Introduction

Malaria is endemic in 99 countries worldwide, and several million people travel from non-endemic countries to malaria-endemic regions each year [1]. Although more than 7000 cases of imported malaria are reported annually both in European countries and in the United States, malaria remains unusual in non-endemic countries and its clinical presentation is often nonspecific [2,3]. Misdiagnosis which remains too frequent, resulting in delays in antimalarial treatment [4]. A careful physical examination and interview, including any recent travel to a malaria-endemic region, are crucial for diagnostic evaluation. More than 90% of cases of imported malaria are due to Plasmodium falciparum, the species associated with severe morbidity and mortality. The remaining cases are due to P. ovale, P. vivax, P. malariae or, rarely, P. knolewsi, alone or in association with P. falciparum [4].

Malaria is a serious disease that must be diagnosed urgently. The reference routine test is microscopic examination of Giemsa-stained blood smears [5]. However, many centers cannot provide reliable round-the-clock smear-based diagnosis. Thus, a simple, sensitive test capable of reliably confirming or ruling out malaria would be a welcome addition to the diagnostic arsenal.

Rapid diagnostic tests (RDTs) that detect malaria parasite proteins by immunochromatography were first developed 20 years ago as a complement to microscopic diagnosis [6]. RDTs detect a variety of proteins, including P. falciparum histidine-rich protein 2 (PfHRP2) and P. falciparum lactate dehydrogenase (PfLDH), both specific to P. falciparum; and also Plasmodium LDH (pLDH) and aldolase, enzymes shared by the 5 human pathogenic Plasmodium species [6]. Such tests are now increasingly used outside of malaria-endemic areas [7–9]. Numerous studies including a Cochrane meta-analysis assessed the accuracy of RDTs for diagnosis of malaria in endemic settings [10–14]. Studies were conducted in Europe among travelers returning from endemic areas [15,16] but results are limited to P. ovale, P. malariae and P. vivax, and shown large differences in performance [9]. Tests available in non-endemic countries bear the CE (European Conformity) label but their performance is extremely variable; in addition, they have mainly been tested in endemic areas and have never been compared in the same study [17].

In 2008, WHO launched a comparative study of some RDTs on selected samples with P. falciparum and P. vivax [18] but studies using clinical samples are the most informative ones regarding test performance in routine use. Previous studies of patients presenting to emergency rooms in Europe with suspected imported malaria were all retrospective and compared only 1 or 2 RDTs with standard microscopic methods [15,16,19–21]. We report the results of a multicenter study of the diagnostic performance of four RDTs selected among those most widely used in Europe to diagnose imported malaria, performed in usual care settings in non-endemic areas. Thick and thin blood smears were considered as the gold standard and discordant results were explored in an attempt to obtain an analytical explanation.

Methods

Results

A total of 1311 patients were included in the study (Table 1). Their mean age was 32.7 years (SD: 17.1) and 54% of patients were male. Although 90.2% of patients were living in France, 51.7% originated from malaria-endemic areas. Most patients had returned from Africa (85.0%, n=1060).

Results of the reference thin blood smear test were missing in 18 cases, and RDT results were missing in five cases (Figure 1); thus, 1288 patients were included in the analysis. Malaria was ruled out in 914 cases (71%) including 4 cases in which only P. falciparum gametocytes were detected. The reference tests confirmed malaria in 378 cases (28.8%). The species distribution was as follows: P. falciparum in 340 cases (89.9%), P. ovale in 17 cases (4.5%), P. vivax in 11 cases (2.9%), P. malariae in 8 cases (2.1%), P. falciparum associated with P. ovale in 1 case (0.3%), and P. falciparum associated with P. malariae in 1 case (0.3%). Median parasite density was 11 250/microliter (p/µl; range 8 to 9 000 000 p/µl). Parasite density was above 2000 p/µl in 59% of positive samples (n=221).

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Figure 1. Flow diagram.

https://doi.org/10.1371/journal.pone.0075486.g001

The kappa value for thin smear readings was 0.92 (0.76–1).

All the RDT results met the validation criteria. Figure 1 shows the number of results available for each of the four tests.

Considering P. falciparum, the sensitivity of the three RDTs targeting PfHRP2 was 96% (Table 3). P. falciparum malaria was missed in 8 of 337 cases with PfHRP2-test₃, 9 of 336 cases with PfHRP2-test₂, and 10 of 336 cases with PfHRP2-test₁. However, the specificity of PfHRP2 detection was 97% whatever the RDT. The PPV and NPV of these 3 tests (PfHRP2-test₁, PfHRP2-test₂, and PfHRP2-test₃) were 90% and 98% respectively. The sensitivity of PfHRP2 detection was related to parasite density and depended on the RDT used. Post-hoc analyses showed that sensitivity at parasite densities lower than 200 p/µl was 85% (28/33) with PfHRP2-test₁, 88% (29/33) with PfHRP₂-test₂ and 94% (31/33) with PfHRP₂-test₃. Sensitivity at parasite densities between 200 and 2000 p/µl was 91% (48/53) with PfHRP2-test₁ and PfHRP₂-test₂, and 92% (49/53) with PfHRP₂-test₃. Sensitivity was 100% with all 3 tests when parasite density was above 2000/µl. Because of the small test-group size, no statistical analysis was possible.

Sensitivity for PfLDH detection was 83%, missing 53 of the 337 cases of P. falciparum malaria. Specificity was 100%. The PPV and NPV of this test were 98% and 80% respectively (Table 3). Post-hoc analyses showed that sensitivity was low (41/85, 48.2%) when parasite density was below 2000 p/µl, but increased to 96.7% (238/246) when parasite density exceeded 2000 p/µl.

The RDTs targeting P. vivax LDH (PvLDH) had a specificity of 99% and a sensitivity of 82% or 91%, depending on the test (Table 3).

The sensitivity of P. falciparum pan-antigen detection was 73.6% for aldolase, and ranged from 69.5% to 81.3% for pLDH, depending on the test (p<0.001) (Table 4).

Regarding non-falciparum species, aldolase was detected as a pan-antigen in 57% of microscopically positive samples, whereas the sensitivity of pLDH detection ranged from 60% to 69%, depending on the test (Table 4). Specificity ranged from 99% to 100%, depending on the test (Table 4).

False-positive results are shown in Table 5. Most occurred with PfHRP2, and PCR was usually negative.

False-negative RDT results (relative to microscopy and PCR) are shown in Table 6. Parasitaemia was between 8 p/µl and 117 000p/µl. Except for the PfLDH test/pLDH-test1 (Optimal-IT), most false-negative results involved P. ovale or P. malariae. Antimalarial drugs were detected in 21.7% to 33.9% of the false-negative samples, with no predominance of a particular drug.

Discussion

We prospectively evaluated the performance of four malaria RDTs on a large panel of samples (n=1288) obtained from travelers returning from malaria-endemic areas to France.

The overall sensitivity of the RDTs for Plasmodium infection (at least one target detected) was higher than 93%, possibly owing to the preponderance of P. falciparum in the samples studied, and to the good sensitivity of PfHRP2 detection for falciparum malaria, as reported by Abba et al. [10].

The PfHRP2-based tests emerged as a reliable alternative to routine microscopy for the diagnosis of P. falciparum malaria, and were more sensitive than the PfLDH-based tests. Taking microscopy as the standard, the sensitivity of the PfHRP2-based tests was 96% (95% CI, 94%-98%), which is higher than the threshold of 95% recommended by the World Health Organization (WHO) [29].

More than 60 RDT brands and 200 different products have already been developed. WHO and the Foundation for Innovative New Diagnostics (FIND) evaluated the sensitivity of 168 RDTs for the diagnosis of P. falciparum and P. vivax malaria [18]. This four-round evaluation showed that the tests submitted to Round 4 performed better, possibly reflecting improvements in test manufacture. The proportion of tests achieving a panel detection rate higher than 75% for a parasite density of >200/µl was higher in the latter study than previously reported. Performance varied widely among the tests at low parasite densities (below 200/µl), but the majority of tests gave high detection rates at densities of 2000 or 5000 parasites/μl [18], as also observed in our study. Only 3 among the 4 tests studied here were part of the WHO panel, namely Binax Now Malaria (PfHRP2-test₁, pan-aldolase test) with performance approximately 87% at 200 parasites/µl and 98% at 2000 p/μl); Core Malaria Pan/Pv/Pf (PfHRP2-test₂, pLDH-test₂), respectively 89% and 98%; and Optimal iT (PfLDH-test, pLDH-test₁), 50% at 200 p/µl and 98% at 2000 p/μl.

In areas where transmission rates are low, parasite density is likely to be lower in patients with symptomatic malaria [9,30]. Thus, test performance at a parasite density of 200/µl is an important criterion. Imported malaria is defined as the presence of Plasmodium sp. in blood, whatever the parasite density. This requires a highly sensitive diagnostic test, as false negativity may lead to a life-threatening delay in treatment. In non-endemic areas, RDTs are mainly useful for confirming infection suggested by low microscopic parasite density, and for determining the species. Nonetheless, despite an NPV of 98% and a sensitivity of 96% obtained here with the PfHRP2-based tests, a negative RDT result does not rule out imported P. falciparum malaria [31]. Of note, all but one of the false-negative PfHRP2 test results in our study involved samples with parasite densities below 2000/µl.

The specificity and NPV of the pLDH-based tests were both 100%. Specificity higher than 90% has been reported in endemic areas [14]. In contrast, the sensitivity of the pLDH-based test was only 83% (95% CI, 79%–87%) in our study, while previous reports have shown sensitivities ranging from 85.1% to 99% among travelers [17,32] and from 85.6% to 98% in endemic countries [12,13], values lower than those of the PfHPR2-based tests for P. falciparum malaria, especially when parasite density was low (<2000/µl) [18].

Thus, RDT performance relies on the choice of monoclonal antibodies [33]. Better sensitivity would allow malaria to be ruled out by a negative RDT result, thus avoiding inappropriate presumptive treatment in areas where this therapeutic strategy is applied [17,34-36].

Although severe malaria, including imported malaria, is almost always due to P. falciparum (and occasionally P. vivax or P. knowlesi, depending on the country visited) [37], species identification can guide the choice of treatment and avoid costly investigations. In our study, 11% of cases were due to non-falciparum species (P. ovale, P. vivax and P. malariae), and no cases of P. knowlesi infection were diagnosed.

The RDTs tested here were unable to identify non-falciparum species, especially P. ovale and P. malariae, as previously reported [38-40]. However, the sensitivity of the PvLDH-based tests was 91% (95% CI, 74%–100%) for P. vivax, especially with the PfHRP2 test₃ – pLDH test₃ (Palutop 4+) assay. Few PvLDH-based RDTs are available, and they have been evaluated in only a handful of studies. Meena et al. reported 76.6% sensitivity and 98.1% specificity for the FalciVax test (Orchid) [41]. Another study reported higher sensitivity (93.4%) for the SD Malaria Antigen P.v. test [39]. WHO reported a wide range of sensitivities for P. vivax detection with specific PvLDH RDTs, ranging from 5.9% to 100% (94.3% for the Core Malaria Pan/Pv/Pf) at higher parasite densities (2000/µl) [18].

False-positive results, representing 3% in our study, may have several causes. Nearly half (12/26) of the samples with positive RDT results and negative blood smears corresponded to PCR-confirmed P. falciparum malaria; these patients received no specific monitoring but were treated with antimalarial drugs in case of high suspicion of malaria. These samples may have been from patients with a suspected relapse of malaria, in which case their false positivity would indicate the persistence of PfHRP2 antigen in the bloodstream after treatment [42]; alternatively, they would confirm that RDTs can diagnose malaria attacks earlier than microscopy. We were unable to test these hypotheses. Cross-reactivity due to self-antibodies such as rheumatoid factor, especially in RDTs in which the conjugate is an IgM antibody [43], is also possible, as the manufacturers of the tests studied here do not specify the conjugate isotypes, and we were unable to screen the samples for rheumatoid factor or HAMA (human antimouse antibodies). A possible impact of gametocytes on RDT specificity [44,45] could not be excluded either, as too few samples contained gametocytes and no asexual forms to draw firm conclusion.

The choice of blood smears as the reference diagnostic test may represent a limitation of our study, as PCR has been proposed as the gold standard [46]. However, clearance of plasmodium DNA from the bloodstream following antimalarial treatment should be further studied to support and validate this option [47].

False-negative results for P. falciparum were observed mainly with the PfLDH-based test. Some of these samples were from patients treated with antimalarial drugs before their inclusion in the study, as shown by plasma drug assays; however, these treatments were not declared by the patients concerned, and their timing could not be determined. Dead parasites were observed on microscopy, but PfLDH production could have been halted by therapy [5,26], possibly contributing to the good specificity of the pLDH test [48].

The low sensitivity of RDT tests may also related to low parasite density, which could also explain the false-negative results obtained with the PfHRP2 detection tests, although some parasites isolated in South America and Africa have been shown not to produce PfHRP2 [49,50]. In such cases PfLDH detection would be of interest [51].

In conclusion, the three PfHRP2-based RDTs tested here showed high sensitivity and acceptable specificity for the diagnosis of imported and uncomplicated P. falciparum malaria, and thus appear to be a reliable, rapid and simple first-line diagnostic option for this potentially life-threatening disease, particularly in emergency settings. However, RDTs alone cannot rule out malaria, meaning that negative results must be confirmed by microscopy or PCR, and patients must be kept under medical supervision until the result is obtained [52,53].

Supporting Information

Acknowledgments

We thank the patients and guardians, and the personnel of the study centers. We also thank Delphine Prieur (Unité de Recherche Clinique Paris Nord) for the implementation and monitoring of the study. We thank David Young for his editorial assistance.