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.

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.

A model for sequestration of the transmission stages of Plasmodium falciparum: adhesion of gametocyte-infected erythrocytes to human bone marrow cells.

With the aim of developing an appropriate in vitro model of the sequestration of developing Plasmodium falciparum sexual-stage parasites, we have investigated the cytoadherence of gametocytes to human bone marrow cells of stromal and endothelial origin. Developing stage III and IV gametocytes, but not mature stage V gametocytes, adhere to bone marrow cells in significantly higher densities than do asexual-stage parasites, although these adhesion densities are severalfold lower than those encountered in classical CD36-dependent assays of P. falciparum cytoadherence. This implies that developing gametocytes undergo a transition from high-avidity, CD36-mediated adhesion during stages I and II to a lower-avidity adhesion during stages III and IV. We show that this adhesion is CD36 independent, fixation sensitive, stimulated by tumor necrosis factor alpha, and dependent on divalent cations and serum components. These data suggest that gametocytes and asexual parasites utilize distinct sets of receptors for adhesion during development in their respective sequestered niches. To identify receptors for gametocyte-specific adhesion of infected erythrocytes to bone marrow cells, we tested a large panel of antibodies for the ability to inhibit cytoadherence. Our results implicate ICAM-1, CD49c, CD166, and CD164 as candidate bone marrow cell receptors for gametocyte adhesion.

The use of direct sequencing of dengue virus cDNA from individual field-collected Aedes aegypti for surveillance and epidemiological studies.

The relative efficiencies of four methods to extract viral RNA from individual dengue-2 virus (D-2V)-infected mosquitoes, Aedes aegypti (L.) (Diptera: Culicidae), were compared. The most efficient of these methods was then used to extract viral RNA for the preparation of cDNA from the abdomens of six engorged D-2V-infected mosquitoes and sera from three dengue fever (DF) patients collected in an isolated rural town in Colombia. Comparisons of viral envelope (E) gene sequences from each of these strongly suggested that the D-2V population which circulated in this study area was a homogeneous genotype which was unrelated to any of the D-2 viruses isolated from elsewhere in the world. When coupled with our rapid method to identify viruses in individual mosquitoes (Romero-Vivas et al. (1998) Medical and Veterinary Entomology, 12, 101-105), the methodology we describe should be useful for epidemiological and surveillance studies of dengue viruses and other arboviruses.

Bilobed oblong porous coated acetabular components in revision total hip arthroplasty.

Thirty-eight oblong bilobed noncustom uncemented, porous-coated titanium acetabular components were used to reconstruct failed hip arthroplasties with large superior segmental acetabular bone deficiencies. No structural bone grafts were used. All patients were followed up for 2 to 5 years (mean, 3 years) after the operation. One patient (whose socket rested primarily on a structural bone graft from a previous procedure) had revision surgery for acetabular loosening. No other patients have had revision surgery or had another ipsilateral hip operation. At latest followup, 35 patients had no or mild pain and two patients had moderate pain. Two implants migrated more than 2 mm in the first year, then stabilized. On the latest radiographs, two implants had bead shedding, but there was no measurable migration or change in position. For selected patients with large superolateral acetabular bone deficiencies, this implant facilitated a complex reconstruction, provided good clinical results, and showed satisfactory stability at early to midterm followup in most patients.

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.

A comparison of intramuscular tenoxicam with intramuscular morphine for pain relief following tonsillectomy in children.

A double blind trial was conducted to evaluate the analgesic efficacy of intramuscular tenoxicam for pain relief following tonsillectomy in children. Fifty children, aged 3-10 years, were randomly allocated to receive intramuscular tenoxicam 0.75 mg.kg-1 or intramuscular morphine sulphate 0.2 mg.kg-1 after induction of anaesthesia. Although the tenoxicam group required significantly more postoperative morphine.(mean 57.8 micrograms.kg-1 compared with 26.9 micrograms.kg-1, P = 0.025), the total morphine dose was significantly reduced after tenoxicam (57.8 micrograms.kg-1 compared with 226.9 micrograms.kg-1, P < 0.0001). There was no difference between the quality of analgesia after discharge from recovery. The incidence of postoperative vomiting was significantly reduced after tenoxicam (20%) compared with morphine (71%).

Preoperative description of severe acetabular defects caused by failed total hip replacement.

PURPOSE: The reconstruction of severe acetabular defects, caused by failed hip prostheses, is difficult and complex. Current radiographic descriptions of the defects are inadequate. We sought to more accurately describe remaining bone and improve surgical planning using 3D physical models based on CT data. METHOD: Nineteen patients (20 hips) with failed total hip replacements and severe acetabular defects were selected. Plain radiography and CT were performed. Solid 3D physical models of each pelvis were constructed using the CT data. Bone loss in structurally important regions was measured from radiographs, models, and intraoperative observation. Comparisons were made of the radiograph-based and model-based bone loss measurements, surgical plans, and interobserver variability. RESULTS: There was no statistical difference between the model-based measurements and the intraoperative measurements. Radiographs statistically underestimated bone loss by at least 20% (p < 0.01). Interobserver bone loss grading was poor for the radiograph-based measurements (kappa = 0.06) but was substantial for the model-based measurements (kappa = 0.73). In only half the cases was prosthesis type correctly selected based on radiographs. Model-based planning selected the correct prosthesis type in all cases. CONCLUSION: The physical models were substantially more accurate than radiographs for measuring acetabular defects and for surgical planning.

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.

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.

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.

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.

Morphine consumption in patients receiving rectal paracetamol and diclofenac alone and in combination.

Paracetamol and diclofenac have different mechanisms of action, and the combination may be more effective than each drug used alone in treating postoperative pain. In a double-blind, controlled design, we studied 60 patients undergoing elective abdominal gynaecological surgery, who received suppositories of paracetamol 1.5 g, diclofenac 100 mg or a combination of the two before the start of surgery. Patients received morphine in the intraoperative period, and cumulative morphine use from a patient-controlled analgesia system was recorded to measure the analgesic effect of the suppositories. Morphine consumption was greatest in the group that received paracetamol alone and lowest in the group given the combination (P < 0.01). There was no difference in the incidence of morphine-related side effects between the groups. We conclude that a diclofenac-paracetamol combination reduced the amount of morphine used compared with paracetamol alone.

Positional cloning of a sequence from the breakpoint of chromosome 9 commonly associated with the loss of cytoadherence.

Isolates of Plasmodium falciparum commonly undergo a large, subtelomeric deletion of the right end of chromosome 9 during in-vitro cultivation. This deletion is usually accompanied by loss of ability to cytoadhere to melanoma cells, loss of a var-gene product from the red-cell surface and a reduction in gametocyte production. However, cytoadherence is stable in the isolate ItG2, remaining after many generations in culture. Deletions in all the non-cytoadherent clones examined have breakpoints within or delete a novel open-reading frame, called the breakpoint open-reading frame (BPORF), that is a unique sequence in the genome. In ItG2, surprisingly, BPORF has been removed by a 15-kb deletion, internal in chromosome 9. These results indicate mechanisms to explain why the deletion of chromosome 9 occurs so frequently and why cytoadherence is stable in ItG2.

Infusions of subhypnotic doses of propofol for the prevention of postoperative nausea and vomiting.

We studied the antiemetic effects of a low dose infusion of propofol for 24 h after major gynaecological surgery in a double-blind, randomised, controlled trial. Fifty women of ASA physical status 1 or 2 undergoing major gynaecological surgery received an infusion of 1% propofol or intralipid at 0.1 ml.kg-1.h-1 for 24 h after surgery. Pain was managed using morphine delivered by a patient-controlled analgesia pump. The degree of postoperative nausea and vomiting was assessed by the nurses using a four-point ordinal scale, by the patients using a visual analogue scale and by the amount of rescue antiemetic given by the nurses. There were no differences between the two groups in any of the measures of postoperative nausea and vomiting during the first 48 h after surgery. Postoperative nausea and vomiting in the control group was less on the second day compared with the first postoperative day, but not in the propofol group. There were no side effects from the propofol infusion.

Artifacts and thresholding in X-ray CT of a cortical bone and titanium composite.

PURPOSE: X-Ray CT has the potential to provide precise and accurate data from which the mechanical properties of bone can be calculated. Such data would be useful in understanding the response of bone tissue to implants. Various artifacts can, however, degrade accuracy of the data. The aim of this study was to measure the artifactual errors produced in CT of a simulated femoral bone-titanium composite and to propose a method to correct for them. METHOD: A composite phantom that simulates cortical bone and a titanium implant was designed and constructed. The phantom was scanned and the image data were analyzed over a range of thresholds with image analysis software developed for this study. The outer (OD) and inner (ID) diameter and the CT number of the cortical bone, with and without titanium, were measured over a range of cortical thicknesses. RESULTS: While ID can be accurately measured by choosing the proper threshold (800 HU), OD, even at optimal threshold, will be underestimated by approximately 2%. If a proper threshold is selected, CT number can also be accurately determined. Errors of up to 5%, however, are produced by titanium unless corrected by proper threshold selection. CONCLUSION: Intramedullary titanium is not a deterrent to obtaining accurate measurements of cortical bone dimensions and properties. Proper choice of thresholds for image analysis of CT scan data can yield accuracy and precision of < or = 2%.

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.