How blood group O protects against malaria

18 05 2015

It has long been known that people with blood type O are protected from dying of severe malaria. In a study published in Nature Medicine, a team of Scandinavian scientists explains the mechanisms behind the protection that blood type O provides, and suggest that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.

Anopheles albimanus mosquito. Credit: James Gathany (Wikimedia Commons).

Malaria is a serious disease that is estimated by the WHO to infect 200 million people a year, 600,000 of whom, primarily children under five, fatally. Malaria, which is most endemic in sub-Saharan Africa, is caused by different kinds of parasites from the plasmodium family, and effectively all cases of severe or fatal malaria come from the species known as Plasmodium falciparum. In severe cases of the disease, the infected red blood cells adhere excessively in the microvasculature and block the blood flow, causing oxygen deficiency and tissue damage that can lead to coma, brain damage and, eventually death. Scientists have therefore been keen to learn more about how this species of parasite makes the infected red blood cells so sticky.

It has long been known that people with blood type O are protected against severe malaria, while those with other types, such as A, often fall into a coma and die. Unpacking the mechanisms behind this has been one of the main goals of malaria research.

A team of scientists led from Karolinska Institutet in Sweden have now identified a new and important piece of the puzzle by describing the key part played by the RIFIN protein. Using data from different kinds of experiment on cell cultures and animals, they show how the Plasmodium falciparum parasite secretes RIFIN, and how the protein makes its way to the surface of the blood cell, where it acts like glue. The team also demonstrates how it bonds strongly with the surface of type A blood cells, but only weakly to type O.


Conceptually simple

Principal investigator Mats Wahlgren, a Professor at Karolinska Institutet’s Department of Microbiology, Tumour and Cell Biology, describes the finding as “conceptually simple”. However, since RIFIN is found in many different variants, it has taken the research team a lot of time to isolate exactly which variant is responsible for this mechanism.

“Our study ties together previous findings”, said Professor Wahlgren. “We can explain the mechanism behind the protection that blood group O provides against severe malaria, which can, in turn, explain why the blood type is so common in the areas where malaria is common. In Nigeria, for instance, more than half of the population belongs to blood group O, which protects against malaria.”

The study was financed by grants from the Swedish Foundation for Strategic Research, the EU, the Swedish Research Council, the Torsten and Ragnar Söderberg Foundation, the Royal Swedish Academy of Sciences, and Karolinska Institutet. Except Karolinska Institutet, co-authors of the study are affiliated to Stockholm University, Lund University, Karolinska University Hospital, and the national research facility SciLifeLab in Sweden, and to the University of Copenhagen in Denmark and University of Helsinki in Finland. Mats Wahlgren is a shareholder and board member of drug company Dilaforette AB, which is working on an anti-malaria drug. The company was founded with support from Karolinska Development AB, which helps innovators with patent-protected discoveries reach the commercial market.



RIFINs are Adhesins Implicated in Severe Plasmodium falciparum Malaria

Suchi Goel, Mia Palmkvist, Kirsten Moll, Nicolas Joannin, Patricia Lara, Reetesh Akhouri, Nasim Moradi, Karin Öjemalm, Mattias Westman, Davide Angeletti, Hanna Kjellin, Janne Lehtiö, Ola Blixt, Lars Ideström, Carl G Gahmberg, Jill R Storry, Annika K. Hult, Martin L. Olsson, Gunnar von Heijne, IngMarie Nilsson and Mats Wahlgren

Nature Medicine, AOP 9 March 2015, doi: 10.1038/nm.3812
 [en línea] Solna (SUE):, 18 de mayo de 2015 [ref. 10 de marzo de 2015] Disponible en Internet:

Technique for early and rapid malaria diagnosis

29 09 2014

Low-cost field detection system can detect malaria infection within minutes with just a drop of blood


Red blood cells from a patient infected with Plasmodium falciparum.
Credit: Osaro Erhabor

 A team of Singapore scientists have invented a new technique to detect malaria within minutes and all that is required is a drop of blood.

Malaria is a mosquito-borne parasite which affects over 60 million people worldwide and could be fatal in serious cases. It is still a huge problem in developing countries because there is no vaccine for malaria while antimalarial drugs are losing their efficacy with increasing drug resistance on the rise.


The research entitled ‘Micromagnetic Resonance Relaxometry for rapid label-free malaria diagnosis’ was published online on 31 Aug 2014 in the prestigious scientific journal Nature Medicine. This innovative technique is developed by the Singapore-MIT Alliance for Research and Technology (SMART) [新加坡-麻省理工学院科研中心] in collaboration with Nanyang Technological University (NTU).

With this disruptive new technology, hospitals may soon have the ability to rapidly screen and monitor hundreds of patients at the point-of-care for malaria, at much lower cost per patient.

Despite technological advances, currently malaria infection is still detected via stained blood smear microscopy. Lab technician will need to spot the tiny parasitized red blood cells among millions of healthy uninfected red blood cells, especially in the case of early infection, which is like finding a needle in a haystack. It is not only time-consuming and labour-intensive but also often not conclusive as it is highly dependent on the subjective judgement of the microscopist. Therefore, a false-positive call is not unusual.

Other malaria diagnostic techniques such as the Polymerase Chain Reaction (PCR) also have limitations as it is not field-deployable and can only provide semi-quantitative analysis.


SMART new technique

The solution developed by the SMART team since 2010, works by detecting the biomarker hemozoin crystallites, the metabolic waste product of malaria parasites during the intra-erythrocytic* cycle As the technique uses miniaturized Magnetic Resonance Relaxometry (MRR) system, a cousin of Magnetic Resonance Imaging (MRI), it is also more sensitive, accurate and faster than traditional methods.

The technique detects malaria infections at a very early stage, even when the amount of parasites in the blood is extremely low. It was successfully proven in mouse studies, where the presence of malaria parasites was detected at the very next day of infection. Moving forward the team is currently working on human study at clinical settings.

At the onset of malaria, the malaria parasites “eat up” large amount of haemoglobin^ and converts them into hemozoin crystallites. These crystallites are basically oxidized iron nanoparticles (Fe3+), making them way more “magnetic” than the healthy red blood cells, which can be easily picked up by the miniaturized MRR developed by SMART.


Professor Han Jongyoon, Principal Investigator from SMART’s BioSystems and Micromechanics (BioSyM) Interdisciplinary Research Group (IRG), said: “This system is more reliable and allows for rapid screening to be conducted. So, given the flux of people moving in and out of developed nations especially, this system has the potential to help prevent mass import of malaria by infected persons. For developing nations, this system, which does not require refrigeration or other extensive infrastructure, is portable enough to be deployed in rural areas, to help rapidly screen for malaria and hence stem the spread of this infectious disease.”

Professor Peter Preiser, SMART co-Investigator and Chair of NTU’s School of Biological Sciences said that the new test has the additional potential to rapidly detect parasites that are resistant to anti-malarial drugs particularly artemisinin thereby providing a valuable tool in trying to prevent the global spread of these resistant parasites.

“Importantly, rapid and accurate diagnosis will reduce the prescription of drugs to non-infected people – one factor that contributes to why we are seeing more malaria parasites developing resistance to anti-malarial drugs,” said Prof Preiser, a renowned expert in malaria.

“With a more accurate and sensitive detection system like the one we developed, doctors can better diagnose malaria infections in patients. We need to ensure that drug resistance is kept to the minimum because these drugs are really our last line of defence in helping malaria-stricken patients.”


SMART Research Scientist Dr Brian Peng Weng Kung and lead author of the paper, added: “The significant part of this research lies in the fact that this system is practically a “mini MRI” system that is much cheaper to produce than the million-dollar MRI machines used by hospitals. We built tiny radiofrequency (rf) coil which is used to apply rf-pulses and receive signal from a drop of blood, and the whole detection process happens in a few minutes. Furthermore, since this technique does not rely on immuno-assay# labelling that requires expensive chemical reagents, we are able to bring down the screening test cost to less than S$0.10 per test.”

SMART is now spinning off a company to commercialise this technology, which could also work for other types of blood disorders. Moving forward, the research team is also setting up field-tests in the South-East Asia region. They are testing if the system can be run on solar power, which will be useful in rural areas, where electricity is scarce. This research is funded by the Singapore National Research Foundation, Prime Minister’s Office, Singapore under its Campus for Research Excellence And Technological Enterprise (CREATE) programme.

*Intra-erythrocytic: Occurring within the red blood cells
^Haemoglobin: A protein in red blood cells that carries oxygen
#Immunoassay: A test that uses antibody and antigen complexes as a means of generating a measurable result [en línea] Memphis, TN (USA):, 29 de septiembre de 2014 [ref. 02 de septiembre de 2014] Disponible en Internet:

La heparina muestra una doble actividad contra la malaria

31 07 2014

Una investigación publicada en Nanomedicine y llevada a cabo por científicos del IBEC, ISGlobal y la UB abre la puerta a mejorar el tratamiento de la malaria mediante heparina.


El estudio publicado en Nanomedicine explora si la heparina, que ha mostrado tener actividad contra la malaria y afinidad de unión específica para los glóbulos rojos infectados por Plasmodium falciparum frente a los glóbulos no infectados, puede mostrar ambas propiedades y unirlas en una estrategia de administración de fármacos contra la malaria. En este caso la heparina tendría un doble papel como antimalárico y como elemento de focalización de las nanopartículas cargadas con fármacos que actuarían al unirse a los glóbulos rojos infectados. Este estudio, llevado a cabo por investigadores del CRESIB, centro de investigación del Instituto de Bioingeniería de Catalunya (IBEC), de ISGlobal y de la Universitat de Barcelona, ha sido publicado en Nanomedicine.


La heparina adsorbida electrostáticamente sobre los liposomas con carga positiva y cargados con primaquina, medicamento antimalárico, fue capaz de triplicar la actividad en cultivos de P. falciparum del fármaco encapsulado. En concentraciones inferiores a las que inducen anticoagulación de la sangre de ratón in vivo, la actividad parasiticida resultó ser la suma de las actividades separadas de heparina libre como antimalárico y de heparina unida al liposoma como elemento vectorizador para la primaquina encapsulada.


Los investigadores observaron mediante imágenes de fluorescencia confocal y de microscopía electrónica que al cabo de 30 minutos de haber tratado glóbulos rojos infectados por Plasmodium con heparina, ésta había penetrado los parásitos intracelulares.


Xavier Fernández-Busquets, investigador IBEC e ISGlobal y coordinador del estudio, comenta que “estos resultados abren la puerta a mejorar el tratamiento con heparina contra la malaria debido a su actividad aditiva como fármaco y como elemento vectorizador específico de otros antimaláricos; sin embargo, será necesario realizar más investigación a nivel clínico para comprobar el papel de la heparina en pacientes infectados por Plasmodium”.


Referencia del artículo: Marques J, Moles E, Urbán P, Prohens R, Busquets MA, Sevrin C, Grandfils C, Fernàndez-Busquets X. (2014). “Application of heparin as a dual agent with antimalarial and liposome targeting activities towards Plasmodium-infected red blood cells.” Nanomedicine, epub ahead of print [en línea] Barcelona (ESP):, 28 de julio de 2014 [ref. 31 de julio de 2014] Disponible en Internet:

Crean el primer ‘bazo-en-un-chip’ humano funcional del mundo

24 03 2014

Investigadores del Instituto de Bioingeniería de Cataluña (IBEC) y del CRESIB, centro de investigación de ISGlobal han realizado un gran avance en el ámbito de la microingeniería de ‘órganos-en-un-chip’



 Los científicos de estos dos institutos han elaborado por primera vez un modelo funcional de bazo en 3D capaz de actuar como este órgano; filtrar los glóbulos rojos de la sangre. Lo han conseguido recreando a microescala las propiedades físicas y las fuerzas hidrodinámicas de la unidad funcional de la pulpa roja del bazo. Este dispositivo puede servir para detectar posibles fármacos contra la malaria y otras enfermedades hematológicas. Este estudio ha sido publicado en Lab on a Chip.


La idea original de crear un bazo-en-un-chip surgió de los grupos del Dr. Hernando A del Portillo, Profesor ICREA del CRESIB centro de investigación de ISGlobal, quien estudia hace varios años el papel del bazo en la malaria y del Dr. Josep Samitier, director del IBEC y catedrático de la Universidad de Barcelona, quien estudia las propiedades reológicas de la sangre, incluyendo aquella parasitada por malaria, para desarrollar sistemas de diagnóstico. ”Debido a las limitaciones éticas y tecnológicas de estudiar el bazo humano, conocido como la “caja negra” de la cavidad abdominal, ha habido muy pocos avances en su estudio”, explica del Portillo. Para romper esta barrera se inició una colaboración para desarrollar un modelo del bazo humano-en-un-chip mediante un proyecto EXPLORA.

“El sistema fluídico del bazo es muy complejo y adaptado evolutivamente para filtrar y destruir selectivamente glóbulos rojos viejos, micro-organismos y glóbulos rojos parasitados por malaria,” explica el Dr. Antoni Homs, investigador del IBEC y coautor del estudio. “El bazo filtra la sangre mediante un método único, haciéndola ‘microcircular’ a través de lechos de filtración formados por la pulpa roja del bazo en un compartimento especial donde el hematocrito (el porcentaje de células rojas de la sangre) se ve aumentado. De modo que los macrófagos especializados pueden reconocer y destruir glóbulos rojos enfermos.” Además, la sangre en este compartimento solo puede viajar en un único sentido a través de ranuras interendoteliales antes de llegar al sistema circulatorio, lo que representa un riguroso segundo test para asegurar la eliminación de las células viejas o enfermas.


Imagen de previsualización de YouTube

Los investigadores de estos dos centros, pertenecientes a la red de centros CERCA, han imitado estas dos condiciones de control en su plataforma de tamaño micro para simular la microcirculación de la sangre a través de dos canales principales (uno lento y uno rápido) diseñados para dividir el flujo. En el canal ‘lento’ la sangre fluye a través de una matriz de pilares simulando el ambiente real donde el hematocrito aumenta y la sangre “enferma” es destruida. El dispositivo ya se ha probado con glóbulos rojos humanos sanos y en infectados por malaria, trabajo realizado mayoritariamente por los investigadores predoctorales Luis G. Rigat-Brugarolas (IBEC) y Aleix Elizalde-Torrent (CRESIB/ISGlobal), coautores también de este trabajo. “Nuestro dispositivo facilitará el estudio de la función del bazo en malaria, e incluso podría proporcionar una plataforma flexible para la detección de posibles fármacos contra ésta y otras enfermedades hematológicas,” dice del Portillo.

“La investigación en órganos-en-un-chip integrando microfluídica con sistema celulares aún está dando sus primeros pasos, pero ofrece enormes perspectivas hacia el futuro de los ensayos de fármacos para diferentes patologías”, especifica Samitier. Estos dispositivos en 3D, que imitan las interrelaciones tejido-tejido y los microambientes únicamente vistos en los órganos vivos, permite una nueva percepción de las enfermedades que no puede obtenerse fácilmente con los estudios convencionales con animales, que son costosos y consumen mucho tiempo. Además, cede el paso a los resultados relacionados con humanos que los modelos animales no pueden predecir.


Artículo de referencia: Rigat Brugarolas, L. G., Elizalde Torrent, A., Bernabeu, M., de Niz, M., Martin Jaular, L., Fernandez Becerra, C., Homs Corbera, A., Samitier, J. & del Portillo, H. A. (2014). Functional microengineered model of the human splenon-on-a-chip. Lab Chip, epub ahead of print
 [en línea] Barcelona (ESP):, 24 de marzo de 2014 [ref. 07 de marzo de 2014] Disponible en Internet:

Finding malaria’s weak spot

17 03 2014

A ground-breaking imaging system to track malarial infection of blood cells in real time has been created by a collaboration catalysed by the University’s Physics of Medicine Initiative.

After over a decade of research into malaria, biologists Dr Teresa Tiffert and Dr Virgilio Lew at the Department of Physiology, Development and Neuroscience found their efforts to observe a key stage of the infection cycle severely hindered by the limits of available technology. An innovative collaboration with physicist Dr Pietro Cicuta at the Cavendish Laboratory and bio-imaging specialist Professor Clemens Kaminski in the Department of Chemical Engineering and Biotechnology is now yielding new insights into this devastating disease.

Under attack

Malaria is caused by parasites transmitted to humans through the bites of infected mosquitoes. According to the World Malaria Report 2011, there were about 216 million cases of malaria causing an estimated 655,000 deaths in 2010. Tiffert and Lew established their malaria laboratory in Cambridge in 1999 to investigate the most deadly form of the parasite, Plasmodium falciparum. Becoming increasingly resistant to available drugs, this species in particular is a growing public health concern.

Their current focus is a mysterious step in the life cycle of P. falciparum occurring inside the infected human’s bloodstream. The parasites, at this stage called merozoites, attach to and enter red blood cells (RBCs) to develop and multiply. After two days, the new merozoites are released and infect neighbouring RBCs. Over several days, this process amplifies the number of parasitised RBCs and causes severe and potentially lethal symptoms in humans.

“A huge amount of research has been devoted to understanding the RBC penetration process,” said Tiffert. “The focus of many vaccine efforts is the molecules on the surfaces of both parasite and red cell that are instrumental in recognition and penetration. Our collaboration with Clemens developed new imaging approaches to investigate what happens in the cells after invasion. But the pre-invasion stage, when a merozoite first contacts a cell targeted for invasion, remained a profound mystery. Our research indicates that this stage is absolutely critical in determining the proportion of cells that will be infected in an individual.”

For invasion to occur, the tip of the merozoite has to be aligned perpendicularly to the RBC membrane. Tiffert and Lew are focusing on how this alignment comes about, which has proved a formidable technical challenge. “The merozoites are only in the bloodstream for less than two minutes, where they are vulnerable to attack by the host’s immune system, before entering a RBC. To investigate what is going on we need to record lots of pre-invasion and penetration sequences at high speed, using high magnification and variable focusing in three dimensions. And the real challenge is to have the microscope on the right settings and to be recording at exactly the time when an infected cell has burst and released merozoites – something that is impossible to predict,” said Tiffert.

Techniques used by previous investigators have produced few useful recordings of this process occurring in culture, but from these an astonishing picture is emerging. “The contact of the merozoite with the RBC elicits vigorous shape changes in the cell, not seen in any other context,” said Lew. “It seems clear that this helps the merozoite orientate itself correctly for penetration, because all movement stops as soon as this happens. The parasite is somehow getting the RBC to help it invade.”

Imagen de previsualización de YouTube

A collaborative approach

Cicuta, a University Lecturer involved in the University’s Physics of Medicine Initiative – which is bringing together researchers working at the interface of physical sciences, life sciences and clinical sciences – met the trio by chance three years ago. He realised he could use his background in fundamental physics to pioneer a new approach to understanding malaria. “It’s been a gradual move for me to apply what I’ve learnt in physics to biology,” he said. “From the physics point of view, RBC membranes are a material. This material is very soft and undergoes deformations and fluctuations, and I was interested in understanding the mechanics involved during infection with malaria.”

Drawing on his expertise in the development of experimental techniques, Cicuta collaborated with Tiffert, Lew and Kaminski to pioneer a completely automated imaging system that pushes the boundaries of live cell imaging, enabling individual RBCs and merozoites to be observed throughout the process of infection. The research was funded by the Biotechnology and Biological Sciences Research Council and the Engineering and Physical Sciences Research Council.

“This microscope can not only run by itself for days, it can perform all the tasks that a human would otherwise be doing. It can refocus, it can find infected cells and zoom in, and when it detects a release of parasites it can change its imaging modality by going into a high frame-rate acquisition. And when the release has finished it can search around in the culture to find another cell to monitor automatically,” said Cicuta. “We also want to integrate a technique called an optical trap, which uses a laser beam to grab cells and move them around, so we can deliver the parasites to the cells ourselves and see how they invade.”

“So far, we’ve been able to gather over 50 videos of infections, which my PhD student Alex Crick has processed to show very clearly that the RBCs undergo large changes in shape when the merozoites touch them. We’ve also seen very strange shape changes just before the parasites come out of the cells, and we want to see whether this has a bearing on the parasites’ ability to infect subsequent cells.”

During the development of the microscope, the team discovered variability in the way the infected RBCs behave before they burst. “It’s important to know that there isn’t just one story. The only way to find this out is to look at many cells, which this system allows,” said Lew. “It’s a new level of data that allows us to get experimentally significant results, and better understand the diversity of the merozoites,” Cicuta added.

Used in conjunction with other tools such as fluorescent indicators and molecular biological tools, the new technology will allow Tiffert and Lew to test their hypotheses about the pre-invasion stage of the disease. They hope to determine the critical steps, which could provide clues as to how to stop an infection. “This microscope is an extraordinary new tool that has potential for use across a huge field of biological problems involving cellular interactions,” explained Lew.

“It may provide a route to designing effective antimalarial drugs, reducing invasive efficiency and decreasing mortality,” said Tiffert. “The automation we have achieved with this microscope will also be very important for future testing of malaria drugs and vaccines,” added Cicuta.


A visionary initiative

“The Physics of Medicine Initiative has been essential to our work,” said Cicuta. The University formally established the Initiative in December 2008 through the opening of a new purpose-built research facility adjacent to the Cavendish Laboratory, funded by the University and The Wolfson Foundation. The goal is to break down traditional barriers that have tended to limit interactions between researchers in the physical and biomedical sciences.

“I met my collaborators through a Physics of Medicine symposium, and the new building is the only place in the University where this type of research can be done,” added Cicuta. “It’s set up for safe handling of hazardous biological organisms like P. falciparum, and also has the facilities to design hardware for our advanced microscopes. This work is exciting because it’s interdisciplinary. By applying physics to the knowledge biologists have been developing for many years, we can make very fast progress.”

For more information, please contact Jacqueline Garget at the University of Cambridge Office of External Affairs and Communications [en línea] Cambridge (UK):, 17 de marzo de 2014 [ref. 06 de febrero de 2013] Disponible en Internet:’s-weak-spot

Un pediatra catalán, entre los diez jóvenes más sobresalientes del mundo 2012

12 11 2012

Barcelona, 30 oct (EFE).- El pediatra catalán Quique Bassat ha sido escogido como uno de los diez jóvenes más sobresalientes del mundo este año por la Joven Cámara Internacional en la categoría de innovación médica por su “extraordinario trabajo en pediatría y en investigación médica en los países en desarrollo”.


Un pediatra catalán, entre los diez jóvenes más sobresalientes del mundo 2012

Un pediatra catalán, entre los diez jóvenes más sobresalientes del mundo 2012

Barcelona, 30 oct (EFE).- El pediatra catalán Quique Bassat ha sido escogido como uno de los diez jóvenes más sobresalientes del mundo este año por la Joven Cámara Internacional en la categoría de innovación médica por su “extraordinario trabajo en pediatría y en investigación médica en los países en desarrollo”.

La Joven Cámara Internacional (JCI) es una organización internacional fundada en 1944 que participa en el sistema de las Naciones Unidas (ONU) y cada año selecciona a los jóvenes que más han destacado en el campo de la innovación e investigación para mejorar la calidad de vida de las personas.

Quique Bassat, que ha destacado por sus trabajos contra la malaria, recogerá su distinción el próximo 20 de noviembre en el Congreso Mundial de la JCI, que este año se celebrará en Taipei (Taiwán).

“Es muy importante que la JCI se fije en el trabajo que se hace en torno a las enfermedades olvidadas, especialmente en el campo de la malaria, ya que supone un empujón a la investigación médica en los países donde más se necesita”, ha declarado Quique Bassat al conocer la noticia.

“Además, este premio supone un reconocimiento a la investigación como motor de la innovación y del desarrollo de los países pobres”.

Doctor en medicina por la Universidad de Barcelona, Quique Bassat es un médico pediatra especializado en medicina tropical y en epidemiología.

Posee una amplia experiencia en la realización de ensayos clínicos en países en vías de desarrollo, entre los que se encuentra los ensayos de la primera vacuna candidata contra la malaria, la RTS,S.

En la actualidad trabaja como investigador en el CRESIB, el centro de investigación del Instituto de Salud Global de Barcelona (ISGlobal), donde coordina la implementación de varios proyectos relacionados con la malaria en países como Mozambique, Brasil, India o Papúa Nueva Guinea.

La JCI premia cada año a diez jóvenes menores de 40 años que, a través de su trabajo y de la innovación, desarrollan cambios positivos que contribuyen a crear un mundo mejor.

Además del doctor Bassat, entre los diez premiados en 2012 se encuentran los filipinos Benigno “Bam” Aquino y Maurice Edsel Salvana, premiados por su trabajo en microfinanzas y en VIH/SIDA respectivamente; el abogado inglés Bobby Kensah por su trabajo social con jóvenes pandilleros en Inglaterra; Fela Mijoro Razafinjato de las islas Maldivas por su trabajo con las personas con discapacidad y la líder ambiental Keneilwe Mosekis de Botswana, entre otros. [en línea] Madrid (ESP):, 12 de noviembre de 2012 [ref. 30 de octubre de 2012] Disponible en Internet: