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A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.

Background Co-infection of the four major species of human malaria parasite Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale sp. (Po) is regularly observed, but there is limited understanding of between-species interactions. In particular, little is known about the effects of multiple Plasmodium species co-infections on gametocyte production. Methods We developed molecular assays for detecting asexual and gametocyte stages of Pf, Pv, Pm, and Po. This is the first description of molecular diagnostics for Pm and Po gametocytes. These assays were implemented in a unique epidemiological setting in Papua New Guinea with sympatric transmission of all four Plasmodium species permitting a comprehensive investigation of species interactions. Findings The observed frequency of Pf-Pv co-infection for asexual parasites (14.7%) was higher than expected from individual prevalence rates (23.8%Pf x 47.4%Pv = 11.3%). The observed frequency of co-infection with Pf and Pv gametocytes (4.6%) was higher than expected from individual prevalence rates (13.1%Pf x 28.2%Pv = 3.7%). The excess risk of co-infection was 1.38 (95% confidence interval (CI): 1.09, 1.67) for all parasites and 1.37 (95% CI: 0.95, 1.79) for gametocytes. This excess co-infection risk was partially attributable to malaria infections clustering in some villages. Pf-Pv-Pm triple infections were four times more frequent than expected by chance alone, which could not be fully explained by infections clustering in highly exposed individuals. The effect of co-infection on parasite density was analyzed by systematic comparison of all pairwise interactions. This revealed a significant 6.57-fold increase of Pm density when co-infected with Pf. Pm gametocytemia also increased with Pf co-infection. Conclusions Heterogeneity in exposure to mosquitoes is a key epidemiological driver of Plasmodium co-infection. Among the four co-circulating parasites, Pm benefitted most from co-infection with other species. Beyond this, no general prevailing pattern of suppression or facilitation was identified in pairwise analysis of gametocytemia and parasitemia of the four species. Trial registration This trial is registered with ClinicalTrials.gov, Trial ID: NCT02143934.

Background The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross-sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome. Methods Plasmodium falciparum ( Pf ) and Plasmodium vivax ( Pv ) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv _18S rRNA genes or the multi-copy targets Pf _varATS and Pv _mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities. Results In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf _18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_ varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv _18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum , at 8.0% (66/828). Use of Pv _mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG. Conclusion The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

Clinical trials monitoring malaria drug resistance require genotyping of recurrent Plasmodium falciparum parasites to distinguish between treatment failure and new infection occurring during the trial follow up period. Because trial participants usually harbour multi-clonal P. falciparum infections, deep amplicon sequencing (AmpSeq) was employed to improve sensitivity and reliability of minority clone detection. Paired samples from 32 drug trial participants were Illumina deep-sequenced for five molecular markers. Reads were analysed by custom-made software HaplotypR and trial outcomes compared to results from the previous standard genotyping method based on length-polymorphic markers. Diversity of AmpSeq markers in pre-treatment samples was comparable or higher than length-polymorphic markers. AmpSeq was highly reproducible with consistent quantification of co-infecting parasite clones within a host. Outcomes of the three best-performing markers, cpmp, cpp and ama1-D3 , agreed in 26/32 (81%) of patients. Discordance between the three markers performed per sample was much lower by AmpSeq (six patients) compared to length-polymorphic markers (eleven patients). Using AmpSeq for discrimination of recrudescence and new infection in antimalarial drug trials provides highly reproducible and robust characterization of clone dynamics during trial follow-up. AmpSeq overcomes limitations inherent to length-polymorphic markers. Regulatory clinical trials of antimalarial drugs will greatly benefit from this unbiased typing method.

Submicroscopic malaria infections contribute to transmission in exposed populations but their extent is underestimated even by standard molecular diagnostics. Sophisticated sampling and ultra-sensitive molecular methods can maximise test sensitivity but are not feasible in routine surveillance. Here we investigate the gains achievable by using increasingly sensitive methods with the aim to understand what diagnostic sensitivity is necessary to guide malaria interventions. Venous blood samples were collected from participants in a cross-sectional survey in two coastal medium-endemic villages in Madang province, Papua New Guinea. Using ultra-sensitive quantitative PCR (us-qPCR) on concentrated high-volume blood samples (2 mL) as reference, we quantified the proportion of Plasmodium falciparum and Plasmodium vivax infections and gametocyte carriers detectable in fingerprick blood volumes (200 μL) by standard 18S rRNA qPCR, us-qPCR, rapid diagnostic test (RDT), and ultra-sensitive P falciparum RDT. We further compared the epidemiological patterns observed with each diagnostic approach in the study population. Venous blood samples were collected from 300 participants between Dec 5, 2016, and Feb 24, 2017 (ie, during peak rainy season). Standard qPCR identified 87 (54%) of 161 P falciparum infections and 73 (52%) of 141 P vivax infections detected by the reference method. us-qPCR identified an additional 11 (7%) P falciparum infections and 14 (10%) P vivax infections. 80 (86%) of 93 P falciparum gametocyte carriers and 75 (91%) of 82 P vivax gametocyte carriers were found among infections detectable by us-qPCR. Ultra-sensitive RDT missed half of P falciparum infections detected by standard qPCR, including high gametocytaemic infections. Epidemiological patterns corresponded well between standard qPCR and the reference method. As the prevalence of P vivax decreased with increasing age, the proportion of P vivax infections undetectable by standard qPCR increased. Almost all potentially transmitting parasite carriers were identified with us-qPCR on fingerprick blood volumes. Analysing larger blood volumes revealed a large pool of ultra-low-density P falciparum and P vivax infections, which are unlikely to be transmitted. Therefore, current RDTs cannot replace molecular diagnostics for identifying potential P falciparum transmitters. Swiss National Science Foundation.

Abstract Background Regulatory clinical trials are required to ensure the continued supply and deployment of effective antimalarial drugs. Patient follow-up in such trials typically lasts several weeks as the drugs have long half-lives and new infections often occur during this period. “Molecular correction” is therefore used to distinguish drug failures from new infections. The current WHO-recommend method for molecular correction uses length-polymorphic alleles at highly diverse loci but is inherently poor at detecting low density clones in polyclonal infections. This likely leads to substantial underestimates of failure rates, delaying the replacement of failing drugs with potentially lethal consequences. Deep sequenced amplicons (AmpSeq) substantially increase the detectability of low-density clones and may offer a new “gold standard” for molecular correction. Methods Pharmacological simulation of clinical trials was used to evaluate the suitability of AmpSeq for molecular correction. We investigated the impact of factors such as the number of amplicon loci analysed, the informatics criteria used to distinguish genotyping ‘noise’ from real low density signals, the local epidemiology of malaria transmission, and the potential impact of genetic signals from gametocytes. Results AmpSeq greatly improved molecular correction and provided accurate drug failure rate estimates. The use of 3 to 5 amplicons was sufficient, and simple, non-statistical, criteria could be used to classify recurrent infections as drug failures or new infections. Conclusions These results strongly endorse the deployment of AmpSeq as the standard for molecular correction in regulatory trials, with its potential extension into routine surveillance once the requisite technical support becomes established. Competing Interest Statement The authors have declared no competing interest.

Background The use of molecular diagnostics has revealed an unexpectedly large number of asymptomatic low-density malaria infections in many malaria endemic areas. This study compared the gains in parasite prevalence obtained by the use of ultra-sensitive (us)-qPCR as compared to standard qPCR in cross sectional surveys conducted in Thailand, Brazil and Papua New Guinea (PNG). The compared assays differed in the copy number of qPCR targets in the parasite genome. Methods Plasmodium falciparum ( Pf ) and Plasmodium vivax ( Pv ) parasites were quantified by qPCR amplifying the low-copy Pf_ and Pv _18S rRNA genes or the multi-copy targets Pf _varATS and Pv _mtCOX1. Cross-sectional surveys at the three study sites included 2252 participants of all ages and represented different transmission intensities. Results In the two low-transmission areas, P. falciparum positivity was 1.3% (10/773) (Thailand) and 0.8% (5/651) (Brazil) using standard Pf _18S rRNA qPCR. In these two countries, P. falciparum positivity by Pf_ varATS us-qPCR increased to 1.9% (15/773) and 1.7% (11/651). In PNG, an area with moderate transmission intensity, P. falciparum positivity significantly increased from 8.6% (71/828) by standard qPCR to 12.2% (101/828) by us-qPCR. The proportions of P. falciparum infections not detected by standard qPCR were 33%, 55% and 30% in Thailand, Brazil and PNG. Plasmodium vivax was the predominating species in Thailand and Brazil, with 3.9% (30/773) and 4.9% (32/651) positivity by Pv _18S rRNA qPCR. In PNG, P. vivax positivity was similar to P. falciparum , at 8.0% (66/828). Use of Pv _mtCOX1 us-qPCR led to a significant increase in positivity to 5.1% (39/773), 6.4% (42/651) and 11.5% (95/828) in Thailand, Brazil, and PNG. The proportions of P. vivax infections missed by standard qPCR were similar at all three sites, with 23%, 24% and 31% in Thailand, Brazil and PNG. Conclusion The proportional gains in the detection of P. falciparum and P. vivax infections by ultra-sensitive diagnostic assays were substantial at all three study sites. Thus, us-qPCR yields more precise prevalence estimates for both P. falciparum and P. vivax at all studied levels of endemicity and represents a significant diagnostic improvement. Improving sensitivity in P. vivax surveillance by us-qPCR is of particular benefit, because the additionally detected P. vivax infections signal the potential presence of hypnozoites and subsequent risk of relapse and further transmission.

Study Design: Retrospective analysis. Level of evidence III. Objectives: Low-energy vertebral compression fractures are an increasing socioeconomic problem among elderly patients. Percutaneous vertebroplasty has been extensively used for the treatment of painful fractures because of its effectiveness. However, some complications have been described; among them, new vertebral compression fractures, whether adjacent or not to the treated vertebra, are commonly reported complications (8% to 52%). Methods: We retrospectively analyzed epidemiological and technical variables presumably associated with new vertebral compression fractures. To determine the relationship between new vertebral compression fracture and percutaneous vertebroplasty, 30 patients (study group) with this complication were compared with 60 patients treated with percutaneous vertebroplasty without this condition (control group). Results: A higher cement percentage was found in the study group (40.3%) compared with the control group (30.5%). Initial vertebral kyphosis was significantly higher in the first group (15°) compared with the control group (9°). Epidemiological factors were similar in both groups. Conclusions: In our study, increased cement percentage injected and a higher kyphosis were associated with new vertebral compression fractures.

Background and AimsMyasthenia gravis (MG) is a T-cell dependent antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen, comprising several T and B cell auto-epitopes. We hypothesized that an efficacious drug candidate for antigen-specific therapy in MG should comprise a broad range of these auto-epitopes and be administered in a noninflammatory and tolerogenic context.MethodsWe used a soluble mutated form of the extracellular domain of the α1 chain of the AChR (α1-ECDm), which represents the major portion of auto-epitopes involved in MG, and investigated, in a well-characterized rat model of experimental autoimmune myasthenia gravis (EAMG) whether its intravenous administration could safely and efficiently treat the autoimmune disease.ResultsWe demonstrated that intravenous administration of α1-ECDm abrogates established EAMG, in a dose and time dependent manner, as assessed by clinical symptoms, body weight, and compound muscle action potential (CMAP) decrement. Importantly, the effect was more pronounced compared to drugs representing current standard of care for MG. The protein had a short plasma half-life, most of what could be recovered was sequestered in the liver, kidneys and spleen. Further, we did not observe any signs of toxicity or intolerability in animals treated with α1-ECDm.ConclusionWe conclude that intravenous treatment with α1-ECDm is safe and effective in suppressing EAMG. α1-ECDm is in preclinical development as a promising new drug candidate for MG.

PurposeTo determine the variation in the global treatment practices for subaxial unilateral cervical spine facet fractures based on surgeon experience, practice setting, and surgical subspecialty.MethodsA survey was sent to 272 members of the AO Spine Subaxial Injury Classification System Validation Group worldwide. Questions surveyed surgeon preferences with regard to diagnostic work-up and treatment of fracture types F1–F3, according to the AO Spine Subaxial Cervical Spine Injury Classification System, with various associated neurologic injuries.ResultsA total of 161 responses were received. Academic surgeons use the facet portion of the AO Spine classification system less frequently (61.6%) compared to hospital-employed and private practice surgeons (81.1% and 81.8%, respectively) (p = 0.029). The overall consensus was in favor of operative treatment for any facet fracture with radicular symptoms (N2) and for any fractures categorized as F2N2 and above. For F3N0 fractures, significantly less surgeons from Africa/Asia/Middle East (49%) and Europe (59.2%) chose operative treatment than from North/Latin/South America (74.1%) (p = 0.025). For F3N1 fractures, significantly less surgeons from Africa/Asia/Middle East (52%) and Europe (63.3%) recommended operative treatment than from North/Latin/South America (84.5%) (p = 0.001). More than 95% of surgeons included CT in their work-up of facet fractures, regardless of the type. No statistically significant differences were seen in the need for MRI to decide treatment.ConclusionConsiderable agreement exists between surgeon preferences with regard to unilateral facet fracture management with few exceptions. F2N2 fracture subtypes and subtypes with radiculopathy (N2) appear to be the threshold for operative treatment.