Antibody responses to surface antigens of Plasmodium falciparum gametocyte-infected erythrocytes and their relation to gametocytaemia.

An essential element for continuing transmission of Plasmodium falciparum is the availability of mature gametocytes in human peripheral circulation for uptake by mosquitoes. Natural immune responses to circulating gametocytes may play a role in reducing transmission from humans to mosquitoes. Here, antibody recognition of the surface of mature intra-erythrocytic gametocytes produced either by a laboratory-adapted parasite, 3D7, or by a recent clinical isolate of Kenyan origin (HL1204), was evaluated longitudinally in a cohort of Ghanaian school children by flow cytometry. This showed that a proportion of children exhibited antibody responses that recognized gametocyte surface antigens on one or both parasite lines. A subset of the children maintained detectable anti-gametocyte surface antigen (GSA) antibody levels during the 5 week study period. There was indicative evidence that children with anti-GSA antibodies present at enrolment were less likely to have patent gametocytaemia at subsequent visits (odds ratio = 0·29, 95% CI 0·06-1·05; P = 0·034). Our data support the existence of antigens on the surface of gametocyte-infected erythrocytes, but further studies are needed to confirm whether antibodies against them reduce gametocyte carriage. The identification of GSA would allow their evaluation as potential anti-gametocyte vaccine candidates and/or biomarkers for gametocyte carriage.

Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.

Disruption of a novel open reading frame of Plasmodium falciparum chromosome 9 by subtelomeric and internal deletions can lead to loss or maintenance of cytoadherence.

Isolates of Plasmodium falciparum commonly undergo a large subtelomeric deletion of the right end of chromosome 9 during in vitro cultivation, accompanied by loss of ability to cytoadhere to melanoma cells and greatly lowered gametocyte production. ItG2, an isolate in which cytoadherence is stable, has undergone a subtelomeric deletion of intermediate length on chromosome 9. We show here that the deletions in all non-cytoadherent clones examined have breakpoints within or delete a novel open reading frame (the breakpoint open reading frame, BPORF) that is a unique sequence in the genome. Surprisingly, in ItG2 BPORF has been removed by an additional 15 kb internal deletion in chromosome 9. These results suggest mechanisms to account for the observed frequent deletion of the right arm of chromosome 9 and for the known stability of cytoadherence in ItG2. However, we were unable to detect var genes in this region of the ItG2 genome. We conclude that the product of a novel gene distinct from the var family is implicated in cytoadherence.

The cytoadherence linked asexual gene family of Plasmodium falciparum: are there roles other than cytoadherence?

The binding of erythrocytes infected with Plasmodium falciparum to the endothelium lining the small blood vessels of the brain and other organs can mediate severe pathology. A region at the right end of chromosome 9 has been implicated in the binding of parasitised erythrocytes to the endothelial receptor CD36. A gene expressed in asexual erythrocytic stage parasites has been identified in this region and termed the cytoadherence linked asexual gene (clag). Antisense RNA production and targeted gene disruption of clag resulted in greatly reduced binding to CD36. Hybridisation to 3D7 chromosomes showed clag to be a part of a gene family of at least nine members. All members analysed so far have a conserved gene structure of at least nine exons, as well as putative transmembrane domains. The possible functions of the gene family are discussed.

Excimer laser effects on human corneal endothelium. Modulation by serum factor(s).

OBJECTIVE: To determine the possibility of endothelial cell damage after excimer laser ablation. METHODS: Endothelial cell densities and morphology of human corneas after photoablations or mechanical keratectomy were compared with those of the untreated mates after 1 week of culture with or without serum. RESULTS: Corneas cultured in serum-free medium after ablation to a depth of 150 microns showed endothelial cell densities reduced to 60% of untreated, mate corneas; ultrastructural analysis showed endothelial cell damage not seen in untreated mates. Corneas ablated to the same depth and cultured in serum-enriched medium showed no endothelial cell density loss, nor did corneas cultured in serum-free medium after an ablation to a depth of 50 microns or mechanical keratectomies averaging 95 microns. CONCLUSIONS: Endothelial cell loss in deep laser resections may be prevented by factor(s) in fetal bovine serum. The apparent lack of cell loss in clinical studies may be related to the protective action of similar factors in aqueous humor.

A ten-year follow-up of one hundred consecutive Müller curved-stem total hip-replacement arthroplasties.

One hundred consecutive Müller curved-stem total hip replacements were reviewed ten years after operation. Twenty patients with twenty-two arthroplasties had died within the ten-year period without having a revision, and twenty-five arthroplasties had been revised for various reasons. Of the remaining fifty-three arthroplasties, thirty-five were classified as good or excellent, with Harris hip scores of 80 points or higher, and eighteen were classified as poor or fair, with scores lower than 80 points. Follow-up radiographs, made for all but six of the fifty-three hips at ten years, showed a 23 per cent incidence of migration of the acetabular component and a 28 per cent incidence of migration of the femoral component. In addition, there was a 15 per cent incidence of bone resorption in the proximal end of the femur without migration of the femoral component and a 4 per cent incidence of osteolytic defects about the femoral component, also without migration. Combining the radiographically loose replacement (migration) with the clinically loose ones (revised), the over-all incidence of aseptic loosening was 29 per cent for the acetabular component and 40 per cent for the femoral component. There was a positive correlation between the incidence of loosening of the femoral component and younger age, heavier weight, male sex, unilateral hip disease, a wide femoral canal, and varus position of the femoral component, whereas the incidence of loosening of the acetabular component was increased only in association with older age. The rate of loosening of the femoral component appeared to be higher during the early follow-up period and to decrease with time, while the rate of loosening of the acetabular component appeared to be lower during the early follow-up period but to increase with time.

Effects of heat-stress on behavior and the pituitary adrenal axis in rats.

Three experiments were performed in order to analyse the behavioral and biochemical correlates of four different intensities of the same stressor. In Experiment 1, rats were exposed to heat stress (hot-plate) of varying temperatures for 30 seconds. Activity was recorded in an open field immediately after stress for 30 minutes. The data revealed that the milder temperatures increased (21, 47, 52 degrees C), while the higher temperature (57 degrees C) decreased activity. Experiment 2 assessed the pituitary-adrenal response to the different temperatures by measuring levels of plasma corticosterone 30 minutes after stress. The four levels of hot-plate temperatures induced differential levels of corticosterone which may best be described as an inverted U-shaped function, with only the extreme temperature (57 degrees) inducing a significant elevation in levels of the steroid. Experiment 3 further manipulated the pituitary adrenal axis by administering dexamethasone 25 hr and 1 hr before stress and ACTH 15 min before stress. Both affected activity levels by depressing locomotion regardless of the stress intensity. These results are compared to other studies that have addressed the question of stress-induced activation and it is suggested that stress is not a unitary concept, but interacts with the performance of certain behaviors to produce both facilatory or inhibitory results.

Practical application of computer-generated three-dimensional reconstructions in orthopedic surgery.

There are some special technical requirements of CT scans used in producing three-dimensional reconstructions. There are few purely diagnostic applications for this technique, but there will be many future applications in the realm of surgical planning once reasonably priced, interactive systems are available.

Use of general purpose mechanical computer assisted engineering software in orthopaedic surgical planning: advantages and limitations.

Two surgical plans were developed for an appropriately complex reconstructive orthopaedic surgery case. One plan was developed with customary methods using two-dimensional (2D) radiographs. The second plan was developed with general purpose mechanical computer assisted engineering (MCAE) software using x-ray computed tomography (CT) data. The limitations of each method are identified. To create a surgical plan using three-dimensional (3D) medical datasets and MCAE software, five necessary steps were identified: (a) data reduction; (b) contour extraction; (c) 3D model creation; (d) extraction of mass properties; (e) model idealization. The principal limitation of general purpose MCAE software is the lack of pre-processing modules with which to address the unique requirements of medical image datasets.

Early experience with eccentric acetabular components in revision total hip arthroplasty.

Osteolysis associated with acetabular component loosening can produce significant acetabular defects. The combined (segmental and cavitary) or type III defect can present a very challenging reconstructive problem. Several methods of reconstruction for type III defects have been described, including the use of oblong or eccentric sockets. These implants are attractive, theoretically, because they allow restoration of the hip center without use of structural bone graft. The results of this method however have not been widely reported. From a series of type III defects reconstructed from 1987 to 1991, six hips were reconstructed with eccentric acetabular components, provided by three different manufacturers. Four of these six implants were custom and two were off-the-shelf. At 4-years minimum follow-up, three of the six sockets had failed and had been revised. All three failures had been custom implants produced by the same manufacturer. The other three sockets had produced good clinical results. The features common to the failed implants were: (1) custom manufacture; (2) small ingrowth pads not integral with the body of the implant; and (3) lack of specific instrumentation (reamers) for insertion of the implant. Eccentric sockets may have limited, although well-defined, indications for use, such as acetabular revision needed in the presence of a well-fixed or monolithic femoral stem. Early experience suggests that custom eccentric acetabular components are not well suited for such indications.

Volumetric 3-D imaging of computerized tomography scans.

Contiguous transaxial high resolution CT scans of more than 100 patients with craniofacial deformities and orthopedic disorders were obtained. The CT scan examinations were used diagnostically in determining the need for surgery and for planning therapy. The serial section data was reconstructed in a three-dimensional form with surface and transparent volumetric computer graphics processing techniques. Real time sequences showing the presence of internal abnormalities in these patients were produced and recorded on video tape. This study demonstrates the feasibility and technical requirements of three-dimensional volumetric visualization for diagnostic evaluation of patients with craniofacial and orthopedic disorders.

Amodiaquine and artemether-lumefantrine select distinct alleles of the Plasmodium falciparum mdr1 gene in Tanzanian children treated for uncomplicated malaria.

The artemisinin-based combination therapies artemether-lumefantrine (AL) and amodiaquine (AQ) plus artesunate have been adopted for treatment of Plasmodium falciparum malaria in many African countries. Molecular markers of parasite resistance suitable for surveillance have not been established for any of the component drugs in either of these combinations. We assessed P. falciparum mdr1 (Pfmdr1) alleles present in 300 Tanzanian children presenting with uncomplicated falciparum malaria, who were enrolled in a clinical trial of antimalarial therapy. Pfmdr1 genotype analysis was also performed with isolates from 182 children who failed AQ monotherapy and 54 children who failed AL treatment. Pfmdr1 alleles 86Y, 184Y, and 1246Y were more common among treatment failures in the AQ group than among pretreatment infections. The converse was found in the AL-treated group. Children presenting with the 86Y/184Y/1246Y Pfmdr1 haplotype and treated with AQ were significantly more likely to retain this haplotype if they were parasite positive during posttreatment follow-up than were children treated with AL (odds ratio, 33.25; 95% confidence interval, 4.17 to 1441; P, <0.001). We conclude that AL and AQ exert opposite within-host selective effects on the Pfmdr1 gene of P. falciparum.

The flip-side of Cytoadherence: immune selection, antigenic variation and the var Genes of Plasmodium falciparum.

In areas where Plasmodium falciparum is endemic, the natural immunity acquired by people exposed to frequent malaria infection is likely to have a differential selective impact upon different parasite genotypes. It has been suggested that the immune response directed against the variant antigen PfEMP1, which is expressed on the infected erythrocyte surface, is a crucial determinant of parasite population structure and favours the existence of distinct strains, or Varotypes. Here, Colin Sutherland summarizes current knowledge of the var multigene family, which encodes the PfEMP1 variants, and suggests that this information may allow certain predictions of the strain hypothesis to be tested directly.

Management of type III acetabular deficiencies in revision total hip arthroplasty without structural bone graft.

Seventeen patients with loose total hip acetabular components and type III osteolytic acetabular defects were prospectively identified and reconstructed with either spherical acetabular components or eccentric acetabular components without use of structural bone graft. A computed tomography (CT) scan and a three-dimensional (3-D) model were obtained in nine patients to determine whether a spherical component or eccentric component should be used. The clinical results were measured using the Harris Hip Score. Hip scores improved from 44 to 73 points for the entire group but were lower in the subgroup with eccentric components. Also, two of the eccentric cups developed loosening. The hip center was better restored in the eccentric implant group. The cost of the prosthesis was higher in the eccentric implant group due to the expenses of the CT scan and the 3-D model.

Treatment of type III acetabular deficiencies in revision total hip arthroplasty without structural bone-graft.

The use of structural bone allografts has been advocated for the reconstruction of type III acetabular defects, but the results reported for this method have shown revision rates from 20 to 30% and failure rates averaging 50% at short-, to intermediate-term follow-up periods. A prospective study was undertaken to determine the results of reconstructing type III defects without structural allograft and to compare the results using spherical components with the results using eccentric components. Quantitative inclusion criteria were developed and applied to all hips undergoing revision arthroplasty between 1987 and 1991. Twenty-one hips (20 patients) were identified and included in this study. An evaluation protocol including radiographs, and for defects greater than 30 mm, computed tomography scans and three-dimensional models, was used. At an average follow-up period of 46 months, 19 hips (18 patients) were evaluated. Hip scores of these patients improved from 46 to 75. The location of the hip center was improved to a greater degree in those hips with revised to eccentric components, but implant stability was poor. Overall, the mechanical failure rate was 26%, the clinical failure rate was 16%, and the revision rate was 11%. These results compare favorably with the results of reconstruction using structural graft at the same length of follow-up time. Good results can be achieved by reconstructing type III defects without structural allograft if a cementless spherical component can be placed in stable position on host-bone.

Radiographic evaluation of acetabular bone stock in failed total hip arthroplasty.

Evaluation of available bone stock in failed hip arthroplasty where significant osteolysis has occurred is often difficult, since information about the osseous integrity of the innominate bone is obscured by methacrylate and metal. A simple, inexpensive, and reliable solution is the use of the cross-table lateral view of the hip, with modifications of technique to demonstrate osseous anatomy. The author describes this technique and the anatomy of the pelvis from this oblique perspective. Illustrative case reports are presented.