1500/75 mg p.o. once)

30 pregnant women 30 nonpregnant women

97 pregnant women 77 postpartum women

Compartme ntal analysis

methodolog y Compartme ntal analysis

CL/F, V/F, t1/2, AUC0∞.

Total dose, Cmax, CL/F, V/F, t1/2, AUC0-∞, day 7 concentratio n.

Kenya (1999-2000)

Piperaquine Author Country (year) (time period)

Green (2007)

Type of study

Clinical trial

Population

non-pregnant women (n=9: falciparum / vivax / malariae parasitaemia; n=21: no parasitaemia). Primi- and secundigravid women with uncomplicated singleton pregnancies with EGA 16-28wks and Hb>8g/dL without symptomatic malaria (n=11: parasitaemic; n=22: aparasitaemic) & same women post partum (2-3 months) without symptomatic malaria (n=1: parasitaemic; n=10: aparasitaemic).

Drug (dose)

SP (1500/75 mg p.o. once)

Compartme ntal analysis

Pharmacok inetic analytic methodolog y

33 pregnant women 16 HIVpositive 17 HIVnegative 11 postpartum women 6 HIVpositive 5 HIVnegative

Number of women

5

Pharmacok inetic variables

CL/F, V/F, t1/2, AUC0∞.

Remarks

Clinical trial

Clinical trail

Papua New Guinee (not reported)

Sudan (Aug 2007-Feb 2008)

Benjamin (2015)

Adam (2012)

Pregnant women in second and third trimester of pregnancy (EGA 15-40wks) with uncomplicated P. falciparum malaria and Hb>7g/dL. & age- and weight-matched nonpregnant women with uncomplicated P. falciparum malaria.

Pregnant women in second and third trimester of pregnancy (EGA>14wks) and age-matched nonpregnant women with uncomplicated with malaria infection. Group 2 PPQ (1280mg p.o. q.d. for 3 days) + SP (25mg/kg once) DHA-PPQ (2.4/20 mg/kg q.d. for 3 days)

Group 1 DHA-PPQ (7/58 mg/kg p.o. q.d. for 3 days)

12 pregnant women 12 nonpregnant women

32 pregnant women 33 nonpregnant women

Noncompart mental analysis

Compartme ntal analysis

Total dose, Cmax (after dose 1, 2 and 3), Tmax (after dose 1, 2 and 3), CL/F, V/F, T1/2, AUC0last, AUC0∞, AUC∞/dose, AUC0-24, AUC24-48,

MTT, NN, CL/F, Vc/F, Q/F, Vp/F, t1/2, AUC0∞.

Based on the same clinical study as Hoglund (2012).

Sudan (Aug 2007-Feb 2008)

Thailand (June 2008Dec 2008)

Thailand (June 2008Dec 2008)

Hoglund (2012)

Tarning (2012-1)

Rijken (2011-2)

Clinical trial

Clinical trial

Clinical trial

Pregnant women in second and third trimester of pregnancy (Ht7g/dL. & age- and weight-matched nonpregnant women with uncomplicated P. falciparum malaria. Pregnant women in second and third trimester of pregnancy (Ht>25%) with uncomplicated P. falciparum malaria & matched nonpregnant women with P. falciparum malaria. DHA-PPQ (6.4/51.2 mg/kg p.o.

DHA-PPQ (6.4/51.2 mg/kg p.o. q.d. for 3 days)

DHA-PPQ (2.4/20 mg/kg q.d. for 3 days)

Compartme ntal analysis

Compartme ntal analysis

Noncompart mental analysis

12 pregnant women 12 nonpregnant women

24 pregnant women 24 nonpregnant women

24 pregnant women 24 non-

5

Cmax, Tmax, CL/F, V/F, t1/2, AUC024, AUC092, day 7 and 28 concentratio n. Total dose, Cmax, Cmax/dose,

AUC48-72, AUC72-∞, day 7 and 14 concentratio n. Cmax, Tmax, t1/2, AUC 48-90, AUC0-90, day 7 and 28 concentratio n.

Based on the same clinical study as

Based on the same clinical study as Rijken (20112).

Based on the same clinical study as Adam (2012).

uncomplicated P. falciparum malaria & matched nonpregnant women with P. falciparum malaria. q.d. for 3 days)

pregnant women

CL/F, V/F, t1/2, AUC0last, AUC0∞, AUC0∞/dose, AUC0-24, AUC24-48, AUC48-72, AUC72-∞, day 7, 14 and 28 concentratio n.

Tarning 20121.

5

173

“The proportion of silt particles in the topsoil (i.e. mineral matter between 0.002 mm and 0.05 mm – USDA classification - or between 0.002 mm and 0.0625 mm - ISO and FAO classification). Silt is too small to see with the naked eye. It is produced by the mechanical weathering of rock, as opposed to the chemical weathering that results in clays. This mechanical weathering can be due to aeolian abrasion (sandblasting by the wind) as well as water erosion of rocks on the beds of rivers and streams. It is good agricultural soil due to high nutrient levels and good water retention in spaces between particles. Easy to cultivate but very prone to erosion.” Adapted from: Soil Atlas of Africa, 2013. European Commission, Publications Office of the European Union, Luxembourg.1

174

Chapter 6 Health workers’ compliance to rapid diagnostic tests (RDTs) to guide malaria treatment: a systematic review and meta-analysis Alinune N. Kabaghe Benjamin J. Visser Rene Spijker Kamija S. Phiri Martin P. Grobusch Michèle van Vugt Malaria Journal 2016 Mar 15;15(1):163 Appendices and supplementary material are available online at: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-016-1218-5

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Abstract Background The World Health Organization recommends malaria to be confirmed by either microscopy or a rapid diagnostic test (RDT) before treatment. The correct use of RDTs in resource-limited settings facilitates basing treatment onto a confirmed diagnosis; contributes to speeding up considering a correct alternative diagnosis, and prevents overprescription of anti-malarial drugs, reduces costs and avoids unnecessary exposure to adverse drug effects. This review aims to evaluate health workers’ compliance to RDT results and factors contributing to compliance.

Methods A PROSPERO-registered systematic review was conducted to evaluate health workers’ compliance to RDTs in sub-Saharan Africa, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Studies published up to November 2015 were searched without language restrictions in Medline/Ovid, Embase, Cochrane Central Register of Controlled Trials, Web of Science, LILACS, Biosis Previews and the African Index Medicus. The primary outcome was health workers treating patients according to the RDT results obtained.

Results The literature search identified 474 reports; 14 studies were eligible and included in the quantitative analysis. From the meta-analysis, health workers’ overall compliance in terms of initiating treatment or not in accordance with the respective RDT results was 83 % (95 % CI 80–86 %). Compliance to positive and negative results was 97 % (95 % CI 94–99 %) and 78 % (95 % CI 66–89 %), respectively. Community health workers had higher compliance rates to negative test results than clinicians. Patient expectations, work experience, scepticism of results, health workers’ cadres and perceived effectiveness of the test, influenced compliance.

Conclusions With regard to published data, compliance to RDT appears to be generally fair in sub-Saharan Africa; compliance to negative results will need to improve to prevent mismanagement of patients and overprescribing of anti-malarial drugs. Improving diagnostic capacity for other febrile illnesses and developing local evidence-based guidelines may help improve compliance and management of negative RDT results.

Trial registration CRD42015016151 (PROSPERO)

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Health workers’ compliance to RDTs to guide malaria treatment: a meta-analysis

Background Plasmodium falciparum malaria is estimated to have caused 528,000 deaths and 163 million clinical episodes in sub-Saharan Africa in 2013.4 Early diagnosis and treatment with appropriate anti-malarial drugs can prevent severe illness and lethal outcome.38, 96 Artemisinin-based combination therapy (ACT) is currently recommended for the treatment of uncomplicated malaria caused by P. falciparum10, 38 and is increasingly used for nonfalciparum malaria.420 Effective case-management of malaria consists of an efficacious treatment, prompt access to treatment and diagnosis, provider compliance to treatment guidelines, and patient adherence to medication (Figure 1). 38, 462

6

Figure 1. Pathway of health systems effectiveness of malaria diagnosis and treatment. (Adapted from MalERA consultative group)462

Presumptive diagnosis and treatment of malaria based on symptoms leads to over- diagnosis of malaria and missed diagnosis for patients without malaria.463, 464 The World Health Organization (WHO) recommends that any suspected malaria case in any epidemiological setting should be parasitologically-confirmed by either microscopy or rapid diagnostic test (RDT) before treatment.38 Lack of trained personnel, equipment,465 and reagents for microscopy in most remote rural areas in Africa466, 467 with high malaria burden makes the RDT the most practically suitable tool to confirm a malaria diagnosis. 4, 468 RDTs are immunochromatographic test kits which confirm the presence of malaria parasites in suspected patients by detecting one or a combination of the following three Plasmodium antigens: Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) for P. falciparum or a ‘pan-specific’ aldolase to detect other species, such as P. vivax or Plasmodium lactate

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Chapter 6

dehydrogenase (LDH) variants (pLDH) (with clonality specific to the various Plasmodium species infecting humans).37, 469 The use of malaria RDT can reduce over-prescribing of anti-malarial drugs (AMD). Studies have shown that in most endemic countries in sub-Saharan Africa, health workers of different cadres do not comply with malaria RDTs; they prescribe AMDs to patients with RDT negative results.468, 470-472 This has implications on resources for patient, family members and health system since some drug combinations are relatively expensive. 473, 474 Non-compliance to malaria negative results by prescribing AMDs neglects underlying cause of fever and expose patients unnecessarily to adverse effects; underlying infections, such as sepsis, pneumonia and meningitis,475-478 present as malaria clinically but are not routinely investigated479 and may not be treated.466, 480 To treat malaria effectively, to reduce costs and avoid unnecessary exposure to drug adverse effects, there is a need to correctly diagnose and comply with malaria treatment guidelines or clinical decision algorithms. Health workers (HWs) need to use the correct treatment based on the RDT results. This systematic review examines data available on HWs compliance to RDT results in subSaharan Africa, and investigates factors associated with compliance to results (HW treating patients according to the RDT result). The primary outcome is the percentage of HWs compliant to overall, positive or negative, test results.

Methods This systematic review was registered in advance in the International prospective register of systematic reviews (PROSPERO; registration number CRD42015016151) which included pre-specified the objectives and inclusion criteria.481 An experienced information specialist (RS) conducted a search without language or time restrictions in the online electronic databases Ovid Medline, Ovid Embase, Cochrane Central Register of Controlled Trials, CINAHL Plus with Full Text, African Index Medicus, and African Journals Online (AJOL). The search used both free text words and medical subject headings for ‘malaria’, ‘RDT’, ‘health worker’ and ‘compliance’. The search was conducted on 3 March 2015 and updated on 12 November 2015. Studies reporting on malaria suspected patients of any age presenting to HWs of any cadre in sub-Saharan Africa were searched. The intervention was the use of a WHO recommended RDT kit for parasitological confirmation of a malaria diagnosis (a list of WHO recommended RDTs is available online482). Bibliographies of relevant studies retrieved from the studies were checked for additional publications. The search strategy is described in Additional file 1. EndNote X7.4 (Thomson

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Health workers’ compliance to RDTs to guide malaria treatment: a meta-analysis

Reuters) was used to manage, de-duplicate and screen the references for eligibility. The inclusion criteria were: studies were conducted in sub-Saharan Africa; RDTs were used to diagnose malaria in symptomatic patients; the RDTs used were WHO-recommended; absolute numbers of RDT result adherence as primary or secondary outcome were reported. Exclusion criteria were: studies using RDT for active case finding and population screening; conference abstracts; no absolute numbers were reported; studies outside sub-Saharan Africa. Eligibility assessment of studies was performed independently in a blinded, standardized way by two reviewers (ANK and BJV). Titles and abstracts were screened first, and the two reviewers screened and selected relevant full-text articles. ANK extracted quantitative data based on the pre-specified criteria into an excel sheet (Additional file 2); factors associated with compliance were also extracted into the same sheet. All the quantitative data was independently checked by BJV. Data extracted included author name, year of publication, place of study, transmission setting, type of RDT, cadre and number of HW, age of patients, number of test results, RDT positives treated and RDT negatives not treated. Both qualitative and quantitative factors were also extracted from included studies which reported them. The risk of bias of studies was not assessed because of the diversity of the study designs included. The primary outcome measure was proportions in percentage of RDT results with appropriate AMD prescription disaggregated to positive and negative results adherence. Appropriate treatment was defined as AMDs prescribed to RDT positive and AMD not prescribed to RDT negative patients (Figure 2). Formulae for these calculations are included in Additional file 3. STATA version 13 (StataCorp, College Station, TX, USA) was used to calculate the pooled estimate of proportions appropriately treated overall and negative and positive compliance using random effects. Random effects analysis was used after an initial fixed effect analysis had I2 above 50 %, suggesting heterogeneity. Pooled estimates were also stratified by health personnel cadre, age of patients and malaria transmission setting. A qualitative synthesis of factors contributing to compliance was also reported for the included studies.

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Chapter 6

Figure 2. Patient pathway for malaria diagnosis and treatment. The shaded areas represent appropriate management (RDT rapid diagnostic test, AMD anti-malarial drug)

Results Study selection The total number of articles after removing duplicates was 474 (Figure 3). After screening title and abstracts for eligibility, 75 full-text articles were examined for eligibility; 14 studies were included in the quantitative analysis.463, 470-472, 483-492 Five of the studies reported on factors associated with compliance to RDT results and were included in the summary of associated factors.463, 471, 483, 484, 489

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6

Figure 3. Study selection flow (PRISMA)111

There were five study designs (Table 1): one randomized control trial,484 four observational,485-487, 491 four cross-sectional,471, 472, 489, 490 four cluster randomized trials463, 470, 483, 488 and pre-post intervention study.492 RDT adherence was a secondary outcome in five out of the 14 studies.471, 484, 488, 490, 492

181

Year

2009

2010 2013

2014

2010

2011

2013

2013

Author

Bisoffi484

Masanja486 Bottieau485

Manyando491

Chinkhumba490

Uzochukwu489

Mubi471

Shakely472

Study setting

Zanzibar

Tanzania

Nigeria

Perennial transmission Low transmission

High transmission

Burkina Faso Stable malaria with seasonal transmission Tanzania Holoendemic Mozambique Perennial transmission with seasonal peaks Zambia Both low and high transmission Malawi Stable malaria with seasonal peak

Country

Table 1. Characteristics of studies included

Cross sectional

Cross sectional

Cross sectional

Cross sectional

Observational

Observational Observational

RCT

Study design

NR

NR

99 NR

All

> 3 months

All

> 5 years

< 5 years

> 5 years All

1050

Sample size

Paracheck Pf

NR

3889

105

ICT malaria pf; 1390 SD Bioline; Paracheck Pf; First Response ICT malaria Pf 280

ParaHIT 10650 Paracheck Pf; 1385 ICT malaria Pf; SD Bioline Pf ICT malaria Pf 1492

Number Age of RDT of HWs study participants NR > 6 months Paracheck Pf

Clinicians, 32 nurses and CHW Clinicians 20 and nurses Clinicians 33 and nurses

Clinicians and nurses

Clinicians

Clinicians Clinicians

Nurses

HW cadre

2011

2012

2014 2011

2015

2011

Batwala483

Mukanga488

Mbacham470 Bastiaens492

Mbonye463

Mukanga487

Uganda

Uganda

Ghana, Uganda Cameroon Tanzania

Uganda

Country

Perennial transmission High transmission

NR NR

Seasonal

14

CHW

198 NR 10

Observational

CRT Before after CRT

CRT

Under years

5

NR

Paracheck Pf; ICT malaria Pf All SD Bioline Below 10 ICT malaria Pf; year olds Paracheck Pf All First response

4-59 months

Number Age of RDT of HWs study participants 30 All Paracheck Pf

Clinical officers and nurses CHW 44

HW cadre

Clinicians and Clinical officers DSV

Study design

Both low and high CRT transmission

Study setting

182

8073

1194 501

1559

44565

Sample size

CHW = Community health worker; CRT = Cluster randomized trial; DSV = Drug shop vendor; NR = Not reported; RCT = randomized control trial;

Year

Author

Health workers’ compliance to RDTs to guide malaria treatment: a meta-analysis

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Chapter 6

Health workers’ compliance to malaria results A pooled meta-analysis using random effects (Fig. 4) for the 14 studies463, 470-472, 483, 484, 486493 shows an overall compliance of 83 % (95 % CI 80–86 %); I2 = 99.9 %, Z = 54.35, p < 0.001. Appropriate malaria treatment based on RDT results (Table 2) was as low as 39.7 % in a Zambian study491 to as high as 99.9 % in Zanzibar.472 The pooled meta-analysis result using random effects for RDT positives prescribed AMDs (Fig. 5) was 97 % (95 % CI 94– 99 %); I2 = 99.2 %, Z = 37.31, p < 0.001. The proportion of positive RDT results prescribed AMDs ranged from 72.1 to 100 %. 12 studies reported appropriate prescription of AMDs to RDT positive patients above 93 %; six of these studies had 100 % RDT positive compliance (Table 2).

Figure 4. Pooled meta-analysis of overall compliance to RDT results

184

Mozambique

Uganda Zambia

Bottiaeu*

Mukanga Manyando

CRT

Cross sectional

Tanzania

Masanja

Observational

Tanzania

Zanzibar

Mubi

Shakely

Uganda

Nigeria

Uzochukwu

Batwala

Malawi

Chinkhumba

Burkina Faso

Bisoffi

RCT

Country

Author

Study design

97.8% 39.7% 86.9% 60.0% 90.5% 99.9% 88.5%

Clinicians and nurses Clinicians, nurses and CHW Clinicians and nurses Clinicians and nurses Clinical officers and nurses

93.4%

95.9%

60.7%

CHW Clinicians

Clinicians

Clinicians

Nurses

Health personnel Appropriate cadre treatment

Table 2: Appropriate treatment overall, RDT positive and RDT negative results.

6

Negatives not treated 19.0% 96.0% 92.8% 95.2% 31.4% 57.9% 25.9% 86.5% 99.9% 76.6%

Positives Treated 97.7% 95.8% 95.1% 98.6% 93.9% 98.0% 100.0% 100.0% 100.0% 100.0%

Clinicians

Cameroon

b

Bastiaens

Mbonye

Mbacham

Tanzania

Uganda

Clinicians

Cameroon

a

Mbacham

CHW

Uganda

Mukanga**

Clinical officers

DSV

CHW

Ghana

Mukanga**

90.4%

98.8%

70.8%

56.1%

99.0%

99.5%

Health personnel Appropriate cadre treatment

Country

Author

100.0%

99.0%

72.9%

72.1%

99.9%

100.0%

Positives Treated

90.0%

98.5

69.4

48.1%

92.4%

96.7%

Negatives not treated

CHW = Community health worker; DSV = Drug shop vendors; * excludes missing data; ** excludes Burkina Faso results. a = basic training; b = enhanced training

Before and after

Study design

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6

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Figure 5. (previous page) Pooled meta-analysis of RDT positive results appropriately prescribed AMDs stratified by HW cadre

Pooled meta-analysis using random effects for RDT negative patients not prescribed AMDs (Fig. 6) was 78 % (95 % CI 66–89 %); I2 = 99.8 %, Z = 14.60, p < 0.001. The proportion of RDT negative patients appropriately not prescribed and AMD was between 19.0–99.9 % (Table 2). Five studies reported less than 60 % compliance to RDT negative results. 470, 484,

489-491

Figure 6. (next page) RDT negative results not prescribed AMD stratified by HW

Community health workers (CHWs) had the highest adherence to negative results (Fig. 6) with a random effects pooled proportion of 95 % (95 % CI 92–98 %); I2 = 86.4 %, Z = 23.26, p < 0.001 than clinicians with a pooled proportion of 75 % (95 % CI 58–89 %); I2 = 99.8 %, Z = 11.30, p < 0.001. There were no differences in compliance when stratified by patient age or transmission setting in pooled meta-analyses.

Compliance factors Six out of the 14 studies included463, 471, 483, 484, 489, 491 reported quantitative or qualitative assessment of factors associated with compliance to RDT. Uzochokwu et al.489 reported that HWs adhered to RDT positive results, as they believed they were more reliable in confirming a malaria diagnosis than presumptive diagnosis or microscopy. Bisoffi et al. 484 compared prescribing behaviour of HWs in the dry compared to rainy seasons and reported improved RDT negative results compliance during the dry season; alternative diagnoses were also made in the dry than the rainy season. Manyando et al.491 reported no association between prescribing of AMD to negative RDTs in children under five, and fever in a Zambian study. There was also no association between community health worker (CHW) or socio-demographic characteristics and classification of malaria based on RDT in a bivariate analysis in Uganda.487 In one study though, 70 % (14/20) of the respondents (HW) believed that RDTs gave inaccurate/false negative results for malaria.471 Persistence of symptoms and patient pressure and demand were other factors reported to contribute to inappropriate AMD prescription in RDT negative cases. 463, 471, 494 HWs would end up prescribing AMDs in these cases to satisfy patients and maintain their reputation. Some HW reported that RDT negative patients improved when they were prescribed AMDs.

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Discussion This is the first systematic review and meta-analysis evaluating the proportion of health workers’ compliance with RDT results. Overall the compliance is fair. However, it also confirms that compliance to RDT negative results compared to positive results was generally low among HWs. Diagnostic accuracy of RDT for both falciparum and non-falciparum malaria is high;37, 469 sensitivity of up to 99.5 % and specificity of up to 90.6 % compared to microscopy for P. falciparum.37 Community health workers can appropriately diagnose and treat malaria using RDT in resource limited settings.468 The use of RDT to guide treatment reduces AMD prescription especially where health workers adhere to results.36 The results show a high proportion of HWs prescribe appropriate treatment based on RDT results. A proportion of patients still remain over- or under-treated, despite policy change of administering ACT to parasitological confirmed cases only. Approximately 17 % of RDT negative patients are inappropriately prescribed AMDs. This estimate, extrapolated to subSaharan Africa means hundreds of thousands of patients are inappropriately diagnosed for malaria and prescribed AMD drugs unnecessarily; unnecessary (=incorrect) AMD prescription leads to drug wastage, unnecessary exposure to drug adverse effects and an increased risk of drug resistance development for current AMDs.495 Where underlying infection is not treated, the patient’s illness prolongs and worsens; the patient or guardian makes multiple visits to seek health services, lose productivity time or income and leads to school absenteeism for school-going children474 leading to a vicious cycle of poverty and malaria.52 Lower cadres of HW showed more compliance to RDT results than trained HWs. The high adherence is likely due to trust in RDT result for confirming malaria diagnosis. Trained HWs on the other hand may trust clinical symptoms and past experience more than RDT result.471, 496

Factors associated with HW compliance from qualitative studies include knowledge of alternative diagnosis, fever during the dry season and a trust in RDT result.483, 484, 497 Trust may be increased by improving diagnostic capacity for other common febrile illnesses, and by developing evidence informed guidelines for treatment of symptomatic RDT negative patients. Such guidelines may not apply in non-endemic areas and therefore should be specific to particular settings. Knowledge of alternative diagnosis is related to the level of training and experience of HW.498 HWs reported they likely made alternative diagnosis during the dry season when malaria transmission is perceived lower in febrile children with negative RDT result compared to the wet season when transmission peaks. For febrile patients, alternative

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diagnoses were made during the dry season while more patients were treated for malaria during the wet season in one study.484 Qualitative studies report pressure on prescribers to satisfy patient expectations as one factor, which contributes to non-compliance of RDT negative results.52, 499 Chandler et al.500 reported patient psychology and prescriber reputation as other factors influencing noncompliance to of HWs to negative RDT. Interventions to improve compliance have not been successful, although they led to a decrease in ACT prescriptions in particular. Some HWs prescribed a non-recommended AMD in malaria negative patients.470, 501 In cases of patients demanding AMDs, community sensitisation on RDTs was reported to improve patient satisfaction.463 At facility level, involvement of patient in discussing malaria results also improved patient satisfaction and reduced patient demand for AMDs.502 Notably, few studies were available which quantified HW’s compliance to malaria RDT results, and even less studies investigated the factors contributing to compliance. Understanding these factors can help design effective strategies to improve compliance of anti-malarial drugs. Chandler et al.500 describe a systematic method of designing an intervention in Tanzania; formative research would be key in designing such an intervention. However, interventions are context-specific and may not be applicable to all settings, and for all HWs. It is essential to investigate factors contributing to non-compliance in specific cadres and settings, exploring impact in a context specific manner before designing and implementing interventions. Although ideal for rural areas in Africa, RDT kits inherently are not 100 % sensitive and specific.36, 37 Clinically diagnosed malaria and positive malaria test may be due to other underlying causes of the fever.480 Crump et al.503 reported only 1.6 % of 820 patients with fever or history of fever actually had malaria infection in a Tanzanian prospective cohort study; bacterial and fungal bloodstream infections were responsible for 9.8 and 2.9 % of the fever, respectively. Resource limited settings lack diagnostic equipment and capacity for some diseases. Diagnostic accuracy of RDTs can be affected further by low and extremely high parasite densities,504, 505 patient-intrinsic factors such as rheumatoid factor positivity,506 user factors such as result interpretation and performance of the test, and environmental storage conditions including high temperatures. It is, therefore, possible, though infrequent, for malaria-infected patients to have a false positive (leading to not-indicated treatment) or more importantly, false negative result, and hence miss malaria treatment if WHO malaria treatment guidelines are followed. A more robust and highly specific test may be useful to rule out malaria. False positives, where malaria parasitaemia is not the cause of the illness (in endemic areas) lead to neglecting of other febrile illnesses. Multidisciplinary research to explore, measure and design interventions for increasing compliance to RDT results in different settings in Africa need to be conducted. More

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innovation in diagnosis of common febrile illnesses in malaria endemic regions needs to be available. There is sparse data on prevalence of other non-malaria febrile illnesses in most malaria endemic regions of Africa. The meta-analysis may have overestimated compliance: studies evaluating diagnostic tests generally report higher compliance when assessed in the study setting compared to a nonstudy setting. Most studies reported higher compliance to positive results compared to negative results. A limitation for the results in the review is that risk of bias and publication bias were not assessed for the studies included; the quality of evidence therefore cannot be reported.

Conclusion HWs compliance to RDT is fair; compliance to positive RDT results is generally higher compared to negative RDT results. Over-treatment of malaria is still a major problem in subSaharan Africa. Both HW and patient factors contribute to inappropriate prescribing of AMDs to RDT negative patients; interventions to improve compliance should target both patients and HWs. Treatment guidelines should be developed for other causes of fever informed by local context and research. Multidisciplinary research will improve compliance of HWs to RDT results.

Acknowledgements This study was supported by the Dioraphte Foundation, The Netherlands. We thank the reviewer for his/her thorough and excellent review.

Electronic supplementary material Additional file 1. Search strategy. Additional file 2. Data extraction form. Additional file 3. Formulae for appropriate treatment.

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Part II: The quality of anti-malarial drugs

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“Calcium carbonate is a compound (actually a salt), with the formula CaCO3. It is a common substance found as rock in all parts of the world and is the main component of shells of marine organisms, snails and eggshells. Calcium carbonate is the active ingredient in agricultural lime and is usually the principal cause of hard water. Calcium carbonate is quite common in soil, particularly in drier areas where it may occur in different forms. Low levels of calcium carbonate enhance soil structure and are generally beneficial for crop production but at higher concentrations they may induce iron deficiency and, when cemented, limit the water storage capacity of soils.” Adapted from: Soil Atlas of Africa, 2013. European Commission, Publications Office of the European Union, Luxembourg.1

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Chapter 7 Assessing the quality of anti-malarial drugs from Gabonese pharmacies using the MiniLab®: a field study Benjamin J. Visser Janneke Meerveld-Gerrits Danielle Kroon Judith Mougoula Rieke Vingerling Emmanuel B. Bache Jimmy Boersma Michèle van Vugt Seldidji T. Agnandji Harparkash Kaur Martin P. Grobusch Malaria Journal 2015 Jul 15;14:273 Appendices and supplementary material are available online at: https://malariajournal.biomedcentral.com/articles/10.1186/s12936-015-0795-z

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Abstract Background Recent studies alluded to the alarming scale of poor anti-malarial drug quality in malariaendemic countries, but also illustrated the major geographical gaps in data on anti-malarial drug quality from endemic countries. Data are particularly scarce from Central Africa, although it carries the highest burden of malaria. The aim of this medicine quality field survey was to determine the prevalence of poor-quality anti-malarial drugs in Gabon.

Methods A field survey of the quality of anti-malarial drugs in Gabonese pharmacies was conducted using the Global Pharma Health Fund Minilab® tests, following the Medicine Quality Assessment Reporting Guidelines. Anti-malarial drugs were purchased randomly from selected pharmacies in Gabon. Semi-quantitative thin-layer chromatography (TLC) and disintegration testing were carried out to measure the concentration of active pharmaceutical ingredients (APIs). The samples failing the TLC test were analysed by high-performance liquid chromatography. Following the collection of anti-malarial drugs, a street survey was conducted to understand where people purchase their anti-malarial drugs.

Results A total of 432 samples were purchased from 41 pharmacies in 11 cities/towns in Gabon. The prevalence of poor-quality anti-malarial drugs was 0.5% (95% CI 0.08–1.84%). Two out of 432 samples failed the MiniLab® semi-quantitative TLC test, of which a suspected artemether-lumefantrine (AL) sample was classified as falsified and one sulfadoxinepyrimethamine (SP) sample as substandard. High performance liquid chromatography with ultraviolet photo diode array detection analysis confirmed the absence of APIs in the AL sample, and showed that the SP sample did contain the stated APIs but the amount was half the stated dose. Of the people interviewed, 92% (187/203) purchased their anti-malarial drugs at a pharmacy.

Conclusion Using the GPHF Minilab®, the prevalence of poor-quality anti-malarial drugs is far lower than anticipated. The findings emphasize the need for randomized and robust sampling methods in order to collect representative data on anti-malarial drug quality.

Trial registration NTR4341 (Dutch Trial Registry)

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Background Plasmodium falciparum malaria is estimated to cause 528,000 deaths and 163 million clinical episodes in Africa.2 Early diagnosis and treatment with appropriate anti-malarial drugs can prevent severe illness and lethal outcome.10, 106, 420 Therefore, it is crucial that the administered anti-malarial drugs are of acceptable quality.507 In Gabon, the majority of antimalarial drugs are purchased directly by the patient or caretaker from the pharmacy (licensed and unlicensed) for self- or home treatment. There is no anti-fake medicine programme, nor an effective drug regulatory system in Gabon (Additional file 1). Gabon does not receive international donor support for anti-malarial medicines. The national malaria control programme of Gabon does not provide anti-malarials for free. Whether quality assured or falsified, anti-malarial drugs have not been reported from the Gabonese markets as from the neighbouring countries. The spread of poor-quality (e.g., counterfeit or falsified) antimalarial drugs may pose an obstacle to effective malaria control. 43, 508 Poor-quality antimalarial drugs have serious consequences for public health.507 Drugs with too little, or devoid of active pharmaceutical ingredients (APIs) may cause increased morbidity and mortality.509 Also, low concentrations of APIs in poor-quality drugs will result in subtherapeutic concentrations of the drug in vivo, which may contribute to the selection of resistant parasites.44 Furthermore, the use of poor-quality anti-malarial drugs leads to financial loss for patients and their families, healthcare systems and pharmaceutical companies producing the genuine product.510 The general public can lose confidence in a pharmaceutical brand, drugs, pharmacies, and healthcare providers.511

A systematic review in 2014 illustrated the alarming scale of poor anti-malarial drug quality in malaria-endemic countries, but also showed major geographical gaps, with no published information on the quality of anti-malarial drugs from 60.6% (63/104) of the malariaendemic countries).45 Using the Worldwide Antimalarial Resistance Network (WWARN) database,512 it was demonstrated that out of 9,348 anti-malarial drugs collected (compiled from 130 publications in total), 30.1% (2,813) failed chemical/packaging quality tests with 39.3% classified as falsified; 2.3% as sub-standard and 58.3% as poor-quality, without evidence available to categorize them as either sub-standard or falsified.45 There are few reports originating from Central Africa. Also for Gabon, systematic data on the geography and epidemiology of poor-quality anti-malarial drugs is scarce. Gabon is a high-endemicity country for malaria.513-515 A study in 2011, assessing the quality of chloroquine tablets in 12 African countries collected two chloroquine samples from the capital of Gabon (Libreville), which were both of good quality.516 The World Health Organization (WHO) investigation in 2003 collected 25 chloroquine samples (29% poor-quality) and ten sulfadoxinepyrimethamine samples (100% good quality) from pharmacies in Libreville. A limited number of reports are available from neighbouring countries Cameroon,507, 516-521 Equatorial Guinea522 and the Republic of Congo .507

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The aim of this study was to determine the prevalence of poor-quality anti-malarial drugs in Gabon, which lacks an effective national product quality monitoring programme (see Additional file 1). Information about the quality of anti-malarial drugs is important for improving malaria treatment and to successfully run malaria control programmes.523, 524

Methods Registration and reporting This medicine quality field survey was registered in advance (30 Dec 2013) in The Netherlands Trial Registry (NTR): NTR4341.525 This report follows, where appropriate, the Medicine Quality Assessment Reporting Guidelines (MEDQUARG).526, 527 Also, the costs of this study are reported (Additional file 2).402

Scientific research and ethical committee statement Scientific clearance was obtained from the Scientific Review Committee (SRC) of the Centre de Recherches de Médicales de Lambaréné (CERMEL), Albert Schweitzer Hospital (SRC number: 2013.11; Additional file 3). The Ethical Committee of CERMEL decided that ethical approval of this study was not required as this study is a quality assurance in healthcare study, no humans having been subjected to it.528

Study area Gabon (an upper-middle income country, GDP $19.34 billion, 2013) straddles the Equator. About 80% of its 267,667 km2 area is covered by dense tropical rainforest. The population of Gabon is estimated to be around 1.6 million inhabitants (6.3 inhabitants/km2), 86.2% of whom live in urban areas. CERMEL is based in Lambaréné, the capital of the MoyenOgooué Province, a semi-urban town of about 30,000 inhabitants surrounded by villages. Gabon is administratively divided into nine provinces, with villages mainly located along roads and rivers. Gabon is a highly malaria-endemic country. The official first-line treatment for uncomplicated falciparum malaria is artesunate + amodiaquine (AS + AQ) or artemetherlumefantrine (AL) and severe falciparum malaria is treated with intravenous quinine. 49 Intramuscular use of artemether or intravascular artesunate is not common in Gabon.

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Primary and secondary outcomes The primary outcome was the proportion (percentage) of poor-quality anti-malarial drugs in pharmacies in Gabon. Secondary outcomes were the proportion of outlets selling poorquality anti-malarial drugs and availability of anti-malarial drugs that are no longer recommended as first- or second-line treatment in Gabon or by WHO. The following secondary outcome was added during the study to assess the external validity of the field survey: to determine where people purchased their anti-malarial drugs.

Definitions The overarching term ‘poor-quality drugs’ is used to describe the different categories: falsified medicines are fake medicines that are designed to mimic real medicines; counterfeit medicines are medicines that do not comply with intellectual-property rights or that infringe trademark law.

Timing and location of the survey The field survey was conducted in January 2014 in Gabon. The six (out of nine) most populated provinces (ISO 3166-2:GA) were selected: Estuaire; Haut-Ogooué, MoyenOgooué, Ngounié, Ogooué-Maritime, and Woleu-Ntem. Selected locations were: Libreville (capital), Franceville, Lambaréné, Mouila, Port-Gentil, Oyem, Bitam, Owendo, Fougamou, Makouke, Bifoun, Gamba, and Lopé (Figure 1, next page).

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Figure 1. Map of sampling sites in Gabon (Source: Google Maps).

Sampling design and sample size There are approximately 75 open and fully functioning pharmacies in Gabon (2013).529 Pharmacies were randomly selected. The randomization procedure was performed by BJV using statistical software (nQuery Advisor® Version 7.0. Statistical Solutions, Cork, Ireland) on the day before the actual sampling. A full list provided by the Health Authorities of Gabon and a list from the National Health Assurance Company (La Caisse Nationale d’Assurance Maladie et de Garantie Sociale du Gabon,530 CNAGMS) with registered/licensed pharmacies and dispensaries was prepared (before sampling) to allow for proper randomization procedures. This list was accomplished with (unlisted) pharmacies by local nurses and fieldworkers. In total, six pharmacies were found which were missing on the CNAGMS list of pharmacies in Gabon. In Lambaréné (Moyen-Ogooué Province), where

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the CERMEL is based, all known pharmacies (n = 7) were sampled during the first week of the sampling period and thus not randomly selected. In all other areas, approximately 50% of the pharmacies were randomly selected in all neighbourhoods. Hospitals (except for the Albert Schweitzer Hospital), markets, grocery shops, and street peddlers were not visited because regulations for drug selling are in place; previsits by local nurses yielded little evidence (if any) of anti-malarial drugs sold there. The appropriate sample size and strategy is challenging, since data of the prevalence of poor-quality anti-malarial drugs are very scarce for Gabon. Thus, the most conservative sample size is given by using an unknown prevalence [hypothesized 50% frequency of outcome factor in the population (p)]. To determine the actual prevalence of poor-quality drugs available in Gabon with a precision of 5% with 95% confidence intervals (z = 1.96), a random sample of 384 was needed. The following equation was used: sample size The sample size was calculated with OpenEpi (Open Source Epidemiologic Statistics for Public Health) version 3.03.531

Sampling procedure A Gabonese ‘mystery shopper’ (JM, nurse, of Gabonese nationality) conducted the actual sampling process in Moyen-Ogooué (177/432 samples) and was trained utilizing standard sampling guidelines. She dressed according to regular Gabonese standards and gave no indication that she was not a regular shopper. A standard scenario was used: she asked which anti-malarial drugs were for sale. Subsequently, she purchased one full child/adult treatment in their original packaging of each of the available anti-malarials and of each available brand, but not of each available batch. Samples included drugs sold in the manufacturer’s original packaging as well as those distributed loose, often in plastic bags. Surprisingly, sellers never asked questions. Although the US Pharmacopeia (USP) recommends 30 dosage units532 for a single tablet of the same lot number from each location, this was not deemed practically feasible in the Gabonese setting, and also too expensive. For logistical reasons the other 255/432 (60%) samples were collected by the investigators. Anti-malarial drugs purchased included: AL, AS + AQ, AS + sulfadoxine, AS-mefloquine, dihydroartemisininpiperaquine, dihydroartemisinin-piperaquine-trimethoprim, artemisinin-piperaquine, artemisinin-naphtoquine, quinine, sulfadoxine-pyrimethamine (SP), mefloquine, proguanil, atovaquone-proguanil, proguanil-chloroquine, pyrimethamine and chloroquine. Other antimalarial drugs were not purchased. Only solid dosage forms were collected (no liquid formulations). To avoid potential bias in subsequent sampling rounds, the exact reason for sampling medicines was not shared with the seller. Results were not reported back to the seller. For every sample collected, the collector completed and signed the sample collection form (including GPS locations) (Additional file 4) as soon as possible after leaving the point of sale and before performing the next purchase. Once purchased, all drugs were stored until testing at room temperature (in an air-conditioned room) with no sunlight. Humidity could

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not be controlled. Tests were completed at the laboratory of the Academic Medical Centre (AMC, The Netherlands) within 3 months of sample collection.

Questionnaire To determine where people purchased their anti-malarial drugs, a questionnaire was presented to food market dwellers in Lambaréné as well as at PK (‘point kilomètre’) 8 Le Marché Bananes (a transportation hub) and Marché du Mont-Bouët in Libreville. The survey was conducted after the purchase of the anti-malarial drugs. The most important question in this survey was: “where do you buy anti-malarial drugs?”.

Storage and shipment of samples Before shipment by air, samples were stored at CERMEL under appropriate storage conditions. The samples arrived within 36 h at AMC in Amsterdam, The Netherlands. Samples were protected by appropriate packaging (primary container and additional packaging) during shipment by air.

Chemical and packaging analysis Samples were analysed at the Internal Medicine Research Laboratory of the AMC between February 2014 and April 2014 by BJV and JMG. The chemical analysis was performed unblinded to packaging. The Global Pharma Health Fund (GPHF) Minilab® (Merck Darmstadt, Germany) was used to run semi-quantitative thin-layer chromatography (TLC) and disintegration tests on each sample to determine the presence and relative concentration of APIs.533 Expired drugs were also tested. The MiniLab® protocols award products a ‘pass’ for TLC if 80% or more of the labelled active ingredient(s) is present. For fixed-dose combinations (e.g., AL) and SP, ‘pass’ was awarded only if both active ingredients met this standard. TLC is an accepted method to assess the quality of drugs.534, 535 The MiniLab protocols have been reviewed by the Promoting the Quality of Medicines (PQM) programme operated by the USP Convention. Each sample/test was run twice on separate days (once by BJV, once by JMG), with the assumption that the result most consistent with the reference was recorded. Thus, every API in every sample was tested twice. Standard operating procedures (SOP) provided with the Minilab were used.533 Quality control of the GPHF MiniLab® was performed daily before the drug testing and consisted of performing TLC on Minilab-reference samples for the anti-malarial drug analysed. In addition, Minilab reagents were quality control tested, using reference samples when a new lot was introduced. Samples were also tested to see if they disintegrated in purified water, following the guidelines of the European Pharmacopeia (EP).536 For this, apparatus A as described in the EP 2.9.1. was used

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at the laboratory of the Pharmaceutical Technology and Biopharmacy Department of Utrecht University (Utrecht, The Netherlands). Since the disintegration test requires six tablets per test, not all samples could be analysed. To include as many samples as possible, it was decided to test the samples per batch number instead of per sample. All samples with the same batch (or LOT) number, were expected to be homogeneous.537 Samples failing TLC were analysed using the high-performance liquid chromatography with ultraviolet photodiode array detection (HPLC–UV-PDA February 2015 in a reference laboratory at the London School of Hygiene and Tropical Medicine)538 to quantify the amount of APIs present in each sample. This was compared with the stated dose on the packaging and the spectra achieved using a quality assured sample. For artemisinin derivatives, the artemisinin derivative screening test (ADST) was conducted according to an earlier published method.535 Not all samples were analysed by HPLC–UV-PDA (the gold standard) due to lack of funding.

The packaging analysis was developed in line with previously published research.507, 526 For the packaging analysis, genuine samples were requested by email from the manufacturers using a standard letter; with two reminders sent 2 and 4 weeks after the first email. Unfortunately no genuine anti-malarial samples were received.

Statistical analysis Descriptive statistics were performed using SPSS 20.0 statistical package (SPSS Inc., Chicago, MI, USA). The confidence interval of the prevalence estimate was calculated using the Wilson procedure with a correction for continuity. Fisher’s exact test was used to calculate the difference between the number of poor-quality drugs of the Gabonese mystery shopper versus two European researchers. Inter-observer reliability (for chemical analysis) was calculated using the Kappa (κ) statistic.539

Sharing data with Medicine Regulatory Agency The results of this field survey were shared with the Medicine Regulatory Agency (MRA) in Gabon, the Director of Health of the Province Moyen-Ogooué and Rapid Alert of the WHO.

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Results The samples purchased in this field survey were readily available over the counter without prescription in all pharmacies. In total, 432 full anti-malarial treatments were collected from 41 pharmacies and one hospital (Albert Schweitzer Hospital, Lambaréné) in 11 cities/towns in Gabon (Table 1; Figure 1). From the collected data, 55% (41/75) of pharmacies in Gabon were surveyed. The ‘class’ of pharmacy and licensing status (e.g., public, private for profit, private not for profit, informal) was not determined and the drug sellers were not interviewed. Of the 432 collected samples, 338 (78%) were artemisinin-based combination therapy (ACT). AS + AQ, the national recommended first-line treatment for falciparum malaria, comprised 10% of the total samples (n = 42). The second-line anti-malarial drug combination is AL and the third-line drug combination is dihydroartemisinin-piperaquine. An ACT was available in every surveyed pharmacy, but AS-AQ was only available in 27 pharmacies (65%). On average, ten full anti-malarial treatments were collected per pharmacy (min–max: 3–20). Two samples were expired at the day of purchase, both collected in a pharmacy in Lambaréné. They were not classified as sub-standard. No chloroquine or artesunate monotherapy was for sale in all the surveyed pharmacies. .

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Other Quinine Sulphadoxine-pyrimethamine Mefloquine Atovaquone-proguanil

ACT Artemether-lumefantrine Artesunate-amodiaquine Artesunate Dihydroartemisinin-piperaquine Artesunate-mefloquine Dihydroartemisinin-piperaquinetrimethoprim Dihydroartemisinin-SP Artemisinin-piperaquine Artemisinin-naphtoquine

43 (10%) 40 (9%) 4 (