Test characteristics of the SD FK80 Plasmodium falciparum/Plasmodium vivax malaria rapid diagnostic test in a non-endemic setting

Background The SD FK80 P.f/P.v Malaria Antigen Rapid Test (Standard Diagnostics, Korea) (FK80) is a three-band malaria rapid diagnostic test detecting Plasmodium falciparum histidine-rich protein-2 (HRP-2) and Plasmodium vivax-specific lactate dehydrogenase (Pv-pLDH). The present study assessed its performance in a non-endemic setting. Methods Stored blood samples (n = 416) from international travellers suspected of malaria were used, with microscopy corrected by PCR as the reference method. Samples infected by Plasmodium falciparum (n = 178), Plasmodium vivax (n = 99), Plasmodium ovale (n = 75) and Plasmodium malariae (n = 24) were included, as well as 40 malaria negative samples. Results Overall sensitivities for the diagnosis of P. falciparum and P. vivax were 91.6% (95% confidence interval (CI): 86.2% - 95.0%) and 75.8% (65.9% - 83.6%). For P. falciparum, sensitivity at parasite densities ≥ 100/μl was 94.6% (88.8% - 97.6%); for P. vivax, sensitivity at parasite densities ≥ 500/μl was 86.8% (75.4% - 93.4%). Four P. falciparum samples showed a Pv-pLDH line, three of them had parasite densities exceeding 50.000/μl. Two P. vivax samples, one P. ovale and one P. malariae sample showed a HRP-2 line. For the HRP-2 and Pv-pLDH lines, respectively 81.4% (136/167) and 55.8% (43/77) of the true positive results were read as medium or strong line intensities. The FK80 showed good reproducibility and reliability for test results and line intensities (kappa values for both exceeding 0.80). Conclusion The FK80 test performed satisfactorily in diagnosing P. falciparum and P. vivax infections in a non-endemic setting.

platform and are available in dipstick or cassette format. Signals are visible as coloured lines, comprising a control line (which indicates that the test has been performed well) and one or two test lines. The so-called two band tests generate a test line that targets P. falciparum by detecting either histidine-rich protein 2 (HRP-2) or P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH). The three band tests include a second target that is common to the four Plasmodium species, such as aldolase or pan-specific parasite lactate dehydrogenase (pan-pLDH). Apart from these common formats, there are also twoband tests detecting Plasmodium vivax-specific pLDH (Pv-pLDH) [6]. The SD FK80 P.f/P.v Malaria Antigen Rapid Test (Standard Diagnostics, Hagal-Dong, Republic of Korea), further referred to as FK80, is a three-band RDT that targets HRP-2 and Pv-pLDH. In areas where both P. falciparum and P. vivax are prevalent, this combination has the advantage to distinguish unequivocally between the two species, whereas a conventional three band P. falciparum/Pan species RDT may not. For instance, in case of a single P. falciparum infection, the FK80 P.f/P.v Malaria Antigen Rapid Test will only show a unique HRP-2 line; in this case the conventional three band P. falciparum/Pan species RDT may show (depending on the parasite density) both the HRP-2 (or Pf-pLDH line) and the pan-species line, a result which is also compatible with a P. falciparum/P. vivax mixed infection. In addition, it can be assumed that the affinity of a dedicated antibody targeting the enzyme of only a single species (Pv-pLDH in the case of P. vivax and the current FK80 RDT) is higher than the affinity of an antibody targeting the different enzymes of the four Plasmodium species (pan-pLDH). The aim of the study was to evaluate the performance of the FK80 in a non-endemic reference setting.

Study design
In this retrospective study, the FK80 was evaluated against a collection of stored samples obtained from international travellers, collected from January 1997 to December 2008. Tests were carried out at the malaria reference laboratory of the Institute of Tropical Medicine (ITM) Antwerp, Belgium. This study complied with the standards for the reporting of diagnostic accuracy studies (STARD) [7].

Patients and samples
Samples were selected from a collection of EDTA-blood samples stored at -70°C and obtained from patients presenting at the outpatient clinic of ITM for clinical suspicion of malaria. The patients were international travellers and, to a lesser extent, immigrants returning from visits to their native countries. In addition, samples sent by Belgian laboratories to ITM in the scope of the national reference function were included. The samples collected at ITM, were aliquoted and frozen at -70°C the day of collec-tion. Between collection and storage at -70°C, the samples remained at room temperature for a maximum of 10 hours, at ambient temperatures below 25°C. The 99 samples sent by Belgian laboratories to ITM for second opinion and confirmation were sent by mail and had been exposed to ambient temperature for the period of shipment, which was generally less than 24 hours and ranged to a maximum of 48 hours. The delays of shipment and processing before storage at -70°C had been validated before and were compliant with routine laboratory procedures. A representative number of Plasmodium-positive samples (n = 376) were selected, including the four malaria species with varying parasite densities and representative geographic distribution. Mixed infections were not included. In addition, samples without malaria parasites (as confirmed by microscopy and PCR) from symptomatic travellers suspected of malaria were included (negative samples, n = 40).

Reference method
Microscopy corrected by polymerase chain reaction (PCR) was used as the reference method. Standard microscopy was performed on thick blood films of all samples to diagnose malaria and to assess parasite density, and on thin blood films of positive samples to define the Plasmodium species. Thick blood films were stained with Giemsa (pH = 8) for 20 minutes, thin films with May-Grünwald Giemsa. The slides were examined by light microscopy using a × 500 magnification, according to the standard procedure at ITM. Parasite density was assessed by counting the number of asexual parasites against 200 white blood cells in a thick film, and converting the value to parasites/μl using the actual count or the standard of 8,000 white blood cell/μl [3]. Malaria diagnosis at Central Laboratory of Clinical Biology is accredited in accordance with the requirements of the ISO 15189:2007 norm. The laboratory technicians have received a detailed training and their performance and agreement are monitored by participation to internal and external quality control assessments. As a standard procedure, all positive slides for malaria and slides showing discordant results between different laboratory tests (microscopy, RDTs or PCR) are confirmed by a blinded second microscopist. Real-time PCR analysis was adapted from Rougemont et al [8] as described previously [9] and performed on all samples used in this study.

Test platform
The FK80 is lateral flow immunochromatographic RDT in a cassette format. Three lines are present, a control line which indicates whether the test is valid, a HRP-2 line and a Pv-pLDH line. According to the manufacturer's instructions, a single HRP-2 line indicates an infection with P. falciparum, a single Pv-pLDH line indicates an infection with P. vivax and a combination of a HRP-2 line and a Pv-pLDH line indicates a mixed infection with P. falciparum and P. vivax. The other Plasmodium species cannot be detected with this test. For the evaluation, test kits of lot number RDT 8002 (expiry date November 2010) were used.

Test procedure
Tests were according to the instructions of the manufacturer except that samples (5 μl) where loaded with a transfer pipette (Finnpipette, Helsinki, Finland) instead of the plastic loop supplied by the manufacturer and that a scoring system was used to assess the intensity of the test lines. In case the control line did not appear, the result was interpreted as invalid and the test was repeated. In order to score test line intensities, the scoring system of Bell and co-workers was adapted [10] and five categories were defined: none (no line visible), faint (barely visible line), weak (paler than the control line), medium (equal to the control line) or strong (stronger than the control line) [9]. To assure timely readings, tests were carried out in timecontrolled batches of ten samples. Readings were carried out at daylight assisted by a standard electricity bulb, between 20 and not beyond 30 minutes after application of the sample and buffer. Readings were performed by three subsequent observers, of whom the one who performed the test procedure was the first. Observers were blinded to the results of microscopy, PCR and to each others' readings. The results of the readings considered were based on consensus agreement, which means that a positive result was defined as a result read positive by at least two out of three different observers. When there was no consensus, results of the first observer were considered. Inter-reader reliabilities were assessed for the test results expressed as positive and negative readings as well as for the intensity readings. To assess test reproducibility, a panel of 19 samples (including nine P. falciparum samples, eight P. vivax samples, one P. ovale sample and one P. malariae sample) was tested on three successive occasions.

Definitions
Samples infected with P. falciparum and P. vivax species were considered separately: Tables 1 and 2 list the definitions of true positive and negatives and those of species mismatches. In the case of P. falciparum, samples with pure gametocytaemia were included among the positive samples.

Statistical analysis
Sensitivity and specificity were calculated for both P. falciparum and P. vivax with 95% confidence intervals (CI) and differences were tested for significance using the Pearson chi-square test or, when this was not possible, the Fisher exact probability test. A p-value of < 0.05 was considered as significant. Reliabilities for positive and negative readings and line intensities were calculated as percentage agreements for all three readers and kappa values for each pair of readers. Associations between line intensity readings and parasite densities were assessed for strength of association with Cramer's V for categorical variables.

Ease of use
Three experienced laboratory technicians scored the ease of use of the FK80 test and the clarity of manufacturer's instructions with a standardized list.

Ethical review
The study was reviewed and approved by the Institutional Review Board of ITM and by the Ethical Committee of Antwerp University, Belgium.

Sample collection
A total of 416 samples were selected, of which 99 samples were sent by Belgian laboratories to ITM for second opinion and confirmation. The samples were collected from January 1997 to December 2008. They were obtained in 416 patients, with a male-to-female ration of 2.19:1, and median age of 38.5 years (range 1 -84 years). Only a minority (eight patients, 1.9%) were children less than five years old. According to microscopy and after correction with PCR analysis, 178 of these samples were positive for P. falciparum, 99 for P. vivax, 75 for P. ovale and 24 for P. malariae. The results of microscopy were corrected in 11 out of the 376 positive samples (2.9%) and were uniquely related to P. vivax -P. ovale mismatches: in the final collection, four out of 99 (4.0%) P. vivax samples and seven out of 74 (9.5%) P. ovale samples had been identified as P. ovale and P. vivax by microscopy. In addition, 40 microscopic and PCR negative samples of symptomatic travellers were included in the panel.

Invalid test results
One of the 416 samples gave an invalid result at initial testing. After 30 minutes, there was no control line visible. Upon repetition, the test performed well.

Sensitivity and specificity
RDTs were performed between January and February 2009. Table 3 lists the test results of all samples. Test characteristics matched with species identification and parasite density for the detection of P. falciparum and P. vivax are listed in tables 4 and 5 respectively. For the detection of P. falciparum the sensitivity was 91.6% and the specificity was 99.2% for all samples combined. Sensitivity was higher at higher parasite densities, but the differences did not reach statistical significance. For the detection of P.
vivax the sensitivity was 75.8% and the specificity was 100% for all samples combined. Sensitivity at parasite densities ≤ 500/μl was significantly lower compared to sensitivity at parasite densities >500/μl (51.6% and 86.8% respectively (p < 0.001)). Eight out of the 416 samples resulted in a species mismatch, with 4/178 (2.2%) P. falciparum samples showing a Pv-pLDH line in addition to the HRP-2 line: three of them had parasite densities higher than 1% (69,953; 87,149 and 1,000,000/μl respectively) and all four patients were upon return from Africa (travel destination were Nigeria (n = 3) and Cameroon). Another four out of 199 (2.0%) non-falciparum samples showed a HRP-2 line (of which two P. vivax samples in addition to the Pv-pLDH line) ( Table 3). None of the P. ovale samples showed positive readings with the Pv-pLDH line.

Line intensities
For the HRP-2 and Pv-pLDH lines, respectively 81.4% (136/167) and 55.8% (43/77) of the true positive results were read as medium or strong line intensities. For HRP-2, faint lines occurred only in three samples: one P. falciparum sample with pure gametocytaemia and two non-falciparum samples. For Pv-pLDH, faint lines occurred only in eight samples: six P. vivax samples (four of them with a parasitemia lower than 500 parasites/μl) and two P. falciparum samples. Line intensity readings for both HRP-2

P. vivax P. falciparum, P. ovale, P. malariae or no parasites detected
Only Pv-pLDH True positive False positive or species-mismatch* None or only HRP-2 or both HRP-2 and Pv-pLDH False negative or species-mismatch** True negative *Non-vivax species diagnosed as P. vivax **P. vivax diagnosed as P. falciparum or as P. falciparum/P. vivax mixed infection

Ease of use
The FK80 was scored as easy to use and the instructions were scored as clear and simple to perform by all three technicians. The test was evaluated as practical and the clearance of the test strip was good. However, the clearance of the test strip was incomplete after the 15 minutes minimal incubation time recommended by the manufacturer. Good clearance was observed only after 20 minutes of waiting time.

Discussion
The present study evaluated the performance of the FK80 against a panel of whole blood samples from international travellers suspected of malaria. The retrospective design of the study has its limitations. For instance, whole blood specimens stored at -70°C were used, and the sta- *Unique HRP-2 line visible **One P. ovale and one P. malariae sample showed a unique HRP-2 line  [11]. However, no obvious differences in test performance were presently found for samples stored for several (> 5) years compared to those stored for a shorter periods (results not shown). In addition, a prospective evaluation of fresh and stored samples revealed similar results in case of the HRP-2 antigen detection [12]. Another limitation is the fact that a calibrated pipette was used for the transfer of the blood; with an expected better accuracy as compared to the kit's application loop. In addition, the diagnosis and evaluation were carried out in a reference setting, which makes extrapolation of the present results to field settings difficult [4,11]. Likewise, the ease of use was checked by an expert team and not by untrained end users in remote areas, and it is known that expert technicians tend to score tests kits more favorably [13].
The performance of the FK80 can best be compared to other tests by considering the HRP-2 and Pv-pLDH lines separately. For the HRP-2 test line in the diagnosis of P. falciparum, the sensitivities were in line with those reported in other HRP-2 tests in returned travellers, with sensitivities ranging from 80% tot 99%, depending on the setting and parasite densities [2,[14][15][16][17][18][19][20]. However, the FK80 did not meet the 95% sensitivity at 100 parasites/μl as recommended by the WHO [21]. Concerning the diagnosis of P. vivax, comparison is more difficult. There are three reports on the performance of the Pv-pLDH detection system in the SD FK70 test, a two-band P. vivax RDT marketed by the same company. Two field studies reported sensitivities of 96.4% and 93.4% respectively, with lower values at low parasite densities [22,23]. In addition, the FK70 was evaluated in the present reference setting on stored whole blood samples from travellers [6]: this study demonstrated an overall sensitivity of 88.0%, with sensitivities at parasite densities below and above 500/μl of 64.3% and 97.2% respectively. Comparison of the sensitivities of the FK80 Pv-pLDH detection with those reported for the pan-pLDH target are even more difficult, as the latter range from 1.5% to 97.0% [24,25]. Compiled sensitivities for the BinaxNOW ® kit for the diagnosis of P. vivax was reported as 69.6% [25]. With regard to specificity, there was no reaction of any of the P. ovale samples with the Pv-pLDH test line, but two P. vivax samples gave positive readings with the HRP-2 line in addition to the Pv-pLDH line. Of note is the Pv-pLDH positive result in four P. falciparum samples: in all cases PCR as well as travel destination argued against P. vivax co-infection, and three of them occurred at higher parasite densities. This is in line with earlier observations, in which occasional cross-reaction of P. falciparum at high parasite densities was observed in the FK70 [6].
The HRP-2 lines generally were of higher intensities as compared to the Pv-pLDH lines. The higher line intensities of the HRP-2 line as compared to the pan-pLDH and aldolase lines have been described previously [9,26]. For both the test lines, the line intensities of true positive results were higher as compared to the previously evaluated FK60 and FK70 tests [6,9].
The intended area of use of the presently evaluated FK80 RDT is confined to regions with both P. falciparum and P. vivax infections, such as Afghanistan or Korea. In such situation, the FK80 has an advantage over the traditional three-band combination RDTs that detect HRP-2 and panspecies specific pLDH of aldolase: unlike the FK80, their design does not allow to exclude a mixed infection in the case of P. falciparum samples that react with both the HRP-2 and the pan-species lines. The FK80 by virtue of its specific Pv-pLDH is able to distinguish accurately between infections caused by P. falciparum, P. vivax and both species combined. Accurate diagnosis of P. vivax is important in view of the need for specific therapy but may also be interesting because of the study of P. vivax malaria, because this infection may be serious [27], requires a specific treatment and is also notorious because of its high transmissibility [28,29]. In other regions where these two species co-exist, the presence of P. ovale and P. malariae can not be excluded and a three-band test with a pan-species pLDH will be a better choice. In the setting of travel medicine, the FK80 may help in distinguishing mixed P. falciparum -P. vivax infections and in the distinction between P. vivax and P. ovale. However, as in this setting cost is not a prohibitive factor, a standard three-band RDT followed by, in case of single pan-pLDH positivity, a twoband Pv-pLDH RDT such as the FK70 is an alternative to support the microscopic diagnosis between P. ovale and P. vivax [6].
In conclusion, the FK80 performed satisfactorily for the diagnosis of P. falciparum and P. vivax infections in a nonendemic setting, especially at higher parasite densities.

List of abbreviations
Ag: Antigen; CI: Confidence interval; DNA: Desoxy-ribonucleic acid; EDTA: Ethylene diamine tetra-acetic Acid; FHML: Faculty of Health Medicine and Life Sciences, Maastricht, The Netherlands; FK60: SD FK60 Malaria Ag P. falciparum/Pan test (Standard Diagnostics), a three-band "one-step" malaria rapid diagnostic test for the detection of P. falciparum and non-falciparum species, targeting the HRP-2 and pLDH antigens; FK70: SD FK70 Malaria Ag. P. vivax test (Standard Diagnostics), a two-band "one-step" malaria rapid diagnostic test for the detection of P. vivax, targeting the Pv-pLDH antigen; FK80: SD FK80 Malaria Ag. P. falciparum/P. vivax test (Standard Diagnostics), a three-band "one step" malaria rapid diagnostic test for the