Background
Travellers from industrialized countries and inhabitants of malaria-endemic regions clearly represent two distinct worlds of malaria [
1]. The global burden of malaria is largely carried by the world's malaria-endemic regions with as many as 500 million cases annually and a death toll of 1 to 3 million children each year. Severe malaria in areas of endemicity is associated with a mortality of 15 to 40% [
2,
3]. In many malaria-endemic regions, strict triage for admission to ICU facilities must be applied because the ICU capacity is usually limited. Recently, a 5-point Coma Acidosis Malaria (CAM) score based on only acidosis (base deficit) and cerebral malaria (measured with Glasgow Coma Scale) was introduced, which could identify adult patients with severe malaria who were at high risk of death [
4].
In striking contrast, in non-endemic industrialized countries malaria is only seen as an occasionally imported disease [
5] and is usually associated with a low case-fatality rate [
6,
7]. Even in the pre-artesunate era, the mortality of severe malaria in non-endemic regions was significantly lower when compared with regions of malaria endemicity [
6‐
8], probably reflecting the availability of adequate supportive care facilities in industrialized countries.
Industrialized countries, however, have to face other -more trivial- problems. For instance, the expertise on diagnosis and treatment of malaria is usually focussed in some specialized hospitals and institutes but many ill-returning travellers may present to non-specialized hospitals or even general practitioners. Making a proper diagnosis of malaria may be troublesome under these circumstances, for instance, by lack of experience in the examination of malaria thick and thin blood smears and in the assessment of parasite load. These non-specialized centres therefore often rely on rapid diagnostic tests for the diagnosis of malaria [
9]. Although sensitive in diagnosing
P. falciparum malaria, these rapid tests do not provide any information about the severity of the infection. Moreover, although artesunate, which is now considered the parenteral drug of choice for treatment of severe falciparum malaria, is available as an orphan drug in The Netherlands, it is currently only in stock in some specialized centres but certainly not available in every Dutch hospital. Some of these general hospitals do not even have any drug in stock for the treatment of malaria [
10]. To prevent unnecessary delay in diagnosis of severe malaria and institution of proper parenteral treatment, a simple, well-validated, laboratory-based biomarker that predicts or excludes severe disease accurately would be of great help for those clinicians occasionally dealing with febrile travellers returning from malaria endemic regions. These clinicians have to decide on subsequent oral anti-malarial treatment or a timely referral to a specialized centre for high-level monitoring and intensified parenteral treatment. In the present study, the diagnostic accuracy of plasma soluble Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), neopterin and procalcitonin was evaluated as potential markers for malaria disease severity in travellers with imported malaria. These bio-substances are all involved in the systemic pro-inflammatory response of the host to invading pathogens. Some of these biomarkers are already in use for the diagnosis and follow-up of sepsis or used in treatment algorithms, resulting in a successful reduction of antibiotic use and duration [
11,
12].
Methods
Study population
The Harbour Hospital is a 161-bed general hospital located in Rotterdam. It also harbours the Institute for Tropical Diseases, which serves as a national reference centre. In the period 1999-2008 almost 500 cases of imported malaria were diagnosed [
13]. For the majority of these cases, demographic, clinical and laboratory data and serum samples were available. For the present study, a representative sample of this cohort was taken and analysed.
Definitions
Patients were classified as having severe
P. falciparum malaria if they met one or more of the WHO criteria for severe malaria, as modified by Hien
et al[
14]:
-
A score on the Glasgow Coma Scale of less then 11 (indicating cerebral malaria).
-
Anaemia (haematocrit < 20%) with parasite counts exceeding 100,000/μl (roughly corresponding to 2% parasitaemia) on a peripheral blood smear.
-
Jaundice (serum bilirubin > 50 μmol/l) with parasite counts exceeding 100.000/μl on a peripheral blood smear.
-
Renal impairment (urine output < 400 ml/24 h and serum creatinine > 250 μmol/l).
-
Hypoglycaemia (blood glucose < 2.2 mmol/l).
-
Hyperparasitaemia (> 10% parasitaemia).
-
Systolic blood pressure < 80 mm Hg with cold extremities (indicating shock).
Study design
In previous studies [
6,
13,
15] these severity criteria were also used to define severe malaria in non-immune travellers. In the present study the occurrence of severe malaria was considered a primary end-point. This contrasts with the design of many studies in patients with severe malaria in regions of malaria endemicity where the severity criteria are used as an entry criterion. In the present study, plasma lactate was used as a surrogate parameter for acid-base dysbalance and reference biomarker. It was evaluated in a previous study in non-immune travellers with imported malaria [
15]. The diagnostic performance of TREM-1, procalcitonin and neopterin for malaria disease severity was compared with that of plasma lactate, which is routinely measured at the Institute for Tropical Diseases in ill-returning travellers.
Procedures
On admission, blood samples were taken for analysis of the red blood cell count, haematocrit, white blood cell count, platelet count, serum electrolytes, total bilirubin, serum creatinine, liver enzymes, and blood glucose. In addition, a serum sample was taken on admission which was stored at -20°C until analysis. For the determination of plasma lactate, a separate blood sample was drawn on admission without congestion and placed on melting ice after which it was immediately analysed after isolation of plasma. Malaria was diagnosed by QBC (Quantitative Buffy Coat) analysis, by a rapid diagnostic antigen test for malaria (Binax NOW® Malaria Test, Binax Inc., Maine, USA) and by conventional microscopy of stained thick and thin blood smears. In case of P. falciparum infections, parasite density was determined. When the parasitaemia was less than 0.5% infected erythrocytes, parasites were counted per 100 leucocytes in thick smears. When the parasitaemia was equal or higher than 0.5% infected erythrocytes, infected erythrocytes were counted in thin blood smear and expressed as a percentage of the total erythrocytes. The number of parasites per microliter was subsequently calculated from these data.
TREM-1 and neopterin levels were determined in serum samples using commercially available ELISA tests (R&D Systems, Abingdon, UK; DRG, Marburg, Germany, respectively). Procalcitonin levels in serum samples were determined using a commercially available EIA test (VIDAS BRAHMS Procalcitonin, bioMérieux, Lyon, France). All tests were performed according to manufacturer's instructions. Detection limits were 3.88 pg/ml for TREM-1, 0.2 ng/ml for neopterin and 0.05 ng/ml for procalcitonin, respectively. According to the manufacturers, normal serum values are < 100 pg/ml for TREM-1, < 3 ng/ml for neopterin and < 0.1 ng/ml for procalcitonin.
Statistical methods
For comparison between groups, the Mann-Whitney U-test was used and p-values of < 0.05 were considered statistically significant. The diagnostic performance of each biomarker was reported as sensitivity, specificity, positive and negative predictive value for severe P. falciparum malaria and their corresponding 95% confidence intervals. Of each test a Receiver Operating Characteristic (ROC) curve, a graphical plot of sensitivity (true positive rate) versus 1-specificity (false positive rate), was constructed as a summary statistic and the area under the ROC curve (AUROC) and its corresponding 95% confidence intervals were calculated. Youden's index J (J = sensitivity+specificity-1) was used to choose the most appropriate cut-off point for each biomarker. All statistical analyses were performed using SPSS 15.0.
Discussion
Severe malaria is disreputable for its high case-fatality rate, but the outcome of severe
P. falciparum infections has significantly improved since the introduction of artesunate as first line treatment of severe malaria, in particular in developing countries [
2]. In industrialized countries such as The Netherlands, the case-fatality rate of imported malaria is low and fatal cases are only occasionally reported. In the present study, in which the biomarkers TREM-1, neopterin and procalcitonin were evaluated for their potential to be used as a marker for severe malaria disease upon admission. This contrasts with the design of many studies in regions of malaria endemicity where severe malaria is usually the entry criterion. For reasons of comparability, the same set of criteria for severe malaria was strictly applied for the diagnosis of severe malaria in this study, even though the study population comprised of presumably non-immune travellers and some authors even suggest a threshold of 5% in stead of 10% parasitized erythrocytes to define hyperparasitaemia in non-immune individuals.
The quantification of soluble TREM-1 levels on admission did not result in proper discrimination of severe
P. falciparum malaria from uncomplicated
P. falciparum malaria and non-
P. falciparum malaria. In contrast, travellers with severe
P. falciparum malaria had significantly higher levels of neopterin and procalcitonin on admission as compared with travellers with uncomplicated
P. falciparum malaria or non-
P. falciparum malaria, respectively. These findings correspond with the results of several other studies performed in semi-immune malaria patients living in malaria-endemic regions [
16‐
18]. When the ROC curve characteristics of neopterin and procalcitonin were compared to that of plasma lactate, the AUROC of neopterin appeared superior whereas the AUROC of procalcitonin appeared inferior to that of lactate, suggesting that neopterin provided the most accurate diagnostic performance for severe
P. falciparum malaria in this cohort of travellers.
Unfortunately, the applicability of these tests in the initial clinical assessment of patients with severe
P. falciparum malaria will probably be limited by the poor positive predictive value of neopterin and procalcitonin indicating that neither test can serve as a valuable tool for the diagnosis of severe
P. falciparum malaria. For illustration, applying a procalcitonin level > 0.9 ng/ml or a neopterin level > 10.0 ng/ml as a guide to intensified monitoring and treatment would result in more than 20 of 64 patients with uncomplicated
P. falciparum malaria receiving more intensive monitoring and treatment than strictly necessary. On the other hand, the high negative predictive value of both neopterin and procalcitonin suggests that these tests can still be of value by providing a tool for exclusion of severe disease. With either a procalcitonin level of less than 0.9 ng/ml or a neopterin level of less than 7.9 ng/ml in serum on admission as a cut-off point for severe
P. falciparum malaria, no patient with severe disease would have been denied access to high-level monitoring and intensive treatment. In a previous study, in which a semi-quantitative 'point-of-care' procalcitonin test as a diagnostic tool for severe
P. falciparum malaria was evaluated prospectively, all 6 patients with severe
P. falciparum malaria had procalcitonin values classified as either "moderate" or "high" (corresponding to a procalcitonin level ≥ 2 ng/ml), but never as "normal" or "low" [
12]. This is compatible with the findings of the current retrospective serum sample-based study in which procalcitonin was measured quantitatively.
Although severe or fatal malaria rarely results from infections with the non-sequestering
Plasmodium species
vivax,
ovale and
malariae, increased neopterin and procalcitonin serum levels were also observed in the majority of these patients, although levels were lower than compared with severe
P. falciparum malaria patients. Although speculatively, these observations suggest that the mechanism whereby neopterin and procalcitonin levels increase in malaria, is not specific for severe
P. falciparum malaria alone. Therefore, it may not accurately reflect the pivotal pathophysiological events in complicated
P. falciparum malaria, such as the sequestration of infected red blood cells in the microcirculation of vital organs and disturbance of microcirculatory flow. Whereas an increased plasma lactate level conceivably reflects a significant reduction in microcirculatory flow in vital organs, the elevated neopterin and procalcitonin levels are probably the result of activation of a common inflammatory host response evoked by infection with the respective
Plasmodium parasites. In fact, some reports even suggest that
P. falciparum malaria per se is not associated with a stronger host response than
P. vivax or
P. ovale malaria, but that the parasite burden of the causative
Plasmodium species may also modulate the extent of the host inflammatory response [
19].
In conclusion, although neither neopterin nor procalcitonin can probably serve as a useful single diagnostic tool for severe P. falciparum malaria, the high negative predictive value of both neopterin and procalcitonin may be helpful for a rapid exclusion of severe P. falciparum malaria on admission. This may be a valuable tool - particularly if available as a rapid diagnostic test - for physicians only occasionally dealing with ill-returned travellers and who need to decide on subsequent oral anti-malarial treatment or a timely referral to a specialized centre for high-level monitoring and intensified parenteral treatment.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RW participated in the design of the study and coordination, performed the experiments and the statistical analyses and drafted the manuscript.
MW participated in the statistical analyses and drafting of the manuscript.
PP participated in the design of the study.
JH participated in the design of the study and revising the manuscript.
RK is responsible for collection of patient materials and database management.
AB participated in the design of the study and revising the manuscript.
PG participated in the design and coordination of the study and in drafting and revising the manuscript.
All authors have seen and approved the final version.