Molecular techniques have been introduced for malaria diagnosis because they offer greater sensitivity and specificity than microscopic examinations. Therefore, DNA isolation methods have been developed for easy preparation and cost effectiveness. The present study described a simple protocol for Plasmodium DNA isolation from EDTA-whole blood. This study demonstrated that after heating infected blood samples with Tris–EDTA buffer and proteinase K solution, without isolation and purification steps, the supernatant can be used as a DNA template for amplification by PCR. The sensitivity of the extracted DNA of Plasmodium falciparum and Plasmodium vivax was separately analyzed by both PCR and semi-nested PCR (Sn-PCR). The results revealed that for PCR the limit of detection was 40 parasites/μl for P. falciparum and 35.2 parasites/μl for P. vivax, whereas for Sn-PCR the limit of detection was 1.6 parasites/μl for P. falciparum and 1.4 parasites/μl for P. vivax. This new method was then verified by DNA extraction of whole blood from 11 asymptomatic Myanmar migrant workers and analyzed by Sn-PCR. The results revealed that DNA can be extracted from all samples, and there were 2 positive samples for Plasmodium (P. falciparum and P. vivax). Therefore, the protocol can be an alternative method for DNA extraction in laboratories with limited resources and a lack of trained technicians for malaria diagnosis. In addition, this protocol can be applied for subclinical cases, and this will be helpful for epidemiology and control.
Cost-Benefit Analysis ; Diagnosis ; DNA ; Edetic Acid ; Endopeptidase K ; Epidemiology ; Heating ; Hot Temperature ; Humans ; Limit of Detection ; Malaria ; Methods ; Myanmar ; Plasmodium falciparum ; Plasmodium vivax ; Plasmodium ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Transients and Migrants

Plasmodium vivax is more challenging to control and eliminate than P. falciparum due to its more asymptomatic infections with low parasite densities making diagnosis more difficult, in addition to its unique biological characteristics. The potential re-introduction of incidence cases, either through borders or via human migrations, is another major hurdle to sustained control and elimination. The Republic of Korea has experienced re-emergence of vivax malaria in 1993 but is one of the 32 malaria-eliminating countries to-date. Despite achieving successful nationwide control and elimination of vivax malaria, the evolutionary characteristics of vivax malaria isolates in the Republic of Korea have not been fully understood. In this review, we present an overview of the genetic variability of such isolates to increase understanding of the epidemiology, diversity, and dynamics of vivax populations in the Republic of Korea.
Asymptomatic Infections ; Diagnosis ; Epidemiology ; Genetic Variation ; Human Migration ; Incidence ; Korea ; Malaria ; Malaria, Vivax ; Parasites ; Plasmodium vivax ; Plasmodium ; Population Characteristics ; Republic of Korea

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.
Amino Acid Sequence ; Antibodies, Protozoan/*blood/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Base Sequence ; Humans ; India ; Malaria, Vivax/*diagnosis/*epidemiology/immunology ; Merozoite Surface Protein 1/genetics/*immunology ; Plasmodium vivax/genetics/immunology ; Protozoan Proteins/genetics/*immunology ; Reagent Kits, Diagnostic ; Recombinant Proteins/diagnostic use/immunology ; Republic of Korea/epidemiology ; Sequence Analysis, DNA ; Seroepidemiologic Studies ; Uganda

Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Coinfection/*diagnosis/epidemiology ; Endemic Diseases ; Enzyme-Linked Immunosorbent Assay/methods ; Female ; Humans ; Malaria, Falciparum/*diagnosis/epidemiology ; Malaria, Vivax/*diagnosis/epidemiology ; Male ; Middle Aged ; Molecular Diagnostic Techniques/*methods ; Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Serologic Tests/methods ; Thailand/epidemiology ; Young Adult

Parasitemia characteristics of Plasmodium vivax malaria in temperate regions may differ from those in tropical zones. However, most parasitological and clinical features of P. vivax malaria have been investigated in the latter. In this study, we investigated 383 malaria patients to clarify the parasitemia characteristics of a P. vivax strain in the Republic of Korea (ROK). The mean parasitemia (8,396/microL) was less than half of tropical P. vivax malaria, and multiple invasions of erythrocytes were not rare (53.5% of the patients, 2.4% of the total investigated RBCs), but less than the observations in tropical zones. The intervals between the first symptom onset and diagnosis were significantly longer in gametocyte (+) patients than in gametocyte (-) patients. Only half of the total patients had both genders of gametocytes (191 of 353), and the male gametocyte density (169/microL) was lower than that of P. vivax strains of a previous study. Multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in P. vivax malaria. The present findings demonstrate the P. vivax strain in ROK reveals relatively low parasitemia and low male to female gametocyte ratio. The low ratio may be related with low transmission efficacy.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Erythrocytes/parasitology ; Female ; Humans ; Malaria, Vivax/*diagnosis/epidemiology/parasitology ; Male ; Middle Aged ; Parasitemia/*diagnosis/epidemiology/parasitology ; Plasmodium vivax/isolation & purification ; Republic of Korea/epidemiology

Parasitemia characteristics of Plasmodium vivax malaria in temperate regions may differ from those in tropical zones. However, most parasitological and clinical features of P. vivax malaria have been investigated in the latter. In this study, we investigated 383 malaria patients to clarify the parasitemia characteristics of a P. vivax strain in the Republic of Korea (ROK). The mean parasitemia (8,396/microL) was less than half of tropical P. vivax malaria, and multiple invasions of erythrocytes were not rare (53.5% of the patients, 2.4% of the total investigated RBCs), but less than the observations in tropical zones. The intervals between the first symptom onset and diagnosis were significantly longer in gametocyte (+) patients than in gametocyte (-) patients. Only half of the total patients had both genders of gametocytes (191 of 353), and the male gametocyte density (169/microL) was lower than that of P. vivax strains of a previous study. Multiple invasions of erythrocytes and gametocytemia were coincident factors of the degree of anemia in P. vivax malaria. The present findings demonstrate the P. vivax strain in ROK reveals relatively low parasitemia and low male to female gametocyte ratio. The low ratio may be related with low transmission efficacy.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Erythrocytes/parasitology ; Female ; Humans ; Malaria, Vivax/*diagnosis/epidemiology/parasitology ; Male ; Middle Aged ; Parasitemia/*diagnosis/epidemiology/parasitology ; Plasmodium vivax/isolation & purification ; Republic of Korea/epidemiology

Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/Pantrade mark, Malaria Ag-Pftrade mark, and Malaria Ag-Pvtrade mark tests, in comparison with the microscopic and PCR methods. A total of 460 blood samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P. vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collected. The sensitivities of Malaria Ag-Pftrade mark and Malaria Antigen Pf/Pantrade mark compared with the microscopic method for P. falciparum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-Pftrade mark, Malaria Ag-Pvtrade mark, and Malaria Antigen Pf/Pantrade mark were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria Ag-Pftrade mark, Malaria Antigen Pf/Pantrade mark, and microscopic method, when PCR was used as a reference method for P. falciparum or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Malaria Ag-Pftrade mark, Malaria Ag-Pvtrade mark, Malaria Antigen Pf/Pantrade mark, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%, respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for application to malaria diagnosis.
Antigens, Protozoan/blood ; Cross-Sectional Studies ; *Diagnostic Techniques and Procedures/instrumentation ; Endemic Diseases/statistics & numerical data ; Humans ; Malaria/*diagnosis/epidemiology/parasitology ; Malaria, Vivax ; Plasmodium falciparum/genetics/immunology/*isolation & purification ; Reagent Kits, Diagnostic ; Thailand/epidemiology

An ELISA was developed for the diagnosis of vivax malaria using multiple stage-specific recombinant antigens of Plasmodium vivax. The DNA from the whole blood of a malaria patient was used as template to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1). Each amplified DNA fragment was inserted into pQE30 plasmid to induce the expression of His-tagged protein in Escherichia coli (M15 strain) by IPTG. His-tagged proteins were purified by Ni-NTA metal-affinity chromatography and used as antigens for ELISA with patient sera that were confirmed previously by blood smear examinations. When applied to patient sera, 122 (80.3%) out of 152 vivax malaria cases reacted to at least one antigen, while no reactions were observed with 128 uninfected serum samples. We applied this ELISA to the screening of 3, 262 civilian residents in endemic regions near the DMZ, which resulted in 236 positively detected (7.2%) cases. This method can be applied to serological diagnosis and mass screening in endemic regions, or can be used as a safety test for transfusion blood in endemic areas.
Animals ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/genetics/*immunology ; *Endemic Diseases ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; Life Cycle Stages ; Malaria, Vivax/*diagnosis/epidemiology/parasitology ; Mass Screening ; Plasmodium vivax/*growth & development/immunology ; Recombinant Proteins/*immunology ; Serologic Tests

PURPOSE: In Korea, vivax malaria re-emerged in 1993 and the outbreak continued in several areas near the DMZ until now. This study was conducted to define the epidemiologic pattern of malaria in Korea and to examine the changes comparing to the one in 1999. METHODS: We collected information about civilian, veteran patients through the National malaria surveillance system and soldier from the Ministry of National Defense. We analyze epidemiological characteristics of malaria by groups (civilian, veteran, soldier). RESULTS: The reported cases of malaria in 2000 were 4,142 that number is an increase of 14% in numbers compared with those of 1999's. Most of cases occured in 17 counties nearby DMZ and from May to October(98.7%) seasonally. The incidence rates (per 100,000) in 2000 by residence were 17.0 in Gangwon-Do, 15.5 in Incheon Metropolitan city, 10.3 in Gyeonggi-Do was dereased. The risk area in 2000 were 17 counties located nearly DMZ and the high risk area were 5 counties where the incidence rate greater than 100. In case of civilian and veteran, the time required to diagnosis from onset of symptom was 8.1 days on the average. CONCLUSION: Epidemiologic pattern of malaria in 2000 did not differ from the one in 1999. Et showed regional spread (increasing risk area) but incidence rate was lowered in the high risk area of 1999. And it is necessary that we pay more attention to Gangwon-Do and Incheon metrocity to reduce the incidence rate in 2001.
Diagnosis ; Epidemiology* ; Gangwon-do ; Gyeonggi-do ; Humans ; Incheon ; Incidence ; Korea* ; Malaria* ; Malaria, Vivax ; Military Personnel ; Seasons ; Veterans

BACKGROUND: Endemic malaria has become increasingly rare since the late 1970s, but since the reemergence of indigenous vivax malaria in 1993, the number of cases of malaria have recently increased. We analyzed the cases of malaria who were treated in Kangnam St. Mary's Hospital with regards to epidemiology, clinical manifestations, and treatment outcome, and made a comparison between indigenous and imported cases. METHODS: We retrospectively analyzed data of 40 confirmed cases of malaria treated in the same hospital. RESULTS: Twenty-one cases were indigenous and 19 were imported malaria. Peripheral blood smear revealed Plasmodium vivax in all indigenous malaria, whereas in imported cases 13 were P. falciparum and 6 were P. vivax. Yeonchon-gun(7 cases) was the most prevalent area in the indigenous cases; Africa and Southeast Asia(7 cases, respectively) were the most prevalent areas in the imported cases. Anemia(hemoglobin <10g/dL) was found more frequently in the imported cases, but the difference was not statistically significant. One patient, who was infected in South America showed initially resistant to primaquine, but was cured after extended treatment with increased dosage of primaquine. CONCLUSION: As cases of indigenous malaria in Korea are increasing, early diagnosis, treament, and prevention of malaria are important. To prevent imported malaria, anyone who travels to endemic areas should receive proper education and chemoprophylaxis, considering the pattern of drug resistance.
Africa ; Chemoprevention ; Drug Resistance ; Early Diagnosis ; Education ; Epidemiology ; Humans ; Korea ; Malaria* ; Malaria, Vivax ; Plasmodium vivax ; Primaquine ; Retrospective Studies ; South America ; Treatment Outcome