1. RESULTS Division of Arboviruses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for disease control, Osong, Korea BACKGROUND METHODS REFERENCES South Korea is not belonging to dengue endemic regions. Recent years imported cases of dengue by Korean travelers from dengue endemic regions globally have been increased. Diagnosis of dengue is required to differentiate from other flavivirus infections due to serological cross reactivity and from other bacterial and parasitic infections such as malaria showing similar clinical symptoms [1]. Some researchers reported other case which is concurrent with dengue and malaria because of similar symptoms such as fever in addition, dengue and malaria overlapped endemic regions [2]. Serum specimens of patients presenting with fever that has developed within less than 2 to 3 weeks after a brief trips to the tropics or subtropics were submitted to Korea National Institute of Health for dengue diagnosis. The specimens has been tested by immunoglobulin M (IgM) ELISA, nonstructural 1 (NS1) antigen ELISA and RT-PCR for laboratory diagnosis of dengue fever [3,4,5,6]. Among the patient with dengue-like symptoms, more than 40 to 60% were negative to dengue in Korea. Thus non-dengue specimens are needed to clarify other infections and cross reaction with dengue virus for determining to make an accurate diagnosis. To give correct etiologic diagnosis of dengue virus infection or other agents in travelers, the specimens were tested to malaria and severe fever with thrombocytopenia syndrome (SFTS) by ELISA and RT-PCR, respectively. LABORATORY DIFFERENTIAL DIAGNOSIS OF DENGUE FEVER FROM MALARIA AND SEVERE FEVER WITH THROMBOCYTOPENIA IN PATIENTS SUSPECTED DENGUE VIRUS INFECTION Serum specimens [1] Concurrent isolation from patient of two arboviruses, chikungunya and dengue type 2. Myers RM et al., Science, 1967, 57, 3792, 1307-1308. [2] Concurrent dengue and malaria, Charrel RN et al., Emerging Infectious Diseases, 2005, 11(7,) 1153-1154. [3] Dengue diagnosis, advances and challenges. Maria G et al., International Journal of Infectious Diseases 2004, 8, 69-80. [4] Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcription PCR. Harris E et al., Journal of Clinical Microbiology, 1998, 2, 2634-2639. [5] Enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. Sophie A et al., Journal of Clinical Microbiology, 2002, 40, 376-381. [6] Identification of dengue type 1 virus (DENV-1) in koreans traveling abroad Jeong YE et al., Public Health and Research Perspectives, 2011, 2, 1, 34-40. [7] Detection of severe fever with thrombocytopenia syndrome virus from haemophysalis longicornis in South Korea. Park SW et al., Emerging Infectious Diseases, 2013 (Submitted) [8] Critical epitopes in the nucleocapsid protein of SFTS cirus recongnized by a panel of SFTS patients derived human minoclonal antibodies, Li Y et al., PLOSONE, 2012, 7(6), e38291.  In this study, the specimens were evaluated co-infection by Malaria and SFTS.  In the previous study we evaluated dengue diagnosis by three assay (RT-PCR, IgM and NS1 antigen ELISA) (data not shown).  Among 456 specimens, 22 showed malaria positive (4.8%) and SFTS virus was detected from the only one serum specimens among 180 specimens of dengue negative (Tables 3 and 4, Figures 2 and 3)  Our results suggest that co-infection of dengue virus with Plasmodium spp. or SFTSV is not generally occurred at least in Korea travelers (Table 3 and 4).  Co-infection of dengue with malaria was suspected in six dengue positive specimens. From this results, differential diagnosis of the diseases has to be considered for international travelers tripped both dengue and malaria endemic regions when they have symptoms of fever.  This study was supported by the intramural grant (#2013-NG53003-00) of the National Institute of Health, Korea. The antibodies to Plasmodium spp. was detected from 22 serum specimens (4.8%) by Malaria EIA kit. Figure 1 showed results of SFTS positive specimens by real-time RT- PCR and C T value (21) of one positive specimen. Malaria ELISA -The dengue negative specimens by three dengue assay (IgM ELISA, NS1 antigen ELISA and RT-PCR) were evaluated by real time RT-PCR (Kogenebiotech, Korea) - The SFTS real-time RT-PCR and conventional RT-PCR targeted S and M segments of the SFTS virus, respectively (Tables 1 and 2). - The S and M segments of the SFTS reported at previous report [7, 8]. SFTS Real time PCR -Malaria antibody was assayed by Malaria EIA test kit (Bio-Rad, USA). - Malaria test kits used four recombinant antigens in a sandwich test. Malaria ELISA - The dengue negative specimens tested by three assays (RT-PCR, IgM and NS1 antigen ELISA) were evaluated by SFTS real-time RT-PCR (Powercheck SFTSV Real-time PCR kit TM, Kogenebiotech) and RT-PCR for the M segment targeting [7]. Real time RT-PCR for SFTS virus Table3. Malaria antibody detection from serum specimens with dengue infection Table 1. Real-Time RT-PCR conditions of SFTS Table4. Detection of SFTS virus by real time RT-PCR Figure 1. Detection of the M segment of SFTS virus by real-time RT- PCR Table 4 presented that SFTS virus by real-time RT-PCR was detected from one specimen among 180 dengue negative specimens. - The specimens of dengue like symptoms between 2011 and 2012 in Division of Arboviruses at Korea National Institute of Health for serologic diagnosis. Among the specimens, 456 were selected for Malaria ELISA and 180 specimens which were negative to dengue were used for SFTS testing. -Viral RNA was extracted from serum by viral RNA extraction kit (Intron, Korea). Table 2. RT-PCR condition of SFTS Figure 2. Detection of the M segment of SFTS virus by RT-PCR Figure 3. Sequence alignment of the M segment of SFTS virus detected from a specimen with SFTS virus HNXY_31 strain P: positive control M: molecular markers S: specimen SUMMARY & CONCLUSION * No. of positive specimens/No. of tested specimens Figure 3 showed the M segment of SFTS virus detected from dengue negative specimen has 99 % similarity with that of SFTS virus HNXY_31.