Evaluation of a rapid diagnostic test specific for Plasmodium vivax

Transcription

1 Tropical Medicine and International Health doi: /j x volume 13 no 12 pp december 2008 Evaluation of a rapid diagnostic test specific for Plasmodium vivax Sun Hyung Kim 1, *, Myung-Hyun Nam 1, *, Kyoung Ho Roh 1,2, Hae Chul Park 3, Deok Hwa Nam 1, Gli Hong Park 4, Eun Taek Han 5, Terry A. Klein 6 and Chae Seung Lim 1 1 Department of Laboratory Medicine, College of Medicine, Korea University, Seoul, Korea 2 Department of Laboratory Medicine, College of Medicine, Konyang University, Daejeon, Korea 3 Laboratory of Neurophysiology, College of Medicine, Korea University, Seoul, Korea 4 Department of Biochemistry, College of Medicine, Korea University, Seoul, Korea 5 Department of Parasitology, College of Medicine, Kangwon University, Chuncheon, Kangwon Province, Republic of Korea 6 Force Health Protection, 18th Medical Command, US Army, Seoul, Korea Summary Plasmodium vivax is the only human malaria indigenous to the Republic of Korea (ROK). A rapid and sensitive diagnostic test (RDT) that detects P. vivax is appropriate for evaluating suspected malaria patients with no travel history abroad. The RDTs, SD Malaria Antigen P.v (SD diagnostic, Kyonggi, ROK) specific for P. vivax and the well documented OptiMAL (DiaMed, Cressier, Switzerland) were compared among 282 volunteers for specificity and sensitivity of P. vivax and Plasmodium falciparum malaria infections against Giemsa-stained blood smears read by an experienced microscopist. A total of 137 volunteers were diagnosed with P. vivax, 45 cases (returned travellers from overseas) were diagnosed with P. falciparum and 100 healthy volunteers were diagnosed as negative for malaria. Correspondingly, the SD Malaria Antigen P.v test identified P. vivax infections in malaria patients (93.4%) and (0%) healthy volunteers. Three patients identified with P. falciparum also were interpreted as P. vivax by the SD Malaria Antigen P.v test; however, these patients were later confirmed as mixed infections of P. vivax and P. falciparum by polymerase chain reaction. OptiMAL interpreted the three mixed infections only as P. falciparum and detected (94.9%) patients with P. vivax. The sensitivity of the SD Malaria Antigen P.v test decreased from 100% (>5000 parasite ll) to 81.3% (1 100 parasites ll) as parasitaemia levels declined. For the regions where P. vivax is the primary malaria parasite, the SD P. vivax-specific rapid diagnostic test may be useful for screening suspected malaria patients when sufficient material and human resources (e.g. trained microscopists) are unavailable for malaria diagnosis. keywords Plasmodium vivax, OptiMAL, parasite lactate dehydrogenase, SD Malaria Antigen P.v Introduction The rapid and accurate diagnosis of malaria is essential for reducing morbidity and mortality. The most reliable and standard method used for detecting malaria infections is by microscopic examination of Giemsa-stained thick and thin blood smears by a highly trained microscopist (Makler et al. 1998; Moody 2002). However, this method is labourintensive, time-consuming and is not the most efficient method, especially for mass screening. Microscopic examination, even conducted by an expert, requires interpretation and its sensitivity falls as malaria parasite densities *Both authors contributed equally to this study. drop. The specificity and sensitivity of rapid diagnostic tests (RDTs) using immunochromatography, such as OptiMAL (DiaMed, Cressier, Switzerland) and ICT (Binax Inc., Scarborough, ME, USA), have been evaluated in various epidemiological settings as an alternative to blood smears for malaria diagnosis (Beadle et al. 1994; Dietze et al. 1995; Craig & Sharp 1997; Pieroni et al. 1998; Iqbal et al. 1999, 2000, 2002, 2003; Cho et al. 2001; Congpuong et al. 2002; Lee et al. 2007; Ratsimbasoa et al. 2007). These tests are relatively costly but effective, quick and easy to use under field conditions. However, they have limitations: false-negative results, for example, which occur when patients have low parasitaemia; or the inability to differentiate Plasmodium vivax from Plasmodium ª 2008 Blackwell Publishing Ltd 1495

2 falciparum and mixed infections. In areas where only falciparum malaria occurs or non-falciparum malaria rarely occurs without co-infection of P. falciparum, RDTs that detect only P. falciparum are generally preferable based on their lower cost. In the Republic of Korea (ROK), P. vivax is the only indigenous malaria vector and accounts for 99.9% of all reported malaria cases where patients had no travel history abroad (Chai 1999; Yeom et al. 2007). Therefore, a RDT that detects only P. vivax or a combination RDT kit that targets both P. falciparum and P. vivax specific antigens is appropriate for suspected malaria patients in ROK who have not been abroad or who travelled to other malaria endemic regions. A recently developed malarial antigen-detecting RDT test kit, SD Bioline Malaria Antigen test, achieved 96.4% sensitivity and 98.9% specificity for P.vivax in ROK (Lee et al. 2007). In this study, we evaluated the sensitivity and specificity of a recently developed P. vivax and P. falciparum specific RDT that was based on parasite lactate dehydrogenase (pldh) and histidine-rich protein 2 (HRP-2), and compared it to the frequently used OptiMAL test. Materials and methods Samples Between April 2003 and August 2007, EDTA blood samples were collected from 182 (123 males, 59 females) Korean patients with microscopy-confirmed malaria, including travellers returning from malarious areas outside of Korea, and from 100 asymptomatic and aparasitaemic healthy volunteers (62 males, 38 females) who were seen at Korea University Ansan Hospital, ROK (Figure 1). Venous blood samples (5 ml) were stored at -80 C until assayed. All malaria patients and healthy volunteers gave informed consent under a human use protocol approved by the Human Use Ethical Committee, Korea University Ansan Hospital. Thick and thin blood films were prepared at the time of the blood draw in accordance with standard procedures, stained with Giemsa, examined by a trained microscopist and the Plasmodium species and parasite density determined. Parasitaemia levels were indirectly estimated using the number of parasites per 200 white blood cells (WBCs) on the blood film and WBC counts ll from an automatic blood cell counter (Cell-Dyn 4000; Abbott diagnostics, Abbott Park, IL, USA). OptiMAL Each blood sample was assayed using the OptiMAL test kit. This test, based on the immunological detection of pldh, differentiates falciparum and non-falciparum malaria and was performed according to the manufacturer s instructions. The immuno-capture assays were independently examined and the results of both assays recorded as positive or negative based on the observation of precipitated bands. SD Malaria Antigen P.f and SD Malaria Antigen P.v Two newly developed malarial antigen-detecting RDT test kits based on immunological detection of the HRP-2 specific to P. falciparum (SD Malaria Antigen P.f) and the pldh specific to P. vivax (SD Malaria Antigen P.v) (SD diagnostic, ROK) were tested. Assays were performed in accordance with the manufacturer s instructions and concurrently with OptiMAL tests. Kit components and blood Sample selection Visitors seen at the clinic with a fever history Visitors seen at the medical examination centre without medical problems Microscopic examination for malaria Malaria-positive (n = 182): Evaluation for species and density of parasites Malaria-negative volunteers (n = 100) Rapid diagnostic tests (RDTs) for malaria PCR was performed on discrepant results Figure 1 Trial profile showing sample selection, microscopic examination, rapid diagnostic tests performed ª 2008 Blackwell Publishing Ltd

3 specimens were used in the tests at room temperature. Whole blood specimens were dropped into the circular end of a test well with a 5 ll capillary pipette (provided), four drops of assay diluent added into the square diluent well, and results interpreted in min. Polymerase chain reaction for Plasmodium vivax and Plasmodium falciparum gene amplification Polymerase chain reaction (PCR) was conducted for three samples with non-concordant results from the RDT assays. Genomic DNA was amplified by PCR using the following oligonucleotide primers, which afforded amplification of a nucleotide region of the polymorphic region of the PvMSP- 1 gene of P. vivax: MSP-1A ( 5-GAGCCCTACTACTTG- ATGGTCC-3, Belem strain 1984) and MSP-1B ( 5-CCT TCTGGTACAGCTCAATG-3, Belem strain 2653), and the circumsporozoite protein gene of P. falciparum; PFCSP-1 ( 5-GGATCCGGAGGAAATGATGATGGAGA- T-3, M15505, 250) and PFCSP-2 ( 5-AAGCTTGTCTTT AGGTTTATCAGCAGA-3, M15505, 1236). Primers were used at a final concentration of 0.1 lm in a 100-ll reaction mixture (10 mm Tris-HCl, ph 8.3, 50 mm KCl, 1.5 mm MgCl 2 and 0.2 mm of each dntp) that contained 10 ll of DNA and 2.5 units of AmpliTaq polymerase (Perkin Elmer, Norwalk, CT, USA). The reaction mixtures were cycled 30 times: denaturation for 1 min at 95 C, annealing for 1 min at 53 C and extension for 3 min at 72 C in a thermal cycler (Perkin-Elmer model 9600, PerkinElmer Inc., Waltham, MA, USA). The amplified products were fractionated by electrophoresis on 1.5% agarose gels containing 0.5 mg ll ethidium bromide. The expected DNA fragment sizes were approximately 670 bp for P. falciparum and 987 bp for P. vivax. Data analysis The sensitivity and specificity of the RDT assays for the detection of P. vivax infections were calculated using microscopy of blood smears as the reference standard. All samples with non-concordant results were re-evaluated by both microscopy and PCR. Mixed infections were confirmed by PCR. The chi-squared test or Fisher s exact test were used to compare the results obtained from the RDT assays, SD Malaria Antigen P.v specific for P. vivax and OptiMAL. P-values <0.05 were considered significant. Results A total of 282 individuals aged years (mean: 31.2 years; male:female ratio of 1.9:1) entered into the study. All participants were examined for malaria parasites by microscopy of Giemsa-stained blood smears; 137 were diagnosed with P. vivax and 45 with P. falciparum; 100 were free of malaria. Of the 45 cases with P. falciparum infections, three cases were confirmed by PCR as mixed infections of P. falciparum and P. vivax (Table 1). The SD Malaria Antigen P.v identified cases (93.4%) from the microscopy-positive P. vivax patients. Three of the 45 P. falciparum patients reacted to SD Malaria Antigen P.v. These cases were diagnosed as P. falciparum infections by microscopic examination; however, they were later confirmed by PCR as mixed infections of P. vivax and P. falciparum. The SD Malaria Antigen P.f identified only the P. falciparum cases without cross reaction to P. vivax. This kit detected (97.6%) of the P. falciparum cases and all three mixed infections. The OptiMAL test identified (94.9%) of the P. vivax cases and (97.6%) of the P. falciparum cases. However, the three mixed infections were interpreted only as P. falciparum by OptiMAL. The healthy control blood smears were negative by microscopy and blood samples negative for both P. vivax and P. falciparum by the RDTs. The positive and negative predictive values for the SD Malaria Antigen kits were 100% and 94.0% for the P.v kit, respectively, and 100% and 99.6% for the P.f kit, respectively. All P. vivax cases with parasitaemia levels >1000 parasites ll tested positive using both OptiMAL and SD Malaria Antigen P.v RDTs (Table 2). The SD Malaria Antigen P.v showed % sensitivity for detecting infections of and parasites ll, compared with % sensitivity, respectively, by the OptiMAL test and were not significantly different (P = 1.000) by chisquared test. There were a total of nine false-negative patients with parasitaemia levels of (n = 1) parasites ll, (n = 5) and (n = 3) in the SD Malaria Antigen P.v test (Table 2). There were only seven false-negative cases among patients with (n = 4) and (n = 3) parasites ll using the OptiMAL assay. These discrepant cases between microscopic examination and two rapid tests were reconfirmed as P. vivax by PCR. Discussion Malaria, especially P. falciparum, is the most common and life-threatening parasitic disease among humans in the tropics and subtropics. Diagnosis by clinical syndrome alone is unreliable as the symptoms often mimic those of other parasitic diseases (Luxemburger et al. 1998). Thus, a rapid and accurate diagnostic tool to replace labourintensive, highly skilled conventional microscopy is necessary to effect rapid and effective treatment for malaria patients. Various kinds of immunochromatographic tests ª 2008 Blackwell Publishing Ltd 1497

4 No. of patients No. (%) of SD Malaria Antigen P.f positive No. (%) of SD Malaria Antigen P.v positive No. (%) of OptiMAL positive No. (%) of PCR positive Table 1 Comparison of SD Malaria Antigen P.f, SD Malaria Antigen P.v, OptiMAL and laboratory microscopy of Giemsastained blood smears for malaria diagnosis Plasmodium vivax (0) 128 (93.4) 130 (94.9) Plasmodium (97.6) 0 (0) 41 (97.6) falciparum Mixed infection* 3 3 (100)* 3 (100)* 3 3 (100)à Negative (0) 0 (0) 0 (0) Total *Detected both P. vivax and P. falciparum mixed infections. Microscopy and OptiMal interpreted it as P. falciparum. àmixed infections of P. vivax and P. falciparum confirmed by PCR. Table 2 Performance of SD Malaria Antigen P.v and OptiMAL compared with expert laboratory microscopy for various Plasmodium vivax parasite densities OptiMAL SD Malaria Antigen P.v Group No. of patients Positive Negative Sensitivity (%) Positive Negative Sensitivity (%) >5000 ll ll ll * ll * * ll * * 81.3 Total * * 93.4 *Confirmed as P. vivax by PCR test. have been recently developed for the rapid diagnosis of P. falciparum and P. vivax. Despite the introduction of these assays over a decade ago, only a few that target malaria parasite antigens have been used. Malaria antigens targeted by the recently developed RDTs are HRP-2, pldh and Plasmodium aldolase (Wongsrichanalai & Gasser 2002; Wongsrichanalai & Miller 2002; Murray et al. 2003). The RDTs use monoclonal antibodies to the HRP-2 or pldh for the detection of P. falciparum, but for the detection of non-falciparum malaria, a second monoclonal antibody against a panmalarial antigen is added. The aldolase enzyme was suggested as a target antigen for the detection of malaria species other than P. falciparum (Moody 2002; Murray et al. 2003). The ideal malaria diagnostic tool should be rapid, highly sensitive, specific, simple to perform and interpret, species discriminating, quantitative, affordable and practical for field use (Wongsrichanalai 2001). However, the current RDTs have several limitations, for example, reduced sensitivity and their inability to detect P. vivax and mixed infections of non-falciparum malaria. In tropical areas, drug resistance is common in P. falciparum populations and the incidence of P. vivax is rising (Congpuong et al. 2002). In these endemic areas, a species-specific RDT kit that discriminates between P. falciparum and P. vivax is necessary to determine the appropriate therapeutic plan. Mason et al. (2001) reported that in malaria endemic areas where P. vivax is not the only common non-p. falciparum species, it cannot be assumed that the samples with positive pan-plasmodium bands positive (anti-plasmodium aldolase) and P. falciparum-negative ICT tests are due only to P. vivax infections. Because of this limitation, a Plasmodium species-specific RDT kit is necessary for field applications, especially where medical resources are limited and light microscopy technicians lacking. To overcome these limitations, we evaluated the SD Malaria Antigen P.v assay, which adapts the monoclonal antibody to the P. vivax-specific lactate dehydrogenase enzyme. This test showed high sensitivity (93.4%) and specificity (100%) for P. vivax malaria on our evaluations; similar sensitivity and specificity were noted in a previous study (Lee et al. 2007), and compared with the approved OptiMAL test, the SD Malaria Antigen P.v assay was equally sensitive. Additionally, three cases with mixed infections, which were microscopically identified only as P. falciparum because of low numbers of P. vivax parasites, were correctly identified by the SD Malaria Antigen P.v and SD Malaria Antigen P.f tests, contrary to 1498 ª 2008 Blackwell Publishing Ltd

5 the OptiMAL test which interpreted these cases only as P. falciparum infections. Rapid results from these RDTs provide public health workers with the necessary information for appropriate chemotherapy, including antirelapse therapy, when required, to malaria patients. Follow-up studies for malaria patients with low parasitaemia levels would most likely be identified as positive when parasitaemia levels exceeded parasites ll. The World Health Organization (WHO) recommends that the sensitivity be 95% at 100 parasites ll for detection of P. falciparum and higher at increased parasite densities. However, lower sensitivity is acceptable for P. vivax in RDTs detecting pan-specific antigens (WHO 2003). Previous studies identified decreased levels of sensitivity for non-p. falciparum (P. vivax) at parasitaemias of 1000 to <5000 parasites ll (Cho et al. 2001; Iqbal et al. 2002; Ratsimbasoa et al. 2007). Compared with these studies, the sensitivity for detecting low-level P. vivax parasitaemias was higher in our evaluations of the SD Malaria Antigen P.v test. The genetic diversity of HRP-2 has been examined and linked to RDT detection sensitivity (Baker et al. 2005). Genetic variability is an unlikely cause of reduced sensitivity for aldolase-detecting RDTs and is more likely to be due to low antigen concentrations to a single small number of epitopes per parasite or other factors affecting RDT quality (Lee et al. 2006). The genetic diversity also is not likely to be the cause of variation in the sensitivity of RDTs detecting pldh (Talman et al. 2007). Considering that P. vivax is the only indigenous malaria in the ROK, a RDT that detects only P. vivax is ideal and is more cost effective compared with the other non-specific assays. The price of the OptiMAL and SD Bioline Malaria Antigen tests (Combo) is about $5.00 when purchased in Korea. The SD Malaria Antigen P.v test costs $3.00, or 60% less than the combo tests [OptiMAL and SD Bioline Malaria Antigen (Combo) tests]. Bought through WHO, SD Bioline Malaria Antigen (Combo) tests kits cost $1.50. In this study, SD Malaria Antigen P.v sensitivity was high and detected nearly all P. vivax infections of ROK malaria patients. The WHO has emphasized the urgent need for simple and cost-effective malaria diagnostic tests that not only overcome the deficiencies of light microscopy and clinical diagnosis, but also are highly sensitive when identifying P. vivax and P. falciparum infections (WHO 1996). These new assays meet the WHO standards and should be considered for further evaluation in countries where only P. vivax infections occur or where P. falciparum is predominant, as well as in epidemiological settings to differentiate between P. vivax species for administering the correct chemotherapy. Acknowledgements The authors would like to thank Dr Dae Won Park and In Keun Choi at the Korea university Ansan hospital. The authors are grateful for the cooperation of the non-malaria and malaria patients who volunteered for this study. This work was supported by the U-health program of the Korea University Medical Center, Seoul R & BD program (2007). SD Malaria Antigen P.v and SD Malaria Antigen P.f kits were donated for evaluation by SD Diagnostic Corporation. Disclaimer The views expressed in this article are those of the authors do not reflect the official policy or position of the Department of Defense, the Department of the Army, or the US Government. References Baker J, McCarthy J, Gatton M et al. (2005) Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests. The Journal of Infectious Diseases 192, Beadle C, Long GW, Weiss WR et al. (1994) Diagnosis of malaria by detection of Plasmodium falciparum HRP-2 antigen with a rapid dipstick antigen-capture assay. Lancet 343, Chai JY (1999) Re-emerging Plasmodium vivax malaria in the Republic of Korea. The Korean Journal of Parasitology 37, Cho D, Kim KH, Park SC, Kim YK, Lee KN & Lim CS (2001) Evaluation of rapid immunocapture assays for diagnosis of Plasmodium vivax in Korea. Parasitology Research 87, Congpuong K, Na-Bangchang K, Thimasarn K, Tasanor U & Wernsdorfer WH (2002) Sensitivity of Plasmodium vivax to chloroquine in Sa Kaeo Province, Thailand. Acta Tropica 83, Craig MH & Sharp BL (1997) Comparative evaluation of four techniques for the diagnosis of Plasmodium falciparum infections. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, Dietze R, Perkins M, Boulos M, Luz F, Reller B & Corey GR (1995) The diagnosis of Plasmodium falciparum infection using a new antigen detection system. The American Journal of Tropical Medicine and Hygiene 52, Iqbal J, Sher A, Hira PR & Al-Owaish R (1999) Comparison of the OptiMAL test with PCR for diagnosis of malaria in immigrants. Journal of Clinical Microbiology 37, Iqbal J, Sher A & Rab A (2000) Plasmodium falciparum histidinerich protein 2-based immunocapture diagnostic assay for malaria: cross-reactivity with rheumatoid factors. Journal of Clinical Microbiology 38, Iqbal J, Khalid N & Hira PR (2002) Comparison of two commercial assays with expert microscopy for confirmation of ª 2008 Blackwell Publishing Ltd 1499

6 symptomatically diagnosed malaria. Journal of Clinical Microbiology 40, Iqbal J, Muneer A, Khalid N & Ahmed MA (2003) Performance of the OptiMAL test for malaria diagnosis among suspected malaria patients at the rural health centers. The American Journal of Tropical Medicine and Hygiene 68, Lee N, Baker J, Bell D, McCarthy J & Cheng Q (2006) Assessing the genetic diversity of the aldolase genes of Plasmodium falciparum and Plasmodium vivax and its potential effect on performance of aldolase-detecting rapid diagnostic tests. Journal of Clinical Microbiology 44, Lee SW, Jeon K, Jeon BR & Park I (2008) Rapid diagnosis of vivax malaria by SD Bioline malaria antigen test when thrombocytopenia is present. Journal of Clinical Microbiology 46, Luxemburger C, Nosten F, Kyle DE, Kiricharoen L, Chongsuphajaisiddhi T & White NJ (1998) Clinical features cannot predict a diagnosis of malaria or differentiate the infecting species in children living in an area of low transmission. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, Makler MT, Palmer CJ & Ager AL (1998) A review of practical techniques for the diagnosis of malaria. Annals of Tropical Medicine and Parasitology 92, Moody A (2002) Rapid diagnostic tests for malaria parasites. Clinical Microbiology Reviews 15, Murray CK, Bell D, Gasser RA & Wongsrichanalai C (2003) Rapid diagnostic testing for malaria. Tropical Medicine and International Health 8, Pieroni P, Mills CD, Ohrt C, Harrington MA & Kain KC (1998) Comparison of the ParaSight-F test and the ICT Malaria Pf test with the polymerase chain reaction for the diagnosis of Plasmodium falciparum malaria in travellers. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, Ratsimbasoa A, Randriamanantena A, Raherinjafy R, Rasoarilalao N & Menard D (2007) Which malaria rapid test for Madagascar? Field and laboratory evaluation of three tests and expert microscopy of samples from suspected malaria patients in Madagascar The American Journal of Tropical Medicine and Hygiene 76, Talman AM, Duval L, Legrand E et al. (2007) Evaluation of the intra- and inter-specific genetic variability of Plasmodium lactate dehydrogenase. Malaria Journal 6, 140. WHO (1996) A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. WHO Informal Consultation on Recent Advances in Diagnostic Techniques and Vaccines for Malaria. Bulletin of the World Health Organization 74, WHO (2003) Malaria Rapid Diagnosis: Making it Work. Meeting report January Manila. World Health Organization, Geneva. Wongsrichanalai C (2001) Rapid diagnostic techniques for malaria control. Trends in Parasitology 17, Wongsrichanalai C & Gasser RA Jr (2002) Current status of malaria rapid diagnostic devices: an update. Trends in Parasitology 18, Wongsrichanalai C & Miller RS (2002) Malaria rapid tests: a public health perspective. Lancet 359, Yeom JS, Kim TS, Oh S et al. (2007) Plasmodium vivax malaria in the Republic of Korea during : changing patterns of infection. The American Journal of Tropical Medicine and Hygiene 76, Corresponding Author Chae Seung Lim, Department of Laboratory Medicine, College of Medicine, Korea University Ansan Hospital, 516, Gojan Dong, Ansan City, Gyeonggi Province, Republic of Korea, Tel.: ; Fax: ; malarim@korea.ac.kr 1500 ª 2008 Blackwell Publishing Ltd