Molecular evidence to suggest pigeon-type Chlamydia psittaci in association with an equine foal loss.

Chlamydia psittaci is an important avian pathogen with spillover from infected wild and domesticated birds also posing a risk to human health. We recently reported a case of C. psittaci equine placentitis associated with further spillover to humans. Molecular typing of this case revealed it belonged to the 6BC clade of C. psittaci, a globally distributed highly virulent set of strains, typically linked to infection spillover from parrots. Equine chlamydiosis associated with C. psittaci infection has previously been reported elsewhere in countries where parrots are not endemic, however, raising questions over the identity of infecting C. psittaci strains and the potential infection reservoirs. In this study, we describe the detection and molecular characterization of C. psittaci in a case of equine abortion in southern Queensland. Equine placenta and fresh liver and lung tissue from the necropsied foetus were positive by C. psittaci-specific qPCR. Chlamydia psittaci-specific multilocus sequence typing and ompA genotyping were used to further characterize the detected equine strains and an additional strain obtained from a dove from a different geographic region presenting with psittacosis. Molecular typing of this case revealed that the infecting equine strains were closely related to the C0sittaci detected in dove, all belonging to an evolutionary lineage of C. psittaci strains typically associated with infections of pigeons globally. This finding suggests a broader diversity of C. psittaci strains may be detected in horses and in association with reproductive loss, highlighting the need for an expansion of surveillance studies globally to understand the epidemiology of equine chlamydiosis and the associated zoonotic risk.

B cell epitope mapping and characterization of naturally acquired antibodies to the Plasmodium vivax merozoite surface protein-3α (PvMSP-3α) in malaria exposed individuals from Brazilian Amazon.

The Plasmodium vivax Merozoite Surface Protein-3α (PvMSP-3α) is considered as a potential vaccine candidate. However, the detailed investigations of the type of immune responses induced in naturally exposed populations are necessary. Therefore, we aim to characterize the naturally induced antibody to PvMSP-3α in 282 individuals with different levels of exposure to malaria infections residents in Brazilian Amazon. PvMSP3 specific antibodies (IgA, IgG and IgG subclass) to five recombinant proteins and the epitope mapping by Spot-synthesis technique to full-protein sequence of amino acids (15aa sequence with overlapping sequence of 9aa) were performed. Our results indicates that PvMSP3 is highly immunogenic in naturally exposed populations, where 78% of studied individuals present IgG immune response against the full-length recombinant protein (PVMSP3-FL) and IgG subclass profile was similar to all five recombinant proteins studied with a high predominance of IgG1 and IgG3. We also observe that IgG and subclass levels against PvMSP3 are associated with malaria exposure. The PvMSP3 epitope mapping by Spot-synthesis shows a natural recognition of at least 15 antigenic determinants, located mainly in the two blocks of repeats, confirming the high immunogenicity of this region. In conclusion, PvMSP-3α is immunogenic in naturally exposed individuals to malaria infections and that antibodies to PvMSP3 are induced to several B cell epitopes. The presence of PvMSP3 cytophilic antibodies (IgG1 and IgG3), suggests that this mechanism could also occur in P. vivax.

MR imaging of progressive enhancement of a bioceramic orbital prosthesis: an indicator of fibrovascular invasion.

The physical properties of bioceramics have made them ideal for a variety of prosthetic devices. Their porous structure allows fibrovascular tissue to invade the implant and secure it and provides a surface for muscular attachment. This process has been well-documented in animal studies; however, this case report describes the periodic imaging changes seen in a 67-year-old man following placement of a bioceramic orbital prosthesis.

Promiscuous T-cell epitopes of Plasmodium merozoite surface protein 9 (PvMSP9) induces IFN-gamma and IL-4 responses in individuals naturally exposed to malaria in the Brazilian Amazon.

Plasmodium vivax merozoite surface protein (PvMSP9) stimulates both cellular and humoral immune responses in individuals who are naturally infected by this parasite species. To identify immunodominant human T-cell epitopes in PvMSP9, we used the MHC class II binding peptide prediction algorithm ProPred. Eleven synthetic peptides representing predicted putative promiscuous T-cell epitopes were tested in IFN-gamma and IL-4 ELISPOT assays using peripheral blood mononuclear cells (PBMC) derived from 142 individuals from Rondonia State, Brazil who had been naturally exposed to P. vivax infections. To determine whether the predicted epitopes are preferentially recognized in the context of multiple alleles, MHC Class II typing of the cohort was also performed. Five synthetic peptides elicited robust cellular responses, and the overall frequencies of IFN-gamma and IL-4 responders to at least one of the promiscuous peptides were 62% and 46%, respectively. The frequencies of IFN-gamma and IL-4 responders to each peptide were not associated with a particular HLA-DRB1 allelic group since most of the peptides induced a response in individuals of 12 out of 13 studied allelic groups. The prediction of promiscuous epitopes using ProPred led to the identification of immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous population exposed to malaria infections. The combination of several such T-cell epitopes in a vaccine construct may increase the frequency of responders and the overall efficacy of subunit vaccines in genetically distinct populations.

Plasmodium vivax DBP binding to Aotus nancymaae erythrocytes is Duffy antigen dependent.

Plasmodium vivax is the second leading cause of malaria worldwide. Invasion of human erythrocytes by P. vivax merozoites is dependent upon the interaction between the parasite Duffy binding protein (PvDBP) and the erythrocyte Duffy antigen receptor. Therefore, disruption of this vital interaction is an attractive target for therapeutic intervention. Although Aotus nancymaae is a commonly used primate model for human P. vivax infections, it has not been confirmed that the interaction between Ao. nancymaae erythrocytes and P. vivax is Duffy antigen dependent. Our results indicate that normal Ao. nancymaae erythrocytes readily bind to PvDBPII and that this binding is completely abolished with chymotrypsin treatment of the erythrocytes. Furthermore, the results of our inhibition assays show a dose-dependent decrease in binding with increasing amounts of anti-PvDBPII polyclonal rabbit sera or anti-Fy6 monoclonal antibody. These data indicate that the interaction between Ao. nancymaae erythrocytes and P. vivax DBPII is Duffy antigen dependent, validating this model system for in vivo studies of anti-PvDBP inhibition.

Naturally acquired humoral and cellular immune responses to Plasmodium vivax merozoite surface protein 9 in Northwestern Amazon individuals.

Antibody and T-cell reactivities to Plasmodium vivax merozoite surface protein 9 (PvMSP9) were evaluated in a cross-sectional study of individuals naturally exposed to malaria infections living in Ribeirinha, a native riverine community and in Colina, a transmigrant community, Rondonia, Brazil. The antibody responses to PvMSP9-RIRIIand PvMSP9-Nt domains in Ribeirinha were higher compared with Colina and correlated with age and time of malaria exposure. IgG2 was most prevalent for PvMSP9-RII in both communities, and IgG1 was the predominant isotype for PvMSP9-Nt and PvMSP9-RIRII in Ribeirinha. IFN-gamma and IL-4 predominated in Ribeirinha, while IFN-gamma predominated in Colina. Variation in exposure to P. vivax likely accounts for the differences observed in cytokine and antibody levels between the two populations studied.

Immunogenicity in rhesus of the Plasmodium vivax mosquito stage antigen Pvs25H with Alhydrogel and Montanide ISA 720.

Pvs25 is an ookinete surface protein from Plasmodium vivax that is the target of transmission-blocking antibodies. Two immunogenicity trials in rhesus monkeys with a recombinant form of the protein, Pvs25H, were undertaken. Monkeys were vaccinated with Pvs25H adsorbed to Alhydrogel or emulsified in Montanide ISA 720 at 0, 4 and 27 weeks (study 1) or in Montanide ISA 720 at 0 and 18 weeks (study 2) with 1.5 or 15 microg Pvs25H in 0.1 or 0.5 mL of emulsion (four combinations). Immunogenicity was assessed by ELISA and by membrane-feeding experiments using P. vivax-infected blood from human volunteers (studies 1 and 2) or from chimpanzees (study 1). Both vaccine trials generated antibodies that blocked transmission of P. vivax to mosquitoes. Antibody titres and transmission blocking were higher with Montanide ISA 720 than with Alhydrogel in the first trial and with the 15 microg Pvs25H/0.5 mL ISA 720 combination in the second trial.

Utility of navigator-prospective acquisition correction technique (PACE) for reducing motion in brain MR imaging studies.

In this retrospective review, we demonstrate the utility of using a commercially available gated navigator sequence (prospective acquisition correction technique [PACE]) to reduce rhythmic breathing motion on brain MR images. For purposes of this report, we studied 2 sedated patients who had marked head rocking, one due to deep breathing and snoring and the other due to ventilator support. Motion degraded the routine images, which, despite a short acquisition time, were nondiagnostic. After application of PACE, a technique commonly used in abdominal studies, the brain images in both patients were judged to be of acceptable diagnostic quality. The use of PACE to diminish head motion is now routine at our institution when sedated or ventilated patients degrade images with involuntary head rocking.

Characterization and application of multiple genetic markers for Plasmodium malariae.

Plasmodium malariae, a protozoan parasite that causes malaria in humans, has a global distribution in tropical and subtropical regions and is commonly found in sympatry with other Plasmodium species of humans. Little is known about the genetics or population structure of P. malariae. In the present study, we describe polymorphic genetic markers for P. malariae and present the first molecular epidemiological data for this parasite. Six microsatellite or minisatellite markers were validated using 76 P. malariae samples from a diverse geographical range. The repeat unit length varied from 2 to 17 bp, and up to 10 different alleles per locus were detected. Multiple genotypes of P. malariae were detected in 33 of 70 samples from humans with naturally acquired infection. Heterozygosity was calculated to be between 0.236 and 0.811. Allelic diversity was reduced for samples from South America and, at some loci, in samples from Thailand compared with those from Malawi. The number of unique multilocus genotypes defined using the 6 markers was significantly greater in Malawi than in Thailand, even when data from single genotype infections were used. There was a significant reduction in the multiplicity of infection in symptomatic infections compared with asymptomatic ones, suggesting that clinical episodes are usually caused by the expansion of a single genotype.

Extensive polymorphism in the plasmodium vivax merozoite surface coat protein MSP-3alpha is limited to specific domains.

Plasmodium merozoites are covered by a complex coat of surface proteins. Several of the Merozoite Surface Proteins (MSPs) that make up this coat have been proposed as vaccine candidates although some of the MSPs are known to be highly polymorphic. We present here the first survey and analysis of the polymorphism in the recently characterized P. vivax surface protein PvMSP-3alpha. Full length or partial sequences were obtained for the Pvmsp-3alpha gene from isolates originating in Central and South America, Asia and the Pacific. The Pvmsp-3alpha sequence is remarkably diverse, but this extensive diversity is largely restricted to certain domains of the encoded protein. An acidic C-terminal domain and a smaller hydrophilic N-terminus are relatively conserved, while a central domain containing coiled-coil heptad repeats is highly polymorphic and in some isolates of P. vivax is partially deleted. Unlike other MSPs, there is no evidence of allelic families of PvMSP-3alpha gene sequences, and no evidence that certain patterns of polymorphism group within isolates of similar geographical origin. The distribution and nature of polymorphism suggest that there are functional restrictions on mutations in this gene, and have implications for inclusion of PvMSP-3alpha as a candidate in a P. vivax vaccine.

A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway.

Invasion of erythrocytes by Plasmodium merozoites is an intricate process involving multiple receptor-ligand interactions. The glycophorins and an unknown trypsin sensitive factor are all erythrocyte receptors used during invasion by the major human pathogen Plasmodium falciparum. However, only one erythrocyte receptor, Glycophorin A, has a well-established cognate parasite ligand, the merozoite protein erythrocyte binding antigen-175 (EBA-175). The involvement of several other parasite proteins during invasion have been proposed, but no direct evidence links them with a specific invasion pathway. Here we report the identification and characterization of P. falciparum normocyte binding protein 1 (PfNBP1), an ortholog of Plasmodium vivax reticulocyte binding protein-1. PfNBP1 binds to a sialic acid dependent trypsin-resistant receptor on the erythrocyte surface that appears to be distinct from known invasion receptors. Antibodies against PfNBP1 can inhibit invasion of trypsinized erythrocytes and two P. falciparum strains that express truncated PfNBP1 are unable to invade trypsinized erythrocytes. One of these strain, 7G8, also does not invade Glycophorin B-negative erythrocytes. PfNBP1 therefore defines a novel trypsin-resistant invasion pathway and adds a level of complexity to current models for P. falciparum erythrocyte invasion.

Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species.

We have undertaken the first comparative pilot gene discovery analysis of approximately 25,000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10,874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.

Accuracy of sentinel node biopsy in predicting nodal status in patients with breast carcinoma.

BACKGROUND AND OBJECTIVES: While sentinel lymph node biopsy is considered by many to have replaced axillary node dissection in the management of breast cancer, concerns remain regarding false-negative results. METHODS: To investigate the accuracy of sentinel node biopsy, we reexamined all sentinel and nonsentinel nodes with multilevel sectioning and immunohistochemical staining in 42 consecutive cases of breast cancer in which sentinel node biopsy was performed and followed by axillary dissection. RESULTS: By routine hematoxylin and eosin (H&E) staining, 34% of patients were found to be node positive, with no cases of false-negative sentinel node biopsy. Reevaluation of 775 negative sentinel and nonsentinel nodes with an additional two levels and immunohistochemistry identified three "node-negative" patients who had micrometastases in the sentinel node, increasing detection in 8% of cases. More important, is the fact however, that there were no cases where additional sections and immunohistochemistry identified metastases in nonsentinel nodes that had bypassed the sentinel node. The accuracy of the sentinel node in predicting the nodal status was 100%. CONCLUSIONS: Cytokeratin immunohistochemistry will identify more patients with nodal micrometastases; however, it was unable to identify any cases where micrometastases were present in nonsentinel nodes when the sentinel node was negative. The status of the sentinel node accurately identifies the status of the axillary basin.

Plasmodium vivax merozoite surface proteins-3beta and-3gamma share structural similarities with P. vivax merozoite surface protein-3alpha and define a new gene family.

The genes encoding two merozoite surface proteins of Plasmodium vivax that are related to PvMSP3 [1] are reported. One of these genes was identified within P. vivax lambdagt11 clone 5.4, which was selected by immunoscreening with a Saimiri monkey antiserum. The insert DNA of this clone was used as a probe to isolate the complete gene from a P. vivax lambdaDASH genomic (g) DNA library. Antibodies to recombinant 5.4 and subsequent fusion proteins produce a pattern of circumferential surface fluorescence by indirect immunofluorescence assays (IFA) on segmented schizonts and free intact merozoites, and recognize a 125 kDa protein via western immunoblots. The gene, however, encodes a protein with a calculated size of 75677 Da, and 3' and 5' RACE analyses were employed to confirm the size of the gene and its coding region. The second related P. vivax gene was isolated by hybridization of a fragment of an orthologous P. knowlesi gene. The encoded proteins of all three related P. vivax genes have putative signal peptides, large central domains that contain >20% alanine residues bound by charged regions, are predicted to form alpha-helices with heptad repeat coiled-coil structures, and do not have a hydrophobic region that could anchor them to the surface of the merozoite. Although the overall identity in amino acid alignment among the three encoded proteins is low (<40%), the shared predicted structural features and motifs indicate that they are members of an intra-species family, which we are designating as the PvMSP-3 family with the reported members being Pvmsp-3alpha, Pvmsp-3beta, and Pvmsp-3gamma. We further demonstrate that this family also includes related proteins from P. knowlesi and P. falciparum.

Genetic diversity and dynamics of plasmodium falciparum and P. vivax populations in multiply infected children with asymptomatic malaria infections in Papua New Guinea.

We describe the dynamics of co-infections of Plasmodium falciparum and P. vivax in 28 asymptomatic children by genotyping these species using the polymorphic loci Msp2 and Msp3alpha, respectively. The total number of Plasmodium spp. infections detected using 3 day sampling over 61 days varied between 1 and 14 (mean 6.6). The dynamics of P. falciparum and P. vivax genotypes varied greatly both within and amongst children. Periodicity in the detection of P. falciparum infections is consistent with the synchronous replication of individual genotypes. Replication synchrony of multiple co-infecting genotypes was not detected. In 4-year-old children P. falciparum genotype complexity was reduced and episodes lasted significantly longer (median duration > 60 days) when compared to children aged 5-14 years (median duration 9 days). P. vivax genotype complexity was not correlated with age but the episode duration was also longer for this species in 4-year-olds than in older children but was not as long as P. falciparum episodes. Recurrence of P. falciparum and P. vivax genotypes over weeks was observed. We interpret these major fluctuations in the density of genotypes over time as the result of the mechanism of antigenic variation thought to be present in these Plasmodium species.

Adjuvant chemotherapy with 5-fluorouracil, L-folinic acid and levamisole for patients with colorectal cancer: non-randomised comparison of weekly versus four-weekly schedules--less pain, same gain. QUASAR Colorectal Cancer Study Group.

BACKGROUND: QUASAR is a large trial of adjuvant chemotherapy for colorectal cancer in which clinicians could choose to deliver a standard adjuvant cytotoxic chemotherapy regimen, 5-fluorouracil (5-FU) and L-folinic acid (L-FA), in either a once-weekly or a four-weekly schedule. We report results of a non-randomised comparison between these schedules with respect to survival, recurrence and differential toxicity. PATIENTS AND METHODS: In a factorial (2 x 2) trial design, QUASAR compared high-dose (175 mg) versus low-dose (25 mg) L-FA and levamisole versus placebo. The dose of 5-FU was fixed at 370 mg/m2 and although the recommended schedule was i.v. bolus delivery, daily for 5 days repeated four-weekly for 6 months, a significant proportion of randomising clinicians were constrained to deliver once-weekly 5-FU-L-FA for 30 weeks. RESULTS: Four thousand nine hundred twenty-seven patients were entered into QUASAR between May 1994 and October 1997, eighteen hundred twenty-nine of whom have recurred and sixteen hundred eighty-nine died. Similar numbers 2370 vs. 2559 were treated with the once-weekly and four-weekly schedules and the demographic features of the 2 groups were well balanced: stage C, 73.3% once-weekly vs. 71.0% four-weekly; colon, 68.0% vs. 68.3%; high-dose FA, 50.1% vs. 49.9%; levamisole, 49.3% vs. 49.3%; females, 40.2% vs. 41.7%; median age (years) 62 vs. 61. The risk of recurrence and survival were similar regardless of schedule: three-year survival was 70.6% once-weekly vs. 71.0% four-weekly; three-year recurrence risk was 35.6% once-weekly vs. 35.5% four-weekly; But, the once-weekly regimen was much less toxic: number of patients for whom toxicity was reported (once-weekly: four-weekly), stomatitis, 37 vs. 337; diarrhoea, 260 vs. 440; neutropenia, 20 vs. 153. CONCLUSIONS: The once-weekly regimen is much less toxic than and, apparently, about as effective as the four-weekly schedule. This suggests that the toxicity of 5-FU-L-FA adjuvant chemotherapy could be reduced substantially by weekly scheduling without compromising efficacy. Alternatively, efficacy might be enhanced with equal toxicity by more dose-intense weekly FU-L-FA regimens. However, this conclusion from a non-randomised comparison needs confirmation in prospective randomised studies.

Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion.

Two related Plasmodium falciparum genes and their encoded proteins have been identified by comparative analyses with Plasmodium vivax reticulocyte binding protein 2 (PvRBP-2). The P. falciparum genes have a structure which suggests that they may be the result of an evolutionary duplication event, as they share more than 8 kb of closely related nucleotide sequence but then have quite divergent unique 3' ends. Between these shared and unique regions is a complex set of repeats, the nature and number of which differs between the two genes, as well as between different P. falciparum strains. Both genes encode large hydrophilic proteins, which are concentrated at the invasive apical end of the merozoite and are predicted to be more than 350 kDa, with an N-terminal signal sequence and a single transmembrane domain near their C termini. Importantly, they also share gene structure and amino acid homology with the Plasmodium yoelii 235-kDa rhoptry protein family, which is also related to PvRBP-2. Together these Plasmodium proteins define an extended family of proteins that appear to function in erythrocyte selection and invasion. As such, they may prove to be essential components of malaria vaccine preparations.

Cross-species interactions between malaria parasites in humans.

The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.