Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes.

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at 10 mg/ml and by 72.5 +/- 3.8% at 1 mg/ml, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell-associated or immobilized CS.

Genetic diversity in the Block 2 region of the merozoite surface protein 1 (MSP-1) of Plasmodium falciparum: additional complexity and selection and convergence in fragment size polymorphism.

Fragment size in the Block 2 repetitive region of merozoite surface protein 1 (MSP1) has commonly been used as a molecular marker in studies of malaria transmission dynamics and host immunity in Plasmodium falciparum malaria. In this study, we further explore the genetic variation in MSP-1 Block 2 underlying potential problems faced while studying the immune responses elicited by this vaccine target and while using it as a molecular marker in epidemiologic investigations. We describe the distribution of a new Block 2 recombinant allele family in samples collected from western Kenya and other malarious regions of the world and provide evidence that this allele family is found worldwide and that all MR alleles most likely originated from a single recombination event. We test whether the number of tandem repeats (i.e. fragment size) can be considered neutral in an area of high transmission in western Kenya. In addition, we investigate the validity of the assumption that Block 2 alleles of the same size and allele family are identical by examining MSP1 Block 2 amino acid sequences obtained from full-length MSP-1 clones generated from infected Kenyan children and find that this assumption does not hold. We conclude that the worldwide presence of a new allele family, the effect of positive natural selection, and the lack of conserved amino acid motifs within alleles of the same size suggest a higher level of complexity that may hamper our ability to elucidate allele family specific immune responses elicited by this vaccine target and its overall use as genetic marker in other types of epidemiologic investigations.

A Plasmodium falciparum isolate with a chromosome 9 deletion expresses a trypsin-resistant cytoadherence molecule.

Sequestration of Plasmodium falciparum infected erythrocytes in the cerebral circulation is strongly implicated in the pathogenesis of cerebral malaria. From previous studies it was postulated that genes essential for cytoadherence were located on the right arm of chromosome 9 as P. falciparum isolates with a deletion in this region lost the capacity to cytoadhere in vitro and no longer expressed Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) on the surface of the infected cells. We have selected a P. falciparum isolate from Papua New Guinea for high levels of cytoadherence to human umbilical vein endothelial cells (HUVECs) and have shown that the cloned parasite has several novel properties related to cytoadherence. The cloned parasite adheres to HUVECs, does not bind to melanoma cells, and expresses a surface molecule with most of the properties of PfEMP-1, despite a deletion in the right arm of chromosome 9. Interestingly, the surface expressed PfEMP-1 in this strain is resistant to trypsin treatment and infected cells continue to cytoadhere after trypsin digestion at a concentration of 100 micrograms ml-1. The receptor on HUVECs for the cloned parasite lines is a molecule different from any previously described, as parasitized cells do not adhere to soluble intercellular adhesion molecule 1, thrombospondin, vascular cell adhesion molecule 1, E-selectin or P-selectin, nor to CD36. Our work, taken together with the results from previous studies, suggest that the ability of parasites to cytoadhere is encoded in at least two distinct genomic locations in the parasite, and the diversity of receptor-ligand interaction is greater than previously described.

Random mating of natural Plasmodium populations demonstrated from individual oocysts.

DNA amplified from individual Plasmodium vivax oocysts, produced by feeding mosquitoes directly on naturally infected humans in Thailand, was used to study cross-mating of 2 polymorphs of the circumsporozoite (CS) gene, VK 210 and VK 247. Alleles were detected in matched blood parasites, sporozoites, and individual oocysts with oligoprobes specific to characteristic repeat units. Oocysts developing from 3 cases in which mixed alleles were present in the blood parasites had genotype frequencies, including hybrids, consistent with the Hardy-Weinberg equilibrium. There was apparently no barrier to hybridization of the 2 alleles nor a bias, as has been found in some laboratory experiments, favoring hybrid formation. These are the first measurements of cross-mating frequencies directly from natural Plasmodium infections and the first observations of genetic hybridization in P. vivax.

Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project.

We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum.

Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate a as a cytoadherence receptor.

The association between cytoadherence of Plasmodium falciparum-infected erythrocytes and the severity of malaria has been evaluated. In this study, we investigate adherence to C32 melanoma cells, CD36, intracellular adhesion molecule-1 (ICAM-1), thrombospondin (TSP), E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and chondroitin sulfate A (CSA) of 36 P. falciparum isolates from patients suffering from acute falciparum malaria. Adherence to purified adhesion molecules varied greatly among different parasite isolates. All isolates but one adhered to CD36, but none bound to E-selectin and VCAM-1 beyond control levels. Some P. falciparum isolates adhered to ICAM-1 and to CSA, a newly identified receptor for adherence. There was no correlation between in vitro binding to any one receptor and the patients' conditions. In addition, we investigated the characteristics of adherence to CSA and to C32 melanoma cells. Infected erythrocytes continued to adhere after trypsin digestion and soluble CSA inhibited adherence to C32 melanoma cells in a dose-dependent manner. The results imply a role for CSA in the natural infection of P. falciparum.

Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand.

Resistance to quinoline-containing compound has been associated with the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene. We analyzed wild P. falciparum isolates with high levels of chloroquine and mefloquine resistance for their macrorestriction maps of chromosome 5 and sequence of pfmdr1. Two types of chromosome 5 amplification were found. Eleven of 62 resistant isolates displayed Bgl 1 fragments larger than 100 kb. Twenty-nine isolates possessed multiple copies of the fragments. We failed to detect any amplification of this region on chromosome 5 in 22 mefloquine-resistant isolates, suggesting that other mechanisms can mediate the mefloquine-resistant phenotype. There was no direct association between pfmdr1 mutations and chloroquine sensitivity. Resistant lines could have Asn-86 and Tyr-184 or Phe-184, the predicted sequence of those chloroquine-sensitive isolates. No mutation at Asn-1042 and Asp-1246 was detected among these chloroquine-resistant isolates. Therefore, a few base substitutions in the pfmdr1 gene may not be sufficient to account for all chloroquine-resistant phenotypes.

Genome fingerprinting by pulsed-field gel electrophoresis of isolates of Burkholderia pseudomallei from patients with melioidosis in Thailand.

A total of 95 isolates of Burkholderia pseudomallei from 53 sporadic cases in Thailand were examined by pulsed-field gel electrophoresis. Digestion of genomic DNA of all isolates by NcoI generated a macrorestriction pattern similar to that of B. pseudomallei which cannot assimilate L-arabinose. Analysis using restriction enzymes SpeI and AvrII demonstrated greater sensitivity than NcoI digestion in the differentiation of B. pseudomallei and could be used for epidemiological groupings. Four cluster groups were evident among 37 isolates tested and the majority of isolates within each cluster displayed more than 65% similarity. Furthermore, multiple isolates from 18 and 35 patients with single and recurrent episodes of melioidosis, respectively, were examined. All patients with a single episode yielded genetically identical isolates and four of 35 patients with recurrent episodes were infected with strains of different genotypic patterns from the primary isolate(s). Hence, most repeated episodes of infection in melioidosis are as a result of the original infecting strains.

Lack of significant association between rosette formation and parasitized erythrocyte adherence to purified CD36.

The ability of Plasmodium falciparum infected erythrocytes from 162 Thai patients with uncomplicated malaria, 82 patients with severe malaria and 19 patients with cerebral malaria to form rosettes in vitro was studied. Of 263 isolates, 62 were evaluated for their adherence to different target molecules. We found that wide variation occurred in isolates from all groups in the level of rosette formation and adherence to CD36, intracellular adhesion molecule-1, thrombospondin and chondroitin sulfate A. No statistically significant correlation between the magnitude of rosette formation and disease severity was found (p > 0.05). In addition, our results from the use of purified CD36 as an adherence receptor showed no association between the degree rosetting and level of cytoadherence (p > 0.05, r = -0.04). Our data provide evidence that rosette formation and cytoadherence involve different molecular mechanisms and both phenomena can occur in all manifestations of the disease.

Production of monoclonal antibody specific to Campylobacter jejuni and its potential in diagnosis of Campylobacter enteritis.

A monoclonal antibody (MAb 3G6) specific for Campylobacter jejuni and Campylobacter coli was produced by immunizing BALB/c mice with a local strain of C.jejuni (28.1). No cross-reactivity was observed with Enterobacteriaceae controls. By immunoprecipitation, MAb 3G6 identified a major protein band of molecular weight 45 kDa and also gave a slight reactivity with 30 and 55 kDa proteins. Using an indirect enzyme-linked immunosorbent assay, MAb 3G6 was able to detect C.jejuni suspended in stool without cross-reactivity to 14 other enteropathogenic bacteria suspended, normal flora in fecal suspension, or fecal debris. In the analysis of fifty clinical specimens, MAb 3G6 detected most positive samples with the exception of one which possessed very low Campylobacter concentration and gave no reactivity to negative samples, demonstrating its high specificity to C. jejuni and C. coli. MAb 3G6 may be suitable as a new tool for the development of diagnostic method for Campylobacter infection.

Production and characterization of monoclonal antibodies reactive with the mycelial and yeast phases of Penicillium marneffei.

A definitive tissue diagnosis of Penicillium marneffei is hampered by a microscopic similarity to the yeast form of Histoplasma capsulatum and Cryptococcus neoformans. In order to obtain a better discrimination for accurate diagnosis, monoclonal antibodies (MAbs) were produced from hybridomas raised from Balb/c mice immunized with mycelial culture filtrate. By indirect immunofluorescent or immunoblot analysis, one immunoglobulin (Ig) G1 (3C2) MAb and three IgM (8B11, 3B9 and 8C3) MAbs were found to react strongly with P. marneffei antigens. In the immunoblots, the MAbs 8B11 and 3B9 reacted most strongly with a high molecular weight component (> 200 kDa) produced during either the mycelial or yeast phase of fungal growth. The immunoreactive epitopes for these two IgM MAbs were most likely associated with carbohydrate moieties, judging from their susceptibility to periodate oxidation and concanavalin A binding. This is in contrast to the immunoreactive epitopes for the MAbs 8C3 and 3C2, which were resistant to destruction by periodate treatment and did not bind to the lectin. Judging from immunofluorescent intensity, the three IgM MAbs could react strongly with the yeast cells present in the tissue biopsies of patients with P. marneffei infection.

Multiple ligands for cytoadherence can be present simultaneously on the surface of Plasmodium falciparum-infected erythrocytes.

A major virulence factor of Plasmodium falciparum is the adherence of parasitized erythrocytes to the wall of postcapillary venules via a specific interaction between parasite-derived erythrocyte surface ligands and receptors on endothelial cells. To study this phenomenon in vitro, we selected a parasite population that expressed at least two different ligands and demonstrated that parasitized cells may coexpress ligands with specificity for multiple receptors. This selected parasite line had several antigenic and cytoadherence characteristics that were different from those of the parent line. Single parasitized erythrocytes were able to adhere to three distinct receptors via at least two separate ligands; a trypsin-sensitive molecule mediated cytoadherence to CD36 and intercellular adhesion molecule 1 and a trypsin-insensitive molecule(s) was responsible for adherence to a third receptor on the surface of melanoma cells. We present evidence that this newly discovered receptor for cytoadherence is an N-linked glycosaminoglycan, as treatment of melanoma cells with endoglycosidase H abolished cytoadherence. These observations emphasize the adaptability of P. falciparum and the complexity of the cytoadherence phenomenon.

Molecular typing of Penicillium marneffei isolates from Thailand by NotI macrorestriction and pulsed-field gel electrophoresis.

Penicillium marneffei is recognized as one of the most frequently detected opportunistic pathogens of AIDS patients in northern Thailand. We undertook a genomic epidemiology study of 64 P. marneffei isolates collected from immunosuppressed patients by pulsed-field gel electrophoresis (PFGE) with restriction enzyme NotI. Among the 69 isolates fingerprinted by PFGE, 17 were compared by HaeIII restriction endonuclease typing. The PFGE method demonstrated a higher degree of discriminatory power than restriction endonuclease typing with HaeII. Moreover, an impressive diversity of P. marneffei isolates was observed, as there were 54 distinct macrorestriction profiles among the 69 isolates of P. marneffei. These profiles were grouped into two large clusters by computer-assisted similarity analysis: macrorestriction pattern I (MPI) and MPII, with nine subprofiles (MPIa to MPIf and MPIIa to MPIIc). We observed no significant correlation between the macrorestriction patterns of the P. marneffei isolates and geographical region or specimen source. It is interesting that all isolates obtained before 1995 were MPI, and we found an increase in the incidence of infections with MPII isolates after 1995. We conclude that PFGE is a highly discriminatory typing method and is well suited for computer-assisted analysis. Together, PFGE and NotI macrorestriction allow reliable identification and epidemiological characterization of isolates as well as generate a manageable database that is convenient for expansion with information on additional P. marneffei isolates.

Differences in genomic macrorestriction patterns of arabinose-positive (Burkholderia thailandensis) and arabinose-negative Burkholderia pseudomallei.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate L-arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara+), whereas the other soil isolates and all clinical isolates could not (Ara-). Only the Ara isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed-field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate L-arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara-) isolates and 9 Ara- soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara+) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed-field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.

Immunoreactivity of a 38-kilodalton Penicillium marneffei antigen with human immunodeficiency virus-positive sera.

Penicillium marneffei produced and secreted a 38-kDa antigen that appeared to be specific for this dimorphic fungus. This component could not be detected in antigenic extracts of Histoplasma capsulatum, Cryptococcus neoformans, Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Candida albicans, and two other species of Penicillium by immunoblot analysis against the sera from patients with culture-confirmed penicilliosis marneffei. Antibody reactive with this antigen was found in a large proportion of human immunodeficiency virus (HIV)-positive patients, indicating a presumptive diagnosis of P. marneffei infection. A small number of asymptomatic HIV-seropositive patients and HIV-seropositive patients with other fungal infections were also found to be positive by this analysis, suggesting that subclinical or mixed fungal infections involving P. marneffei are not uncommon.