Early detection to enable timely therapeutic intervention is crucial for improved outcome in patients with sepsis, but diagnosis is difficult, as the clinical signs associated with the condition commonly occur in patients with systemic inflammatory response syndrome (including sterile SIRS). This article discusses the current and emerging PCR-based technologies for the diagnosis of sepsis.
by Dr Satyanarayana Maddi, Dr Paul Dark and Dr Geoffrey Warhurst
Sepsis and issues for its early diagnosis
Sepsis is the clinical syndrome resulting from the host’s response to infection and represents a major international healthcare problem being a major cause of mortality and morbidity as well as a massive burden on resources [1]. The clinical signs associated with sepsis, such as changes in respiration, pulse, temperature and circulating immune cell counts, are non-specific and commonly seen in patients with a systemic inflammatory response syndrome (or SIRS) as well as in other insults such as tissue injury, where there is no infective cause. Early identification of sepsis and the ability to differentiate it from sterile SIRS is an important diagnostic goal in international medical practice. Evidence suggests that giving the most appropriate antimicrobial therapy at the earliest opportunity to patients with severe forms of sepsis saves more lives than any other medical intervention [1]. The Surviving Sepsis Campaign, which promotes early goal-directed management of sepsis, recommends initiation of antimicrobial therapy within one hour of clinical suspicion of sepsis [1]. Ideally, this requires rapid confirmation that infection is present and identification of the organism(s) involved. The guidelines advocate taking a whole blood sample and, where possible, other supporting clinical samples for microbiological culture prior to antibiotic administration. The problem facing clinicians is that blood cultures routinely take two to three days to confirm the presence of pathogens in the bloodstream (‘pathogenaemia’) and up to five days to either rule it out or to obtain a complete profile of the pathogen including its antibiotic susceptibility/resistance pattern. Also, since viable organisms are needed for culture, the tests can be compromised if the patient has received antimicrobial therapy prior to sampling, which is common in this clinical field.
In the face of this lack of time-critical information on the infection status of the patient coupled with the knowledge that delaying antimicrobial therapy will impair the survival chances of those patients that have infection, current opinion favours the early use of broad-spectrum and high potency antibiotics with focussing to specific organisms when microbiological evidence becomes available [1, 2]. This ‘safety first’ approach is currently the best available but does have negative consequences, particularly in terms of the overuse of antibiotics. The widespread use of broad-spectrum antibiotics is implicated in the emergence of antibiotic resistant pathogens and increasing rates of infection with Clostridium difficile and fungi. In addition many patients who will subsequently be shown to have had no infection are exposed to unnecessary treatment with powerful and potentially toxic drugs.
Application of PCR to diagnosis of pathogenaemia in suspected sepsis
While microbiological culture is likely to remain the gold standard for infection diagnosis, there is growing interest in the potential of PCR technology to provide early, time critical information based on the detection and recognition of bacterial or fungal pathogen DNA in blood [1, 2]. Platforms based on real-time PCR have proved to be the most effective in this field allowing continuous monitoring of amplicon production with either fluorescent dyes that bind non-specifically to double stranded DNA or fluorescently labelled probes that bind to specific sequences. In real-time PCR, the whole process of amplification, product detection and analysis is achieved in a single reaction vessel. Furthermore, several sequence-specific probes with different fluorescent reporters can be added to the reaction, allowing simultaneous determination of multiple products. This process is therefore ideally suited to sepsis diagnosis in which a variety of pathogen species could be involved. In terms of its application to infection diagnosis in blood (and other clinical samples), PCR offers a number of potential advantages; results are available in a matter of hours rather than days, the extreme sensitivity facilitates detection of even minute amounts of pathogen DNA in clinical samples and the test is not significantly affected by prior administration of antibiotics.
Two basic approaches to assay design have been used, either using specific primers to detect a particular organism or, more commonly, universal primers that bind to conserved sequences in bacterial but not human DNA and can detect a broad range of organisms [1]. The latter approach is ideally suited to sepsis diagnosis which can be caused by a variety of pathogen species. For bacteria, the most favourable targets are sequences in the 16S and 23S rRNA genes which are ubiquitous in bacteria and therefore ideally suited for universal detection of bacterial pathogens. More recently, the gene sequence between the 16S and 23S regions, the so-called internally transcribed region (ITS), has been targeted because it contains additional hypervariable regions that allow even better discrimination between bacterial species. Fungal pathogens can be detected by targeting analogous regions in the fungal genome [1].
Following PCR of these regions, pathogen species present can be identified by (a) specific binding of fluorescent hybridisation probes to the amplified target (b) sequencing of the amplified DNA (c) hybridisation to microarrays (d) melting temperature profiling of the amplified products.
Commercial PCR platforms for bloodstream infection diagnosis
Based on these approaches, a number of commercial systems are now available for detection of bacterial/fungal DNA in blood. Lightcycler SeptiFast, the first real-time PCR system to receive a European CE-mark (2006) for use in diagnosis of bloodstream infection, is manufactured by Roche Diagnostics (Basel, Switzerland) [Figure 1]. SeptiFast is a multiplex assay for detection and identification of a defined panel of 25 bacterial and fungal pathogens known to cause the majority of bloodstream infections in critical care. The assay can be completed in 6-8 hours and has a reported sensitivity of between 3 and 30 colony forming units (CFU) per mL of blood. SeptiFast is currently the most studied commercial PCR-based test for sepsis-associated blood-stream infection with numerous clinical validity studies published to date. At the time of writing, the author’s laboratory is hosting the first independent multicentre systematic validity study comparing SeptiFast with culture for the diagnosis of suspected healthcare-associated bloodstream infection [2]. Based on the results of this study, independent recommendations will be made to the UK’s Department of Health as to whether this real-time PCR technology has sufficient clinical diagnostic accuracy to move forward to efficacy testing during the provision of routine clinical care.
SepsiTest (Molzym GmbH & Co. KG, Bremen, Germany) [Figure 2], which was awarded a CE mark in 2008, uses universal primers to detect bacterial or fungal DNA in blood and other clinical samples but relies on post-test sequencing of the products for subsequent species identification [1, 3]. Studies evaluating the use of SepsiTest in a clinical setting are beginning to appear in the literature [3]. A third CE marked commercial platform, VYOO PCR identification test from SIRS-Lab GmbH, Germany is a semi-automated method combining broad range PCR with multiplex detection plus microarray hybridisation. In addition to detecting 34 bacterial and six fungal species covering 99% of sepsis-associated pathogens, it also detects five resistance markers i.e. mecA for Methicillin Resistance Staphylococcus aureus, vanA and vanB for vancomycin resistance in enterococci and blaCTX-M15 and blaSHV for extended spectrum β-lactamases in gram negative bacilli. To date no published clinical validity studies of this product are available.
Future/emerging approaches and technologies
High resolution melting analysis (HRMA) is a post PCR amplification method of analysing DNA that does not require multiple expensive fluorescent probes, and is solely dependent on intercalating dye chemistry for its results. Using universal primers, the target regions are amplified and then melting curve analysis is performed in high resolution (high resolution with HRM analysis) after the PCR. Thanks to the advances in instrumentation which can delineate minute shifts in the melting temperatures, the species are identified using shifts in the melting profile of the amplicons [4]. HRM analysis is quick and cost effective. The HRMA is still in developmental stage but the future looks encouraging.
Other approaches under various stages of developments for diagnosis of sepsis are FilmArrays (Idaho Technology Inc. USA) [5] [Figure 3], and ‘‘Lab-on-a-Chip’’ using microfluidic-technology i.e. taqMan Low-density array (TLDA), which overcomes limitations in multiplex PCR assays, namely the narrow range of probe regions needed for multiplexing and the inability of the PCR instrument to detect more than six fluorophores simultaneously [6].
Conclusions and future
Widespread technology adoption of these PCR systems will not occur in healthcare until clinical effectiveness has been proven. No adequately powered systematic clinical effectiveness studies have been performed to date in the field of sepsis, resulting in the absence of data that would support optimal pricing of the available technologies alongside health service adoption. There is clearly an unmet need in the field of sepsis diagnostics, but a more coordinated approach to health technology assessment and adoption in this field is urgently required to help patients benefit from the elegant technologies currently available and from those under development.
References
1. Dark PM, Dean P, Warhurst G. Bench-to-bedside review: the promise of rapid infection diagnosis during sepsis using polymerase chain reaction-based pathogen detection. Crit Care 2009;13(4):217.
2. Dark P, Dunn G, Chadwick P, Young D, Bentley A, Carlson G et al. The clinical diagnostic accuracy of rapid detection of healthcare-associated bloodstream infection in intensive care using multipathogen real-time PCR technology. BMJ Open 2011 Jan 1;1(1):e000181.
3. Kühn C, Disqué C, Mühl H, Orszag P, Stiesch M, and Haverich A. Evaluation of Commercial Universal rRNA Gene PCR plus Sequencing Tests for Identification of Bacteria and Fungi Associated with Infectious Endocarditis. J Clin Microbiol. 2011 August; 49(8): 2919–2923.
4. Ozbak H, Dark P, Maddi S, Chadwick P, Warhurst G. Combined molecular gram typing and high-resolution melting analysis for rapid identification of a syndromic panel of bacteria responsible for sepsis-associated bloodstream infection. J Mol Diagn 2012 Mar;14(2):176-184.
5. Caliendo AM. Multiplex PCR and emerging technologies for the detection of respiratory pathogens. Clin Infect Dis 2011; 52 (4): S326-30
6. Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ et al. Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol 2011 Jun;49(6):2175-2182.
The authors
Satyanarayana Maddi, Paul Dark, Geoffrey Warhurst
Infection Inflammation Injury Research Group (3IRG)
Salford Royal NHS Foundation Trust, UK. School of Translational Medicine
The University of Manchester, UK.
Asthma patients who smoke report more pronounced symptoms, an attenuated response to inhaled corticosteroids and more frequent attacks. Furthermore, diagnosing asthma in smokers can be difficult, as smoking impacts on the results of frequently used diagnostic and monitoring tools for asthma, including exhaled NO (eNO) and airway challenges.
by Dr Christian G. Westergaard, Professor Vibeke Backer and Dr Celeste Porsbjerg
Clinical background
Asthma is one of the most frequent chronic diseases worldwide, with an estimated global prevalence of 300 million people. The disease is characterised by respiratory symptoms, airway hyperresponsiveness (AHR) and bronchopulmonary inflammation. Asthma symptoms can be triggered by different agents, including exposure to allergens, physical activity and unspecific irritants such as air pollution, perfume, air humidity and tobacco smoke. The prevalence of asthma varies considerably between countries, however, in general, the prevalence has been increasing during recent decades, and ranges from a few percent to more than 15% in some countries [1].
It is estimated by the WHO that 1.25 billion people in the world are smokers. The global tobacco consumption was in 2000 estimated to be 15 billion cigarettes per day. This number is not expected to decrease until 2030, because the total number of smokers will become higher due to a larger world population [2].
Tobacco smoking has very damaging impacts on the asthmatic disease. Asthma patients who smoke report more pronounced symptoms, an attenuated response to inhaled corticosteroids, more frequent exacerbations and a higher mortality rate from asthma. Furthermore, these patients suffer from an accelerated decline in lung function, where both the highly reactive tobacco smoke and the chronic asthmatic inflammation in combination contribute to airway tissue destruction. Unfortunately, tobacco smoking is common among asthma patients, with a frequency of smokers at least as high as found in the rest of the population. In most countries, smokers constitute 15–40% of the population.
In the clinical setting, spotting the asthma patients among smokers can be challenging, due to the overlap of airway symptoms between true asthma and smoking-induced manifestations such as productive cough as well as breathlessness during exercise. In patients with a significant smoking history, an element of early chronic obstructive pulmonary disease (COPD) can also blur the clinical picture.
The diagnosing and monitoring of asthma has traditionally been based on the evaluation of symptoms in combination with spirometric measurements, which to date remain key elements in the clinical handling of asthma patients. However, as asthma is basically an inflammatory disease, many new diagnostic approaches focusing on airway inflammation have emerged, such as exhaled nitric oxide (eNO), sputum induction and airway challenges, of which the most recently approved is the mannitol test. All of these newer tests contribute to the understanding of the underlying pathophysiological mechanisms of the disease as well as expanding our diagnostic possibilities.
However, it appears that tobacco smoke may attenuate the clinical utility of many of the tests. In the following section, the focus will be on the effect of smoking on inflammation markers, AHR and spirometry, respectively.
Inflammation markers: eNO and induced sputum
Smoking has a considerable impact on the measurement of eNO. Several studies have reported a pronounced reduction of eNO in smokers compared to non-smokers, as much as 40–60% in current smokers [3]. Even passive smoking seems to have an effect on eNO values. Moreover, in a study from 2009 it was shown that eNO could only discriminate asthmatics from healthy controls in never-smokers, and not in either current or former smokers [4]. However, we have recently reported data from large sample, demonstrating that in adults with symptoms suggestive of asthma, eNO was equally good at differentiating between asthma and non-asthma, albeit with a lower cut-off for an abnormal eNO in smokers than in ex- and never smokers [5].
An eNO value of 17–22 ppb has been proposed for diagnosing asthma in current smokers [5, 6], supported by others who suggested 18 ppb as a cut-off for smokers without allergic rhinitis [6]. These similar cut-off values represent quite different sensitivity values, from about 40 to 100 %, when preserving a high specificity of at least 90%.
It would seem that eNO can also be applied in disease monitoring of the smokers when used in sequential measurements, because even in smokers, relative changes in eNO have been shown to reflect the dynamics of disease activity [7]. It has been demonstrated that, similar to non-smokers, a decrease in eNO of <20% precludes asthma control improvement, and that an increase in eNO of <30% is not associated with loss of control [7].
An important issue is the lack of knowledge regarding cut-off values for predicting steroid-response. In non-smokers, the effect of treatment with steroids has been found to be associated with airway eosinophilia, which again correlates well with eNO. Hence, a cut-off value for eNO predicting a sputum eosinophil count >3% and a high likelihood of a positive steroid response has been investigated. In smokers, this value was 28 ppb, ranging from 15 to 33 ppb, depending on atopy and high dose ICS usage [8], compared to 24 to 58 ppb in non-smokers. Such cut-off values are, however, not easy to determine, due to many factors of importance for the level of eNO, including atopy with rhinitis, life tobacco consumption and respiratory tract infections.
Several underlying mechanisms for the decreased eNO in smokers have been demonstrated, including increased arginase expression leading to reduced amounts of iNOS substrate, attenuated eosinophilic and enhanced neutrophilic inflammation as well as the impact on exogenous NO from cigarette smoke leading impairment of NO synthesis.
Another way of characterising the inflammation in the airway tissue is through sputum induction. This technique is rarely used in the diagnosis of asthma. In smoking asthmatics, it seems that the cell distribution is altered into a less eosinophilic and more neutrophilic direction. This may partially explain why smokers are less responsive to steroids.
Bronchial challenges: mannitol and methacholine
Another important approach in asthma diagnostics is measurement of airway hyperresponsiveness (AHR) using the mannitol challenge, which is an indirect bronchial provocation. In non-smokers, this test can be successfully applied for both diagnostic and monitoring purposes. It has been shown that the mannitol challenge is useful in confirming a diagnosis of asthma (specificity close to 100%), unfortunately, however, the sensitivity is considerably more moderate, around 60% [9] and thereby lower than that of the methacholine challenge [9]. Being a relative recent invention, the diagnostic properties of the mannitol test have not yet been evaluated in a smoking asthmatic population. However, in non-asthmatic smokers, a study has indicated that as much as one quarter of the subjects expressed a positive mannitol test [10]. Thus, until investigated properly in smoking asthma patients, the mannitol challenge test should be interpreted with caution and be accompanied with other tests in order to account for false positives.
AHR can also be assessed through direct challenges such as inhaled methacholine. The higher sensitivity for the methacholine test (69%) compared to the mannitol test is, unfortunately, not accompanied by an equivalently higher specificity, which has been reported to be 80% [9]. In non-asthmatics, previous studies have indicated increased AHR to methacholine in smokers. But as is the case with the mannitol test, the diagnostic properties of the methacholine test have not yet been investigated in a smoking asthmatic population, which is surprising considering that the test has been applied for decades. However, a few studies have documented that smoking does appear to affect AHR to both direct and indirect challenges. In COPD patients, it has been shown that one year of smoking cessation is associated with improvement in AHR to methacholine as well as to AMP; this finding has been supported later in a study primarily of healthy subjects, but also a few asthma patients.
Spirometry with reversibility test
Increased bronchial muscular tonus is a key feature in persistent asthma, which is the reason that measurements of lung function, including the reversibility test, have been widely used in asthma diagnostics and monitoring for decades. For some smoking asthma patients, this will continue to be a corner stone in confirming the diagnosis, but in smoking asthmatic subjects with a baseline normal FEV1 or patients with very severe asthma and hence attenuated airway compliance, reversibility testing may not be the best diagnostic test. Many studies of asthma and COPD patients have shown improvement in FEV1 after smoking cessation, indicating an airway narrowing effect of tobacco smoke. However, a study of 134 asthma patients with airway reversibility showed no difference in baseline FEV1 between smokers and non-smokers, and the salbutamol reversibility was similar [11]. This latter finding has also been confirmed in a few other studies, but, in general, our knowledge of the effect of smoking on the β2-agonist reversibility of airway resistance is sparse.
Conclusion
Smoking affects the results of most of the different clinical asthma tests available, and test results should be interpreted with smoking status in mind. Clinicians should be aware of potential limitations of each test, especially eNO, which decreases in smokers but remains useful, and the mannitol test, which may give false positive in smokers. It remains crucial to obtain an explorative anamnestic interview, involving clarification of symptom triggers, seasonal variation, presence of wheezing, concomitant rhinitis, night symptoms, familiar dispositions, symptom debut, allergies and of course, smoking history.
References
1. Masoli M, Fabian D, Holt S, Beasley R. Allergy. 2004; 59(5): 469-478.
2. Annual global cigarette consumption. http://www.who.int/tobacco/en/atlas8.pdf
3. Alving K, Malinovschi A. Eur Respir Mon 2010; 49: 1-31.
4. Malinovschi A, Janson C, Högman M, Rolla G, Torén K, Norbäck D, Olin AC. Allergy. 2009; 64(1): 55-61.
5. Malinovschi A, Backer V, Harving H, Porsbjerg C. Respir Med. 2012; 106(6): 794–801.
6. Matsunaga K, Hirano T, Akamatsu K, Koarai A, Sugiura H, Minakata Y, Ichinose M. Allergol Int. 2011; 60(3): 331-337.
7. Michils A, Louis R, Peché R, Baldassarre S, Van Muylem A. Eur Respir J. 2009; 33(6): 1295–301.
8. Schleich FN, Seidel L, Sele J, Manise M, Quaedvlieg V, Michils A, Louis R. Thorax. 2010; 65(12): 1039-1044.
9. Sverrild A, Porsbjerg C, Thomsen SF, Backer V. J Allergy Clin Immunol. 2010; 126(5): 952–958.
10. Stolz D, Anderson SD, Gysin C, Miedinger D, Surber C, Tamm M, Leuppi JD. Respir Med. 2007; 101(7): 1470-1476.
11. Chaudhuri R, McSharry C, McCoard A, Livingston E, Hothersall E, Spears M, Lafferty J, Thomson NC. Allergy. 2008; 63(1): 132-135.
The authors
Christian G. Westergaard MD*,
Vibeke Backer MD, DMSc and
Celeste Porsbjerg MD, PhD
Bispebjerg Hospital
Respiratory Research Unit
Bispebjerg Bakke 23, Entrance 66
DK-2400 Copenhagen NV, Denmark
*Corresponding author:
e-mail: cgwestergaard@hotmail.com
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
At the end of the 19th century the Scottish pathologist William Russell published an article in The Lancet titled ‘The Parasite of Cancer’ [The Lancet 1899; 3984: 1138-1141]. The response by the physicians of the day ranged from scepticism to sheer disbelief, largely because cancer is considered to be a non-communicable disease. With the exception of a few eccentric scientists, this incredulity persisted into the latter half of the 20th century, when the discovery of the Epstein-Barr virus from Burkitt lymphoma cells in 1964 led to recognition that there may be a few cancer-causing viruses. However in the last fifty years there has been a steady increase in the number of infectious agents found to cause cancer, and in May this year a relevant article was published in The Lancet Oncology. The authors used data from 2008 and found that of the 12.7 million new cancer cases occurring in that year, around two million were caused by infectious agents.
The cancer-causing microbes discovered so far include viruses, bacteria and parasites. Currently hepatitis B and C viruses, which can cause hepatocellular carcinoma, human papilloma viruses, strains of which are the aetiological agents of cervical cancer, and the bacterium Helicobacter pylori, which can cause gastric cancer, are considered to account for more than 80% of the cancers caused by infectious agents. Other carcinogenic viruses discovered so far include human T-lymphotropic virus, Kaposi’s sarcoma-associated herpes virus and Merkel cell polyomavirus. Well established bacterial associations with cancer include Salmonella typhi and gallbladder/hepatobiliary carcinoma and Chlamydia pneumoniae and lung carcinoma. In addition there is evidence for the association of Streptococcus bovis and colorectal cancer. There are also some established relationships between parasitic infections and cancer. The association of long-term infection with the parasitic fluke Schistosoma haematobium and bladder cancer is well documented, as is the association of long-term infection with the Far Eastern liver fluke Opisthorchis viverrini and cholangiocarcinoma. Some evidence also links the common protozoan parasites Crytosporidium parvum to gastrointestinal cancer and Toxoplasma gondii to the development of brain tumours.
All these organisms are able to evade the host’s immune system and establish persistent infections of many years’ duration, ultimately initiating abnormal cell growth followed by tumour development. So couldn’t efforts to lighten the global burden of cancer put more emphasis on timely diagnosis of these infections followed by suitable therapy, or even better the development and widespread use of effective vaccines? And whilst there isn’t a single ‘parasite of cancer’, it is likely that there are many other cancer-causing pathogens to uncover!
November 2024
Beukenlaan 137
5616 VD Eindhoven
The Netherlands
+31 85064 55 82
info@clinlabint.com
PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.