Correlation of Pfg377 ortholog gene expression of Plasmodium vivax and mosquito infection.

OBJECTIVE: To determine the expression of Pfg377 ortholog gene in Plasmodium vivax, and examine its correlation with mosquito infection. METHODS: Seventy clinical blood samples positive for P. vivax by microscopy, were used for the mosquito infectivity assay. Infectivity to female Anopheles dirus was determined from oocyst counts. The transcripts of Pfg377 ortholog gene of P. vivax from blood samples infective and non-infective to mosquitoes were examined using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Of 70 P. vivax positive blood samples, 50 (71.4%) samples were mosquito-infective and 20 (28.6%) were not. In infective samples, the expression level of Pfg377 ortholog gene was significantly higher than in the non-infective group (P<0.05). In infective samples, the expression level of Pfg377 ortholog gene at ≥100 copies/ml of blood cut-off point correlated with ≥10 oocysts/mosquito cut-off point of average oocyst numbers and with ≥50% cut-off point of per cent infected mosquitoes (Pearson's chi-square correlation, P=0.014 and P=0.026, respectively). CONCLUSION: The cut-off point of the expression level of Pfg377 ortholog gene could be used to predict the infectiousness of P. vivax gametocytes leading to mosquito infection and parasite transmission in the field.

Plasmodium falciparum-infected erythrocytes form spontaneous erythrocyte rosettes.

Erythrocytes infected with trophozoites or schizonts of Plasmodium falciparum bind uninfected erythrocytes, leading to rosette formation. Both established laboratory strains and fresh isolates from patients form such rosettes, but at widely different frequencies. IgG preparations from the serum of some P. falciparum-immune donors and heparin inhibited rosette formation. The results indicate that cytoadherence of infected erythrocytes to endothelial cells and rosetting represent distinct genetic traits.

Immune response to Plasmodium vivax has a potential to reduce malaria severity.

Plasmodium falciparum infection causes transient immunosuppression during the parasitaemic stage. However, the immune response during simultaneous infections with both P. vivax and P. falciparum has been investigated rarely. In particular, it is not clear whether the host's immune response to malaria will be different when infected with a single or mixed malaria species. Phenotypes of T cells from mixed P. vivax-P. falciparum (PV-PF) infection were characterized by flow cytometry, and anti-malarial antibodies in the plasma were determined by an enzyme-linked immunosorbent assay. We found the percentage of CD3+delta2+-T cell receptor (TCR) T cells in the acute-mixed PV-PF infection and single P. vivax infection three times higher than in the single P. falciparum infection. This implied that P. vivax might lead to the host immune response to the production of effector T killer cells. During the parasitaemic stage, the mixed PV-PF infection had the highest number of plasma antibodies against both P. vivax and P. falciparum. Interestingly, plasma from the group of single P. vivax or P. falciparum malaria infections had both anti-P. vivax and anti-P. falciparum antibodies. In addition, antigenic cross-reactivity of P. vivax or P. falciparum resulting in antibodies against both malaria species was shown in the supernatant of lymphocyte cultures cross-stimulated with either antigen of P. vivax or P. falciparum. The role of delta2 +/- TCR T cells and the antibodies against both species during acute mixed malaria infection could have an impact on the immunity to malaria infection.

Human monoclonal antibodies to Pf 155, a major antigen of malaria parasite Plasmodium falciparum.

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

Monoclonal antibodies to a synthetic peptide corresponding to a repeated sequence in the Plasmodium falciparum antigen Pf155.

Mouse monoclonal antibodies were prepared against a synthetic peptide (EENVEHDA) corresponding to a tandemly repeated sequence in the C-terminus of the Plasmodium falciparum antigen Pf155. One antibody (IgG1) producing hybridoma was studied in detail. The specificity of the antibody was determined by enzyme-linked immunosorbent assays using bovine serum albumin-conjugated or free peptides as solid phase antigens and various synthetic peptides for inhibition. The antibody reacted with Pf155 as detected by immunofluorescence and immunoblotting. It was also an efficient inhibitor of merozoite invasion in P. falciparum in vitro cultures indicating that it defines a biologically important epitope present on the native Pf155 molecule.

Rosette formation by Plasmodium coatneyi-infected red blood cells.

Animal models are needed for the study of cytoadherence in falciparum malaria. Red blood cell (RBC) rosette formation is one type of cytoadherence and appears to be associated with knob formation, endothelial cell adhesion and sequestration of Plasmodium-infected RBCs. Since Plasmodium coatneyi-infected RBCs develop knobs and sequester, we hypothesized that they also form rosettes. RBCs from P. coatneyi-infected rhesus monkeys (Macaca-mulatta) were collected, allowed to mature overnight in vitro and found to form rosettes as hypothesized. This observation adds to the known falciparum-like characteristics of P. coatneyi, and suggests that the Macaca mulatta-P. coatneyi model may be appropriate for pathophysiologic studies of cytoadherence.

Plasmodium coatneyi ring-infected erythrocyte surface antigens.

Plasmodium coatneyi produced ring-infected erythrocyte surface antigen (RESA) during infection of the rhesus monkey. This antigen was immunogenic and elicited an antibody response that was not persistent but was boosted by repeated infections in a manner similar to that seen in P. falciparum infections in humans. Preliminary data showed that the appearance and increasing titer of antibodies to P. coatneyi RESA-like antigen were associated with prolongation of intervals from inoculation to patency and with control of parasitemia. Studies using both immunofluorescence assay and Western blot analysis showed that P. coatneyi-immune rhesus serum cross-reacted with P. falciparum antigens, but P. falciparum immune human serum did not recognize P. coatneyi antigens in either assay. These results show that P. coatneyi expresses RESA-like antigen that elicits an antibody response similar to that observed for human antibody to P. falciparum RESA. However, antibodies to P. coatneyi did not cross-react with P. falciparum RESA in erythrocyte membrane immunofluorescence assay and dot immunoblot analysis, suggesting that different immunogenic epitopes are present on the two molecules. Our observations support the use of this primate model in RESA-based vaccine development.

The effects of hemoglobin genotype and ABO blood group on the formation of rosettes by Plasmodium falciparum-infected red blood cells.

The mechanisms by which the hemoglobin genotype AS protect against severe malaria are not fully understood. We have investigated the possibility that protection might be achieved through an inability of red blood cells (RBC) with the AS genotype to form rosettes with RBC infected by Plasmodium falciparum. No evidence was obtained to support this hypothesis because RBC with the AS genotype formed rosettes with wild isolates of P. falciparum as readily as RBC with the AA genotype. However, the previous finding that parasitized RBC form rosettes more readily with RBC belonging to group A or B than with RBC belonging to group O was confirmed even in fresh clinical isolates.

Short report: Rosette formation in Plasmodium ovale infection.

Red blood cells infected by mature stages of Plasmodium ovale obtained from a 56-year-old Thai patient formed rosettes readily with uninfected erythrocytes. Ex vivo, the ring stage-infected erythrocytes matured well under the in vitro conditions used for P. falciparum culture, and the infected erythrocytes formed rosettes when the parasites became mature trophozoites. These rosettes were stable and remained intact until completion of schizogony. Plasmodium ovale rosettes were similar to those formed by P. falciparum- and P. vivax-infected erythrocytes. Rosette formation appears to be a common property of three species of human plasmodia.

Lymphocyte responses to Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA) peptides in individuals with naturally acquired Plasmodium falciparum malaria.

Antibody titers and lymphocyte responses to synthetic peptides corresponding to repeated amino acid sequences of the 3' and 5' regions of Pf155/ring-infected erythrocyte surface antigen (RESA) were studied in two groups of Thai subjects, soldiers (Rangers), and villagers who differed in their history of malaria exposure. The frequency of Pf155/RESA seropositivity was similar in the two groups while the frequency of high titer antibody was significantly greater in villagers than in Rangers. Lymphocyte responsiveness in vitro to all Pf155/RESA peptides was infrequent for both groups although half of the subjects studied responded to crude Plasmodium falciparum asexual blood stage malaria antigen (MA). Among responders, Pf155/RESA peptides elicited lymphocyte responses in which proliferation and interferon-gamma (IFN-gamma) production were not associated, whereas with MA, the two responses were associated. The MA-stimulated lymphocyte proliferation and IFN-gamma production for both groups of volunteers appeared to be independent of antibody titer. In this study, antibody, but not lymphocyte, responses to Pf155/RESA peptides were shown to reflect differences in prior exposure and levels of acquired immunity to falciparum malaria.

Involvement of cytokines in the histopathology of cerebral malaria.

Histopathologic and immunohistologic studies were performed in two cases of fatal cerebral malaria. On admission, both patients were in unarousable coma with hyperparasitemia. Examination of the tissue sections from various organs showed parasite sequestration in both cases with more extensive area of sequestration in case 1 than in case 2. A panel of monoclonal antibodies against cytokines applied to these tissues clearly detected tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), interleukin-1beta (IL-1beta), and IL-10 in the tissues from brain and liver of case 1. A different cytokine profile, IL-4 and IL-10, was found in the brain tissues of case 2; no TNF alpha nor IFN gamma was detected. There was no cytokine detected in the tissues of other organs in either case. Results of the study suggest that histopathology in the brain of fatal cerebral malaria may be associated with focal accumulation of cytokines. Additionally, the type of cytokines produced locally in a particular tissue during malaria infection may be regulated by the degree of regional parasite sequestration.

Characteristics of Plasmodium vivax-infected erythrocyte rosettes.

To investigate the rosette formation properties of Plasmodium vivax, blood was sampled from 26 adult Thai patients admitted with acute P. vivax malaria and a predominance of trophozoite and schizont stages in their peripheral blood smears. In each case, P. vivax-infected cells formed spontaneous rosettes with two or more uninfected red blood cells. Rosette formation of P. vivax was dependent on the divalent cations (Ca2+/Mg2+) and was highly sensitive to trypsin and heparin, but, unlike P. falciparum, rosettes of P. vivax did not reform after removal of heparin. Plasma taken from patients with either acute uncomplicated P. falciparum or P. vivax malaria reversed rosette formation of all P. vivax isolates whereas plasma from uninfected controls had no effect. There was a small but significant increase in rosette-reversing activity in plasma taken during the convalescent period (P < 0.001). The increment in reversal activity was significantly greater in plasma taken following recovery from P. vivax malaria compared with P. falciparum malaria. This suggests that P. vivax rosette reversal activity is antibody mediated and has both species-specific and cross-species components.

Antibodies to Pf155, a major antigen of Plasmodium falciparum: longitudinal studies in humans.

Antibodies to Pf155, a major Plasmodium falciparum antigen detected in the membrane of glutaraldehyde-fixed and air-dried erythrocytes infected with P. falciparum, were studied in serum samples collected from patients treated for neurosyphilis by induced P. falciparum infection. In 3 patients with no previous documented exposure to malaria, the antibodies were detected late and reached low titers. In 5 patients with extensive previous malaria infections, the antibodies appeared rapidly and reached high titers. The immunofluorescence findings were confirmed by immunoblots. No correlation was observed between antibodies to Pf155 and antibodies detected by standard immunofluorescence with whole parasite antigen.

Seroepidemiologic studies of humoral immune response to the Plasmodium falciparum antigens in Thailand.

We have investigated seroreactivity against Plasmodium falciparum crude parasite antigens, the P. falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), as well as against two synthetic peptides (EENV)6 and (EENVEHDA)3 that represent important epitopes of Pf155/RESA. The study population consisted of 421 children and adult Thais living in an area with moderate malaria transmission. We related these serologic findings to some important epidemiologic baseline data collected in the study area. The parasite rate in study subjects was 18.76%. Sixty-two percent were seropositive to crude P. falciparum antigens, 30.3% to the Pf155/RESA antigen, 23.05% to (EENV)6, and 20.17% to (EENVEHDA)3. Antibody responses to crude P. falciparum antigens and to Pf155/RESA were age dependent and increased with exposure. There was evidence that Pf155/RESA antibodies might play a role in protective immunity in this population. Since Pf155/RESA is a potential vaccine candidate antigen, the information obtained from these field studies will provide some seroepidemiologic baseline data for subsequent vaccine trials.

Ex-vivo short-term culture and developmental assessment of Plasmodium vivax.

A simple reproducible method for short-term ex-vivo Plasmodium vivax culture is presented in which glucose, ascorbic acid, thiamine, hypoxanthine, and 50% human AB+ serum are added to the standard P. falciparum in-vitro culture medium. Culture of freshly obtained blood samples from patients with acute vivax malaria with > 0.5% parasitaemia resulted in > 95% complete schizogony. Culture could be continued for 5-6 cycles without the addition of red cells. Criteria for staging the erythrocytic development of P. vivax in the first schizogonic cycle based on synchronous ex-vivo culture are presented. The asexual cycle was divided into 7 morphological stages: tiny ring (0-6 h), small ring (6-12 h), large ring (12-18 h), early trophozoite (18-28 h), late trophozoite (28-36 h), early schizont (36-42 h) and mature schizont (42-48 h). This simple method of culturing P. vivax ex vivo is suitable for antimalarial susceptibility and immunoparasitology studies.

Enzyme-linked staining method for light microscopic detection of antibodies to parasite antigens on the membrane of Plasmodium falciparum infected erythrocytes.

Indirect erythrocyte membrane immunofluorescence (EMIF), a standard method for detection of antibodies to the P. falciparum blood stage vaccine candidate antigen Pf155/RESA, has been adapted to light microscopy by enzyme-linked immunostaining of the erythrocyte membrane, using alkaline phosphatase and chromogenic substrate. This method gives a dark blue staining of the membranes of ring infected erythrocytes. Results obtained with 70 African sera in EMIF and in enzyme immunostaining correlated well although the enzyme based method sometimes resulted in higher antibody titers and appeared to be slightly more sensitive. Similar results were obtained when comparing immunofluorescence with enzyme immunostaining for detection of antibodies to intraerythrocytic parasite antigens. The enzyme linked immunostaining described is simple and fast and does not require expensive equipment and should, thus, be well suited for use in laboratories with limited resources or under field conditions.

An immunofluorescence study of cerebral malaria. A correlation with histopathology.

Histopathologic and immunopathologic features of cerebral malaria have been defined in a study of six autopsy cases with severe Plasmodium falciparum infection. In five cases, immunofluorescent studies demonstrated intense deposition of P falciparum antigen, IgG, and fibrin in cerebral vessels associated with the histopathologic finding of hemorrhage in the white matter of cerebrum and cerebellum regardless of the presence of parasitized erythrocytes in the cerebral vessels. Immunofluorescent study also demonstrated the extravascular deposits of P falciparum granular antigen associated with acute inflammatory lesion in cerebral tissue in one case. These findings suggested that the immunopathogenic mechanism may in some way play a role in the pathogenesis of cerebral malaria.

Lack of association between CSF nitrate and sera nitrate in falciparum malaria infection.

Nitrate levels in CSF and sera from 16 coma and 19 noncoma falciparum malaria patients were determined using nitric oxide colorometric assay. The medians (range lower, upper limits) of nitrate in sera of comatose and noncomatose patients were 0.28 (0.11, 1.24) and 0.23 (0.05, 0.87) microM, respectively. The medians of nitrate level in CSF of coma and noncoma cases were 0.09 (0.01, 0.28) and 0.15 (0, 1.18) microM, respectively. There was no difference of nitrate level in sera and CSF from comatose or noncomatose patients compared to that in normal sera and CSF. The amount of nitrate in sera and CSF of both groups was not significantly correlated with coma depth, parasitemia, parasite clearance time and time to recovery. Contrast to our in vitro study using immunoperoxidase staining, we found inducible nitric oside synthase production by brain endothelial cells during 4-24 hours of coculturing with late stage of P. falciparum infected red blood cells. These results suggests that malaria severity can not be differentiated by nitrate level in body fluid.

The effects of quinine and artesunate treatment on plasma tumor necrosis factor levels in malaria-infected patients.

Tumor necrosis factor-alpha (TNF-alpha) is an endogenous mediator of shock and inflammation including malaria. Many lines of evidence suggest that cytoadherence, the life-threatening pathology associated with complicated and cerebral malaria, results from the overproduction of TNF in response to malarial parasite. Quinine has been shown to inhibit TNF synthesis and cytoadherence in vitro suggesting an additional beneficial effect of quinine on its anti-TNF action. On the other hand, artesunate inhibits cytoadherence better than quinine does not suppress TNF production in vitro. The present study compares the effect of artesunate and quinine on TNF levels of malaria-infected patients. Surprisingly, plasma TNF levels increased dramatically after quinine administration but did not increase after artesunate administration. This difference may be explained by previous observations showing that artesunate kills parasites in vitro and clears parasitemias in vivo for more rapidly than quinine. The rapid clearance of plasma TNF in quinine treated patients might be due to the drug's TNF-suppressive activity.

Epidemiological studies of malaria at Pong Nam Ron, eastern Thailand.

Malaria is still a serious health problem in Thailand. Present attempts at controlling the disease by drug treatment and other means remain unsatisfactory. Thus, development of vaccination against malaria is a major research goal of malaria immunology. The objective of this study was to acquire epidemiological base line data for subsequent vaccine trials. A cross-sectional descriptive survey was conducted among 451 local inhabitants during the beginning of the transmission season in June 1989 at Pong Nam Ron District, Chanthaburi Province, Eastern Thailand where malaria transmission was likely to be high. Following the cross-sectional survey weekly morbidity surveillance was started to detect new cases of malaria by using active and passive case detection at the district hospital, local health centers and at neighboring malaria clinics. Entomological observations were made monthly to determine inoculation rates. Forty-six percent of the population were male and 54% female; one third were under the age of 15 and 14% under the age of 5 years. Eighty percent of the adults were married. Sixty percent of the subjects interviewed gave a history of malarial illness in the past. Malaria, malnutrition, abnormal hemoglobin diseases and parasitic infestation were the main health problems in the study area. The annual parasite incidence of malaria was 149.6/1,000 population and two-thirds of them were asymptomatic indicating a semi-immune condition among these subjects. It was difficult to interpret the results of entomological studies due to low density of the malaria vector.