Markers for population genetic analysis of human plasmodia species, P. falciparum and P. vivax

Present report deals with the genetic diversity existing among the field isolates of Plasmodium falciparum and P. vivax in India. Isoenzymes and molecular markers were used to analyse field isolates of P. falciparum and P. vivax. High level of length polymorphism was observed in repeat nucleotide sequences of MSP-1, MSP-2 and GLURP in P. falciparum isolates and CSP, GAM-1 and MSP-3 alpha in P. vivax isolates. In study populations a high proportion of isolates (up to 60%) were comprised of more than one genetically distinct parasite type--multiclonal. Presence of identical allelic forms of enzyme and DNA variations in different geographical areas and in different years suggest that isolates belong to a single random mating population of P. vivax and P. falciparum. Observed random combination of alleles in the field isolates suggest the unlinked nature of loci studied. Study supports the feasibility of using molecular markers for the identification of recrudescence in P. falciparum from fresh infection.     

Nest-PCR和PCR-RFLP在恶性疟原虫地理株基因分型及多态性研究中的应用

目的 分析套式PCR（nest-PCR）和限制性内切酶片段长度多态性（PCR-RFLP）方法在恶性疟原虫地理株裂殖子表面蛋白（MSP2）基因多态性研究中的分型效率及特异性。 方法 分别在海南省三亚市和云南省腾冲县等地通过静脉采血法采集疟疾患者血样98份,其中恶性疟88份,间日疟10份。另从上海地区的健康人群中抽取10份血样作为阴性对照。用nest-PCR和PCR-RFLP方法分别对疟原虫地理株进行MSP2等位基因分型,并对分型结果进行归纳和比较分析。 结果 常规nest-PCR方法对于恶性疟原虫地理株MSP2等位基因的总检出效率为79.8％（166/208）,其中,对于FC27家族等位基因的检出效率为92.7％（101/109）,但对3D7家族等位基因的检出率仅为65.7％（65/99）,且无法鉴定具体的等位基因。PCR-RFLP分型法检出效率比nest-PCR法提高了20.2%,且特异性好。 结论 PCR-RFLP相对于常规的nest-PCR分型法具有检出效率、分辨率和特异性皆高的优点,可以鉴定地理株中具体的等位基因类型。     

Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand

Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.     

2006和2008年度安徽蒙城间日疟原虫群体遗传结构研究

目的 对2006年和2008年采自安徽蒙城的间日疟原虫群体遗传结构及其与发病率之间的关系进行研究.方法 以间日疟原虫裂殖子表面基因蛋白3α(Plasmodium vivax merozoite surface protein genes-3α,PvMSP-3α)为分子标志,利用PCR-RFLP技术对不同年度间日疟原虫群体遗传多态现象进行分析,用卡方检验比较年度间等位基因频率是否存在差异.结果 共发现了3种类型的MSP-3α基因.2006年的样本仅发现两个型:多数为A型(91.30%),少数为C型(4.35%),并有2例A和C的混合感染(4.35%);2008年样本中,仅发现A型(88.60%)和B型(11.40%),无混合感染.PCRRFLP分析MSP-3α基因存在高度多态性.卡方检验显示2006年和2008年间的差异无统计学意义.结论 安徽蒙城的间日疟原虫存在较高的遗传多态性,且遗传多态性未随疟疾疫情的下降而改变.     

Demographic risk factors for severe and fatal vivax and falciparum malaria among hospital admissions in northeastern Indonesian Papua

Between January 1998 and December 2000, the Jayapura Provincial Public Hospital in northeastern Indonesian New Guinea (Papua) admitted 5,936 patients with a diagnosis of malaria. The microscopic diagnosis at admission was Plasmodium falciparum (3,976, 67%), Plasmodium vivax (1,135, 19%), Plasmodium malariae (8, < 1%), and mixed species infections (817, 14%). Approximately 9% (367) of patients were classified as having severe malaria (277 P. falciparum, 36 P. vivax, 53 mixed infections, and 1 P. malariae) and 88 died (79 P. falciparum/mixed infections and 9 P. vivax). Risk of fatal outcomes among severe malaria patients was indistinguishable between those with falciparum versus vivax malaria (OR = 0.89; P = 0.771). Compared with non-pregnant women, pregnant women showed no higher risk of severe malaria (P = 0.643) or death caused by severe malaria (P = 0.748). This study compares admissions per population (based on census data), parasitemia, morbidity, and mortality among children versus adults, pregnant versus non-pregnant women, and urban/suburban versus rural residents.     

Ni-NTA蛋白芯片技术检测间日疟原虫感染的体液免疫应答

目的 建立检测间日疟感染的Ni-NTA蛋白芯片技术.方法 采用无细胞蛋白合成体系表达问日疟原虫重组蛋白,建立原位纯化重组蛋白和检测间日疟原虫感染患者血清中抗体反应的Ni-NTA蛋白芯片技术.并对3个裂殖子表面蛋白(merozoite surface proteins,MSPs)MSPl-42、MSP8和MSP10的免疫应答进行分析.结果 应用Ni-NTA蛋白芯片技术检测问日疟原虫感染患者血清抗体,鉴定出具有免疫原性的15个间日疟原虫蛋白,主要包括10个MSPs、2个Cys6蛋白以及其他3个未知蛋白,结果与以往报道的抗体芯片类似.MSPl-42、MSP8和MSP10依次识别出100.0%(20/20)、90.0%(18/20)和70.0%(14/20)的间日疟原虫感染患者血清,特异性均为100%(10/10),且曲线下面积(area under the curve.AUC)达到0.87～1.00.结论 成功建立了检测间日疟原虫感染的Ni-NTA蛋白芯片技术.该方法有助于从间日疟原虫基因组中快速筛选鉴定具有免疫原性的蛋白以及应用于功能蛋白质组学研究.

Abstract：

Objective To develop Ni-NTA surface based chips to detect the humoral immune response to Plasmodium vivax infection.Methotis A set of recombinant P.vivax proteins was expressed by wheat germ cell-free system and IgG immune responses to these recombinant P. vivax proteins including 3 merozoite surface proteins(MSPl-42,MSP8 and MSPIO)were measured with sera from P.vwax-infected patients and healthv individuals using Ni-NTA chips.Results Ni-NTA surface based chips were successfully applied to detect immune responses to P.vivaz infection.Fifteen high immunoreactive recombinant P.vivax proteins were identified using Ni-NTA chips,including 10 MSPs,2 Cys6 lipid raft-associated proteins and 3 hypothetical proteins.MSPI-42.MSP8 and MSPIO recognized by IgG immune response to 100.0%(20/20),90.0%(18/20),and 70.0%(14/20)serum samples from P.vivax infected patients,respectively.Moreover,no crossreactivity to MSPs was found in serum samples from healthy individuals with area under the curve(AUC)values of 0.87-1.00.Conclusions In this report,Ni-NTA chips have been applied to investigate blood stage-specific immunogenic proteins from vivax malaria.The method may aid in determining the immunogenicity of candidate antigens and the functional identification of the large number of unknown and hypothetical proteins in P.vivax genome.     

Reactivity of sera from cases of Plasmodium vivax malaria towards three recombinant antigens based on the surface proteins of the parasite

Sera collected in South Korea, from 61 cases of Plasmodium vivax malaria and, as controls, 40 healthy volunteers, were tested in ELISA for IgG or IgM reacting with any of three recombinant P. vivax proteins. The antigens used, representing the parasite's major merozoite surface protein (MSP), circumsporozoite surface protein (CSP) and Duffy-binding protein (DBP), had all been expressed in an Escherichia coli system and purified. The ELISA results were recorded as optical densities (OD). The highest ratio observed between the mean OD for a malaria serum and that for a control serum was that for IgG against MSP, although CSP gave a higher ratio than MSP or DBP in the IgM ELISA. In the ELISA for IgG, the OD for MSP were found to be correlated with those for DBP (r = 0.53; P < 0.5) but the OD for CSP were not correlated with those for MSP or DBP. As the most intense reactions observed were those between the IgG from the malaria sera and the recombinant MSP, the latter antigen may be useful in diagnostic tests and as a component of any vaccine used to protect against P. vivax malaria.     

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.     

快速诊断疟疾胶体金免疫层析试条方法的建立与评价

目的 建立一种能区分恶性疟的快速、简便诊断疟疾的胶体金免疫层析试条方法，并对其进行评价。 方法 筛选基于恶性疟原虫乳酸脱氢酶制备的单克隆抗体对，采用柠檬酸三钠还原法制备胶体金颗粒，标记筛选到的单克隆抗体F4H12、G4C9和D8F7，并将其吸附于样品垫；将单克隆抗体B2G10（针对恶性疟原虫与间日疟原虫）和D6A7（只针对恶性疟原虫）分别划线包被于同一硝酸纤维素膜适当位置，制成免疫层析检测试条。用该试条检测疫区非疟疾发热病人血样（107份）和内脏利什曼病患者血样（17份）以评价其特异性，检测确诊的疟疾患者血样（间日疟110份， 恶性疟54份）以评价其敏感性。均用单盲法检测。 结果 检测107份疫区非疟疾发热病人血样和17份内脏利什曼病患者血样，有119份显示为阴性，特异性约为96.0%；其中17份内脏利什曼病患者血样全部为阴性。检测164份疟疾患者血样，阳性153份，敏感性为93.3%，其中间日疟检出率为92.7%（102/110），恶性疟检出率为94.4%（51/54）。 结论 研制出的快速诊断疟疾胶体金免疫层析试条敏感性、特异性均较高。     

间日疟原虫MSP1 C端编码基因的克隆及序列分析

目的 克隆间日疟原虫MSP1C端编码基因,并进行序列测定和分析。方法 根据间日疟原虫MSP1C端编码基因设计1对引物,采用PCR技术从深圳间日疟患者血样(编号为PvSZ1)的核酸提取物中扩增出MSP1C端编码基因,回收纯化后,与T载体连接构建重组子pMD/MSP1,并转化大肠杆菌JM109。阳性克隆以限制性酶切分析与PCR法鉴定后,双脱氧链末端终止法双向测定序列并分析。结果 从间日疟血样提取的DNA中扩增出1119bp的基因片段,所构建的pMD/MSP1阳性克隆重组子经双酶切和PCR鉴定与预期结果一致。所测定的间日疟原虫PvSZ1C端基因序列与国外Sal-1株比较,碱基数相同,未发现碱基缺失,相同的核苷酸占96.7％,序列中第542位碱基变化为同义突变(GTC→GTT,均编码缬氨酸),第375～542区间的碱基变化数占整个序列变化碱基总数的92.9％(34/37)。所推测的氨基酸序列与Sal-1株比较,同源性为92.5％。结论 成功克隆了间日疟原虫PvSZ1株MSP1C端编码基因,该基因在不同间日疟原虫地理株间相对保守,所克隆基因序列的第375～542核苷酸区域为高频变化区。     

Humoral immune responses against Plasmodium vivax MSP1 in humans living in a malaria endemic area in Flores, Indonesia

The aim of this study was to evaluate the relationship among age, parasitemia status, spleen size, hematocrit, and antibody levels to Plasmodium vivax merozoite surface protein 1 (MSP1) in individuals chronically exposed to P. vivax. Subjects were recruited from the population of three adjacent villages on the Island of Flores in Indonesia where malaria transmission is hyperendemic and tropical splenomegaly syndrome is highly prevalent. Subjects were evaluated for spleen size, hematocrit, presence of parasitemia, and presence of antibodies to a recombinant peptide consisting of 90 amino acids from the carboxy terminus of MSP1. Fifty-seven percent of 2-4 year olds, 45% of 5-9 years old, and 7% of > or = 15 years old were parasitemic; 99% of the > or = 15 years old had splenomegaly, and 31% of them had Hackett 4 or 5 spleens. The frequency of antibody positivity to MSP1 antigen in ELISA increased with age reaching a maximum of 89% in > or = 20 years old. The frequency of antibody positivity to MSPI also increased with spleen size, and with a decline in the prevalence of parasitemia.     

Polymerase chain reaction and molecular genotyping to monitor parasitological response to anti-malarial chemotherapy in the Peruvian Amazon

Over the past decade, anti-malarial drug resistance has rapidly become a major public health problem in the Peruvian Amazon. This study compared polymerase chain reaction (PCR) to light microscopy for diagnosing and monitoring the parasitological response of malaria patients to anti-malarial chemotherapy in the Peruvian Amazon region of Iquitos. Typing of P. falciparum using MSP1, MSP2, and glutamine-rich protein distinguished among infecting parasites. Most (73%) P. falciparum patients were parasitologically resistant to sulfadoxine-pyrimethamine (RI = 10, RII = 1). Sensitivity of microscopy was lower than PCR (69% for P. vivax and 78% for P. falciparum), but parasite clearance times were comparable between microscopy and PCR. PCR sensitively and specifically detected mixed infections and low-level parasitemia indicative of drug resistance, making this approach of practical use for the control of malaria at the public health level. Genotyping malaria parasites will be useful to distinguish drug failure from new infections in clinical trials of anti-malarial drugs in the Peruvian Amazon region.     

Circumsporozoite protein gene diversity among temperate and tropical Plasmodium vivax isolates from Iran

To date, there is no information on the genetic diversity of the circumsporozoite protein (CSP), a leading vaccine candidate, in Plasmodium vivax populations circulating in Iran. The gene for this protein, Pvcsp, was amplified from 374 P. vivax isolates collected in the temperate northern, and in the tropical southern endemic areas. PCR-RFLP analysis of the repeated central region revealed that the parasites collected in the northern area were almost exclusively of the VK210 type. Parasites collected in the south-eastern areas were of both VK210 and VK247 types. We detected VK210 parasite in 70.5% of the samples, VK247 parasites in 17.5% and mixed type infections in 12% of the isolates. Sequence analysis of 137 isolates obtained from both areas identified a total of 25 distinct genotypes. The degree of genetic diversity was generally higher for the tropical (21 genotypes) than the temperate (7 genotypes) P. vivax populations, a difference possibly reflecting the high cross-border exchanges between Afghanistan and Pakistan and southern Iran. Interestingly, all but two VK210 type isolates sequenced harboured a 36-bp post-repeat insert previously only observed in North Korea and China. This large-scale survey of parasite diversity in the Eastern Mediterranean Region provides a set of baseline data suitable for future molecular epidemiological studies of P. vivax.     

西藏林芝地区间日疟原虫PvMSP-3α多态性研究

【摘要】 目的 分析西藏林芝地区间日疟原虫遗传多态性。 方法 以间日疟原虫裂殖子表面蛋白3α（Plasmodium vivax merozoite surface protion genes 3α,PvMSP-3α）为分子标志,利用PCR-RFLP技术对西藏林芝间日疟原虫多态性进行分析。 结果 共发现2种类型的MSP-3α基因,多数为A型（90.3%）,B型较少（9.7%）,无混合感染。PCR-RFLP分析MSP-3α基因存在较低多态性。 结论 西藏林芝地区间日疟原虫的遗传多态性较低。     

Comparative features and outcomes of malaria at a tertiary care hospital in Karachi, Pakistan.

OBJECTIVES: A comparison of clinical and laboratory features, diagnostic methods, drug treatment, and outcomes for patients hospitalized with malaria by Plasmodium species. METHODS: Records of 521 patients hospitalized during the four and half-year study period were analyzed. RESULTS: Infections were caused by Plasmodium vivax (51.8%), Plasmodium falciparum (46.5%), P. vivax plus P. falciparum (1.3%), and Plasmodium malariae (0.4%). Vomiting (odds ratio (OR)=1.86, p=0.001) and abdominal pain (OR=1.60, p=0.024) occurred more frequently in patients infected with P. falciparum compared to P. vivax; this was also the case for hepatomegaly, splenomegaly and jaundice. Low hemoglobin levels were common but were significantly lower with P. falciparum, and creatinine levels were significantly higher with P. falciparum. Treatment regimens consisted of single drug therapy (61.5%), appropriate combination therapy (15.8%), and inappropriate combination therapy (22.7%). Antimalarials given alone included chloroquine (38.7%), quinine (19%) and doxycycline (1.5%). The overall mortality was 1.7% (n=9) and nearly 56% of patients developed disease complications, most commonly thrombocytopenia (36.4%), anemia (23.4%), and thrombocytopenia plus anemia (32.7%). CONCLUSIONS: Despite resistance, chloroquine was prescribed in patients with malaria requiring hospitalization. We found a high proportion of single antimalarial drug use as well as inappropriate combination therapy (22.7%), and inadequate use of primaquine terminal prophylaxis. Physicians need to be acquainted with malaria treatment guidelines in an endemic zone.     

Genetic diversity of Plasmodium vivax Pvcsp and Pvmsp1 in Guyana, South America

Approximately 55% of malaria infections in the Guyana Amazon region are attributed to Plasmodium falciparum while the other 45% are attributed to non-falciparum, mostly Plasmodium vivax. However, little is known about the P. vivax strain types circulating in the region. Using PCR for Plasmodium detection and two genetic markers specific to P. vivax to detect the polymorphic circumsporozoite protein (CSP) and the conserved 19-kDa region of the merozoite surface protein-1 (MSP-1), we investigated the overall Plasmodium strain distribution and population diversity within P. vivax in isolates collected from the blood of infected individuals in the interior Amazon region of Guyana, South America. Out of a total of 250 samples positive for Plasmodium, P. vivax was detected in 30% (76/250) and P. falciparum was detected in 76% (189/250). Mixed infections containing both P. falciparum and P. vivax constituted 6% (15/250) of the total positive samples. Further analysis of P. vivax strains showed that 92% (56/61) of the P. vivax samples hybridized with a probe specific to type VK210, 39% (24/61) hybridized with a probe specific for type VK247, and 25% (15/61) hybridized with a probe specific for the P. vivax-like CS genotype. DNA sequencing of the 19-kDa C-terminal domain in block 13 of MSP-1 amplified from 61 samples from patients infected with P. vivax demonstrated that this region is highly conserved, and all samples were identical at the nucleotide level to the Belem and Salvador-1 types. No synonymous or nonsynonymous mutations were observed in this region of the gene, indicating that current vaccine-development efforts based on the MSP-1(19) fragment would be applicable in Guyana.     

Natural Plasmodium infections in Brazilian wild monkeys: reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N=140), semideciduous Atlantic forest (N=257) and Cerrado (a savannah-like habitat) (N=51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the S?o Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N=30) and Cebus apella (N=39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C. apella specimens were negative. Parasitological examination of the samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P. falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions.     

Identification of the Plasmodium vivax mdr-like gene (pvmdr1) and analysis of single-nucleotide polymorphisms among isolates from different areas of endemicity

Because of the lack of methods for continuous in vitro culture of Plasmodium vivax, little is known about drug-resistance mechanisms in this malaria-causing parasite. Therefore, identification of all the genes potentially involved in drug resistance and of molecular markers related to drug resistance would provide a framework for studying the incidence and spread of drug-resistant P. vivax strains. We have identified the P. vivax orthologue of the pfmdr1 gene (pvmdr1), which was shown to have a role in the drug resistance of Plasmodium falciparum. Comparison of the alignments of both nucleotide and amino acid sequences of pvmdr1 with those of other Plasmodium multidrug-resistance genes revealed an open-reading frame of 4392 base pairs encoding a deduced protein of 1464 amino acids. Nucleotide polymorphisms at 2 codons of the pvmdr1 gene--Y976F and F1076L--were found in 14 of 23 P. vivax isolates from different areas of endemicity, including Thailand, Indonesia, Turkey, Azerbaijan, and French Guyana.     

Possible role of PGD_2 in malaria infections

Objective:To preliminarily investigate the possible role of prostaglandin D_2(PGD_2) in malaria infections.Methods:Blood and urinary samples(n=120 each) were collected from Thai patients with Plasmodium falciparum(P.falciparum) with moderate(n=26) and high(n=4) parasitemia,patients with Plasmodium vivax(P.vivax)(n=30),patients with fever associated with other infections(n=30),and healthy subjects(n=30).PGD_2 concentrations in plasma and urinary samples of healthy subjects,patients with fever associated with other infections and patients with malaria were determined using Prostaglandin D2-MOX express EIA kit(Cayman Chemical,USA).Results:The possible association between PGD_2 and malaria infections is clearly demonstrated with PGD_2 concentration in urine.The urinary PGD_2 concentrations were relatively high(about 5-fold) in patients with P.falciparum with moderate parasitemia and P.vivax infections compared with other groups.Furthermore,the concentration in patients with P.falciparum with moderate parasitemia and P.vivax infection were significantly higher than that in healthy subjects and patients with fever associated with other infections.Conclusions:Urinary PGD_2 concentrations may offer a more dependable and useful tool for predicting malaria severity.Confirmation is this preliminary finding is required with a larger sample size.