Corresponding Author Chae Seung Lim, Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Guro 2 Dong, Guro Gu, Seoul, 152-703, Republic of Korea. Tel.: +82 2 26263245; Fax +82-2-26261465; E-mail: firstname.lastname@example.org
Plasmodium vivax malaria is the indigenous strain in the Republic of Korea (ROK). Plasmodium vivax can be transmitted through the transfusions of various blood components, which became a severe problem with the safety of blood transfusions and blood-related products in ROK. We evaluated a P. vivax-specific enzyme-linked immunosorbent assay (Genedia Malaria Ab ELISA 2.0, Green Cross, ROK) with blood samples from four groups: 251 samples from P. vivax-infected patients, 39 samples from post-treatment patients upon follow-up, 200 samples from healthy volunteers and 421 samples from domestic travellers to and from high endemic areas of ROK. The positive cases from the ELISA test were confirmed by both Giemsa microscopic and polymerase chain reaction methods. The clinical sensitivity and specificity of detecting P. vivax with ELISA test were 94.4% and 99.0%, respectively. Thirteen of 421 domestic travellers (3.0%) to endemic areas tested positive. The results indicate the effectiveness of detecting antibodies against P. vivax in blood with Genedia Malaria Ab ELISA 2.0 test in a large blood screen setting.
Évaluation de l’ELISA de Plasmodium vivax pour le dépistage sanguin
Plasmodium vivax (P. vivax) est la souche indigène de la malaria dans la République de Corée (RDC). P. vivax peut être transmis par des transfusions de divers composants sanguins, ce qui est devenu un grave problème de la sécurité des transfusions sanguines et des produits sanguins associés dans la RDC. Nous avons évalué un test immuno-enzymatique spécifique de P. vivax (Genedia Malaria Ab ELISA 2.0, Green Cross, RDC) sur des échantillons de sang de quatre groupes: 251 échantillons provenant de patients infectés par P. vivax, 39 échantillons provenant de patients suivis post-traitement, 200 échantillons de volontaires sains et 421 échantillons provenant de voyageurs en provenance de et vers les zones hautement endémiques de la RDC. Les cas positifs de l’ELISA ont été confirmés à la fois par des méthodes de microscopie Giemsa et par la réaction en chaîne par polymérase (PCR). La sensibilité et la spécificité clinique de la détection de P. vivax avec les tests ELISA étaient de 94,4% et 99,0%, respectivement. 13 sur 421 (3,0%) voyageurs domestiques vers des zones endémiques ont été testés positifs. Les résultats indiquent l’efficacité de la détection d’anticorps contre P. vivax dans le sang avec le test Genedia Malaria ELISA Ab 2,0 dans un large cadre de dépistage sanguin.
Mots-clés: Plasmodium vivax, ELISA, test d’anticorps, République de Corée
Evaluación del ELISA de Plasmodium vivax para el tamizaje en sangre
Plasmodium vivax es el agente etiológico de la malaria en la República de Corea (RC). P.vivax puede transmitirse a través de transfusiones de varios componentes de la sangre, lo cual se convierte en un problema severo de seguridad para transfusiones de sangre y productos relacionados en la RC. Hemos evaluado un ensayo por inmunoabsorción ligado a enzimas (Genedia Malaria Ab ELISA 2.0, Green Cross, RC) específico para P.vivax con muestras de sangre de cuatro grupos: 251 muestras de pacientes infectados con P.vivax, 39 muestras de pacientes que habían recibido tratamiento y estaban en seguimiento, 200 muestras de voluntarios sanos, y 421 muestras de viajeros domésticos, hacia y desde áreas de alta endemicidad en la RC. Los casos positivos por ELISA fueron confirmados mediante dos métodos: microscopía tras tinción con Giemsa y la reacción en cadena de la polimerasa (PCR). La sensibilidad y especificidad clínicas de detectar P.vivax mediante ELISA eran del 94.4% y 99.0%, respectivamente. 13 de 421(3.0%) viajeros hacia áreas endémicas dieron positivo. Los resultados indican la efectividad de detectar anticuerpos frente a P.vivax en sangre utilizando la prueba de Genedia Malaria Ab ELISA 2.0 en un amplio tamizaje de muestras de sangre.
Palabras clave: Plasmodium vivax, ELISA, prueba anticuerpos, República de Corea
Blood transfusions are essential to save lives of patients in emergency situations. On the other hand, many infectious diseases, such as malaria, hepatitis virus, syphilis and AIDS are transmitted through blood transfusions (Mollison et al. 1997). Among these infectious diseases, transfusion-transmitted malaria was not investigated extensively in non-tropical countries because of low incidences, except in certain geographical regions. With frequent international travel and/or re-emergence of malaria in some malaria-free countries, the risk of transmitting malaria through blood transfusion should be reconsidered (Dodd 1998; Mungai et al. 2001; Kitchen et al. 2005a).
Despite much progress made in controlling Plasmodium vivax malaria (P. vivax) in the Republic of Korea (ROK), transmission remains epidemic in the northern parts of ROK near the Demilitarized Zone (DMZ). Malaria transmission levels are not uniformly distributed in ROK, especially in many locations near DMZ (Korea National Institutes of Health 2000). After the re-emergence of malaria in 1993, the transmission of P. vivax malaria through blood transfusions has posed a real threat since 2000. By 2010, ten cases of transfusion-transmitted malaria had been reported in ROK (Cho et al. 2001; Lee et al. 2001).
Presently, most countries use blood donor deferral criteria based on the medical history elicited by interview, to prevent transfusion-transmitted malaria. However, the current donor exclusion guidelines have been called in doubt (Purdy et al. 2004) to address the need for an effective and evidence-based malaria blood screening test. Donor antibody screening tests for malaria have been used in a number of countries. However, none of the P. vivax malaria antibody tests have been implemented to screen donated blood, because of the lack of a reliable and sensitive screening test against P. vivax. Because P. vivax is the only indigenous malaria in ROK, quick and enhanced P. vivax malaria blood screening test would prevent its spread by transfusion in blood banks. Recently, several successful pre-transfusion malarial screening strategies for countries with low-prevalence of malaria were reported (Kitchen et al. 2004; Garraud et al. 2008; Seed et al. 2010).
The aim of this study was to evaluate a P. vivax malaria-specific enzyme-linked immunosorbent assay (ELISA) antibody test for screening for P. vivax in the blood and to estimate the risks of malaria infection in ROK.
Material and methods
A total of 921 samples was collected between April of 2002 and December of 2007 at Korea University Ansan Hospital, ROK for this study. These samples were divided into four groups: 251 of patients with acute malaria (Group 1 –P. vivax patients with parasitaemia; samples were collected within a week after the appearance of the symptoms, which were confirmed by manual Giemsa stain), 39 of post-treatment patients (Group 2 – confirmed malaria patients without parasitaemia presently, followed up at least monthly), 200 of normal healthy controls for clinical specificity (Group 3 – patients with no prior history of malaria or trip to a malaria-endemic area in the past 3 years) and 421 samples from donors (Group 4 – donors who had travelled to endemic areas within the past 6 months). The Ethical Committee of the Korea University Ansan Hospital approved this study.
For the malaria diagnosis, thick and thin blood films were prepared with the standard protocol. Following the Giemsa stain of the blood films, a malaria expert diagnosed the species and density of plasmodial parasites through microscopic examination. Parasitaemia was indirectly calculated using the parasite numbers per 200 WBCs in the blood film, where the WBC counts were determined by the automatic blood cell counter (Cell-Dyn 4000; Abbott diagnostics, Abott Park, IL, USA). Infected patients were treated with hydroxychloroquine and primaquine. Twenty-six patients were followed up for 6 and 12 months for their post-treatments. These samples were used for evaluating the period of antibody persistency.
Polymerase Chain Reaction (PCR)
Circumsporozoite protein (CSP) genes of P. vivax were amplified by nested-PCR as previously documented (Oh et al. 2008). Primers were used at a final concentration of 0.1 μm in 100 μl reaction mixture (10 mm Tris–HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, 0.2 mm each dNTP), containing 10 μl of DNA and 2.5 units of AmpliTaq polymerase (Perkin Elmer Co., Norwalk, CT, USA). Reaction mixtures were cycled 30 times: 1 min denaturation at 95 °C, 1 min annealing at 53 °C and 3 min extension at 72 °C in a DNA thermal cycler (Perkin-Elmer Model 9600). Amplified products were size-fractionated by electrophoresis on 1.5% agarose gels containing ethidium bromide (0.5 mg/ml).
Genedia Malaria Ab ELISA 2.0 test (Green cross co., ROK) for P.vivax antibodies
The Genedia Malaria Ab ELISA 2.0 test was kindly donated by Green Cross Co for this purpose. It is based on binding anti-P. vivax antibodies in a serum sample to recombinant P. vivax antigens immobilized on 96-well plates. The mixture of recombinant P. vivax antigens consisted of merozoite surface protein (MSP-1) and CSP from Korean P. vivax strain. The ELISA test was performed as recommended by the manufacturer. Briefly, 50 μl of sera was added to the wells with 100 μl of diluent. After 1 h of incubation at 37 °C, unbound materials were washed away with PBS-Tween-20, and the peroxidase-conjugated goat anti-human IgG (1:40 000 dilution) was added to each well. After another hour of incubation at 37 °C, the excess labelled antibody was washed away with PBS-Tween-20, and the chromogenic signal was developed with ortho-phenylene diamine (Sigma Aldrich, St. Louis, Mo, USA) substrate system. The plates were read at 490 nm. Each serum was tested in duplicate, and the optical density (OD) values at 490 nm were averaged. For the data analysis of dichotomous groups, OD cut-off value for the positive ELISA signals was set by recommendation (0.2 + 3 times of mean OD of the unexposed normal healthy control) by the manufacturer. The test did not distinguish antibody types between IgG and IgM, detecting all antibodies against P. vivax. The assay was performed with an automatic ELISA processor (Bio-Rad CODA System, Bio-Rad, Hercules, CA, USA).
Clinical sensitivity was calculated based on the proportion of positive antibody test results among samples of patients with acute malaria, which were confirmed positive of malaria parasites by Giemsa stain microscopy analysis. Clinical specificity was calculated based on the proportion of negative test results among samples, confirmed by thick blood films.
Clinical sensitivity in confirmed P.vivax malaria samples
Genedia Malaria Ab ELISA 2.0 test gave positive results in 94.4% (237/251) of samples obtained from subjects with confirmed P. vivax malaria. Thirty-nine malaria-infected cases were from post-treatment patients upon follow-up, where 66.7% (27/39) of patients tested positive for P. vivax antibody. Among these 39 samples, 29 samples were collected within 3 months after treatment, because they tested positive by Genedia Malaria Ab ELISA 2.0 test with cut-off OD value of 0.516. However, PCR failed to detect parasite DNA in all follow-up cases (Table 1). The mean OD value from the post-treatment patients upon follow-up is shown in Table 1.
Table 1. Genedia Malaria Ab ELISA 2.0 test and microscopy results were analysed and compared with patients confirmed of Plasmodium vivax, healthy controls, visitors to malaria-endemic areas and the post-treatment patients upon follow-up
Confirmed cases of P. vivax
Visitors to malaria-endemic areas*
*Absence of parasite was confirmed by both PCR and microscopic examination.
OD, Optical density.
2.19 ± 1.02
0.74 ± 0.33
0.97 ± 0.32
0.33 ± 0.09
0.37 ± 0.05
0.17 ± 0.07
0.18 ± 0.18
1.61 ± 0.93
2.09 ± 1.08
0.17 ± 0.09
0.13 ± 0.23
1.22 ± 0.98
Clinical specificity from the normal healthy controls
From 200 non-exposed normal healthy controls, two samples (1.0%) were reactive, even though the parasitaemia was not detected by both microscopic examination and PCR. Hence, the clinical specificity was 99.0% with mean OD value of 0.17 ± 0.09 in normal healthy volunteers.
Antibody positivity rate among donors who had visited endemic areas
We enrolled 421 patients who had travelled to highly endemic areas in ROK. Plasmodium vivax parasite was not identified in any by either microscopic examination of blood films or PCR. Thirteen cases (3.0%) revealed positive antibody reactivity to P. vivax by Genedia Malaria Ab ELISA 2.0 test. The mean OD value from the healthy visitors to the endemic areas was 0.13 ± 0.23 (Figure 1).
Plasmodium vivax is the only indigenous malaria reported in the Korean Peninsula (Korea Center for Disease Control and Prevention 2006). After the resurgence of P. vivax in ROK, 10 cases of malaria transmitted by blood transfusions were reported between 1997 and 2006, which corresponded to 0–2.85 cases per million transfused units during each year (Cho et al. 2001; Lee et al. 2001). Hence, malaria became a serious threat, affecting the blood donation policy of ROK, as an emerging transfusion-transmitted disease in ROK. Because of cross-proximity between residential areas of donors and malaria-endemic areas in Korea, the blood banks of ROK reached shortages of some blood groups every winter.
Careful questioning of donors is still widely practiced to identify prospective donors for reducing the risk of malaria transmission by blood transfusion worldwide (Dodd 1998; Kitchen et al. 2005b; Korean Society of Blood Transfusion 2007). According to KCDC classifications, ROK maintained donor deferral criteria for the cellular (RBC and platelet) products, restricting donors and residents for periods of 12 or 36 months preceding domestic travels to malaria-endemic areas (Korea Center for Disease Control and Prevention 2006, Korean Society of Blood Transfusion 2007). These rules also included donors who returned to ROK after the exclusion to endemic areas by WHO-classifications, and the permanent exclusion was placed on candidate donors with a prior history of malaria. Considering the lower rate of malaria prevalence in ROK than other tropical countries, the current deferral criteria in ROK seemed to be too strong. Hence, the deferral policies, designed to exclude all potential donors on the basis of their travel history to endemic areas in ROK, would not be practical. Adding a suitable donor screening test to separate the travel-based exclusions for the donation of cellular components would be an alternative way to prevent transfusion-transmitted malaria with minimal loss of donors.
Diagnosing malaria in clinics and blood banks should have different objectives for testing patients and blood donors. The main objective of blood banks in screening malaria should focus on developing a rapid and robust diagnosis to rule out the donors with risk of transmitting malaria by transfusion. Therefore, key criteria in screening donors were as follows: the prevalence of the region and malaria immunity in the donor population, test sensitivity, cost, reliability, speed and complexity. On the other hand, the objectives of malaria diagnosis in the clinics should emphasize the accuracy of the test for each individual patient. Serological and nucleic acid testing (NAT) offers an accurate diagnosis of malaria in the clinical setting. However, the NAT usually takes longer than the rapid ELISA test. Currently, with many advances, NAT could be available for screening blood donors, which could shorten the deferral period. Malarial antibodies are usually produced within 1–2 weeks after the initial infection and are detectable for months to years, even after clearance of the parasite. But the peak of antibody production occurs in a time window of not more than 7–14 days (Contreras et al. 1999, Seed et al. 2005, 2010). Hence, most combined donor screening strategies could reduce the embargo period up to 4 months without increasing the risk of transmitting malaria by transfusion. Although NAT offers higher accuracy and sensitivity for detecting malaria-infected blood than serologic tests, it presents many limitations in pre-transfusion malaria screening, such as limited sensitivity against all potentially infectious strains; inability to differentiate individuals with prior exposure to malaria from those with malarial antibody from transient parasitaemic episodes; its complex nature; and high comparative cost (Seed et al. 2005).
Currently, malaria antibody ELISA test is not approved for screening blood donors with probable malaria exposure on a large scale by the blood processing centres in the world. The standard Giemsa stained microscopic method would not be useful for such purpose. Hence, quick and enhanced tests are needed by the blood processing centres. Use of antibody-based screening depends primarily on the antibody prevalence in the donor population. For example, the seroprevalence ranges from 2.4% to 46.1% in endemic areas (Razakandrainibe et al. 2009), reducing the availability of donors. In a restricted area with low malaria prevalence, a malaria antibody test would detect only a small number of donors with risk of malaria transmission; hence, a larger pool of donors would be available. Malaria blood screening test should also focus on P. vivax infection in temperate areas, especially with P. vivax as only malaria species.
Until now, the sensitivity of antibody assays for P. vivax was unsatisfactory compared to the P. falciparum test. The sensitivity of antibody assays for P. falciparum ranged between 76% and 100% in acute samples, while the reported sensitivity for P. vivax varied between 50% and 100% (Chiodini et al. 1997; Seed et al. 2005; Doderer et al. 2007). Previously, a malaria antibody test kit (ELISA malaria antibody test, DiaMed, Switzerland) revealed a clinical sensitivity of 53.0% with a specificity of 94.0% (Oh et al. 2008) with ROK samples, suggesting clinical ineffectiveness for screening P. vivax among donors. Loss of donated blood because of false-positive results was estimated to be 6%.
In this study, Genedia Malaria Ab ELISA 2.0 test revealed its effectiveness in identifying potential blood samples with P. vivax infection. The sensitivity of the ELISA test was better than those of other screening methods. Moreover, the mean OD value of samples from patients with exposure to P. vivax was much higher than the values from normal healthy volunteers, demonstrating clear differentiation between suspected donors and normal healthy donors by Genedia Malaria Ab ELISA test. Our results supported the implementation of such a test to improve the detection of P. vivax malaria antibody and prevent the transfusion-transmitted malaria in the blood processing centres. The sensitivity of malaria antibody tests is known to be strongly influenced by the length of time after the malaria invasion (Jelinek et al. 1995, 1998). Accordingly, the correlations observed in our study would have been influenced by the time intervals after the exposure. The OD values against the malaria antibody seemed to be decreasing over time, but still remained above the cut-off value of normal healthy controls (Park et al. 2000). All samples with P. vivax parasitaemia in our study were collected within 1 week after the appearance of the symptoms, and the period of asymptomatic parasitaemia of vivax malaria was 6–10 days (Park et al. 2001), which was not significantly different from falciparum malaria (Seed et al. 2005, 2010).
Genedia Malaria Ab ELISA 2.0 test failed to detect 14 samples from confirmed cases of P. vivax infection by Giemsa stain microscopy and PCR analyses. The false-negative rate was significantly reduced in comparison with previous study (Oh et al. 2008), which suggested that the usage of antigens specific for Korean-strain malaria in the ELISA kit improved the detection rate of P. vivax malaria in Korean samples. However, if the ELISA kit was used as the pre-transfusion screening test, these false-negative results should not be overlooked because the transfusion-transmitted malaria infection could occur. The genetic diversity of Korean P. vivax strain could be a possible cause of the failure of detection. MSP-1 and CSP antigens used in the ELISA kit were reported to be highly polymorphic in the P. vivax strain in Guyana, and recent studies also revealed the increased diversity of P. vivax strain in Korea (Bonilla et al. 2006; Choi et al. 2010).
For the definition of healthy control group in our study, we adopted the recommendations for deferral of donors for malaria risk by the Food and Drug Administration and American Association of Blood Banks, proposed in 1994. When the above criterion was applied between 1993 and 1998, the incidence rate of transfusion-transmitted malaria ranged between 0 and 0.18 case per million transfused units in the United States (Mungai et al. 2001). However, considering the chance of clinically undiscovered malarial parasitaemia and the transfusion-transmitted malaria, the current donor deferral policy of 3 years without travel to the endemic zones for the normal healthy control group may not be enough to detect the transfusion-transmitted malaria (Kitchen et al. 2005a,b). Hence, two positive cases from the Genedia Malaria Ab ELISA 2.0 test in the healthy control group in our study could have been overdiagnosed, because the PCR test of the cases showed negative results, suggesting false reactive results of antibody testing. For the pre-transfusion antibody screening of malaria, these false-positive cases should be confirmed by supplementary tests such as PCR method or antigen testing (Seed et al. 2010).
In conclusion, our results suggest the effectiveness of Genedia Malaria Ab ELISA 2.0 test for screening blood donors and detecting P. vivax in ROK. In addition, this assay would be useful for screening blood in countries with increasing P. vivax infections.
This work was supported by a National Research Foundation of Korea grant (KRF-2009-0076070) from the Korean Government.