Rapid diagnostic tests (RDTs) for malaria using antibodies against pan-Plasmodium antigen lactate dehydrogenase (pLDH) are commonly used for malaria diagnosis. The level of malaria parasitemia determined by peripheral blood smears (PBS) correlates with the pLDH concentration in most cases. We report a case of malaria recurrence associated with false-negative RDT results. A 22-year-old male patient was admitted to the Armed Forces Capital Hospital with fever and chills, and was diagnosed with malaria infection. Four days after antimalarial treatment, these symptoms recurred. After admitting to our hospital, doxycycline was administered for 4 days. Even after administration of doxycycline, the malaria parasites in blood smears remained positive, but RDT showed negative results. Therefore, for patients receiving doxycycline, serial blood smear testing should be performed to exclude false-negative malaria RDT results.
Antibodies ; Arm ; Chills ; Diagnosis ; Diagnostic Tests, Routine ; Doxycycline ; Fever ; Humans ; L-Lactate Dehydrogenase ; Malaria ; Male ; Parasitemia ; Parasites ; Recurrence ; Young Adult

Malarial infection induces tissue hypoxia in the host through destruction of red blood cells. Tissue hypoxia in malarial infection may increase the activity of HIF1α through an intracellular oxygen-sensing pathway. Activation of HIF1α may also induce vascular endothelial growth factor (VEGF) to trigger angiogenesis. To investigate whether malarial infection actually generates hypoxia-induced angiogenesis, we analyzed severity of hypoxia, the expression of hypoxia-related angiogenic factors, and numbers of blood vessels in various tissues infected with Plasmodium berghei. Infection in mice was performed by intraperitoneal injection of 2×10⁶ parasitized red blood cells. After infection, we studied parasitemia and survival. We analyzed hypoxia, numbers of blood vessels, and expression of hypoxia-related angiogenic factors including VEGF and HIF1α. We used Western blot, immunofluorescence, and immunohistochemistry to analyze various tissues from Plasmodium berghei-infected mice. In malaria-infected mice, parasitemia was increased over the duration of infection and directly associated with mortality rate. Expression of VEGF and HIF1α increased with the parasitemia in various tissues. Additionally, numbers of blood vessels significantly increased in each tissue type of the malaria-infected group compared to the uninfected control group. These results suggest that malarial infection in mice activates hypoxia-induced angiogenesis by stimulation of HIF1α and VEGF in various tissues.
Angiogenesis Inducing Agents ; Animals ; Anoxia ; Blood Vessels ; Blotting, Western ; Erythrocytes ; Fluorescent Antibody Technique ; Immunohistochemistry ; Injections, Intraperitoneal ; Malaria ; Mice ; Mortality ; Parasitemia ; Plasmodium ; Plasmodium berghei ; Vascular Endothelial Growth Factor A

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.
Animals ; Anopheles ; Antibodies ; Clone Cells ; Coinfection ; Communicable Diseases ; Culicidae ; Humans ; Immunity, Humoral ; Life Cycle Stages ; Malaria ; Merozoite Surface Protein 1* ; Mice ; Mice, Inbred ICR ; Parasitemia ; Parasites ; Plasmodium yoelii* ; Plasmodium* ; Rodentia ; Sporozoites ; Vaccines

In recent years, rapid diagnostic tests (RDTs) have been widely used for malaria detection, primarily because of their simple operation, fast results, and straightforward interpretation. The Asan EasyTest(TM) Malaria Pf/Pan Ag is one of the most commonly used malaria RDTs in several countries, including Korea and India. In this study, we tested the diagnostic performance of this RDT in Uganda to evaluate its usefulness for field diagnosis of malaria in this country. Microscopic and PCR analyses, and the Asan EasyTest(TM) Malaria Pf/Pan Ag rapid diagnostic test, were performed on blood samples from 185 individuals with suspected malaria in several villages in Uganda. Compared to the microscopic analysis, the sensitivity of the RDT to detect malaria infection was 95.8% and 83.3% for Plasmodium falciparum and non-P. falciparum, respectively. Although the diagnostic sensitivity of the RDT decreased when parasitemia was < or =500 parasites/microl, it showed 96.8% sensitivity (98.4% for P. falciparum and 93.8% for non-P. falciparum) in blood samples with parasitemia > or =100 parasites/microl. The specificity of the RDT was 97.3% for P. falciparum and 97.3% for non-P. falciparum. These results collectively suggest that the accuracy of the Asan EasyTest(TM) Malaria Pf/Pan Ag makes it an effective point-of-care diagnostic tool for malaria in Uganda.
Adolescent ; Adult ; Antigens, Protozoan/blood/*isolation & purification ; Child ; Child, Preschool ; Humans ; Malaria, Falciparum/*diagnosis/epidemiology ; Parasitemia ; Point-of-Care Systems ; Predictive Value of Tests ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Uganda/epidemiology ; Young Adult

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.
Animals ; Azure Stains ; Biological Assay ; Brain/parasitology ; DNA, Protozoan/*blood/genetics ; Lung/parasitology ; Mice ; Nucleic Acid Amplification Techniques/*veterinary ; Parasitemia ; Real-Time Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity ; Swine ; Swine Diseases/*diagnosis/parasitology ; Toxoplasma/genetics/*isolation & purification ; Toxoplasmosis, Animal/*diagnosis/parasitology

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.
*Flow Cytometry ; Fluorescent Dyes/chemistry ; Humans ; *Microscopy ; Organic Chemicals/chemistry ; Parasitemia/*diagnosis ; Plasmodium falciparum/*isolation & purification ; Reproducibility of Results ; Ribonucleases/metabolism ; Signal-To-Noise Ratio

OBJECTIVETo compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210.

METHODSThe study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays.

RESULTSA total of 30 out of 64 blood samples collected from patients with P. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210.

CONCLUSIONSOn the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.

Antimalarials ; pharmacology ; Chloroquine ; pharmacology ; Humans ; Inhibitory Concentration 50 ; Malaria, Vivax ; parasitology ; Organic Chemicals ; Parasitemia ; parasitology ; Parasitic Sensitivity Tests ; Plasmodium vivax ; drug effects ; isolation & purification ; Schizonts ; drug effects

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

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.
Animals ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/*blood ; Enzyme-Linked Immunosorbent Assay/methods ; Immunoblotting/methods ; Immunoglobulin G/*blood ; Immunoglobulin M/*blood ; Mice ; Parasitemia ; Rabbits ; Time Factors ; Toxoplasma/*immunology ; Toxoplasmosis, Animal/*immunology/parasitology