Stereotactic guidance for navigated percutaneous sacroiliac joint fusion.

Arthrodesis of the sacroiliac joint (SIJ) for surgical treatment of SIJ dysfunction has regained interest among spine specialists. Current techniques described in the literature most often utilize intraoperative fluoroscopy to aid in implant placement; however, image guidance for SIJ fusion may allow for minimally invasive percutaneous instrumentation with more precise implant placement. In the following cases, we performed percutaneous stereotactic navigated sacroiliac instrumentation using O-arm® multidimensional surgical imaging with StealthStation® navigation (Medtronic, Inc. Minneapolis, MN). Patients were positioned prone and an image-guidance reference frame was placed contralateral to the surgical site. O-arm® integrated with StealthStation® allowed immediate auto-registration. The skin incision was planned with an image-guidance probe. An image-guided awl, drill and tap were utilized to choose a starting point and trajectory. Threaded titanium cage(s) packed with autograft and/or allograft were then placed. O-arm® image-guidance allowed for implant placement in the SIJ with a small skin incision. However, we could not track the cage depth position with our current system, and in one patient, the SIJ cage had to be revised secondary to the anterior breach of sacrum

Functional Evaluation of Plasmodium Export Signals in Plasmodium berghei Suggests Multiple Modes of Protein Export

The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms

The use of genotyping in antimalarial clinical trials: a systematic review of published studies from 1995–2005

BACKGROUND: The use of genotyping to distinguish recrudescent from new infections is currently recommended for all clinical antimalarial efficacy trials by the World Health Organization. However, genotyping-adjusted drug efficacy estimates may vary between trials due to the use of different genotyping methods and to the different settings in which these methods are applied. METHODS: A systematic review of all clinical antimalarial efficacy trials published from 1995–2005 was performed to characterize the use of genotyping, including the methods used and the effect of these methods on estimates of drug efficacy. RESULTS: In a multivariate analysis, the method of interpretation of genotyping results, the studied therapy, the location of the trial, and the duration of study follow-up all had statistically significant effects on the percent of genotyped outcomes classified as new infections. CONCLUSION: Criteria for defining appropriate, standardized genotyping methods for use in different settings are needed to enable more accurate estimates of antimalarial drug efficacy and better comparison between trials. The advantages and disadvantages of different genotyping methods and their potential impact in various settings are discussed

Structures of falcipain-2 and falcipain-3 bound to small molecule inhibitors: implications for substrate specificity.

Falcipain-2 and falcipain-3 are critical hemoglobinases of Plasmodium falciparum, the most virulent human malaria parasite. We have determined the 2.9 A crystal structure of falcipain-2 in complex with the epoxysuccinate E64 and the 2.5 A crystal structure of falcipain-3 in complex with the aldehyde leupeptin. These complexes represent the first crystal structures of plasmodial cysteine proteases with small molecule inhibitors and the first reported crystal structure of falcipain-3. Our structural analyses indicate that the relative shape and flexibility of the S2 pocket are affected by a number of discrete amino acid substitutions. The cumulative effect of subtle differences, including those at "gatekeeper" positions, may explain the observed kinetic differences between these two closely related enzymes

Drug resistance mediating Plasmodium falciparum polymorphisms and clinical presentations of parasitaemic children in Uganda.

BackgroundPlasmodium falciparum genetic polymorphisms that mediate altered drug sensitivity may impact upon virulence. In a cross-sectional study, Ugandan children with infections mutant at pfcrt K76T, pfmdr1 N86Y, or pfmdr1 D1246Y had about one-fourth the odds of symptomatic malaria compared to those with infections with wild type (WT) sequences. However, results may have been confounded by greater likelihood in those with symptomatic disease of higher density mixed infections and/or recent prior treatment that selected for WT alleles.MethodsPolymorphisms in samples from paired episodes of asymptomatic and symptomatic parasitaemia in 114 subjects aged 4-11 years were followed longitudinally in Tororo District, Uganda. Paired episodes occurred within 3-12 months of each other and had no treatment for malaria in the prior 60 days. The prevalence of WT, mixed, and mutant alleles was determined using multiplex ligase detection reaction-fluorescent microsphere assays.ResultsConsidering paired episodes in the same subject, the odds of symptomatic malaria were lower for infections with mutant compared to WT or mixed sequence at N86Y (OR 0.26, 95% CI 0.09-0.79, p = 0.018), but not K76T or D1246Y. However, symptomatic episodes (which had higher densities) were more likely than asymptomatic to be mixed (for N86Y OR 2.0, 95% CI 1.04-4.0, p = 0.036). Excluding mixed infections, the odds of symptomatic malaria were lower for infections with mutant compared to WT sequence at N86Y (OR 0.33, 95% CI 0.11-0.98, p = 0.046), but not the other alleles. However, if mixed genotypes were grouped with mutants in this analysis or assuming that mixed infections consisted of 50% WT and 50% mutant genotypes, the odds of symptomatic infection did not differ between infections that were mutant or WT at the studied alleles.ConclusionsAlthough infections with only the mutant pfmdr1 86Y genotype were associated with symptomatic infection, this association could primarily be explained by greater parasite densities and therefore greater prevalence of mixed infections in symptomatic children. These results indicate limited association between the tested polymorphisms and risk of symptomatic disease and highlight the value of longitudinal studies for assessing associations between parasite factors and clinical outcomes

Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary electrophoresis from randomized trials of artemether-lumefantrine (AL) vs. dihydroartemisinin-piperaquine (DP) performed in two areas of Uganda: Kanungu, where transmission is moderate, and Apac, where transmission is very high. Both gel and capillary methods evaluated polymorphic regions of the merozoite surface protein 1 and 2 and glutamine rich protein genes. Results Capillary electrophoresis detected more alleles and provided higher discriminatory power than agarose gel electrophoresis at both study sites. There was only moderate agreement between classification of outcomes with the two methods in Kanungu (kappa = 0.66) and poor agreement in Apac (kappa = 0.24). Overall efficacy results were similar when using gel vs. capillary methods in Kanungu (42-day risk of treatment failure for AL: 6.9% vs. 5.5%, p = 0.4; DP 2.4% vs. 2.9%, p = 0.5). However, the measured risk of recrudescence was significantly higher when using gel vs. capillary electrophoresis in Apac (risk of treatment failure for AL: 17.0% vs. 10.7%, p = 0.02; DP: 8.5% vs. 3.4%, p = 0.03). Risk differences between AL and DP were not significantly different whether gel or capillary methods were used. Conclusions Genotyping with gel electrophoresis overestimates the risk of recrudescence in anti-malarial trials performed in areas of high transmission intensity. Capillary electrophoresis provides more accurate outcomes for such trials and should be performed when possible. In areas of moderate transmission, gel electrophoresis appears adequate to estimate comparative risks of treatment failure

Principal role of dihydropteroate synthase mutations in mediating resistance to sulfadoxine-pyrimethamine in single-drug and combination therapy of uncomplicated malaria in Uganda.

Antimalarial resistance to sulfadoxine-pyrimethamine (SP) is mediated by mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes. However, the relative importance of different mutations is incompletely understood and has not been studied with combination therapy. Samples from 812 patients treated for uncomplicated malaria in Kampala, Uganda were tested for the presence of mutations commonly found in Africa. The dhps Glu-540 mutation was the strongest independent predictor of treatment failure. The dhfr Arg-59 mutation was only predictive of treatment failure in the presence of the dhps Glu-540 mutation. Comparing combination regimens with SP monotherapy, the addition of chloroquine to SP did not improve efficacy, the addition of artesunate lowered the risk of treatment failure only for infections with both the dhfr Arg-59 and dhps Glu-540 mutations, and the addition of amodiaquine lowered this risk for all dhfr/dhps mutation patterns. The dhps Glu-540 mutation played a principal role and the dhfr Arg-59 mutation a secondary role in mediating resistance to SP alone and in combination

Short report: Dynamics of Plasmodium falciparum malaria after sub-optimal therapy in Uganda.

We followed parasite genotypes of 75 patients for 42 days after treatment of uncomplicated malaria with chloroquine + sulfadoxine-pyrimethamine in Kampala, Uganda. Infections were complex (mean, 2.88 strains) and followed three patterns: 27% of patients eliminated all strains and remained parasite-free, 48% had a long aparasitemic interval followed by reappearance of original strains after 3-33 days (mean, 9.2 days), and 25% failed to clear original strains and required therapy after 3-35 days (mean, 17 days). These results highlight the complexity of malaria in Africa and have implications for efficacy trials, because missing late reappearances of strains could lead to misclassification of outcomes