ELISA detection of vivax malaria with recombinant multiple stage-specific antigens and its application to survey of residents in endemic areas

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.     

Current status of vivax malaria among civilians in Korea

A result of national malaria surveillance in Korean civilians was described. Since a case of indigenous vivax malaria was detected in 1993, a total of 2,198 cases was confirmed by blood smear up to 1997. Of them, 1,548 cases were soldiers serving in the demilitarized zone (DMZ), while 650 cases were civilians. Number of civilian cases was 3 in 1994, 19 in 1995, 71 in 1996, and 557 in 1997. Of them, 239 were ex-soldiers who discharged after military service in the prevalent areas such as Paju, Yonchon, Kimpo, Kangwha, Tongduchon in Kyonggi-do and Chorwon in Kangwon-do while 308 patients were civilian residents in the prevalent areas. Seventy-two patients, living nationwide, had a history of visiting the prevalent areas during transmission season. Only 32 civilian patients denied any relation with the prevalent areas. As a whole, a half of the civilian cases was diagnosed when living in non-prevalent areas. Male patients in their twenties was the highest in number. Annual parasite index is steadily elevated in residents living in the prevalent areas. Monthly incidence showed an unimodal distribution, forming a peak in August. Ex-soldiers exhibited a delayed incubation ranging from 153 to 452 days (279 ± 41 days). The time required for diagnosis was shortened from 23.6 days in 1995 to 13.7 days in 1997. Although the current epidemic of vivax malaria started as a border malaria, it seems highly probable that vivax malaria is established in the local areas and responsible for at least a part of transmission.     

A case of symptomatic splenic infarction in vivax malaria

Splenic infarction is a rare complication in malaria cases, and is caused primarily by Plasmodium falciparum. Recently in South Korea, only P. vivax has prevailed since 1993. Although the probability that symptomatic splenic infarction may occur in vivax malaria cases is considered relatively high, there have never been any case reports describing the occurrence of symptomatic splenic infarction in cases of vivax malaria. A 34-year-old man presented with fever that had persisted for 5 days. P. vivax infection was verified using a peripheral blood smear, and chloroquine was utilized to treat the fever successfully. Six days later, the patient developed pain in the left upper abdomen, which was diagnosed as splenic infarction by computed tomography.     

Fourteen polymorphic microsatellite DNA markers for the human malaria parasite Plasmodium vivax

We have optimized a set of 14 polymorphic microsatellite markers for the human malaria parasite Plasmodium vivax, all of them consisting of either tri‐ or tetranucleotide repeats. These markers, whose polymerase chain reaction amplification conditions are identical, were used to screen 25 parasite isolates from malaria‐endemic areas in Sri Lanka. The total number of alleles per locus ranged between 6 and 13 (average, 7.8), and expected heterozygosity ranged from 0.627 to 0.913 (average, 0.790). These markers are now being used to characterize the population structure of P. vivax in other endemic areas.     

Western blot analysis of cerebrospinal fluid for detection ofAspergillus antigens

Western-blot immunoassay of cerebrospinal fluid (CSF) specimens of patients with central nervous system (CNS) aspergillosis (3), CNS candidosis (1) and bacterial meningitis (2) was carried out using pooled serum from histopathologically proven deep-seated aspergillosis cases to detect unique antigenic fractions for aspergillosis in CSF. No reactivity was observed in patients with non-fungal meningitis. Four cross-reactive bands (40, 90, 200 and >200 KD) were detectable in CSF from patients with both aspergillosis and candidosis of the CNS. Four additional bands (90–200 KD) were consistently present only in patients with aspergillosis. One prominent band (110 KD) was found only in the patient with aspergillosis who had a fatal outcome and raised the possibility of being a poor prognostic marker.     

Incidence patterns of vivax malaria in civilians residing in a high-risk county of Kyonggi-do (province), Republic of Korea.

The characteristics of vivax malaria epidemics along the demilitarized zone (DMZ) in the Republic of Korea has been established by the early surveillance data. To further characterize the epidemic, data of civilian patients microscopically diagnosed with malaria from 1995 through 2000 were analyzed in Yonchon-gun (county). Malaria incidence was greater in male civilians > 30 years of age (p < 0.05). The annual parasite index was significantly higher in those living in the administrative areas (Myeon) traversed by DMZ than those living in Myons not traversed by DMZ (p < 0.05). Analysis according to the distance (4 to 14 km) from DMZ showed that people living in villages close to DMZ had higher annual parasite indices than those living in villages remote from DMZ (p for trend < 0.05). Civilians living in Myeons with plains and located in northwestern part of the county had higher annual parasite indices than those living in hilly Myeons located in southeastern part of the county (p for trend < 0.05). These findings suggest that the contraction of vivax malaria is related with night-time outdoor activities, and that the distance from DMZ is a risk factor. In this area, the flying distance of infected vector mosquitos can explain the annually repeating occurrence of civilian cases.     

Slow clearance of Plasmodium vivax with chloroquine amongst children younger than six months of age in the Brazilian Amazon

Plasmodium vivax is the most widespread parasite causing malaria, being especially prevalent in the Americas and Southeast Asia. Children are one of the most affected populations, especially in highly endemic areas. However, there are few studies evaluating the therapeutic response of infants with vivax malaria. This study retrospectively evaluated the parasitaemia clearance in children diagnosed with vivax malaria during the first five days of exclusive treatment with chloroquine (CQ). Infants aged less than six months old had a significantly slower parasitaemia clearance time compared to the group of infants and children between six months and 12 years old (Kaplan-Meier survival analysis; Wilcoxon test; p = 0.004). The impaired clearance of parasitaemia in younger children with vivax malaria is shown for the first time in Latin America. It is speculated that CQ pharmacokinetics in young children with vivax malaria is distinct, but this specific population may also allow the detection of CQ-resistant parasites during follow-up, due to the lack of previous immunity.     

Reemerging vivax malaria: changing patterns of annual incidence and control programs in the Republic of Korea

Changing patterns of the reemerging Plasmodium vivax malaria in the Republic of Korea (South Korea) during the period 1993 to 2005 are briefly analyzed with emphasis on the control measures used and the effects of meteorological and entomological factors. Data were obtained from the Communicable Diseases Monthly Reports published by the Korea Center for Disease Control and Prevention, and webpages of World Health Organization and United Nations. Meteorological data of Kangwon-do (Province) were obtained from local weather stations. After its first reemergence in 1993, the prevalence of malaria increased exponentially, peaking in 2000, and then decreased. In total, 21,419 cases were reported between 1993 and 2005 in South Korea. In North Korea, a total of 916,225 cases were reported between 1999 and 2004. The occurrence of malaria in high risk areas of South Korea was significantly (P > 0.05) correlated with the mosquito population but not with temperature and rainfall. Control programs, including early case detection and treatment, mass chemoprophylaxis of soldiers, and international financial aids to North Korea for malaria control have been instituted. The situation of the reemerging vivax malaria in the Republic of Korea is remarkably improving during the recent years, at least in part, due to the control activities undertaken in South and North Korea.     

Border malaria characters of reemerging vivax malaria in the Republic of Korea.

Since 1993, the number of vivax malaria cases has increased every year in the northern part of the Republic of Korea (ROK). This study was designed to characterize factors related to the reemergence of malaria in the ROK. A total of 21 cases diagnosed in 1993 and 1994 distributed sporadically in the narrow zone along the demilitarized zone (DMZ). Of total 317 civilian inhabitant cases reported in 1994-1997, 287 cases were studied and 80.8% of them resided within 10 km from the southern border of the DMZ. The frequency distribution of anti-Plasmodium vivax antibody titers using indirect fluorescent antibody test was compared in three villages in relation with distance from the DMZ. The number of inhabitants with high antibody titers was larger in the village nearest to the border than that in more distant villages. The present results highly suggested that the reemerging vivax malaria start in the border area, most possibly caused by infected mosquitoes which flew across the border. This pattern of transmission repeated year after year.     

Analysis of stage-specific and shared antigens derived from Rhipicephalus sanguineus by electrophoresis and Western blotting

e carried out an SDS-PAGE analysis of antigens of Rhipicephalus sanguineus using extracts of eggs (EE), larvae (LE), nymphs (NE), male salivary glands (MSGE), male midguts (MME), female salivary glands (FSGE) and female midguts (FME). Under non-reducing conditions a common band of about 205 kDa was observed. EE, LE and NE extracts showed groups of bands between 150 and 75 kDa. A protein pattern was observed in FSGE extract with a group of bands between 75 and 50 kDa and four bands between 15 and 6.5 kDa. In this case an apparently exclusive band of molecular weight about 25 kDa was observed. Under reducing conditions similarities between LE and NE extracts increased, separating from the EE pattern. On the other hand, we have determined the presence of stage-specific and common antigens on EE, LE, NE, MSGE, MME, FSGE and FME extracts of R.sanguineus by means of immunoblots using polyclonal sera of rabbits infested with larvae, nymphs or adults of this tick. EE extract was only recognized by the anti-larva sera. Higher reactivity was observed when the extracts were tested with anti-adult sera. In these experiments a very prominent band of molecular weight about 45 kDa was detected. This band was not observed under reducing conditions. Higher reactivity with anti-adult sera was observed against FSGE extract.     

Target antigens of transmission blocking immunity of Plasmodium vivax malaria. Characterization and polymorphism in natural parasite isolates.

A panel of 20 anti-Plasmodium vivax female gamete mAb has been established and was characterized with respect to their transmission-blocking properties in membrane-feeding experiments and their target Ag identified. Seven mAb suppressed the infectivity of P. vivax parasites to Anopheles tesselatus mosquitoes. The m.w. of the Ag recognized by these mAb were ascertained by SDS-PAGE and Western blots. Three sets of polypeptides of low Mr--20, 24, and a doublet of 37/42 kDa--have been defined as target Ag of transmission-blocking antibodies of P. vivax. All epitopes of these target Ag were found to be dependent on the tertiary conformational structure of the Ag. Polymorphism of target Ag of transmission-blocking immunity was investigated in over 30 natural isolates of P. vivax in Sri Lanka based on the reactivity of a mAb with an isolate as assessed by the indirect immunofluorescent test with the use of live extracellular female gametes, and in Western blots with the use of extracted gametes. The functional consequences of antigenic polymorphism on immunity was investigated in transmission-blocking assays by using membrane-feeding experiments. A majority of target Ag of transmission-blocking immunity were found to be polymorphic, exhibiting size as well as epitope polymorphism. Results indicate that failure of a mAb to affect the infectivity of a parasite isolate of P. vivax to mosquitoes can be caused by polymorphism of the target Ag among isolates.     

Analysis of vivax malaria cases in Gangwon-do (province), Korea in the year 2000.

A total of 827 malaria cases were reported in the Gangwon-do in the year 2000. There were 18.2 cases per 100,000 inhabitants. There were 283 cases among civilians and 544 cases among the military. 90.6% of cases were reported in Cheorwon--(531), Hwacheon--(152), and Goseong--(66) gun (county), which bordered the demilitarized zone (DMZ). A distinct feature pertaining to the malaria cases in Gangwon-do is that the number of cases has increased about two times over the last year. The mean time from the beginning of symptoms to malaria diagnosis was five days. Control systems for malaria by public health organizations and military organizations are well maintained, but were not able to reduce the malaria prevalence rate. The cause for the increase in pattern of the malaria cases in Gangwon-do may be caused by the spreading of prevalent areas of malaria to the east. Continuous endeavor such as early detection of cases, early treatment, education on clinical symptoms and prevention of mosquito bites with repellent and mosquito nets will help to reduce the infection rate of malaria in Gangwon-do.     

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.     

Saccharide anions as inhibitors of the malaria parasite

The asexual erythrocytic stage of Plasmodium falciparum was grown in culture in the presence or absence of glycoconjugate polyanions of varying structure, size and substitutions. Heparin, dextran sulfate, fucoidan and pentosan polysulfate had antimalarial IC50 values between one and 11 μg ml−1. Constituent heparin disaccharides were ineffective against the malaria parasite and desulfation from either the O- or N-substitution sites of heparin or reduction of the uronic acid carboxyl group neutralized the antimalarial response to varying degrees. Immobilization of heparin onto agarose beads still permitted antimalarial activity suggesting that parasite uptake of the glycoconjugate is not required for inhibition. Accordingly, it is concluded that invasion of free parasites into the erythrocytes was inhibited rather than parasite maturation within the red cell. Merozoite surface antigen-1 was apparently prevented from binding to human erythrocytes in the presence of highly sulfated polyanions and, in a dose-dependent fashion, heparin. Abbreviations: MSA-1, merozoite surface antigen-1 This revised version was published online in November 2006 with corrections to the Cover Date.     

Safety and tolerability of elubaquine (bulaquine, CDRI 80/53) for treatment of Plasmidium vivax malaria in Thailand

We conducted a study to compare the safety and tolerability of anti-relapse drugs elubaquine and primaquine against Plasmodium vivax malaria. After standard therapy with chloroquine, 30 mg/kg given over 3 days, 141 patients with P. vivax infection were randomized to receive primaquine or elubaquine. The 2 treatment regimens were primaquine 30 mg once daily for 7 days (group A, n = 71), and elubaquine 25 mg once daily for 7 days (group B, n = 70). All patients cleared parasitemia within 7 days after chloroquine treatment. Among patients treated with primaquine, one patient relapsed on day 26; no relapse occurred with elubaquine treatement. Both drugs were well tolerated. Adverse effects occurred only in patients with G6PD deficiency who were treated with primaquine (group A, n = 4), whose mean hematocrit fell significantly on days 7, 8 and 9 (P = 0.015, 0.027, and 0.048, respectively). No significant change in hematocrit was observed in patients with G6PD deficiency who were treated with elubaquine (group B, n = 3) or in patients with normal G6PD. In conclusion, elubaquine, as anti-relapse therapy for P. vivax malaria, was as safe and well tolerated as primaquine and did not cause clinically significant hemolysis.     

Computer modeling of small heat-shock metalloprotease of the human malaria parasite Plasmodium vivax.

We present here computer generated model of N-terminal fragment, amino acids (aa) 36-245, of a Plasmodium vivax heat shock metalloprotease called PVHSP28, whose gene was cloned and characterised earlier. The fragment showed homology with HSPs from many organisms, including Escherichia coli and Haemophilus influenzae. PVHSP28 had the signature sequence 'HEXXH' and 'EXXXD' of Zinc metalloproteases. Being the first malarial HSP possessing metalloprotease activity, PVHSP28 is an ideal target for the design of new anti-malarial drugs. However, except for a small region (aa 62-132) which had 24.6% sequence similarity with 1TAQ (a DNA polymerase), it did not show sequence similarity with any published structures in protein data bank. Hence it could not be modelled using any automated modeling programs. We modelled 36-245 aa of PVHSP28 using predicted secondary structure as well as experimentally determined and predicted properties of the protein on the basis of its amino acid sequence, using various Internet tools and in-house package MODEL. The model was energy minimised using Sander's module of AMBER 5.0, working on a Silicon Graphics machine, with all atom force field.     

Alterations in the permeability of mouse erythrocytes infected with the malaria parasite, Plasmodium berghei

Alterations in the permeability of erythrocytes from mice infected with the malaria parasite Plasmodium berghei were demonstrated with l-¹⁴C-glucose. This sugar rapidly entered erythrocytes from infected mice but not those from normal mice. The results obtained were not due to contamination either by white cells, which were removed beforehand, or by immature cells, as judged by comparable investigations on blood from phenylhydrazine treated mice. Uptake was proportional to the concentration in the suspending medium, and was affected neither by relatively high concentrations of d-glucose nor by N-ethyl maleimide, and this suggests that the rapid uptake was due to an increased rate of diffusion through the membrane of the erythrocytes.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Crystal structure of S-adenosyl-L-homocysteine hydrolase from the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.     

Western blot analysis of stray cat sera against Toxoplasma gondii and the diagnostic availability of monoclonal antibodies in sandwich-ELISA

A total of 198 sera from stray cats was assayed against Toxoplasma gondii antigen by western blot. Out of 198 sera assayed, 26 sera (13.1%) showed typical blot patterns against T. gondii. When spotted by ELISA absorbance and indirect latex agglutination test (ILAT) titer, all 26 cases were distributed over the cut-off value of ELISA whereas 24 cases (92.3%) were in the positive range of 1:32 or higher and 2 cases in negative range by ILAT. Among western blot negative 172 sera, 162 cases were negative in both ILAT and ELISA while 10 cases were reactive falsely such that three cases were ILAT positive with 1:32 titer and 9 cases were ELISA positive (2 cases overlapped). These 10 cases reacted peculiarly without typical binding pattern in Western blot. Sandwich-ELISA was performed with monoclonal antibodies (mAbs) of Tg563 (30 kDa, SAG1), Tg505 (22 kDa, SAG2), Tg605 (43 kDa, SAG3), Tg556 (28 kDa. GRA2), Tg737 (32 kDa, GRA6), Tg695 (66 kDa, ROP2), Tg786 (42 kDa, ROP6), and Tg621 (32 kDa, anonymous but cytosolic) clone, respectively. All western blot-positive cases were in the positive range and negative cases in the negative range clearly. Among the 10 false reactive cases, 3 cases were in the positive range with one or more mAbs. All mAbs used in this study were confirmed to be specific to T. gondii infection as a standardized sandwich-ELISA to differentiate it from other pathogens.