Characteristics of natural antibody responses to the circumsporozoite protein of Plasmodium vivax

The antibody response to the prototype circumsporozoite (CS) protein of Plasmodium vivax (CSPV) was studied in Thai soldiers experiencing occupational malaria. Seventy-four (65%) of 114 men followed during assignment to a malaria transmission area developed blood-stage infection with P. vivax. IgG antibodies against the central repeat region of the CSPV protein were quantitated by ELISA using the recombinant protein, NS181V20, as the capture antigen. One quarter of the subjects had detectable anti-CSPV antibodies at the beginning of the study. CSPV antibody seroconversion was documented in 16 of 26 subjects assessed during their first observed episodes of vivax malaria. This antibody response was of moderate magnitude, fell off after the first week post-diagnosis and appeared, at the low levels observed, to be unassociated with protection. Continued assessment of anti-CSPV antibody after subjects left the transmission area found no increase associated with release of P. vivax. These findings indicate that CS antibody responses to P. vivax during occupational malaria share many characteristics with responses to P. falciparum.     

Occurrence of a common epitope in circumsporozoite proteins of Plasmodium falciparum isolated from different areas in Thailand

Fifteen isolates of P. falciparum sporozoites obtained from patients with acute falciparum malaria from various malaria endemic areas in Thailand were tested for the presence of a common antigenic determinant in their CS protein molecules. SDS-PAGE and Western blot analysis using MAB or human serum antibodies specific to the CS proteins of the parasites revealed a common epitope shared in the CS proteins of all strains of P. falciparum tested. However, the CS proteins exhibited M.W. variation when different strains of the parasites were compared. A similar result was obtained when the human serum antibodies were used. The present study clearly indicated the occurrence of the common epitope in phenotypically different CS proteins among isolates of P. falciparum sporozoites and supported the notion that antigens containing these repetitive epitopes could be used as the candidates for the sporozoite vaccine against P. falciparum infection.     

Development and evaluation of a rapid diagnostic test for Plasmodium falciparum, P. vivax, and mixed-species malaria antigens

Plasmodium falciparum and P. vivax malaria are endemic to many parts of the world and humans can be co-infected with both species. Because each Plasmodium species has different biological and clinical characteristics, accurate differentiation of the infecting species is essential for effective treatment. Therefore, we produced three monoclonal antibodies that recognize the lactate dehydrogenase of P. falciparum, P. vivax, or both to develop the first P. falciparum, P. vivax, and mixed-species infections malaria antigen detection kit. The detection limits of this kit were 150 and 250 parasites/μL for P. falciparum and P. vivax, respectively, and the kit was able to detect mixed-species infections. The sensitivity and specificity of this kit was assessed with 722 clinical specimens. Our results showed that its sensitivities for P. falciparum, P. vivax, and mixed-species infection were 96.5%, 95.3%, and 85.7%, respectively. In addition, its specificity was high (99.4%).     

Low CD8+ T lymphocyte response to P. falciparum circumsporozoite protein in naturally-exposed malaria endemic populations in Thailand

Cytotoxic T lymphocytes (CTLs) specific for epitope(s) within the circumsporozoite (CS) protein of malaria sporozoite have been shown to play an important role in protective immunity against malaria. Human CTLs against the potential epitope at the carboxy terminal region of CS protein of Plasmodium falciparum 7G8 strain (Pf7G8CS 368-390) were determined in thirty-six falciparum malaria patients and ten healthy controls. Four of 36 individuals and none of the healthy controls developed Pf7G8CS 368-390 specific CTL activity. The CTL activity was antigen specific and CD8+ T cell dependent. Although low CTL response has been determined, the study suggested that there was a correlation between initial parasitemia and the specific Pf7G8CS 368-390 CTL activity. A correlation between such CTL activity and anti-R32tet32 antibody levels among individuals with previous malaria experience was found, which was in contrast to those among individuals with recent malaria infection. All these 4 CTL positive individuals had at least two episodes of clinical malaria experience while all 25 individuals who were exposed to malaria for the first time did not have such a specific CTL response. These results showed that individuals with a history of natural endemic exposure to P. falciparum sporozoite developed low specific CTL responses to Pf7G8CS 368-390, so that previous but recent sporozoite exposure might be a prerequisite for generation of such CS protein specific CTL response.     

Evaluation of a rapid diagnostic test specific for Plasmodium vivax

Plasmodium vivax is the only human malaria indigenous to the Republic of Korea (ROK). A rapid and sensitive diagnostic test (RDT) that detects P. vivax is appropriate for evaluating suspected malaria patients with no travel history abroad. The RDTs, SD Malaria Antigen P.v (SD diagnostic, Kyonggi, ROK) specific for P. vivax and the well documented OptiMAL (DiaMed, Cressier, Switzerland) were compared among 282 volunteers for specificity and sensitivity of P. vivax and Plasmodium falciparum malaria infections against Giemsa-stained blood smears read by an experienced microscopist. A total of 137 volunteers were diagnosed with P. vivax, 45 cases (returned travellers from overseas) were diagnosed with P. falciparum and 100 healthy volunteers were diagnosed as negative for malaria. Correspondingly, the SD Malaria Antigen P.v test identified P. vivax infections in 128/137 malaria patients (93.4%) and 0/100 (0%) healthy volunteers. Three patients identified with P. falciparum also were interpreted as P. vivax by the SD Malaria Antigen P.v test; however, these patients were later confirmed as mixed infections of P. vivax and P. falciparum by polymerase chain reaction. OptiMAL interpreted the three mixed infections only as P. falciparum and detected 130/137 (94.9%) patients with P. vivax. The sensitivity of the SD Malaria Antigen P.v test decreased from 100% (>5000 parasite/microl) to 81.3% (1-100 parasites/microl) as parasitaemia levels declined. For the regions where P. vivax is the primary malaria parasite, the SD P. vivax-specific rapid diagnostic test may be useful for screening suspected malaria patients when sufficient material and human resources (e.g. trained microscopists) are unavailable for malaria diagnosis.     

Cross-reactive cellular immune response to circumsporozoite proteins of Plasmodium vivax and P. falciparum in malaria-exposed individuals

Acquired immunity against the recombinant circumsporozoite protein of P. falciparum (rPfCS) or P. vivax (rPvCS) was studied in two malarious areas of the Brazilian Amazon. Cellular responsiveness, evaluated by proliferative assays, was detected in about 45% of individuals who had recovered from recent acute malaria infections. Peripheral blood mononuclear cells of individuals whose last malaria infection was by P. vivax responded more to the rCS proteins than those who had P. falciparum . Since in P. vivax infections hypnozoites in the liver retain CS antigen, this stage may have contributed to the increased cellular response. The unexpected result was that in primoinfections by P. falciparum or P. vivax the proliferative response did not correspond to the rPfCS and rPvCS, respectively. Furthermore, among the malaria-exposed individuals, there was a positive correlation between the intensity of the responses to the two rCS proteins. Our results suggest that cross-reactive epitopes exist in the CS protein of P. falciparum and P. vivax . In the areas studied, the frequency of antibodies against rPvCS and/or rPfCS ranged from 43% to 11%. Species-specific antibodies against the CS protein were detected in the primoinfected individuals. Some individuals living in the endemic area but with no clinical history of malaria were positive by serology (8%) or by in vitro proliferation (21%). However, antibodies and cellular responses against rCS were detected only in malaria-exposed individuals, since those living outside the endemic area were all negative.     

Antibodies against circumsporozoite proteins of Plasmodium falciparum induced by natural infection

Sera from 10 individuals who lived in a malaria endemic area, 10 patients with acute uncomplicated falciparum malaria and 10 patients with cerebral malaria and hyperimmune mouse serum were tested for their reactivities against Plasmodium falciparum sporozoite antigens by Western blot analysis using 125I-labeled staphylococcal protein A as the detecting reagent. These sera were shown by indirect immunofluorescence and/or circumsporozoite precipitation test to have antibodies reacting against the parasites. It was found that all serum antibodies from the three groups of individuals and the mouse serum reacted in a similar pattern with circumsporozoite (CS) proteins of P. falciparum. Ten sera from normal individuals were negative in all reactions. Monoclonal antibody (MAB) specific against CS proteins of the parasites showed that the proteins exhibited as four different molecular weight (MW) polypeptides, i.e., 67,000, 65,000, 60,000, and 58,000 daltons. These CS proteins of P. falciparum were found to be species and stage specific. Radioimmunoprecipitation using 35S-methionine-labeled parasites and sera of individuals from the various categories or MABs gave a similar result. Another protein antigen of P. falciparum sporozoites had a MW of 80,000 daltons. This antigen was not species specific, probably not membrane associated and was present in a minute quantity in the parasite's extract.     

Seroconversion to circumsporozoite antigen of Plasmodium falciparum demonstrates a high risk of malaria transmission in travelers to East Africa.

Circumsporozoite (CS) antibodies have been shown to be reliable indicators of malaria transmission in endemic areas. Their prevalence in travelers can indicate the degree of exposure to plasmodial infection. Two hundred sixty-two short-term travelers to Kenya were recruited to a prospective study to determine the incidence of CS antibody conversion. All travelers were receiving malaria chemoprophylaxis. Serum samples were drawn before departure and 4-6 weeks after their return to Germany. Sera from 310 volunteers who did not leave Germany served as controls. Serum specimens from 13 (4.96%) of the 262 travelers were found to be positive after return. None of the travelers developed symptoms of clinical malaria or antibodies against the blood stages of Plasmodium falciparum. All 310 control samples tested negative. These data demonstrate a considerable risk of malaria transmission for short-term travelers to East Africa.     

Characterization of naturally acquired antibodies to the non-repetitive flanking regions of the circumsporozoite protein of Plasmodium falciparum.

Antibody responses to the circumsporozoite (CS) protein of Plasmodium falciparum have previously been reported against the central repeating tetrapeptides of this protein. Segments of the protein flanking the repeat region also contain B-cell epitopes, but specific antibody responses have not been previously characterized. Longitudinal serum sets from 16 Thai adults who developed acute falciparum malaria were selected to represent a spectrum of antibody response to the repeat region (R32). These sera were assayed by enzyme-linked immunosorbent assay using as capture antigen a recombinant fusion protein, NS1(81)RLF, which contains both flanking regions, but lacks the NANP and NVDP repeats of the P. falciparum CS protein. Antibody responses to the repeatless flanking (RLF) regions were observed in all subjects, including five individuals who lacked detectable anti-R32 antibody responses. Anti-RLF antibody responses induced by natural infection appear to be short-lived and of low-to-moderate magnitude. Thus, if anti-RLF antibodies prove to be protective, derived vaccine candidates may require presentation of these epitopes with adjuvants or delivery systems that enhance immunogenicity.     

Malaria anticircumsporozoite antibodies in dutch soldiers returning from sub-saharan africa

One hundred and twenty-five Dutch servicemen returning from central Africa after a short deployment were enrolled in a study aimed at assessing the effectiveness of malaria prevention measures. None of the persons developed an episode of clinically overt malaria during or after deployment, and no antibodies against blood stages of Plasmodium falciparum could be found. However, antibodies against the circumsporozoite protein (CS) of P. falciparum were demonstrable in 14 persons (11.2% of the study population) by an ELISA test using the recombinant CS-antigen R32tet_{32, while one person only was positive in an IFA test based on schizonts of P. fieldi as antigen. We concluded that the anti-CS-positive servicemen were probably bitten by mosquitoes carrying P. falciparum parasites while the IFA-positive person was possibly infected by P. vivax , P. ovale or P. malariae parasites. There was no significant association between the different antimalaria preventive measures and the development of anti-CS antibodies. Therefore mefloquine prophylaxis as the single most widely used preventive measure in this group of servicemen was possibly a major contributing factor in averting development of overt malaria.}     

The Duffy blood group and resistance to Plasmodium vivax in Honduras.

To test the hypothesis that the Duffy blood group negative genotype is a factor in resistance to Plasmodium vivax, we determined the Duffy blood group, the malaria antibodies, and the slide-demonstrated infection rates with P. vivax and P. falciparum of 420 persons living in Nueva Armenia, Honduras. In all, 247 persons were Duffy negative. Demonstrated infections with P. falciparum were almost equally distributed between Duffy-positive (5,8%) and Duffy-negative (4.9%) persons. Similarly, Duffy-positive (25.6%) and Duffy-negative (28.2%) persons had equal proportions of indirect fluorescent antibody test titers suggestive of past or present P. falciparum infection. In contrast, all 14 P. vivax infections were found in Duffy-negative persons. There was no evidence suggesting that Duffy-positive and Duffy-negative persons had different exposures to malaria. The Duffy negative genotype FyFy appears to be a factor in resistance to P. vivax.     

恶性疟原虫重组乳酸脱氢酶胶体金免疫层析法检测试纸条的研制

目的 用氯金酸标记抗恶性疟原虫乳酸脱氢酶（LDH）单克隆抗体（McAb），研制用于诊断恶性疟原虫和间日疟原虫感染的胶体金免疫层析（GICA）试纸。方法 采用柠檬酸盐还原法制备胶体金颗粒、标记抗恶性疟原虫重组乳酸脱氢酶单克隆抗体，制成免疫层析检测试纸条。用该试纸条检测已知疟疾病人及健康人血样，鉴定方法的敏感性和特异性。结果 检测重组LDH抗原的最小量为5ng／ml；对30例恶性疟病人血样及40例间日疟病人血样进行检测，敏感性分别为83．33％和57．50％，检测100例健康者血样特异性为98．00％。结论 初步建立了同时诊断恶性疟原虫和间日疟原虫感染的GICA检测法。     

Multicentre study, in patients with imported malaria, on the sensitivity and specificity of a dipstick test (ICT Malaria P.f./P.v.) compared with expert microscopy

A prospective, multicentre study was carried out in Italy to assess the sensitivity and specificity of a rapid dipstick test (ICT Malaria P.f./P.v.) in the diagnosis of imported malaria caused by Plasmodium falciparum and other Plasmodium spp. The test is based on the detection of histidine-rich protein-2 (HRP-2) from P. falciparum and 'panmalarial' antigen in peripheral blood. The 241 subjects were international travellers or immigrants from areas where malaria is endemic. When compared with the microscopical examination of bloodsmears (used as the 'gold standard'), the dipsticks were found to be 94.4% sensitive and 94.5% specific for pure infections with P. falciparum. The performance of the tests when used on patients infected with species other than P. falciparum or more than one Plasmodium spp. showed a high degree of variability. Although the dipsticks represent a very simple, rapid, and valuable diagnostic aid, they should not be considered a complete substitute for direct microscopical diagnosis using stained bloodsmears.     

Evaluation of a dipstick test for the rapid diagnosis of imported malaria among patients presenting within the network TropNetEurop.

Lack of experience on the part of involved laboratory personnel frequently complicates swift diagnosis of imported falciparum malaria in non-endemic areas. Diagnostic tools based on the dipstick principle for the detection of plasmodial histidine-rich protein 2 have been marketed for several years and have been extensively evaluated. Recently, a test kit capable of detecting antigen of Plasmodium falciparum and P. vivax has been introduced. In order to evaluate this newly available tool, specimens from 664 patients were screened during the course of a prospective multicentre study within the European Network on Imported Infectious Disease Surveillance (TropNetEurop). Among the screened specimens, samples from 82 patients (12.3%) were positive for falciparum malaria using expert microscopy. A further 17 samples were positive for vivax malaria. The evaluated test kit performed with a sensitivity of 87.8% and a specificity of 99% for detection of falciparum malaria. Respective values for vivax malaria were 76.5% and 100%. Dipstick tests have the potential of improving the speed and accuracy of the diagnosis of falciparum malaria, especially if non-specialized laboratories are involved. However, decreased values of sensitivity and specificity, in comparison with expert microscopy, still impose a clear limit on the usefulness of the currently available kits.     

Antibodies to Plasmodium falciparum and Plasmodium vivax merozoite surface protein 5 in Indonesia: species-specific and cross-reactive responses

BACKGROUND: Merozoite surface protein (MSP) 5 is a candidate antigen for a malaria vaccine. In cross-sectional and longitudinal studies, we measured MSP5 antibody responses in Papuans with acute Plasmodium falciparum malaria, Plasmodium vivax malaria, and mixed P. falciparum and P. vivax malaria and in those with past exposure. METHODS: Enzyme-linked immunosorbant assay (ELISA) was used to quantitate antibody responses to P. falciparum MSP5 (PfMSP5) and P. vivax MSP5 (PvMSP5) in 82 subjects with P. falciparum infection, 86 subjects with P. vivax infection, 85 subjects with mixed infection, and 87 asymptomatic individuals. Longitudinal responses through day 28 were tested in 20 persons. Cross-reactivity was tested by competition ELISA. RESULTS: PfMSP5 or PvMSP5 immunoglobulin (Ig)Gwas detected in 39%-52% of subjects, and IgM was detected in 44%-72%. IgG responses were distributed equally between IgG3 and IgG1 for PfMSP5 but were predominantly IgG3 for PvMSP5. Although IgG responses were generally specific for PfMSP5 or PvMSP5, cross-species reactivity was found in 7 of 107 dual-positive responders. No significant difference was seen in the magnitude, frequency, or subclass of PfMSP5 or PvMSP5 IgG antibodies between groups. There was no significant association between antibody responses and therapeutic response. CONCLUSION: PfMSP5 and PvMSP5 were frequently recognized by short-lived, species-specific antibodies. Although infrequent, the cross-reactive MSP5 antibodies indicate that an appropriately formulated vaccine may elicit and/or enhance cross-species recognition, which may be very useful in areas where both parasites are endemic.     

Naturally acquired circumsporozoite antibodies and their role in protection in endemic falciparum and vivax malaria

The role of naturally acquired circumsporozoite (CS) antibodies in protection against falciparum and vivax malaria was evaluated in a group of Thai endemic villagers using a prospective cohort and a case-control study design. There was no evidence of protection by either the presence of positive CS antibody levels at the presumed time of sporozoite exposure or in individuals who persistently had measurable levels of the antibodies. The study defined levels of CS antibodies that were not protective in natural infection.     

Antibodies against malaria sporozoites in patients with acute uncomplicated malaria and patients with cerebral malaria

Serum samples from 95 patients with acute uncomplicated falciparum malaria (AM) and 95 patients with cerebral malaria (CM) were tested by the indirect immunofluorescent assay (IFA) for IgG and IgM antibodies against Plasmodium falciparum and P. vivax sporozoites. Forty-six (48%) CM patients were positive for antibodies against P. falciparum sporozoites whereas only 23 (24%) were positive for antibodies against P. vivax sporozoites (P less than 0.002). A similar result was obtained in AM patients. However, CM patients had significantly lower mean IgG anti-sporozoite titer for P. falciparum than did AM patients (P less than 0.05), especially when only anti-sporozoite antibody-positive CM and AM patients were compared (P less than 0.0005), suggesting that CM patients had relatively less exposure and were probably less immune to malaria than were AM patients. The persistence of anti-sporozoite antibodies also was investigated in paired sera taken 63 days apart from 108 patients with acute falciparum malaria. There were significant decreases in the mean antibody titers in the follow-up sera during the period of stay in the malaria-free area. It was proposed that determination of anti-sporozoite antibody be made as a substitute for, or in addition to, anti-blood stage antibody for seroepidemiological study of malaria, especially in the monitoring of the success of the malaria control program.     

Interaction of Malaysian sera with Plasmodium vivax sporozoite antigen

A seroepidemiologic survey of Plasmodium vivax and Plasmodium falciparum transmission was conducted in 94 Orang Asli children and adults. The prevalence of malaria was 46% in this population, and infections due to P. vivax and P. falciparum occurred with equal frequency. Multi-species infection was common, particularly in children less than 10 years of age. Circumsporozoite (CS) antibodies to P. vivax were detected by ELISA, using the recombinant protein NS181V20, in sera from 53-95% of all subjects in this study. The specificity of reactivity to NS181V20 was confirmed by immunofluorescence using air-dried sporozoites. CS antibodies to P. falciparum were present in less than 50% of the population less than 30 years of age. These data support further testing of this protein as a candidate vivax vaccine.     

艾博混合疟/恶性疟快速诊断试剂效果评价

目的 评价艾博混合疟/恶性疟(Malaria Pan./p.f..)快速诊断试剂的检测效果.方法 采集疟疾、疑似疟疾、不明原因发热及感冒“四热”患者血样,以显微镜镜检方法为金标准,比较艾博混合疟/恶性疟试剂检测效果结果 共采集584份血样,艾博试剂检测恶性疟原虫277份,灵敏度为98.9％,特异性为97.7％,与显微镜符合率为98.3％.艾博试剂检测混合疟377份的灵敏度为99.5％,特异性为99.0％,与显微镜符合率为99.3％,总符合率为98.8％,上述2方面的检测结果经Kappa一致性检验,Kappa值均大于0.97.结论 艾博混合疟/恶性疟试剂盒实验室检测敏感性和特异性高,能检测混合疟并区分恶性疟,结果与血涂片吉氏染色镜检结果高度一致,适用于疟疾流行的基层使用.     

Relative utility of dipsticks for diagnosis of malaria in mesoendemic area for Plasmodium falciparum and P. vivax in northeastern India

For diagnosis of malaria, popular brands of rapid test kits collectively termed as "dipsticks" were subject to field evaluation in northeastern India for their comparative sensitivity and specificity vis-à-vis conventional microscopic results. Dipsticks based on Plasmodium falciparum-specific histidine-rich protein (Pf HRP-2) antigen capture assay revealed 100% sensitivity and high specificity (94-100%); thus, they were concluded to be reliable tools for confirmed diagnosis of malarial infection. However, an advanced version of the same kit, having incorporated additional pan-malarial monoclonal antibody, was found to be less sensitive (71%) for non-falciparum infections. Besides, Pf HRP-2-based kits continued to show positive results up to day 7, even after clearance of parasitemia on account of persistent antigenemia. This very limitation seemed to have been overcome by parasite-specific lactate dehydrogenase (pLDH) enzyme-based kit. This kit was observed to have high sensitivity (81-89%) and specificity (100%) for both falciparum and non-falciparum malaria, but cannot distinguish mono-infection from mixed infections. It is concluded that the rational use of these kits would accord health benefits in terms of early detection and prompt treatment, reduce drug pressure, and possibly delay the emergence and spread of multi-drug-resistant strains of malarial parasites.