World Malaria Day is April 25th. Research for malaria vaccines, drugs and diagnostics is needed. The launch of the ‘End Malaria – Blue Ribbon’ in 2006 by the Malaria Foundation International and the ‘Eradication Goal’ by Bill and Melinda Gates in 2007 exemplify a global commitment. Since, research and development efforts have reached an all-time high.
by Prof. M.R. Galinski and Dr E. VS Meyer
There has been a steady rise in attention on malaria, and the political will to eradicate this disease. About half the world’s population lives at risk of malaria infection, in about 100 countries. The world has woken up to the fact that malaria remains a persistent and recurrent scourge to hundreds of millions of people each year. The rapidity with which this change has occurred is quite astounding and, if continued, brings promise for the countless victims, suffering each year from malaria attacks. With today’s stepped up efforts to eliminate and ultimately eradicate malaria, the number of reported malaria cases is beginning to decline [1]. While cause for optimism, persistent dedication, continued strategic research and heightened surveillance are critical. Still today, about a million people develop severe disease and succumb to their infections each year. World Malaria Day represents a time of reflection and a public opportunity to inform and educate.
The stakes are high. About 60 species of Anopheline mosquitoes transmit the disease to humans. Vaccines, new therapeutics, mosquito control tools and diagnostics are needed, along with enhanced monitoring and clinical responsiveness. This armamentarium is needed to fight five species of the Plasmodium parasite that cause malaria in humans: Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi.
The production and validation of malaria vaccines, drugs and diagnostics is a major task for one species, let alone five, each with their biological differences [2-4, Table 1]. Malaria is in effect a very complex parasitic disease which can differ depending on the infecting species, transmission dynamics and host characteristics. Interventions aim to target the various life stages, as the parasite transforms from an infectious sporozoite, to a thriving parasite in hepatocytes, followed by red blood cells and back to an Anopheles mosquito vector.
Plasmodium – the causative agent of malaria
Malaria has often been synonymous with the most deadly species, P. falciparum, which has received the vast majority of research and development support. While present in many parts of the world, P. falciparum is most prevalent in Sub Saharan Africa, causing upwards of 90% of all malaria illness and death on the African continent. Plasmodium vivax, on the other hand, is most widespread, particularly from a global perspective looking outside Africa, and current publications are bringing awareness to the fact that P. vivax causes severe illness and potentially death, in addition to morbidity and socioeconomic problems [2]. Plasmodium knowlesi, originally known as a parasite of macaque monkeys in South East Asia, has also become a concern, since hundreds (and perhaps thousands) of clinical cases of P. knowlesi malaria have been diagnosed in Malaysia and surrounding countries, with a number of reported deaths [3].
Plasmodium is a eukaryotic parasite with multiple life-cycle stages requiring the timely regulation and expression of 5,000 to 6,000 genes [5]. The disease is propagated each time a female anopheles mosquito injects infective sporozoites while taking a blood meal. If even one sporozoite successfully traverses the skin and blood vessels and manages to gain access to and grow in liver cells, the disease has the chance to progress. Sporozoites invade and develop in hepatocytes forming multinucleated schizonts, carrying tens of thousands of merozoite progeny. When released into the blood stream (6-15 days later, depending on the species) the cyclical process of infection and development within erythrocytes begins, with classic rigors and illness, unless sufficient immunity sets in to suppress the parasite’s multiplication, the host symptoms, or both. Immediate treatment is critical, particularly for non-immune individuals. The asexual forms (rings, trophozoites and schizonts) growing in erythrocytes produce clinical manifestations, while erythrocytes harbouring sexual stage gametocytes develop to perpetuate transmission of the parasite to the vector. Each Plasmodium species has unique characteristics that call for differentiation in clinical tests [4]. Research on P. falciparum has been facilitated by long-term in vitro culture. However P. vivax, which invades reticulocytes, does not thrive in vitro unless these young cells are provided; and robust long-term cultures are currently not feasible [2]. Thus, P. vivax research has been limited to samples from human or non-human primate infections, and the use of related simian species such as P. cynomolgi in macaques [2, 6].
In addition to primary liver-stage schizonts, P. vivax and P. ovale produce dormant liver forms, called hypnozoites, which can relapse and initiate repeated erythrocytic cycles of infection as soon as two weeks, or years after an initial blood-stage infection and treatment. The molecular make-up and biology of hypnozoites remain unknown. Knowing what triggers their activation could lead to new clinical tests to detect the presence of these forms, and guide treatment.
Diagnostics
Effective, rapid and timely diagnosis of malaria is of utmost importance. Clinical symptoms appear when the parasite destroys red blood cells during the erythrocytic cycle; symptoms include fever, chills and malaise that initially can be confounded with other infections. It is critical that when healthcare personnel suspect malaria infection a clinical history is taken, including travel to malaria endemic areas, and that light microscopy analysis of a blood film stained with Giemsa is performed, the gold standard for malaria diagnosis. Documentation of the species or multiple species is important for determining the proper treatment.
Few laboratories are equipped to process blood samples and run polymerase chain reaction (PCR) tests to confirm the Plasmodium species in specimens. However, Rapid Diagnostic Tests (RDTs) – the craze of the past decade, and wave of the future – are commercially available kits that detect Plasmodium species-specific antigens. Recently, RDTs have been positioned in malaria-endemic regions to ‘fill the gap’ of effective, reliable and timely diagnosis. Yet, implementation in the field has had to overcome challenges related to quality assurance, and interpretation of results [7, 8]. According to the Centers for Disease Control and Prevention, Binax NOW is the only RDT approved for use in the United States; it detects the histidine rich protein II (HRPII) from P. falciparum and another antigen present in all five species (Pf, Pv, Pm, Po, Pk). Yet, definitive diagnosis should be confirmed by standard microscopy.
Treatment
Currently primaquine is the only drug available that eliminates the dormant hypnozoites, and it faces resistance and contraindications. Alternatives are urgently needed and more emphasis on P. vivax research is likely to lead to such discoveries [2, 9]. Drug development research has brought about new options to treat blood-stage infections, with Artemisinin Combination Therapies (ACTs) taking centre stage since 2004. But recent examples of artemisinin resistance emphasise the need for intense surveillance [10]. Complete treatment of the blood-stage parasites is important to ward off the development of drug resistance, and the possible recrudescence of blood-stage parasitaemia and illness.
Vaccine development
Vaccines have been in the pipeline for 30 years, and yet the potential is at an all-time high to make them a reality – someday. Gone are the days of optimistically predicting a malaria vaccine will be available within five years or so, but the potential within a matter of decades is considered a reality by many today. Current vaccine candidates have been based on one or a few proteins discovered 10, 20 or more years ago [11]. While there have been signs of protective effects in clinical trials, up to about 50%, it is clear that an all-encompassing malaria vaccine is still not within reach. The ideal vaccine would provide complete protection against infection and disease for all species. Current vaccines being tested are predominantly to protect against P. falciparum.
Genomic and post-genomic advances
Malaria genome sequences have been completed for P. falciparum, P. vivax and P. knowlesi, and additional laboratory strains and patient isolates are being sequenced from around the world for comparative purposes [5]. Sequence data are providing the means for understanding the parasite’s ‘omics’: genome, transcriptome, proteome, metabolome, etc. High throughput omics technologies also enable global understandings; e.g. of the mechanisms of parasite variation and drug resistance. Investigations have advanced from a one-gene, one-protein at a time approach to the large-scale potential of comparative studies and systems biology approaches to help develop vaccines, drugs or diagnostics [Figure 2]. With this comes the recognition of the intricacies of each species and host-pathogen interactions. Systems biology approaches can lead to the identification of host and parasite factors, or biomarkers, that are associated with the various clinical presentations seen in patients with malaria. Human or non-human primate clinical samples, obtained in experimental settings, can be studied to generate mathematical models, and incorporate computational biology predictions into the iterative design of experimental plans to better understand the disease state, and pinpoint novel targets for clinical testing and interventions.
Education
With today’s capability for widespread communications and expansion of educational tools comes the responsibility of propagating accurate knowledge. Malaria education can expand, and become as widespread as cell phones, evident in the far reaches of villages around the world. Twenty years ago the Malaria Foundation International was founded with the belief that ‘no one would want to be left out’ of the process of fighting malaria and making it history. We have seen incremental developments in this direction, and social networking is a tool unforeseen in those days that makes it possible for the broadest participation. If malaria education of young children, especially in malaria-stricken countries, is emphasised today, knowledge would become widespread, and the push for eradication would benefit from a constant boost of new supporters.
Malaria is preventable and treatable, but current tools are inadequate to eradicate this disease. Continued research and development for diagnostics, new drugs and vaccines are necessary in parallel with epidemiological and clinical surveillance programmes to break the cycle of transmission, illness and death. The current post-genomics era brings hope for currently unpredictable solutions, particularly with regards to systems biology approaches, which may enable the identification of host or parasite factors that can become clinical indicators for future laboratory tests.
References
1. WHO. World Malaria Report. Edited by Program WGM. Geneva: World Health Organization; 2011.
2. Mueller I, Galinski MR, Baird JK, Carlton JM, Kochar DK, Alonso PL, del Portillo HA. Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite. Lancet Infect Dis 2009; 9(9): 555-566.
3. Galinski MR, Barnwell JW. Monkey malaria kills four humans. Trends Parasitol 2009; 25(5): 200-204.
4. Coatneyi GR, Collins WE, Warren M, Contacos PG. The Primate Malarias. Washington, DC; 1971.
5. Volkman SK, Ndiaye D, Diakite M, Koita OA, Nwakanma D, Daniels RF, Park DJ, Neafsey DE, Muskavitch MA, Krogstad DJ et al. Application of genomics to field investigations of malaria by the international centers of excellence for malaria research. Acta Trop 2012; 121(3): 324-332.
6. Galinski MR, Barnwell JW: Malaria Infections in Non-Human Primates and Model Systems for Research. In: Nonhuman Primates in Biomedical Disease. Edited by Christian R. Abee KM, Suzette D. Tardif, Timothy Morris, 2 edn: Elsevier; 2012.
7. Bell D, Wongsrichanalai C, Barnwell JW: Ensuring quality and access for malaria diagnosis: how can it be achieved? Nat Rev Microbiol 2006; 4(9): 682-695.
8. malERA Consultative Group on Drugs. A research agenda for malaria eradication: diagnoses and diagnostics. PLoS Med 2011; 8(1): e1000396.
9. Baird JK. Elimination Therapy for the Endemic Malarias. Curr Infect Dis Rep 2012.
10. WHO. Global plan for artemisinin resistance
containment (GPARC). Geneva: World Health Organization; 2011.
11. Schwartz L, Brown GV, Genton B, Moorthy VS. A review of malaria vaccine clinical projects based on the WHO rainbow table. Malar J 2012; 11: 11.
The authors
Professor Mary R. Galinski and Dr Esmeralda VS Meyer
International Center for Malaria Research Education, and Development
Emory University School of Medicine
Division of Infectious Diseases
Emory Vaccine Center
Yerkes National Primate Research Center
Emory University
Atlanta, GA 30329 USA
mary.galinski@emory.edu
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, /in Featured Articles /by 3wmediaWidening the focus of malaria control
, /in Featured Articles /by 3wmediaAlthough, according to the most recent World Malaria report, there were still an estimated 655,000 deaths from the disease in 2010, the majority occurring in African children, major investments in malaria control by the international community in the past decade have yielded excellent returns, with mortality rates falling by more than 25% globally. However as we approach this year’s World Malaria Day (25th April), it may be prudent to consider widening the focus of these international control efforts.
Of the five distinct species of Plasmodium causing human malaria, two species, namely P. malariae and P. ovale, have low prevalence and normally only cause mild disease. The thrust of global control efforts has been directed at P. falciparum, one of the two highly prevalent species, because it causes the highest mortality. However the former name of ‘benign tertian malaria’ given to P. vivax, the other highly prevalent species that is also the most widely distributed, is certainly a misnomer; infection with P. vivax is anything but mild. It has been estimated that this species, endemic in South and Central America, the Middle East, Africa and Asia, and found in temperate as well as tropical areas, causes over two hundred million cases of malaria per year. While fatal infections, usually resulting from a ruptured spleen, are infrequent, P. vivax is harder to diagnose than P. falciparum as it infects immature red blood cells and parasitaemias are thus lower. It is also harder to treat because the life cycle includes dormant liver stages (hypnozoites) that cause periodic relapse infections, accompanied by severe anaemia, respiratory distress and poor obstetric outcomes. And it affects all age groups rather than predominantly children, so as well as the human suffering endured, the economic impact is huge.
The fifth species of Plasmodium that can cause human malaria, P. knowlesi, was previously only thought to infect certain species of macaque monkeys, but has now been recognised as a clinically significant zoonosis. It has been reported from several South East Asian countries, including Thailand, Malaysia, Vietnam, Myanmar, Singapore, Indonesia and the Philippines, and causes up to 70% of the malaria cases in some of these areas. As with P. falciparum, infection with P. knowlesi is potentially fatal if it is not diagnosed and treated promptly; unfortunately microscopically it is very similar to the much less serious P. malariae and is frequently misdiagnosed. And a major concern is that deforestation and increasing human settlement in P. knowlesi endemic areas may result in humans, rather than macaques, becoming the preferred host, and thus the dissemination of P. knowlesi to neighbouring countries where there are no suitable simian hosts, but where the vector mosquitoes (predominantly Anopheles leucophyrus group) breed.
International investment and efforts to control malaria in the last decade have been truly laudable, but it is now time to look outside the P. falciparum box.
World Malaria Day 2012 – incites hope
, /in Featured Articles /by 3wmediaWorld Malaria Day is April 25th. Research for malaria vaccines, drugs and diagnostics is needed. The launch of the ‘End Malaria – Blue Ribbon’ in 2006 by the Malaria Foundation International and the ‘Eradication Goal’ by Bill and Melinda Gates in 2007 exemplify a global commitment. Since, research and development efforts have reached an all-time high.
by Prof. M.R. Galinski and Dr E. VS Meyer
There has been a steady rise in attention on malaria, and the political will to eradicate this disease. About half the world’s population lives at risk of malaria infection, in about 100 countries. The world has woken up to the fact that malaria remains a persistent and recurrent scourge to hundreds of millions of people each year. The rapidity with which this change has occurred is quite astounding and, if continued, brings promise for the countless victims, suffering each year from malaria attacks. With today’s stepped up efforts to eliminate and ultimately eradicate malaria, the number of reported malaria cases is beginning to decline [1]. While cause for optimism, persistent dedication, continued strategic research and heightened surveillance are critical. Still today, about a million people develop severe disease and succumb to their infections each year. World Malaria Day represents a time of reflection and a public opportunity to inform and educate.
The stakes are high. About 60 species of Anopheline mosquitoes transmit the disease to humans. Vaccines, new therapeutics, mosquito control tools and diagnostics are needed, along with enhanced monitoring and clinical responsiveness. This armamentarium is needed to fight five species of the Plasmodium parasite that cause malaria in humans: Plasmodium falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi.
The production and validation of malaria vaccines, drugs and diagnostics is a major task for one species, let alone five, each with their biological differences [2-4, Table 1]. Malaria is in effect a very complex parasitic disease which can differ depending on the infecting species, transmission dynamics and host characteristics. Interventions aim to target the various life stages, as the parasite transforms from an infectious sporozoite, to a thriving parasite in hepatocytes, followed by red blood cells and back to an Anopheles mosquito vector.
Plasmodium – the causative agent of malaria
Malaria has often been synonymous with the most deadly species, P. falciparum, which has received the vast majority of research and development support. While present in many parts of the world, P. falciparum is most prevalent in Sub Saharan Africa, causing upwards of 90% of all malaria illness and death on the African continent. Plasmodium vivax, on the other hand, is most widespread, particularly from a global perspective looking outside Africa, and current publications are bringing awareness to the fact that P. vivax causes severe illness and potentially death, in addition to morbidity and socioeconomic problems [2]. Plasmodium knowlesi, originally known as a parasite of macaque monkeys in South East Asia, has also become a concern, since hundreds (and perhaps thousands) of clinical cases of P. knowlesi malaria have been diagnosed in Malaysia and surrounding countries, with a number of reported deaths [3].
Plasmodium is a eukaryotic parasite with multiple life-cycle stages requiring the timely regulation and expression of 5,000 to 6,000 genes [5]. The disease is propagated each time a female anopheles mosquito injects infective sporozoites while taking a blood meal. If even one sporozoite successfully traverses the skin and blood vessels and manages to gain access to and grow in liver cells, the disease has the chance to progress. Sporozoites invade and develop in hepatocytes forming multinucleated schizonts, carrying tens of thousands of merozoite progeny. When released into the blood stream (6-15 days later, depending on the species) the cyclical process of infection and development within erythrocytes begins, with classic rigors and illness, unless sufficient immunity sets in to suppress the parasite’s multiplication, the host symptoms, or both. Immediate treatment is critical, particularly for non-immune individuals. The asexual forms (rings, trophozoites and schizonts) growing in erythrocytes produce clinical manifestations, while erythrocytes harbouring sexual stage gametocytes develop to perpetuate transmission of the parasite to the vector. Each Plasmodium species has unique characteristics that call for differentiation in clinical tests [4]. Research on P. falciparum has been facilitated by long-term in vitro culture. However P. vivax, which invades reticulocytes, does not thrive in vitro unless these young cells are provided; and robust long-term cultures are currently not feasible [2]. Thus, P. vivax research has been limited to samples from human or non-human primate infections, and the use of related simian species such as P. cynomolgi in macaques [2, 6].
In addition to primary liver-stage schizonts, P. vivax and P. ovale produce dormant liver forms, called hypnozoites, which can relapse and initiate repeated erythrocytic cycles of infection as soon as two weeks, or years after an initial blood-stage infection and treatment. The molecular make-up and biology of hypnozoites remain unknown. Knowing what triggers their activation could lead to new clinical tests to detect the presence of these forms, and guide treatment.
Diagnostics
Effective, rapid and timely diagnosis of malaria is of utmost importance. Clinical symptoms appear when the parasite destroys red blood cells during the erythrocytic cycle; symptoms include fever, chills and malaise that initially can be confounded with other infections. It is critical that when healthcare personnel suspect malaria infection a clinical history is taken, including travel to malaria endemic areas, and that light microscopy analysis of a blood film stained with Giemsa is performed, the gold standard for malaria diagnosis. Documentation of the species or multiple species is important for determining the proper treatment.
Few laboratories are equipped to process blood samples and run polymerase chain reaction (PCR) tests to confirm the Plasmodium species in specimens. However, Rapid Diagnostic Tests (RDTs) – the craze of the past decade, and wave of the future – are commercially available kits that detect Plasmodium species-specific antigens. Recently, RDTs have been positioned in malaria-endemic regions to ‘fill the gap’ of effective, reliable and timely diagnosis. Yet, implementation in the field has had to overcome challenges related to quality assurance, and interpretation of results [7, 8]. According to the Centers for Disease Control and Prevention, Binax NOW is the only RDT approved for use in the United States; it detects the histidine rich protein II (HRPII) from P. falciparum and another antigen present in all five species (Pf, Pv, Pm, Po, Pk). Yet, definitive diagnosis should be confirmed by standard microscopy.
Treatment
Currently primaquine is the only drug available that eliminates the dormant hypnozoites, and it faces resistance and contraindications. Alternatives are urgently needed and more emphasis on P. vivax research is likely to lead to such discoveries [2, 9]. Drug development research has brought about new options to treat blood-stage infections, with Artemisinin Combination Therapies (ACTs) taking centre stage since 2004. But recent examples of artemisinin resistance emphasise the need for intense surveillance [10]. Complete treatment of the blood-stage parasites is important to ward off the development of drug resistance, and the possible recrudescence of blood-stage parasitaemia and illness.
Vaccine development
Vaccines have been in the pipeline for 30 years, and yet the potential is at an all-time high to make them a reality – someday. Gone are the days of optimistically predicting a malaria vaccine will be available within five years or so, but the potential within a matter of decades is considered a reality by many today. Current vaccine candidates have been based on one or a few proteins discovered 10, 20 or more years ago [11]. While there have been signs of protective effects in clinical trials, up to about 50%, it is clear that an all-encompassing malaria vaccine is still not within reach. The ideal vaccine would provide complete protection against infection and disease for all species. Current vaccines being tested are predominantly to protect against P. falciparum.
Genomic and post-genomic advances
Malaria genome sequences have been completed for P. falciparum, P. vivax and P. knowlesi, and additional laboratory strains and patient isolates are being sequenced from around the world for comparative purposes [5]. Sequence data are providing the means for understanding the parasite’s ‘omics’: genome, transcriptome, proteome, metabolome, etc. High throughput omics technologies also enable global understandings; e.g. of the mechanisms of parasite variation and drug resistance. Investigations have advanced from a one-gene, one-protein at a time approach to the large-scale potential of comparative studies and systems biology approaches to help develop vaccines, drugs or diagnostics [Figure 2]. With this comes the recognition of the intricacies of each species and host-pathogen interactions. Systems biology approaches can lead to the identification of host and parasite factors, or biomarkers, that are associated with the various clinical presentations seen in patients with malaria. Human or non-human primate clinical samples, obtained in experimental settings, can be studied to generate mathematical models, and incorporate computational biology predictions into the iterative design of experimental plans to better understand the disease state, and pinpoint novel targets for clinical testing and interventions.
Education
With today’s capability for widespread communications and expansion of educational tools comes the responsibility of propagating accurate knowledge. Malaria education can expand, and become as widespread as cell phones, evident in the far reaches of villages around the world. Twenty years ago the Malaria Foundation International was founded with the belief that ‘no one would want to be left out’ of the process of fighting malaria and making it history. We have seen incremental developments in this direction, and social networking is a tool unforeseen in those days that makes it possible for the broadest participation. If malaria education of young children, especially in malaria-stricken countries, is emphasised today, knowledge would become widespread, and the push for eradication would benefit from a constant boost of new supporters.
Malaria is preventable and treatable, but current tools are inadequate to eradicate this disease. Continued research and development for diagnostics, new drugs and vaccines are necessary in parallel with epidemiological and clinical surveillance programmes to break the cycle of transmission, illness and death. The current post-genomics era brings hope for currently unpredictable solutions, particularly with regards to systems biology approaches, which may enable the identification of host or parasite factors that can become clinical indicators for future laboratory tests.
References
1. WHO. World Malaria Report. Edited by Program WGM. Geneva: World Health Organization; 2011.
2. Mueller I, Galinski MR, Baird JK, Carlton JM, Kochar DK, Alonso PL, del Portillo HA. Key gaps in the knowledge of Plasmodium vivax, a neglected human malaria parasite. Lancet Infect Dis 2009; 9(9): 555-566.
3. Galinski MR, Barnwell JW. Monkey malaria kills four humans. Trends Parasitol 2009; 25(5): 200-204.
4. Coatneyi GR, Collins WE, Warren M, Contacos PG. The Primate Malarias. Washington, DC; 1971.
5. Volkman SK, Ndiaye D, Diakite M, Koita OA, Nwakanma D, Daniels RF, Park DJ, Neafsey DE, Muskavitch MA, Krogstad DJ et al. Application of genomics to field investigations of malaria by the international centers of excellence for malaria research. Acta Trop 2012; 121(3): 324-332.
6. Galinski MR, Barnwell JW: Malaria Infections in Non-Human Primates and Model Systems for Research. In: Nonhuman Primates in Biomedical Disease. Edited by Christian R. Abee KM, Suzette D. Tardif, Timothy Morris, 2 edn: Elsevier; 2012.
7. Bell D, Wongsrichanalai C, Barnwell JW: Ensuring quality and access for malaria diagnosis: how can it be achieved? Nat Rev Microbiol 2006; 4(9): 682-695.
8. malERA Consultative Group on Drugs. A research agenda for malaria eradication: diagnoses and diagnostics. PLoS Med 2011; 8(1): e1000396.
9. Baird JK. Elimination Therapy for the Endemic Malarias. Curr Infect Dis Rep 2012.
10. WHO. Global plan for artemisinin resistance
containment (GPARC). Geneva: World Health Organization; 2011.
11. Schwartz L, Brown GV, Genton B, Moorthy VS. A review of malaria vaccine clinical projects based on the WHO rainbow table. Malar J 2012; 11: 11.
The authors
Professor Mary R. Galinski and Dr Esmeralda VS Meyer
International Center for Malaria Research Education, and Development
Emory University School of Medicine
Division of Infectious Diseases
Emory Vaccine Center
Yerkes National Primate Research Center
Emory University
Atlanta, GA 30329 USA
mary.galinski@emory.edu
Malaria: rapid and precise diagnosis saves lives
, /in Featured Articles /by 3wmediaMalaria is an acute and life threatening infection in individuls with no previous immunity. Symptoms are nonspecific and cannot be distinguished from those of influenza or severe bacterial infections. All febrile patients should thus be asked if they have been travelling over the past six months and if so whether the journey was to a malaria endemic area.
Microscopic examination of Giemsa stained thick and thin blood films remains the gold standard, but rapid tests using antigen-capture assays are increasingly used where access to expert microscopy is not available. The appropriate use of rapid tests and their limits are discussed.
by Dr Eskild Petersen
Malaria is caused by a protozoan parasite and five species can infect humans: Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. Humans are infected by bites of Anopheles mosquitoes and humans are the reservoir hosts except in the case of P. knowlesi, which is transmitted from monkeys and is only seen in South East Asia. Infection with P. falciparum shows the highest mortality and drug resistance is much more common in P. falciparum compared to P. vivax, and not a problem in the other malaria species. P. ovale and P. vivax have persistent liver forms, hypnozoites, which may reactivate, usually within six months after infection, and give rise to a malaria attack.
Malaria in Europe
Malaria was endemic in Europe up to the middle of the 20th century [Table 1]. Presently malaria is almost exclusively imported, although a number of Plasmodium vivax cases were seen in Greece in 2011, probably introduced with migrant workers from endemic areas [1]. It is estimated that between 10,000 and 15,000 cases of malaria are imported into Europe every year, which makes the recognition of symptoms and knowledge of appropriate diagnosis important.
A special risk group is immigrants resident in Europe who visit their home countries where malaria is found. The proportion of imported malaria cases in immigrants in Europe has increased from a reported 14% more than 10 years ago to 86% in more recent studies [2]. More than five million African immigrants could be living in Europe, one third of whom are from Sub-Saharan Africa [3], and children of immigrants are particularly at risk [4].
Mortality of imported malaria
The mortality from imported Plasmodium falciparum malaria cases varies from 0.4% in a large cohort from France [5], up to 5% in a recent cluster of cases imported from The Gambia [6]. Malaria infection in non-immunes is an emergency which requires prompt diagnosis and treatment while asymptomatic malaria in immigrants raises other public health issues.
Clinical symptoms
Individuals without immunity, i.e. persons who have not lived in malaria endemic countries for a long time, normally have a febrile illness with an acute onset. The symptoms include fever, malaise, muscle and joint pains, headache and rarely respiratory distress and diarrhoea. Malaria infection can be complicated by bacterial septicaemia. As the infection progresses there can be drowsiness, coma, kidney failure, disseminated intravascular coagulation and low blood pressure, and in the non-immune the mortality of untreated P. falciparum malaria is probably more than 50%.
P. falciparum in non-immunes does not usually follow a regular cyclic pattern and the fact that fever is not cyclic with a 48 or 72 hour cycle cannot be used to exclude malaria. Malaria in non-immunes is a medical emergency and diagnosis should be performed without delay.
In semi-immunes the clinical symptoms may be much more discrete and the development more subtle. Immunity to malaria is not a sterile immunity and a low level parasitaemia is seen in semi-immune individuals, ie. individuals from malaria endemic areas [7]. A special risk group is pregnant women from malaria endemic areas who are at greater risk of clinical malaria during pregnancy [8].
Malaria parasites may persist in asymptomatic immigrants long after their arrival in the host country, and malaria can be transmitted, for instance by blood transfusion or organ transplantation.
Who should be tested for malaria?
Diagnostic tests for malaria infection should be performed in any febrile patients who have a history of exposure, which includes patients with a history of travel in malaria endemic areas, as defined by the WHO.
However, rare modes of transmission mean that patients with fever but without a travel history to endemic areas should be tested. This includes so called ‘airport malaria’ where Anopheles mosquitoes carrying malaria parasites are transported in an airplane, leave the destination and take a blood meal from someone living close to the airport [9,10]. Malaria parasites can be transmitted in blood when sharing instruments used for intravenous drug abuse [11]. Transmission of malaria by blood transfusions from asymptomatic carriers is a huge problem in tropical Africa [12] and febrile patients with a history of receiving blood transfusion from a donor in a malaria endemic area should be suspected of having malaria until it is proven otherwise.
Diagnostic procedures for detecting malaria parasites
Traditionally malaria diagnosis rests on the microscopic examination of thick and thin blood films, but over the past decades, rapid tests based on antigen capture are increasingly used. However, rapid test have pitfalls and parasite density must be measured and followed to monitor the response to treatment. Thus microscopy is still a mandatory skill in institutions taking care of malaria patients.
Microscopic examination of Giemsa stained thick blood films remains the gold standard because it is rapid, easy to perform and sensitive [13] with a sensitivity down to five parasites per microlitre of blood [14]. Microscopy and counting of malaria parasites in patients are mandatory to assess the response to treatment and must be available at centres managing patients with malaria.
Rapid test are available which show a 100% sensitivity down to a parasite density level of 200 parasites per microlitre, equivalent to a parasitaemia of approximately 0.004% [15]. Molecular diagnosis by polymerase chain reaction (PCR) can detect parasites down to a density of 0.01 parasites per microlitre after a lysis procedure, and 1 parasite per microlitre without lysis [16]. However, PCR analysis is not instantly available around the clock so in practice diagnosis relies on rapid diagnostic tests and microscopy of Giemsa stained thick blood films.
Rapid tests are increasingly used in medical centres with limited access to experienced microscopists. However, a rapid test cannot determine the parasite density and rapid tests have limitations. False negative results in patients with very high parasite densities have been described, probably due to the so called ‘pro-zone’ phenomena known from other diagnostic tests [17, 18]. The problem seems to be limited to tests based on detection of the Histidine Rich protein 2, HRP2, and not tests based of detection of Plasmodia LDH, Lactate Dehydrogenase [15, 17]. Mutations in the HRP2 gene may also result is false negative results [19, 20]. All species ie. P. falciparum, vivax, ovale and malariae and P. knowlesi, will be found with tests based on the detection of pan-malarial aldolase antigen aldolase and LDH antigens [21]. P. ovale can be divided in variant and classic P. ovale [22], and variant P. ovale is not picked up in HRP2 based rapid diagnostic tests [23].
Thus clinicians using rapid tests should be instructed that no test so far is 100% reliable. In order to reduce the risk of false negative results, testing should be performed at least twice with 24 hours in between and preferable three times within a 24 hours interval. Variant P. ovale and P. knowlesi infections will be detected by rapid tests, which include those incorporating the pan plasmodia antigens Aldolase or Lactate Dehydrogenase antigens [15, 24]. The latest results of the WHO multicentre evaluation of different rapid diagnostic tests showed that the best performance was found in tests based on a combination of the HRP2 and PLDH proteins [15].
References
1. Danis K et al. Euro Surveill 2011;16:19993.
2. Jelinek T et al. Clin Infect Dis 2002; 34:572-576.
3. Eurostat. European Commission. Katya Vasileva. Population and social conditions. 34/2011. Available at: http://epp.eurostat.ec.europa.eu/cache/ITY_OFFPUB/KS-SF-11-034/EN/KS-SF-11-034-EN.PDF
4. Stäger K et al. Emerg Infect Dis 2009; 15:185–91.
5. Bruneel F et al. PLoS One 2010; 5(10):e13236.
6. Jelinek T et al. Euro Surveill.- 2008;13:19077.
7. Wertheimer ER et al. Emerg Infect Dis 2011;17:1701-3.
8. D’Ortenzio E et al. Emerg Infect Dis 2008;14:323-6.
9. Thang HD et al. Neth J Med 2002;60:441-3.
10. Tatem AJ et al. Malar J 2006;5:57.
11. Chau TT et al. Clin Infect Dis 2002;34:1317-22.
12. Noubouossie D et al. Transfus Med 2012;22:63-7
13. Bowers KM et al. Malar J 2009;8:267.
14. Petersen E et al. Am J Trop Med Hyg 1996; 55:485-489.
15. WHO. Rapid Diagnostic Tests. Results of round 3.http://www.who.int/tdr/publications/tdr-research-publications/rdt_round3/en/index.html Geneva 2011 (Accessed 17th March 2012).
16. Mahajan B et al. Transfusion 2012 Feb 10. doi: 10.1111/j.1537-2995.2011.03541.x. [Epub ahead of print].
17. Luchavez J et al. Malar J 2011;10:286.
18. Gillet P et al. Malar J 2011;10:166.
19. Koita OA et al. Am J Trop Med Hyg 2012;86:194-8.
20. Baker J et al. PLoS One 2011;6:e22593.
21. Chiodini PL et al. Trans R Soc Trop Med Hyg 2007; 101:331-337.
22. Sutherland CJ et al. J Infect Dis 2010;201:1544-1550.
23. Tordrup D et al. Malar J 2011;10:15.
24. Hellemond JJ van et al. Emerg Infect Dis 2009;15:1478–1480.
25. Bruce-Chwatt LJ, Zulueta J de. The rise and fall of malaria in Europe. Oxford University Press. 1980.
The author
Dr Eskild Petersen
Department of Infectious Diseases
Aarhus University Hospital
Skejby
Aarhus
Denmark
Tel +45 7845 2817
e-mail: joepeter@rm.dk
Malaria: a global threat
, /in Featured Articles /by 3wmediaMalaria threatens the existence of large numbers of children in tropical and subtropical areas of the world. Increasing malaria parasite drug-unresponsiveness and insecticides-unresponsive mosquitoes lead to emergence of new malaria foci. Insecticide-impregnated bed nets and case detection/prompt treatment with artesunate-based drug combinations offer the most effective control measures. Counterfeit antimalarial drugs pose a serious threat to malaria control. No effective vaccine has been introduced into clinical practice to date.
by Prof. E.A.G Khalil and Dr M.E.E. Elfaki
Malaria is a febrile parasitic disease that is transmitted by female mosquitoes with no known intermediate host except in the case of Plasmodium knowlesi. Malaria is prevalent over most areas of Asia, Africa, eastern Europe, south America and South Pacific. Hot climate and low socio economic conditions make malaria prevalent in these areas. Malaria affects 300 to 700 million people annually with 1-2 million deaths, mostly of children [1]. The malaria parasite can infect all age groups, but children and pregnant women are at an increased risk for developing the severe form of the disease. The red blood cells are the principal cells affected, the parasite usually affects red blood cells of all ages. There are five species of malaria parasite that cause human disease: Plasmodium falciparum, Plasmdium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi.
Malaria can present in a mild uncomplicated form that is characterised by fever, headache, arthralgia, vomiting, malaise, sweating and splenomegaly. On the other hand a severe and complicated form exists that presents as severe anaemia, pulmonary oedema, seizures, coma and renal/respiratory effects. Brain effects can result in the death of 20% of even optimally treated individuals with residuals brain damage in some surviving children. Large numbers of individuals in endemic areas may harbour the parasite without obvious symptoms (subclinical infection); these individuals represent a reservoir during the dry season [2,3].
Under-nutrition is an underlying cause of malaria morbidity in children under the age of five [4]. Nutritional supplementation with vitamin A, zinc, selenium, iron and folate are reported to reduce malaria morbidity in children, probably through their effects on the immune system [5].
Immunity against malaria
The ability of humans to fight malaria relies on the presence of specialised immune cells that produce antibodies against malaria parasite proteins expressed on the surface of infected red blood cells (humoral immunity). Human immunity also relies on the production of cytokines, specialised proteins that arm immune cells and make them more capable of killing malaria parasites. In addition, specialised T-lymphocytes, namely CD8+ cells help the body to eliminate the parasite through destruction of infected cells (cellular immunity) [6,7].
Treatment of simple and complicated malaria
Chloroquine was the drug of choice for malaria treatment for some time, but this has dramatically changed due to the emergence of resistance in different parts of the globe. The same problem has occurred with other antimalarial drugs such as mefloquine, quinine and sulphadoxine [7,8]. Artesunate-based combinations are now used as first line treatment of simple malaria by many control programmes [10]. In addition, fixed-combination anti-malarials such as Dihydroartemsinin-piperaquine (DP) can effectively treat uncomplicated, multidrug-resistant falciparum malaria [11].
Intermittent preventive malaria treatment (IPT) using sulphadoxine/pyrimethamine has been shown to reduce the burden of malaria effectively in children in areas of seasonal transmission [12]. Supportive treatment is an important adjunct to antimalarial treatment (antipyretic, anticonvulsant and exchange blood transfusion) in severe P. falciparum malaria [13,14].
Control of malaria
Malaria morbidity and mortality can be markedly reduced with a sum of money not exceeding $ 3.0/adult. At the present time case detection and prompt treatment with artesunate-based combination drugs and the use of insecticide-treated bed nets (ITN) are the most effective control measures. ITN have proven to reduce malaria morbidity and mortality [10,15,16].
Counterfeit drugs
Counterfeit drugs present a major obstacle to malaria control programmes by prolonging morbidity and increasing mortality. About a third to one half of drugs sold in Africa and Asia are counterfeit drugs. There is some evidence that the problem of counterfeit drugs is increasing, especially in countries where regulatory authorities do not have the will to investigate and take action or do not have the necessary resources. However there is a lot of pressure not to publicise the issue of counterfeit anti-malaria drugs [17,18,19,20].
Vaccines against malaria
The ability of the malaria parasite evade the immune system is the main reason that no really effective vaccine has been produced to date. A number of the parasite molecules have been targeted as vaccine candidates in vain. Recently, the RTS,S/AS01 vaccine has been shown to provide protection against clinical and severe malaria in African children [21,22].
Conclusion
Better use of ITN, rapid and accurate diagnostic tests and the use of artesunate-based drug combinations can effectively control malaria. Counterfeit anti-malarials are a serious and under-estimated problem that could definitely cripple malaria control programmes in Africa and Asia.
References
1. WHO 2005. World Malaria Report.
2. Looareesuwan S et al. Lancet 1985; 2: 4-8
3. Reuben R. Soc Sci Med 1993; 37: 473–480.
4. Caulfield LE et al. Am J Trop Med Hyg 2004; 71 suppl 55-63.
5. Shankar AH. J Infect Dis 2000 182 (Supplement 1): S37-S53. doi: 10.1086/315906.
6. Goodhttp MF & Doolan DL. Curr Opin Immunol 1999; 11, 4, 412–419.
7. Stevenson M & Riley EM. Nature Rev Immunol 2004; 4, 169-180.
8. al-Yaman F et al. P N G Med J 1996; 39 :16-22.
9. Le Bras J & Durand R. Fundam Clin Pharmacol 2003; 17 :147-53.
10. WHO/MAL/94.1067. The role of artemisinin and its derivatives in the current treatment of malaria (1994-1995): report of an informal consultation convened by WHO in Geneva, 27-29 September 1993. Geneva: WHO 1994.
11. Ashley EA et al.. Clin Infect Dis 2005; 41 : 425-432. doi: 10.1086/432011.
12. Dicko A et al. Mal J 2008; 7:123 doi:10. 1186/ 1475-2875-7-123.
13. World Health Organization, Division of Control of Tropical Diseases. Severe and complicated malaria. Trans R Soc Trop Med Hyg 1990; 84: Suppl 2:1-65.
14. Hien TT et al. Trans R Soc Trop Med Hyg 1992; 86:582-583
15. Guerin PJ et al. Lancet Infect Dis 2002; 2 :564-573.
16. Frey C et al. Mal J 2006; 5:70.
17. World Health Organization. Report of the International Workshop on Counterfeit Drugs. 1998; WHO/DRS/CFD/98.1. Geneva: WHO.
18. Newton PN et al. BMJ 2002; 324: 800–801.
19. Dondorp AM et al. Trop Med Int Health 2004; 9: 1241–1246.
20. Rudolf PMM & Bernstein IBG. N Engl J Med 2004; 350: 1384–1386.
21. Plassmeyer ML et al. J Biol Chem 2009; 284 : 26951–63.
22. Agnandji ST et al. N Engl J Med 2011; 365: 1863-1875.
The authors
Prof. E.A.G. Khalil and Dr M.E.E. Elfaki
Department of Clinical Pathology
& Immunology
Institute of Endemic Diseases
University of Khartoum
Khartoum
Sudan
Rapid diagnostic tests for malaria
, /in Featured Articles /by 3wmediaTogether with HIV/AIDS and TB, malaria is one of the major public health challenges of the developing world. Prompt diagnosis is a priority. Rapid diagnostic tests are readily available, quick to yield results and can be effectively used in resource-limited settings.
by Meghna Patel
Malaria is a tropical disease caused by parasites of the genus Plasmodium and transmitted by Anopheles mosquitoes. Being endemic in more than 100 countries, half the world’s population is at risk for malaria. Children are at particular risk, accounting for most malaria deaths globally [1]. Each year roughly 250 million people are infected and nearly a million people die from the disease [2]. Malaria causes significant morbidity and mortality, particularly in resource-poor regions. Sub-Saharan Africa is the hardest hit region in the world and parts of Asia and Latin America also face significant malaria epidemics [3]. Four major species of malarial parasite infect humans: Plasmodium falciparum, P. vivax, P. ovale and P. malariae. The first two species cause the most infections worldwide. On the continent of Africa, P. falciparum malaria predominates, whereas in parts of Asia and Latin America, P. vivax is more prevalent. Two other species, P. ovale and P. malariae, are also capable of causing human disease. A fifth species, Plasmodium knowlesi, is found in Southeast Asia; it mainly causes malaria in simians but it can also infect humans.
Since malaria is preventable and treatable, such high incidences point to inappropriate management of the condition in some cases, with incorrect or inefficient diagnosis and/or treatment. Rapid and accurate diagnosis of malaria before treatment is essential for effective and timely treatment of patients and to minimise the spread of drug resistance and thus the requirement of more expensive drugs, frequently unaffordable for resource-poor countries [4]. This review discusses the currently available techniques for malaria diagnosis
focusing on rapid diagnostic tests (RDT).
Diagnosis
As in other pathological conditions malarial diagnosis is based on clinical investigations and pathological laboratory analysis. Diagnosis based on clinical symptoms is the least expensive, most commonly used method in resource poor conditions. However, the overlapping of malaria symptoms with other tropical diseases impairs its specificity and therefore encourages the indiscriminate use of anti-malarials for managing febrile conditions in endemic areas.
Laboratory diagnosis of malaria includes identifying malarial parasites or their antigens/products in patient blood. Although this may seem simple, diagnostic efficacy depends on various factors such as stage and forms of the various malarial species, endemicity of different species, density of parasitaemia etc.
In the laboratory, malaria is diagnosed using different techniques e.g. conventional microscopic diagnosis by examining stained thin and thick peripheral blood smears, other concentration techniques, e.g. quantitative buffy coat (QBC), rapid diagnostic tests and molecular diagnostic methods, such as PCR. The pros and cons of these methods have also been described, chiefly related to sensitivity, specificity, accuracy, precision, time consumed, cost-effectiveness, labour intensiveness, the need for skilled microscopists etc.
Malaria is conventionally diagnosed by microscopic examination of stained blood films using Giemsa, Wright’s or Field’s stains [5]. Even though microscopic examination is considered to be the gold standard method, the most important limitation is its relatively low sensitivity, thus the generation of false negative results, particulary when microscopy is carried out using a low quality microscope and/or by less experienced personnel, and with low parasitaemias as in asymptomatic malaria. Furthermore the technique is laborious and not really suitable for remote rural settings, with no electricity or health facility resources.
The QBC technique was designed to enhance microscopic detection of malaria parasites [6]. This technique utilises micro-haematocrit tubes, fluorescent dyes and an appropriate fluorescence microscope for detection. Although simple, reliable and user-friendly, QBC is not widely applicable as it is costly, requires specialised instrumentation and is far from ideal for determining species and numbers of parasites.
Serological methods to diagnose malaria usually target antibodies against asexual blood stage malarial parasites. Immunofluorescence antibody testing (IFA) has proved a reliable serological test for malaria [7]. Although IFA is sensitive and specific, it is time-consuming and subjective. Furthermore the reliability greatly depends on the use of standardised reagents, in turn dependent on the expertise of laboratory workers.
Recent developments in malaria diagnosis suggest the use of PCR-based techniques. These techniques have proven to be one of the most specific and sensitive diagnostic methods, especially in malaria cases with low parasitaemia or mixed infections [8]. PCR was found to be more sensitive than QBC and some RDTs [9,10]. Compared with the gold standard method for malaria diagnosis, PCR has exhibited higher sensitivity and specificity [8]. Moreover, PCR can also help detect drug-resistant parasites, and is compatible with automation so that large numbers of samples can be processed. Some modified PCR methods e.g., nested PCR, real-time PCR and reverse transcription PCR are reliable and appear to be useful second-line techniques. Although PCR appears to offer the paramount sensitivity and specificity, its adoption in labs is limited due to the complex methodology, high cost and the demand for specialised instruments, the complex quality control and the difficulty of recruiting trained technicians especially in resource-poor conditions.
As the majority of malaria cases are found in countries where cost-effectiveness is an especially important factor and the ease of diagnostic test performance and training of personnel are also major considerations, new technology has given due attention to these points and utilised techniques that comply with diagnostic need without being very demanding. This has mainly resulted in the
development of RDTs.
Rapid diagnostic tests
RDT are largely based on the principle of immunochromatograpy, in which either monoclonal or polyclonal antibodies against the parasite antigen are immobilised to capture the parasite antigens from the peripheral blood. Currently, immunochromatographic tests target the histidine-rich protein-II of P. falciparum, a pan-malarial Plasmodium aldolase and the parasite-specific
lactate dehydrogenase.
Histidine-rich protein II of P. falciparum (PfHRP-II) is a water soluble protein that is produced by the asexual stages and young gametocytes of P. falciparum. It is abundantly expressed on the red cell membrane surface [11].
Parasite lactate dehydrogenase (pLDH) is a soluble glycolytic enzyme produced by the asexual and sexual stages of the live malarial parasites [9]. It is present in and released from the parasite-infected erythrocytes. It has been found in all four major species causing malaria in humans as their respective isoforms.
Plasmodium aldolase is an enzyme of the glycolytic pathway expressed by sexual and asexual stages of malaria parasites. RDTs have been developed in different test formats such as dipstick, card, well and cassette. The test procedure varies between different test kits. In general, the blood sample is mixed with a buffer solution that contains a haemolysing compound and a specific antibody that is labelled with a visually detectable marker such as colloidal gold. If the target antigen is present in the blood, a labelled antigen-antibody complex is formed and it migrates forward in the test strip and is captured at the test line. It is essential to include a control line to check on test validity. A washing buffer is then added to clear the background for easy
visualisation of the coloured lines.
RDTs are available in kit form with all the necessary reagents so they can be utilised even in remote places by less skilled personnel to generate results within a short period of time, usually within 15-20 minutes.
WHO recommended a few desirable characteristics for RDTs regarding their accuracy and sensitivity (WHO/MAL/2000.1091). According to this RDTs should be at least as accurate as results derived from microscopy performed by an average technician under routine field conditions, the sensitivity should be above 95% compared to microscopy, and the detection of parasitaemia should be such that levels of 100 parasites /µL (0.002% parasitaemia) should be detected reliably with a sensitivity of 100%. One product received U.S. FDA clearance in June 2007.
Today most RDTs have achieved this goal for P. falciparum, but not for other species. Roughly, RDT sensitivity declines at parasite densities < 500/µL blood for P. falciparum and < 5,000/µL blood for P. vivax [12]. RDT consumption, especially in developing countries, has increased over the past few years.
SPAN diagnostics offers RDTs i.e. ParaHIT-Total and ParaHIT-f in both dip stick, as well as in device format, for rapid and reliable diagnosis of malaria. ParaHIT-f is intended to diagnose malaria caused by P. falciparum with the use of P. falciparum specific HRP-II, wheareas ParaHIT-Total explores HRP-II and pan malarial species specific aldolase, as separate lines to screen malaria and for
differential determination of P. falciparum.
References
1. WHO, World Malaria Report 2010; December 2010.
2. WHO 10 facts on malaria
3. CDC, Malaria
4. Barnish G et al. Newer drug combinations for malaria. BMJ 2004; 328: 1511–1512
5. Warhurst DC et al. Laboratory diagnosis of malaria. J Clin Pathol 1996; 49: 533-538
6. Clendennen TE 3rd et al. QBC and Giemsa stained thick blood films: diagnostic performance of laboratory technologists. Trans R Soc Trop Med Hyg 1995; 89: 183-184
7. She RC et al. Comparison of immune fluorescence antibody testing and two enzyme immunoassays in the serologic diagnosis of malaria. J Travel Med 2007; 14: 105-111
8. Morassin B et al. One year’s experience with the polymerase chain reaction as a routine method for the diagnosis of imported malaria. Am J Trop Med Hyg 2002; 66: 503- 508
9. Makler MT et al. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 1998; 92: 419-433
10. Rakotonirina H et al. Accuracy and reliability of malaria diagnostic techniques for guiding febrile outpatient treatment in malaria-endemic countries. Am J Trop Med Hyg 2008; 78: 217-221
11. Rock EP et al. Comparative analysis of the Plasmodium falciparum histidine-rich proteins HRP1, HRP2 and HRP3 in malaria diagnosis of diverse origin. Parasitology 1987; 95: 209–227.
12. Wongsrichanalai C et al. A Review of Malaria Diagnostic Tools: Microscopy and Rapid Diagnostic Test (RDT). Am J Trop Med Hyg 2007; 77: 119–12.
The author
Meghna Patel
SPAN Diagnostics Ltd
Udhna, Surat, India
HCC biomarker, a proteomics approach: the journey from bench to bedside
, /in Featured Articles /by 3wmediaA goal of clinical proteomics is to find a disease indicator (biomarker) to identify the presence of, or monitor, a disease. It may be surprising that approximately one-third of all cancer cases could be effectively treated if detected at an early enough stage. As a heterogeneous disease, cancer evolves via multiple pathways and is a culmination of a variety of genetic, molecular and clinical events. Given that there is significant variation in the risk of developing cancer and that early detection often results in increased survival, developing technologies capable of identifying patients at highest risk and detecting tumours in the earliest stages of development is a pressing need.
by Dr Gul M. Mustafa, Prof. Cornelis Elferink and Prof. John R. Petersen
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, ranking sixth among cancers in incidence worldwide and is the 3rd leading cause of cancer death. Despite some significant improvements in diagnosis and treatment of human liver diseases over the last decade, the HCC mortality rate has not changed to any extent. Currently there are approximately 20,000 new case in the US annually with millions world-wide [1]. The projected rise in the new HCC cases in the US and the world is mainly due to latent hepatitis C virus (HCV) infections in the general population, accounting for approximately 80% of HCC cases several decades after initial infection. The less than 5% survival rate of patients with HCC is primarily due to the disease eluding early detection and diagnosis, when options for effective treatment still remain. Surveillance of patients at highest risk for developing HCC, notably patients with cirrhosis, would benefit greatly from a biomarker assay capable of accurately detecting HCC in its earliest stages when it is still possible to intervene. One of the most widely used markers for HCC is alpha fetoprotein (AFP) although it is non-specific, providing low sensitivity and poor specificity, especially for early detection of HCC [2]. The false-negative rate with AFP level can be 40% for tumours < 3 cm in diameter. More reliable methods such as triple phase Computed Tomography (CT) imaging and liver biopsies exist, but these are expensive and not conducive to long-term surveillance. Therefore, the identification of superior biomarkers will be of huge clinical significance to
at-risk populations.
The ideal biomarker for this type of application would be one where HCC is detected with a high sensitivity and specificity in easily obtained biological samples in a non-invasive, or minimally invasive, manner. Blood represents the best source for detection of HCC related biomarkers, as every cell in the body leaves a record of its physiological state by the products it sheds into the blood, either as a waste or as a signal to neighbouring cells. What some may view as cellular refuse in is reality a diagnostic gold mine. Because of its easy accessibility from patients on a regular basis and because it is in contact with all the tissues in the body, it is an excellent choice for a proteomics approach as it may reveal when changes, such as development of HCC, occur. The systematic analysis of the whole serum or plasma proteome may thus provide a functional meaning to the information provided by genome expression studies. Expression of proteins, their isoforms or post-translational modifications, can be detected by proteomic analysis and these data can provide precious information to better understand the pathologic/molecular basis of HCC [3]. Proteomic analysis may also allow monitoring of the course of the disease process from cirrhosis to HCC, eventually leading to earlier diagnosis which is essential in determining the best course of treatment options and possible outcomes. In addition to earlier diagnosis proteomic analysis may also be useful in measuring the efficacy/progress of treatment or detecting tumour reoccurrence both of which are missing in HCC treatment.
Proteomics analysis
Proteomics analysis is currently considered to be the best tool for the global evaluation of protein expression, and has been widely applied in the analysis of diseases, especially cancer research. For us the approach was to compare the serum/plasma protein profile from patients infected with HCV against the sera from patients with confirmed HCC. Proteins found to be consistently altered between the two patient populations can then be identified and further characterised to determine if they can be used as biomarkers of HCC. While on the surface this sounds simple, due the complexity of the proteome and the wide dynamic concentration range (9 orders of magnitude from pg/mL to mg/mL) of constituent protein/peptide species it is an extremely challenging task. Because the serum/plasma proteome is predominated by high abundance proteins such as albumin and immunoglobulins, extensive fractionation prior to analysis is required. To reduce the few over-represented (i.e. abundant) proteins, without losing any valuable information, existing fractionation methodologies often discard the high abundance carrier proteins, such as albumin, and thus fail to capture the information associated with this valuable resource. We have used aptamer-based technology (Bio-Rad) a technology that reduces the dynamic range and thus retains the complexity of the serum peptidome, in contrast to strategies that just deplete carrier proteins.
Quantitative protein expression profiling
Because proteins entering the blood from surrounding tissue are much less abundant, it is this fraction that is likely to contain most of the undiscovered biomarkers. Quantitative protein expression profiling is a crucial part of proteomics, and such profiling requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. Thousands of different protein species present in the biological fluid or tissue must be separated, identified and characterised, which cannot be accomplished by a single experimental approach. An effective approach is two-dimensional differential in gel electrophoresis (2D-DIGE) and mass spectrometry [4]. While two-dimensional electrophoresis (2DE) has been widely used for proteomics research, the inter-gel variation along with excessive time/labour costs are major problems. Two-dimensional differential in gel electrophoresis (2D-DIGE) is a modification of 2DE and is considered as one of the most significant advances in quantitative proteomics. Using 2D-DIGE, two samples that are to be compared are pre-labelled with mass- and charge-matched fluorescent cyanine dyes and co-separated on the same 2D gel. The use of internal standards in every gel minimises problems associated with technical variability. Moreover with the great sensitivity and dynamic range that is afforded by the fluorescent dyes, 2D-DIGE can give greater accuracy of quantitation than traditional silver staining. The data captured from these gels using the Imagers, such as the Typhoon trio, along with and proprietary (Decyder) software can be configured to give inter-gel and intra-gel statistical analysis providing both a quantitative and qualitative analysis. We and others are using this approach to identify differentially expressed proteins for differential expression between the pre-cancerous and cancerous patient groups.
Stable isotope labelling
Another technique that can be useful in the analysis of the whole serum proteome is stable isotope labeling using O16/O18. This is a quantitative proteomic technique that distinguishes individual peptides during LC-MS/MS on the basis of a 4 Dalton m/z change after differential O16/O18 labelling that takes place at the C-terminal carboxyl group of tryptic fragments [5]. It is then possible to determine the ratio of individual protein expression levels between the two samples. Alternatively it is possible to use O16/O18 stable isotope labeling to determine the differential expression between two patient groups. In this way the low molecular weight serum peptidome (<20kDa), suspected of harbouring metabolites and degradation products reflecting HCC, can also be interrogated Selected reaction monitoring
Selected reaction monitoring (SRM), which is used to monitor a precursor and its product ion m/z, is another powerful proteomic tool using tandem mass spectrometry to monitor target peptides within a complex protein digest. The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, renders it amenable to biomarker discovery and validation proteomics. Using the selectivity of multiple stages of mass selection of tandem mass spectrometers, these targeted SRM assays are the mass spectrometry equivalent of a Western blot. An advantage of using a targeted mass spectrometry-based assay over a traditional Western blot is that it does not rely on the creation of highly selective immunoaffinity reagents. Thus, targeted SRM assays using heavy isotope-labelled internal standards can be multiplexed in quantitative assays that can be directly applicable to clinical settings. A targeted proteomics workflow based on SRM on a triple Quadrupole mass spectrometry platform shows the potential of fast verification of biomarker candidates reducing the gap between discovery and validation in the biomarker pipeline. Although useful, due diligence needs to be exercised in developing and validating SRM assays.
Sample handling
Biomarker research necessitates a clear, rational framework. Technologically, the platform needs to be able to detect low abundant plasma/serum proteins and reproducibly measure them in a high throughput manner. Conceptually, the choice of the technological platform and availability of quality samples should be part of an overall study design that integrates basic and clinical research. Sample preparation is an important and very critical part of clinical proteomics as the collection, sample handling and storage can have a significant impact on the integrity of the proteins being detected. It is so important that a standard operating procedure outlining the steps that should be followed in collecting and storing clinical samples was recently published [6]. In addition to a standardised collection procedure, biological samples need to be carefully chosen based on well-established guidelines either for candidate discovery in the form of controls and the disease being detected or for validation of the candidate biomarkers using well characterised samples.
Most importantly, the samples should be representative of the target population and directly address the clinical question. A conceptual structure of a biomarker study can be provided in the form of sequential phases, each having clear objectives and predefined goals [Figure 1]. Furthermore, guidelines for reporting the outcome of biomarker studies are critical to adequately assess the quality of the research, interpretation and generalisation of the results. By being attentive to and applying these considerations, biomarker research should become more efficient and lead to biomarkers that are translatable into the clinical arena.
Aknowledgements
This research was supported by a pilot grant from the Clinical Translational Sciences Award (5UL1RR029876) and the Mary Gibb Jones endowment.
References
1. Kim WR. The burden of hepatitis C in the United States. Hepatology 2002; 36: 30-34.
2. Sterling RK, Wright EC, Morgan TR, Seeff LB, Hoefs JC, Di Bisceglie AM, Dienstag JL, Lok AS. Frequency of elevated hepatocellular carcinoma (HCC) biomarkers in patients with advanced hepatitis C. Am J Gastroenterol 2012; 107(1): 64-74.
3. Maria P, Laura ML, Antonio RA, Jose LM, Javier B, Ruben C, Jordi M and Manuel de la Mata. Proteomic analysis for developing new biomarkers of hepatocellular carcinoma. World J Hepatol 2010; 2(3): 127-135.
4. Sun W, Xing B, Sun Y, Du X, Lu M, Hao C, Lu Z, Mi W, Wu S, Wei H, Gao X, Zhu Y, Jiang Y, Qian X, He F. Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues. Mol Cell Proteomics 2007; 6(10): 1798-808.
5. Miyagi M, Rao KC. Proteolytic 18O-labeling strategies for quantitative proteomics. Mass Spectrom Rev 2007; 26(1):121-36.
6. Tuck MK et al. Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res 2009; 1: 113-117.
The authors
Gul M. Mustafa, Ph.D. Postdoctoral Fellow, Department of Pharmacology
Cornelis Elferink, Ph.D., Professor, Department of Pharmacology, Director Sealy Center Environmental Health and Medicine
John R. Petersen, Ph.D., Professor and Director Victory Lakes Clinical Laboratory, Department of Pathology,
University of Texas Medical Branch
301 University Boulevard
Galveston, Texas 77555, USA
e-mail: jrpeters@utmb.edu