Together 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
There are a huge number of peer-reviewed papers covering sepsis, and it is frequently difficult for healthcare professionals to keep up with the literature. As a special service to our readers, CLI presents a few key abstracts from the clinical and scientific literature chosen by our editorial board as being particularly worthy of attention.
Predictors of survival in sepsis: what is the best inflammatory marker to measure?
Beyond the widely used acute-phase proteins C-reactive protein (CRP) and procalcitonin (PCT) in sepsis manegement, many new molecules have been studied deriving from different organs or cells affected, due to the systemic nature of sepsis. Cytokines, coagulation factors/characteristics, vasoactive hormones and several others have recently proved to be relevant in sepsis syndrome and probably useful for outcome prediction. However, single time point measurements may be less predictive than consideration of the time-dependent course of parameters. Many biomarkers display relevant correlation with the clinical outcome of patients with severe sepsis and septic shock. Consideration of their time courses may be more reliable than absolute levels. Clinical decision should not only be based on biomarkers but organ dysfunctions, for example, should also be taken into account.
Cytokine profiles of preterm neonates with fungal and bacterial sepsis
Information on cytokine profiles in fungal sepsis (FS), an important cause of mortality in extremely low birthweight infants (ELBW), is lacking. The authors hypothesised that cytokine profiles in the 1st 21 days of life in ELBW with FS differ from those with bacterial sepsis (BS) or no sepsis (NS). In a secondary analyses of the NICHD Cytokine study, three groups were defined – FS (≥1 episode of FS), BS (≥1 episode of BS without FS) and NS. Association between 11 cytokines assayed in dried blood spots obtained on days 0-1, 3±1, 7±2, 14±3, and 21±3 and sepsis group was explored.Of 1066 infants, 89 had FS and 368 had BS. Compared to BS, FS was more likely to be associated with lower birthweight, vaginal delivery, patent ductus arteriosus, postnatal steroids, multiple central lines, longer respiratory support and hospital stay, and higher mortality (p<0.05). Analyses controlling for covariates showed significant group differences over time for IFN-γ, IL-10, IL-18, TGF-β and TNF-α (p<0.05). These differences, which may have implications for diagnosis and treatment, require validation in rigorously designed prospective studies.
Prognostic value of proadrenomedullin in severe sepsis and septic shock patients with community-acquired pneumonia
Midregional proadrenomedullin (proADM) is a novel biomarker with potential prognostic utility in patients with community-acquired pneumonia. The aim of this study was to investigate the value of proADM levels for severity assessment and outcome prediction in severe sepsis and septic shock due to CAP. The prospective observational study included 49 patients admitted to ICU with both a clinical and radiologic diagnosis of pneumonia and fulfilling criteria for severe sepsis or septic shock. The prognostic accuracy of proADM levels was compared with those of pneumonia severity index and of procalcitonin (PCT) and C-reactive protein (CRP). Forty-nine patients with severe sepsis or septic shock due to CAP were included in the study. Mortality was 24.5% for ICU and 34.7% for hospital mortality. In all cases proADM values at ICU admission were pathological (considering normal proADM levels <4 nmol/L). ProADM consistently rose as PSI class advanced from II to V (p = 0.02). Median proADM levels were higher (p <0.01) in hospital non-survivors 5.0 (1.9-10.1) nmol/L vs. survivors 1.7 (1.3-3.1) nmol/L. These differences were also significant with respect to ICU mortality. The receiver-operating characteristic curve for proADM yielded an AUC of 0.72; better than the AUC for PCT and CRP (0.40 and 0.44 respectively) and similar to PSI (0.74). In this study MR-proADM levels correlated with increasing severity of illness and death. High MR-proADM levels thus offer additional risk stratification in high-risk CAP patients.
As with much else in healthcare, change is the driver of clinical lab technologies today. Rapid advances in genetics, especially the game-changing promise of biomarkers and personalized medicine, have dramatically extended the traditional spectrum of clinical lab technologies. A snapshot of the specialties within a modern clinical lab and the key drivers of change within each is provided below.
Blood banking: Over the past decade, automation has halved serological testing times. Nevertheless, enduring safety concerns have led to new technologies such as CAT (Column Agglutination), along with remote, real-time and secure monitoring of equipment by technical service providers.
Clinical chemistry and microbiology: The choice and sequencing of chromatography, mass spectrometry, electrophoresis, thermocycling or radioisotopes have become quicker and more reliable due to the widespread use of testing protocols. New tools for microbiologists include phosphoimaging and fluorescence activated cell sorters. One of the most promising fields at present consist of new DNA-based techniques.
Cytotechnology: Still focused largely on cancers, cytotechnology has expanded its scope from diagnosis to prognosis. The key drivers here are molecular diagnostics and FISH (fluorescence in situ hybridization), with data warehousing support for tissue correlation. FISH is used to track specific DNA sequences on chromosomes, by using probes which bind only with specific fragments of the chromosome; these are then identified by fluorescence microscopes. FISH has proved to be indispensable in diagnosing rare diseases such as Cri-du-chat, certain kinds of childhood leukemias, as well as syndromes like Prader-Willi and Angelman.
Histotechnology: Traditionally associated with cutting and staining tissue specimens for the study of diseases at a microscopic level, histotechnicians are now branching out into one of the fastest growing areas of clinical lab technology, namely immunohistochemistry. This is the localization of antigens via the use of labelled antibodies, with antigen-antibody interactions subsequently visualized by markers. The 1950s era technology of using fluorescent dye was followed by enzyme labelling in the 1960s and 1970s (respectively peroxidase and phosphatase). Colloidal gold permitted electron microscopes to be deployed for multi-level staining, since gold particles can be manufactured in a vast range of sizes. Other techniques include autoradiography, using radioactive elements as labels for visualizing immunoreactions.
Immunology: Rather than the painstaking, bottom-up process of examining individual cells under a microscope, immunology is now becoming top-down. Fuelled by the Human Genome Project, studies of tissues and organs and the molecular pathways of the immune system have led to a host of new waypoints in mapping the progress of a disease (e.g signal transduction mechanisms), along with innovative tools such as custom-built peptide probes, supermagnetic nanobeads, hybridomas, epitopes and tetramer assays, in brief – the new science of proteomics.
Molecular biology: Automated cell counting equipment and ultra-sophisticated electron microscopes have buttressed the arsenal of tools to conduct protein and nucleic acid tests, above all the identification of anomalies and abnormalities. Precision remains a key driver in a field where a margin of 1/1,000 can be a serious error, and destroy the integrity of a unique sample. Another enduring concern is sterility, especially RNAse contamination.
Blood doping benefits endurance athletes (notoriously, but not only, cyclists) by raising the red blood cell (rbc) count or haematocrit, and so increasing the oxygen supply to the muscles. It is one of the most difficult types of drug abuse to detect. Awareness of blood doping was raised in the popular press recently when comments were made about the impressive nature of China’s Ye Shiwen’s Olympic gold medal wins and with Lance Armstrong’s (cycling’s famous winner of seven Tours de France after surviving advanced testicular cancer) sudden decision to drop his fight against the US Anti-Doping Agency’s drug charges. Hematocrit levels can be raised by a variety of methods ranging from legal altitude training, to the banned use of autologous blood transfusions and erythropoietin (EPO) injections.
Detection of these banned methods is extremely difficult and the fight against them is being waged in a number of ways. The UCI’s (cycling’s governing body) lines of defence include simply demonstrating possession of banned substances and monitoring hematocrit levels, with a limit set at 50% (normal being 41–50% for men).
Some early success was had with testing urine to distinguish pharmaceutical EPO from the nearly identical natural hormone by isolectric focusing, though its accuracy has been questioned with claims that it is not possible to distinguish pharmaceutical EPO from other unrelated proteins that are present in urine after strenuous exercise or as the result of sample degradation and bacterial contamination.
At present, tests that provide indirect evidence of autologous blood transfusion (where the athlete withdraws and then re-injects his own blood) are under development and involve looking at the ratio of immature to mature red blood cells and might also include the measurement of 2,3-bisphophoglycerate (2,3-BPG). As 2,3-BPG degrades over time, stored blood used for autologous transfusions would have less than fresh blood and so levels of 2,3-BPG lower than normal may then indicate blood doping by this method. The presence of plasticizers in the blood (from the IV bags in which blood is stored) has also been used as evidence of blood doping.
While these advances in the detection of blood doping are being made it is tempting to think that we have got there, that the cheats will be caught. However, in the high-stakes world of elite athletes this would be a naive hope: the possibility of athletes subjecting themselves to EPO gene therapy – so called gene doping – has been suggested and methods for the detection of transgenic DNA following in vivo gene transfer are already being developed.
Malaria 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
Sepsis frequently results in acute kidney injury (AKI). Although AKI markedly contributes to mortality in sepsis, its diagnosis is frequently delayed due to limitations of current biomarkers of renal impairment. Neutrophil-gelatinase-associated lipocalin (NGAL) has been demonstrated to be a biomarker of early AKI. This review analyses the potential use of NGAL in sepsis.
by Dr W. Huber, Dr B. Saugel, Dr R. M Schmid and Dr A. Wacker-Gussmann
Pathophysiology, definition and epidemiology of sepsis
Sepsis is a clinical syndrome characterised by systemic inflammatory response to infection [1-2]. Incidence of sepsis has increased by a factor of four within the last three decades, with an estimated incidence of 650,000 cases per year in the USA. SIRS (systemic inflammatory response syndrome) describes a similar inflammatory reaction to non-infectious aetiologies such as poly-trauma, acute pancreatitis and burns. Apart from different aetiology, sepsis and SIRS share a common definition requiring two or more of four criteria of systemic inflammation (fever/hypothermia, tachycardia >90/min, tachypnoe >20/min or paCO2<32mmHg and leukocytosis (>12G/L) or leukopenia (<4G/L) [Table 1], [1-2].
Pathophysiology of sepsis is mainly attributed to imbalanced and generalised release of pro-inflammatory mediators resulting in impaired circulation, tissue injury and organ failures up to multiple-organ-dysfunction-syndrome (MODS). Despite strong evidence for therapeutic efficacy of early causative therapy (treatment of infection source), antibiotics and several supportive strategies, mortality from severe sepsis and septic shock remained up to 20-50% in recent sepsis trials [1-2]. There is an ongoing debate on the benefits of supportive strategies such as hydrocortisone, intensified-insulin-therapy and immunomodulation. However, there is strong consensus about the paramount importance of early sensitive diagnosis and staging of sepsis (severe sepsis and septic shock) in order to initiate appropriate monitoring and therapy as early as possible. In general, patients with severe sepsis will require intensive care and haemodynamic monitoring to optimise circulation.
Diagnoses of severe sepsis and septic shock are mainly based on the evidence of organ failure and emphasize the impact of circulatory failure. The impact of different organ failures on outcome of ICU-patients is substantiated by numerous studies [1-5)]. Interestingly, renal and liver failure were among the organ failures with the most pronounced impact on outcome in several studies [3-5]. At first glance, this might be surprising. However, circulatory and respiratory failure can be easily detected at early stages of severe sepsis, and symptomatic therapy of these organ failures is the main target of intensive care. By contrast, renal and liver failure remain underrated and 'late-stage-diagnosed losses of organ function' in the development of MODS. Difficulties in early detection of renal and hepatic failure by traditional markers has probably also resulted in their under-representation in scoring-systems:
Regarding renal failure, APACHE-II and the SOFA-score are mainly based on absolute serum creatinine values. However, the use of serum creatinine as a marker in these scores and particularly as an early marker of septic renal failure is limited by a number of drawbacks: serum levels of creatinine are dependent on age, gender, muscle mass and race. Furthermore, in case of impaired glomerular filtration, serum creatinine levels can be lowered by tubular secretion, which contributes to the phenomenon of the 'creatinine-blind-range' of renal failure: glomerular-filtration rate (GFR) can decrease to about 50% with serum creatinine levels staying within the normal range. Numerous formulae for GFR estimation slightly improve this drawback. However, GFR formulae are neither part of sepsis definitions nor are they included in SAPS-II, SOFA- and APACHE-II-score. Even the more recent Acute-Kidney-Injury-Network (AKIN) definition of AKI rejected GFR, which has been included in the previous RIFLE-classification (RIFLE: Risk, Injury, Failure; Loss, End-Stage Renal Disease). RIFLE and AKIN as well as the new KDIGO-definition (KDIGO: Kidney Disease: Improving Global Outcomes) are mainly based on changes in serum creatinine compared to baseline values, which are 'known or presumed to have occurred within the prior seven days' [6]. Comparison with a baseline value which is not known in a substantial percentage of patients remains a major problem of these definitions. In general, their usefulness is substantiated as consensus definitions for acute changes in renal function within 2-7 days after the first measurement of serum creatinine rather than being highly sensitive for early AKI. This also relates to the fact that increased serum creatinine on ICU admission of a septic patient might result from constant chronic renal impairment as well as acute renal failure in a patient with previously normal renal function. Both, acute and chronic renal impairment have been demonstrated to significantly influence outcome, albeit to a different degree, with patients with AKI more frequently requiring mechanical ventilation [4, 7].
In the context of sepsis, specification of renal impairment is particularly important: acute septic renal impairment results in markedly worse prognosis, classification as severe sepsis and intensified monitoring in an ICU. By contrast, stable chronic renal impairment in a patients just fulfilling two of the four sepsis criteria would be a minor risk factor contributing to outcome similar to older age. Being a marker of function rather than of injury remains the major drawback of serum creatinine for differentiation of renal impairment.
Approaches to early detection of AKI
Systematic efforts have therefore been made to characterise markers of early renal injury. Using several established animal models of acute renal injury (e.g. ischaemia, nephrotoxic medication including contrast-medium), up-regulation of a number of potential genes has been demonstrated as a short-term reaction to experimental acute renal injury [8]. Among those up-regulated genes and a number of other biomarkers, NGAL, Kidney-Injury-Molecule-1 (KIM-1), interleukin-18 (IL-18) and cystatin C have been most intensively studied. Cystatin C provides characteristics most similar to creatinine: this marker is a cysteine proteinase inhibitor synthesised in all nucleated cells and freely filtered by the glomerulus. The major adavantages over serum creatinine are that cystatin C is not secreted by the tubulus and that it is not affected by age, gender, muscle mass and race. However, with increased levels of cystatin C resulting from accumulation due to decreased glomerular filtration, cystatin C remains as a marker of decrease in renal function rather than a biomarker of early kidney injury. Several studies suggest its slightly earlier (within 24h?) detection of AKI compared to serum creatinine.
Another ‘candidate molecule’ for early detection of AKI is IL-18, a pro-inflammatory cytokine that is induced in the proximal tubule and detected in urine after AKI. In clinical settings, increase in urinary levels within 6h and peak-values within 12h have been demonstrated in cardiopulmonary bypass patients with AKI after 48h according to serum creatinine.
KIM-1 is a transmembrane protein that is markedly over-expressed in the proximal tubule after ischaemic or toxic AKI. A number of clinical studies suggest earlier detection of AKI by KIM-1 compared to serum creatinine, e.g. with elevated urinary KIM-1 levels 12h after paediatric cardiac surgery and prediction of renal replacement therapy (RRT) and mortality in AKI.
NGAL
The most promising biomarker for early acute kidney injury at present is NGAL, which is the profuct of one of seven genes markedly up-regulated in a ischaemia-reperfusion mouse model [8]. NGAL is a 178 amino-acids polypeptide expressed by neutrophils and other epithelial cells including the proximal tubule. NGAL provides several physiological functions including bacteriostatic (depriving bacteria of iron essential for growth), antioxidant (stops free and reactive iron from producing oxygen free radicals) and growth-factor properties (regulates cell proliferation, apoptosis, differentiation). Furthermore, there is a possible rescue role in other epithelia (breast, uterus), and NGAL also is overexpressed in some epithelial tumours.
Regarding its potential clinical use, NGAL has been validated as an early biomarker of AKI induced by cisplatin, contrast-media and cardiac surgery as well as a screening marker for patients at risk in the emergency department (ED) and ICU. In these settings, urinary and plasma levels of NGAL after 2h-12h were significant predictors of AKI defined by later increases in serum creatinine within 24-48h. Depending on setting and methodology, best predictive capabilities of NGAL were found for cut-off values between 50 and 150 µg/L. In a large ED study in 635 patients, urinary NGAL levels clearly differentiated between acute (markedly elevated NGAL) and chronic (not elevated NGAL) renal impairment, whereas there was substantial overlap of serum creatinine values for both groups ([9].
These abilities to discriminate between acute and chronic renal impairment might be particularly useful in patients with sepsis [Figures 1 and 2]. With assessment of renal function in ICU patients based on serum creatinine, normal creatinine levels might be ‘false negative’ and will increase as late as after 48h. On the other hand, increased values of creatinine can result from stable chronic renal impairment. Misinterpretation of these values – ‘false positive’ for septic renal failure – might result in inappropriate allocation of resources, e.g. efficacy for most of the supportive measures in sepsis has been demonstrated predominantly for patients at high risk and with severe sepsis, whereas side effects might outweigh the benefits in patients with less pronounced sepsis.
A potential role for NGAL in sepsis has been suggested in several clinical studies. In 143 paediatric ICU patients Wheeler et al. demonstrated that septic shock, but not SIRS, resulted in a significant elevation of NGAL compared to controls [10]. Furthermore, NGAL on admission was significantly higher in children developing AKI within seven days after admission compared to children without AKI. Serum levels of NGAL and creatinine did not correlate on day one after admission.
A study in 971 ICU patients investigated the predictive capabilities of nine biomarkers on admission regarding severe sepsis within 72h. The best predictive capabilities were found for NGAL, whereas D-dimer, BNP and CRP were of limited use. A score based on NGAL, IL-1-receptor-antagonist and protein C levels significantly distinguished four groups of patients developing no sepsis, severe sepsis, septic shock and death [11]: the area under the curve for the score derived from these three biomarkers was 0.80 for severe sepsis, 0.77 for septic shock and 0.79 for death.
Another recent study found significantly elevated plasma NGAL levels within
4 hours after admission in septic as well as non-septic-patients with AKI according to RIFLE-criteria compared to patients without AKI [12]. Increases in NGAL were even more pronounced in septic compared to non-septic AKI patients. Similarly, urinary NGAL-levels were higher in septic compared to non-septic patients without AKI [13], suggesting that cut-off-values for NGAL to predict AKI might be higher than for non-septic patients.
In summary, clinical applications of NGAL in sepsis comprise early detection of AKI in patients with normal serum creatinine (NGAL+, crea-) compared to patients without renal impairment (NGAL-, crea-). Furthermore, NGAL might be useful to distinguish patients with stable chronic renal impairment (NGAL-, crea+) from patients with ongoing or ‘acute on chronic’ renal injury (NGAL+, crea+) [Figure 1].
With regard to sepsis, more sensitive detection of AKI (NGAL+, crea-) would result in staging as ‘severe sepsis’ instead of sepsis in patients without other organ failures [Figure 2]. Early detection of AKI might help to allocate additional causative (antimicrobial therapy, intervention) and supportive measures for sepsis as well as specific measures to prevent further renal damage. These attempts include intensified haemodynamic monitoring to optimise fluid load, avoidance of further nephrotoxic medications and procedures (contrast-application) or at least prophylactic approaches such as hydration or administration of theophylline or acetylcysteine [13]. Allocation of these resources according to significant predictors and avoidance of further renal impairment carries a high potential for cost effectiveness as emphasised by a number of studies.
Further studies are required to validate that early determination of NGAL improves diagnosis and outcome in septic and non-septic patients at risk of AKI. Future studies should also investigate if including NGAL into scoring (APACHE-II, SOFA, SAPS-II) systems improves their predictive capabilities.
References
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2. German Sepsis Society. German Interdisciplinary Association of Intensive Care and Emergency Medicine. Prevention, diagnosis, therapy and follow-up care of sepsis: 1st revision of S-2k guidelines of the German Sepsis Society (Deutsche Sepsis-Gesellschaft e.V. (DSG)) and the German Interdisciplinary Association of Intensive Care and Emergency Medicine (Deutsche Interdisziplinäre Vereinigung für Intensiv- und Notfallmedizin (DIVI)). Ger Med Sci. 2010 Jun 28;8:Doc14.
3. Chertow GM et al. J Am Soc Nephrol 2005 Nov;16(11):3365-70.
4. Metnitz PG et al. Crit Care Med 2002 Sep;30(9):2051-8.
5. Kramer L et al. Crit Care Med 2007 Apr;35(4):1099-104.
6. KDIGO Clinical Practice Guideline Acute Kidney Injury. Kidney International Supplements 2012; 2: 1-138.
7. Walcher A et al. Ren Fail 2011;33(10):935-42.
8. Mishra J et al. J Am Soc Nephrol 2003 Oct;14(10):2534-43.
9. Nickolas TL et al. Ann Intern Med 2008 Jun 3;148(11):810-9.
10. Wheeler DS et al. Crit Care Med 2008 Apr;36(4):1297-303.
11. Shapiro NI et al. Ann Emerg Med 2010 Jul;56(1):52-59.e1.
12. Lentini P et al. Crit Care Res Pract 2012: 2012:856401. Epub 2012 Feb 14.
13. De Geus HR et al. Am J Respir Crit Care Med 2011 Apr 1;183(7):907-14. Epub 2010 Oct 8
14. Huber W et al. Radiology 2006; 239(3):793-804.
The authors
Wolfgang Huber MD, Bernd Saugel MD, Roland M Schmid MD
II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Ismaningerstr. 22, D-81675 München, Germany
and
Annette Wacker-Gussmann MD
Universitätsklinik Tübingen, Kinderheilkunde und Jugendmedizin, Abteilung für Neonatologie, Calwerstr. 7, D72076 Tübingen, Germany
Correspondence to Wolfgang Huber
e-mail: wolfgang.huber@lrz.tu-muenchen.de; Tel: +0049 (0) 89 4140-5478
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