An Immunohistochemical Study of the Pathology of Fatal Malaria: Evidence for Widespread Endothelial Activation and a Potential Role for Intercellular Adhesion Molecule-1 in Cerebral Sequestration

The sequestration of parasitized erythrocytes in the microvasculature of vital organs is central to the pathogenesis of severe Plasmodium falciparum malaria. This process is mediated by specific interactions between parasite adherence ligands and host receptors on vascular endothelium such as intercellular adhesion molecule-1 (ICAM-1) and CD36. Using immunohistochemistry we have examined the distribution of putative sequestration receptors in different organs from fatal cases of P.falciparum malaria and noninfected controls. Receptor expression and parasite sequestration in the brain were quantified and correlated. Fatal malaria was associated with widespread induction of endothelial activation markers, with significantly higher levels of ICAM-1 and E-selectin expression on vessels in the brain. In contrast, cerebral endothelial CD36 and thrombospondin staining were sparse, with no evidence for increased expression in malaria. There was highly significant co-localization of sequestration with the expression of ICAM-1, CD36, and E-selectin in cerebral vessels but no cellular inflammatory response. These results suggest that these receptors have a role in sequestration in vivo and indicate that systemic endothelial activation is a feature of fatal malaria.     

In vitro efficacy of antimalarial drugs against Plasmodium vivax on the western border of Thailand

The susceptibility of 20 isolates of Plasmodium vivax on the Thailand-Myanmar border to seven antimalarial drugs was evaluated using the schizont maturation inhibition technique. The geometric mean 50% inhibition concentration (IC(50)) values were quinine = 308 ng/mL, amodiaquine =14 ng/mL, chloroquine =50 ng/mL, mefloquine = 127 ng/mL, sulfadoxine/pyrimethamine (80:1) = 800/10 ng/mL, pyrimethamine = 8 ng/mL, and artesunate = 0.5 ng/mL. Compared with P. falciparum in this area, P. vivax was more sensitive to chloroquine and artesunate, equally sensitive to quinine, and more resistant to mefloquine.     

Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae

Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).     

Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand

The dhps sequences of 55 Plasmodium vivax isolates (39 from Thailand and 16 from elsewhere) revealed mutant Pvdhps at codons 383 and/or 553 (A --> G) in 33 isolates, all from Thailand. Mutations of Pvdhps and Pvdhfr were correlated. Multiple mutations were associated with high-grade sulfadoxine-pyrimethamine resistance.     

Relapses of Plasmodium vivax infection usually result from activation of heterologous hypnozoites

BACKGROUND: Relapses originating from hypnozoites are characteristic of Plasmodium vivax infections. Thus, reappearance of parasitemia after treatment can result from relapse, recrudescence, or reinfection. It has been assumed that parasites causing relapse would be a subset of the parasites that caused the primary infection. METHODS: Paired samples were collected before initiation of antimalarial treatment and at recurrence of parasitemia from 149 patients with vivax malaria in Thailand (n=36), where reinfection could be excluded, and during field studies in Myanmar (n=75) and India (n=38). RESULTS: Combined genetic data from 2 genotyping approaches showed that novel P. vivax populations were present in the majority of patients with recurrent infection (107 [72%] of 149 patients overall [78% of patients in Thailand, 75% of patients in Myanmar {Burma}, and 63% of patients in India]). In 61% of the Thai and Burmese patients and in 55% of the Indian patients, the recurrent infections contained none of the parasite genotypes that caused the acute infection. CONCLUSIONS: The P. vivax populations emerging from hypnozoites commonly differ from the populations that caused the acute episode. Activation of heterologous hypnozoite populations is the most common cause of first relapse in patients with vivax malaria.     

Spurious Amplification of a Plasmodium vivax Small-Subunit RNA Gene by Use of Primers Currently Used To Detect P. knowlesi

The PCR primers commonly used to detect Plasmodium knowlesi infections in humans were found to cross-react stochastically with P. vivax genomic DNA. A nested primer set that targets one of the P. knowlesi small-subunit rRNA genes was validated for specificity and for sensitivity of detection of <10 parasite genomes.In 2004, it was reported that infections (in ca. 60% of the malaria cases) recorded initially as being caused by Plasmodium malariae in the Kapit division of Malaysian Borneo were in fact caused by P. knowlesi (8), a parasite of long-tailed (Macaca fascicularis) and pig-tailed (M. nemestrina) macaques. More recent studies have described P. knowlesi infections in several southeast Asian countries: 5 in the Philippines (5), 11 in Thailand (3, 7), and 1 in Singapore (6). Most cases have been reported to occur in Sarawak and the neighboring state of Sabah in Borneo (2), with some also recorded in peninsular Malaysia (11). Most cases have been detected in forest dwellers living in close proximity to the natural monkey hosts. In Sarawak, Anopheles latens is the principal vector (12). The role of human-to-human transmission is uncertain. The parasite''s potential to disseminate far from its zone of endemicity was highlighted by the detection of P. knowlesi in a traveler returning to China from Myanmar (14) and in two travelers returning to Finland (4) and Sweden (1) from Borneo. P. knowlesi has a quotidian cycle and can reach high parasite densities rapidly in humans, and infection is potentially fatal (2). Accurate identification in cases of human malaria is essential.Upon microscopy analysis, young ring stages of P. knowlesi resemble P. falciparum, but older forms are similar to the band forms of P. malariae. The recorded P. knowlesi infections in humans were discovered and confirmed by a PCR assay using a set of oligonucleotide primers (Pmk8 and Pmkr9) that target one of the parasite''s small-subunit rRNA (ssrRNA) genes (8). Most Plasmodium parasites have two, and some species have three, distinct ssrRNA genes that are differentially expressed during the parasite''s life cycle (13), and the Pmk8-Pmkr9 primers target the gene (ssrRNA-S) expressed during the sexual stages (8). In the course of nested PCR screening of Thai isolates for the presence of P. knowlesi by using the Pmk8-Pmkr9 primers, we noted an unexpected number of positive patient samples that had been identified microscopically as P. vivax and in which parasites resembling P. knowlesi were not observed. This raised the possibility that the Pmk8-Pmkr9 primers could be cross-reactive with some P. vivax isolates.In order to assess the specificity of Pmk8-Pmkr9, we carried out nested amplification (8) with a broad panel of genomic DNA samples from the four human malaria parasites, including isolates from Thai patients infected with P. falciparum (n = 30), P. vivax (n = 30), P. malariae (n = 19), and P. ovale (n = 4). The diagnoses were confirmed by a species-specific nested PCR assay (10). We also included samples of DNA obtained from MR4 (Malaria Research and Reference Resource Center [http://www.mr4.org/]) which were purified from Aotus monkeys infected with strains of P. vivax collected from Nicaragua, Panama, and Thailand (MRA340G, MRA343G, and MRA342G) or from P. knowlesi Malayan, H, Philippine, and Hackeri strains (MRA487F, MRA456G, MRA457, and MRA547) and with other malarial species that infect primates: P. cynomolgi ceylonensis (MRA484F), P. cynomolgi bastianellii (MRA350G), P. cynomolgi Smithsonian and Cambodian strains (MRA351G and MRA597G), P. inui OS (MRA486F), P. simiovale Sri Lanka (MRA488F), P. brasilianum (MRA349G), P. fragile (MRA352G), and P. simium (MRA353G).Nested PCR revealed that the Pmk8-Pmkr9 primers yielded positive amplification not only for the 4 P. knowlesi control strains but also for 8 P. vivax isolates (6 of 30 from Thailand and the isolates from Nicaragua and Panama). Amplification was negative for all the other parasite samples described above. When the P. knowlesi and P. vivax ssrRNA-S gene sequences from GenBank were aligned, the sequences of the region targeted by the Pmkr9 primer were found to be identical (Fig. ​(Fig.1A);1A); in the region corresponding to the Pmk8 primer, the P. vivax sequence showed dissimilarity at the 3′ end but presented only two mismatches in the first 19 bases (Fig. ​(Fig.1A).1A). Thus, it seems unlikely that the Pmk8-Pmkr9 pair would support efficient priming for the P. vivax ssrRNA-S gene. In order to establish whether the positive amplification observed for some of the P. vivax samples was due to stochastic cross-reactivity or to a hitherto unknown polymorphism in the P. vivax ssrRNA-S gene, the rPLU3-Pmkr9 primer fragments (Fig. ​(Fig.1A)1A) bearing the Pmk8 regions from three P. vivax samples that gave positive amplification with Pmk8-Pmkr9 were cloned into the Topo vector (Invitrogen, The Netherlands). Bacterial clones harboring the rPLU3-Pmkr9 fragment were then screened with Pmk8-Pmkr9, and in each case, a positive clone and a negative clone were picked and the insert was sequenced. All the sequences obtained were aligned with the published P. vivax ssrRNA-S gene sequences, and no sequence variations in the region that corresponds to the Pmk8 primer were observed. Moreover, when the six sequenced plasmids were subjected to PCR analysis using Pmk8-Pmkr9, positive amplification was observed randomly. These results demonstrate that stochastic cross-reaction with the Pmk8-Pmkr9 primers led to false-positive amplification when P. vivax genomic DNA was used.Open in a separate windowFIG. 1.(A) Alignments of the target sequences for Pmk8 and Pmkr9 primers in the ssrRNA-S genes of P. vivax (n = 2; accession numbers {"type":"entrez-nucleotide","attrs":{"text":"U03080","term_id":"425270","term_text":"U03080"}}U03080 and {"type":"entrez-nucleotide","attrs":{"text":"U07368","term_id":"501040","term_text":"U07368"}}U07368) and P. knowlesi (n = 8; accession numbers {"type":"entrez-nucleotide-range","attrs":{"text":"DQ350256 to DQ350262","start_term":"DQ350256","end_term":"DQ350262","start_term_id":"86155920","end_term_id":"86155926"}}DQ350256 to DQ350262) and schematic representation of the Plasmodium ssrRNA-S gene. The Pmkr9 sequence differs from the published sequences by a single base close to the 3′ end of the primer. (B) Alignments of the target sequences for the PkF1060, PkF1140, and PkR1550 primers in the ssrRNA-A genes of P. vivax (n = 2; accession numbers {"type":"entrez-nucleotide","attrs":{"text":"U03079","term_id":"425269","term_text":"U03079"}}U03079 and {"type":"entrez-nucleotide","attrs":{"text":"U07367","term_id":"501039","term_text":"U07367"}}U07367) and P. knowlesi (n = 11; accession numbers L07560AY580317 and {"type":"entrez-nucleotide-range","attrs":{"text":"AY327549 to AY327557","start_term":"AY327549","end_term":"AY327557","start_term_id":"37962820","end_term_id":"37962828"}}AY327549 to AY327557) and schematic representation of the Plasmodium ssrRNA-A gene. The relative positions of the different primers employed for nested PCR amplification, Topo cloning, and screening and those of the amplified fragments are indicated above (dotted lines, primary reaction) and below (solid lines, secondary reaction) the gene representations. It should be noted that the reverse primers (indicated by the arrows pointing to the left below the sequences) are presented as their reverse complemented sequences.It is important to be clear that the unsuspected cross-reactivity of the Pmk8-Pmkr9 primers described here does not put into question the results published to date for P. knowlesi infections discovered in humans by using these primers. In all such cases, the presence of P. vivax in the tested samples was convincingly excluded and/or confirmation of P. knowlesi was obtained through amplification and sequencing of another P. knowlesi gene.Although cross-reactivity of Pmk8-Pmkr9 with P. vivax may be reduced by further optimization of the amplification conditions, it was felt that a new set of primers truly specific for P. knowlesi would be a better alternative. Three primers suitable for seminested PCR amplification of a fragment of the P. knowlesi ssrRNA gene expressed during the asexual stages (ssrRNA-A) were designed (Fig. ​(Fig.1B)1B) to target regions that differ in the corresponding related P. vivax gene. These primers were tested using the same panel of genomic DNA described above. All amplification reactions were carried out with a total volume of 20 μl, in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 250 nM (each) oligonucleotide primers, MgCl₂ at the concentration suitable for each primer pair (1 mM for PkF1060-PkR1550 and 2 mM for PkF1140-PkR1550), 125 μM (each) deoxynucleoside triphosphates, and 4 U of Taq polymerase (Invitrogen). The cycling conditions were an initial denaturation step at 95°C for 5 min, followed by cycles of annealing at 55°C for PkF1060-PkR1550 and 50°C for PkF1140-PkR1550 for 1 min, extension at 72°C for 1 min, and finally, denaturation at 94°C for 1 min; 30 cycles for the primary amplification reaction and 35 for the secondary amplification reaction were carried out before a final annealing step, followed by 5 min of extension. When the primers were used for seminested amplification or when PkF1140-PkR1550 primers were used in the secondary reaction after primary amplification with rPLU1-rPLU5 (8, 9), the primers were found to be specific to P. knowlesi, with no amplification observed for any of the other parasite species. This result confirmed that the eight P. vivax samples for which cross-reactivity with Pmk8-Pmkr9 was noted were indeed free of P. knowlesi. The sensitivity of the nested reaction was found to be equivalent to that obtained using the rPLU3-rPLU4 primers, which consistently detect 1 to 10 parasite genomes per sample (9).     

Activities of artesunate and primaquine against asexual- and sexual-stage parasites in falciparum malaria

The activities of primaquine in combination with quinine or artesunate against asexual- and sexual-stage parasites were assessed in 176 adult Thai patients with uncomplicated Plasmodium falciparum malaria. Patients were randomized to one of the six following 7-day oral treatment regimens: (i) quinine alone, (ii) quinine with tetracycline, (iii) quinine with primaquine at 15 mg/day, (iv) quinine with primaquine at 30 mg/day, (v) artesunate alone, or (vi) artesunate with primaquine. Clinical recovery occurred in all patients. There were no significant differences in fever clearance times, rates of P. falciparum reappearance, or recurrent vivax malaria between the six treatment groups. Patients treated with artesunate alone or in combination with primaquine had significantly shorter parasite clearance times (mean +/- standard deviation = 65 +/- 18 versus 79 +/- 21 h) and lower gametocyte carriage rates (40 versus 62.7%) than those treated with quinine (P < or = 0.007). Primaquine did not affect the therapeutic response (P > 0.2). Gametocytemia was detected in 98 patients (56% [22% before treatment and 34% after treatment]). Artesunate reduced the appearance of gametocytemia (relative risk [95% confidence interval] = 0.34 [0.17 to 0.70]), whereas combinations containing primaquine resulted in shorter gametocyte clearance times (medians of 66 versus 271 h for quinine groups and 73 versus 137 h for artesunate groups; P < or = 0.038). These results suggest that artesunate predominantly inhibits gametocyte development whereas primaquine accelerates gametocyte clearance in P. falciparum malaria.     

Risk factors and clinical features associated with severe dengue infection in adults and children during the 2001 epidemic in Chonburi, Thailand

OBJECTIVES: Dengue haemorrhagic fever (DHF) is an important cause of morbidity in South-east Asia and used to occur almost exclusively in young children. In recent years, there has been a progressive shift in age-distribution towards older children and adults. We investigated an outbreak in 2001 in both children and adults, in an endemic area of Thailand. METHODS: Retrospective study of 347 patients with serologically confirmed dengue infection admitted to Chonburi Hospital during an epidemic in 2001. RESULTS: A total of 128 (37%) patients had dengue fever (DF) and 219 (63%) had DHF. Patients with DHF were significantly older than patients with DF (11 years vs. 8 years). Clinical bleeding was noted in 124 individuals, both with DF (n = 24) and DHF (n = 100), and significantly more frequently in adults. Twenty-nine (13.2%) of all DHF cases were caused by primary infection. Secondary dengue infection was associated significantly with the development of DHF in children, OR (95% CI) = 3.63 (1.94-6.82), P < 0.0001, but not in adults, OR (95% CI) = 0.6 (0.02-6.04), P = 1. Unusual clinical manifestations were observed in 23 patients: three presented with encephalopathy and 20 with highly elevated liver-enzymes. In the latter group, four patients were icteric and nine had gastrointestinal bleeding. CONCLUSION: These results indicate that DHF in South-east Asia is common in both children and adults. In dengue-endemic countries, dengue should be considered as a differential diagnosis in patients with clinical gastrointestinal bleeding in association with increased liver enzymes.     

Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure

Mutations in the dihydrofolate reductase (dhfr) genes of Plasmodium falciparum and P. vivax are associated with resistance to the antifolate antimalarial drugs. P. vivax dhfr sequences were obtained from 55 P. vivax isolates (isolates Belem and Sal 1, which are established lines originating from Latin America, and isolates from patient samples from Thailand [n = 44], India [n = 5], Iran [n = 2], and Madagascar [n = 2]) by direct sequencing of both strands of the purified PCR product and were compared to the P. vivax dhfr sequence from a P. vivax parasite isolated in Pakistan (isolate ARI/Pakistan), considered to represent the wild-type sequence. In total, 144 P. vivax dhfr mutations were found at only 12 positions, of which 4 have not been described previously. An F-->L mutation at residue 57 had been observed previously, but a novel codon (TTA) resulted in a mutation in seven of the nine mutated variant sequences. A new mutation at residue 117 resulted in S-->T (S-->N has been described previously). These two variants are the same as those observed in the P. falciparum dhfr gene at residue 108, where they are associated with different levels of antifolate resistance. Two novel mutations, I-->L at residue 13 and T-->M at residue 61, appear to be unique to P. vivax. The clinical, epidemiological, and sequence data suggest a sequential pathway for the acquisition of the P. vivax dhfr mutations. Mutations at residues 117 and 58 arise first when drug pressure is applied. Highly mutated genes carry the S-->T rather than the S-->N mutation at residue 117. Mutations at residues 57 and 61 then occur, followed by a fifth mutation at residue 13.