Strategies for Understanding and Reducing the Plasmodium vivax and Plasmodium ovale Hypnozoite Reservoir in Papua New Guinean Children: A Randomised Placebo-Controlled Trial and Mathematical Model

Background

The undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children.

Methods and Findings

From 17 August 2009 to 20 May 2010, 524 children aged 5–10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes.

Conclusions

These results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission.

Trial registration

ClinicalTrials.gov NCT02143934     

Off-site primary percutaneous coronary intervention in a new centre is safe: comparing clinical outcomes with a hospital with surgical backup

Objectives

To evaluate the procedural and clinical outcomes of a new primary percutaneous coronary intervention (PPCI) centre without surgical back-up (off-site PCI) and to investigate whether these results are comparable with a high volume on-site PCI centre in the Netherlands.

Background

Controversy remains about the safety and efficacy of PPCI in off-site PCI centres.

Methods

We retrospectively analysed clinical and procedural data as well as 6?month follow-up of 226 patients diagnosed with ST-elevated myocardial infarction (STEMI) who underwent PPCI at VieCuri Medical Centre Venlo and 115 STEMI patients who underwent PPCI at Catharina Hospital Eindhoven.

Results

PPCI patients in VieCuri Medical Centre had similar procedural and clinical outcomes to those in Catharina Hospital. Overall there were no significant differences. The occurrence of procedural complications was low in both groups (8.4?% VieCuri vs. 12.3?% Catharina Hospital). In the VieCuri group there was one procedural-related death. No patients in either group needed emergency surgery. At 30 days, 17 (7.9?%) patients in the VieCuri group and 9 (8.1?%) in the Catharina Hospital group had a major adverse cardiac event.

Conclusion

Performing PPCI in an off-site PCI centre is safe and effective. The study results show that the procedural and clinical outcomes of an off-site PPCI centre are comparable with an on-site high-volume PPCI centre.

     

Characterization of extremely halophilic Archaea isolated from the Ayvalik Saltern,Turkey

Seven extremely halophilic strains were isolated from the Ayvalik Saltern in the north-eastern part of Turkey. Chemical analyses of the brine and salt samples were performed to measure their salt content, hardness and pH. Isolated strains were tested for their antibiotic sensitivities; cell and colony morphologies; hydrolysis of casein, starch, gelatin, Tween 20 and Tween 80; and oxidase and catalase activity. All strains were found to belong to the domain Archaea. Characterization of polar lipids by thin layer chromatography indicated that all isolates contained phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidyl glycerophosphate (PGP-Me), and phosphatidylglycerosulphate (PGS). Four isolates had triglycosyl diether (TGD-2) as glycolipid, and the other three contained a sulphated diglycosyl diether instead. All isolates were examined for the presence of plasmids by agarose gel electrophoresis. Four strains were found to harbour plasmids ranging in size from 13.8 to 15.3 kbp. Correlation between the protein profiles in SDS–PAGE and the phenotypic properties of the strains was poor. The data presented here provide the first published account of the microbiota of the Ayvalik saltern, which provides a large part of the salt produced in Turkey.     

β1 Integrin Expression Increases Susceptibility of Memory B Cells to Epstein-Barr Virus Infection

Epstein-Barr virus (EBV) uses nasal mucosa-associated lymphoid tissue (NALT) as a portal of entry to establish life-long persistence in memory B cells. We previously showed that naïve and memory B cells from NALT are equally susceptible to EBV infection. Here we show that memory B cells from NALT are significantly more susceptible to EBV infection than those from remote lymphatic organs. We identify β₁ integrin, which is expressed the most by naïve B cells of distinct lymphoid origin and by memory B cells from NALT, as a mediator of increased susceptibility to infection by EBV. Furthermore, we show that BMRF-2-β₁ integrin interaction and the downstream signal transduction pathway are critical for postbinding events. An increase of β₁ integrin expression in peripheral blood memory B cells provoked by CD40 stimulation plus B-cell receptor cross-linking increased the susceptibility of non-NALT memory B cells to EBV infection. Thus, EBV seems to utilize the increased activation status of memory B cells residing in the NALT to establish and ensure persistence.Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is transmitted via saliva and infects more than 90% of the world''s population (21). Much of EBV''s medical importance relates to its association with B-cell malignancies, including Burkitt''s lymphoma, Hodgkin''s lymphoma, and posttransplant lymphoproliferative disease (21). The oncogenic potential of EBV is clearly illustrated by its unique capability to transform B cells in vitro (21).In the current paradigm, EBV infects naïve B cells in tonsils in vivo (32). EBV is present mainly as a latent virus; upon infection, EBV expresses distinct patterns of its latency genes depending upon distinct B-cell differentiation stages, varying from expression of all 10 known EBV latency genes in naïve B cells to the complete absence of EBV mRNA expression in resting memory B cells. This has led to the model that EBV, by virtue of expression of its latency genes, provides cell survival signals in naïve B cells (32). In particular, recent data suggest that EBV expedites the antigen-driven somatic hypermutation and selection of B cells taking place in germinal centers (GC) (26). Chaganti et al. challenged the current paradigm by showing for patients with primary EBV infection that EBV avoids GC transit and directly infects memory B cells (6). This report is consistent with in vitro experiments showing that EBV is able to infect memory B cells (9, 10), in addition to the well-accepted susceptibility of naïve and GC B cells to EBV.Irrespective of which B-cell subset is the primary target of EBV, its propagation within the host is linked to proliferation of infected B cells, which deliver latent EBV to daughter cells, or, more rarely, to switching of EBV to lytic infection (21). The latter process can eventually be triggered by the differentiation of infected memory B cells into plasma cells and results in the release of virions that may subsequently infect new B cells (17). Importantly, transmission of EBV to naïve hosts is thought to occur via droplets loaded with virions (21). Thus, lytic replication of EBV takes place best in nasal mucosa-associated lymphoid tissue (NALT), which will release EBV into the saliva, generating infectious droplets. Therefore, the NALT is the point of EBV transmission, i.e., the portal of entry of EBV as well as a shedding organ for further transmission (21).The attachment of EBV to B cells is mediated by the direct interaction of EBV glycoprotein gp350/220 with cellular CD21, initiating receptor-mediated endocytosis. After binding to CD21, EBV gp42 can interact with host HLA class II molecules, leading to a conformational change in the viral glycoproteins and triggering fusion with the host cell membrane (12, 28). Nevertheless, experimental data suggest that CD21 and HLA class II molecules are dispensable for the infection of B cells (14). Notably, in polarized oropharyngeal epithelial cells, which lack CD21, interactions between β1 integrin and the EBV glycoprotein BMRF-2 via its Arg-Gly-Asp (RGD) motif are critical for infection (34, 38, 39). The role of β₁ integrin in mediating EBV infection of memory B cells from NALT or non-NALT is unknown.We recently demonstrated that tonsillar memory B cells are much more susceptible to EBV infection than those from the peripheral blood, originating from various lymphoid tissues (9). Thus, tonsillar memory B cells seem to express properties which render them more susceptible to EBV infection than their counterparts of other lymphatic origin.Here we hypothesized that memory B cells from the NALT exhibit specific properties rendering them highly susceptible to EBV infection. Indeed, in this work, we found that memory B cells from the NALT are distinguishable from memory B cells of other lymphoid tissue by their β₁ integrin expression levels, and thus their activation status, and that this higher expression level is a critical factor in their greater susceptibility to EBV infection.     

The first year of the Venlo percutaneous coronary intervention program: procedural and 6-month clinical outcomes

Objectives

Analysis of the first results of off-site percutaneous coronary interventions (PCI) and fractional flow reserve (FFR) measurements at VieCuri Medical Centre for Northern Limburg in Venlo.

Background

Off-site PCI is accepted in the European and American Cardiac Guidelines as the need for PCI increases and it has been proven to be a safe treatment option for acute coronary syndrome.

Methods

Retrospective cohort study reporting characteristics, PCI and FFR specifications, complications and 6-month follow-up for all consecutive patients from the beginning of off-site PCI in Venlo until July 2012. If possible, the data were compared with those of Medical Centre Alkmaar, the first off-site PCI centre in the Netherlands.

Results

Of the 333 patients, 19 (5.7 %) had a procedural complication. At 6 months, a major adverse cardiovascular event (MACE) occurred in 43 (13.1 %) patients. There were no deaths or emergency surgery related to the PCI or FFR procedures. There was no significant difference in occurrence of a MACE or adverse cerebral event between the Alkmaar and Venlo population in the 30-day follow-up.

Conclusion

This study demonstrates off-site PCI at VieCuri Venlo to have a high success rate. Furthermore, there was a low complication rate, low MACE and no procedure-related mortality.     

How Does Spatial Study Design Influence Density Estimates from Spatial Capture-Recapture Models?

When estimating population density from data collected on non-invasive detector arrays, recently developed spatial capture-recapture (SCR) models present an advance over non-spatial models by accounting for individual movement. While these models should be more robust to changes in trapping designs, they have not been well tested. Here we investigate how the spatial arrangement and size of the trapping array influence parameter estimates for SCR models. We analysed black bear data collected with 123 hair snares with an SCR model accounting for differences in detection and movement between sexes and across the trapping occasions. To see how the size of the trap array and trap dispersion influence parameter estimates, we repeated analysis for data from subsets of traps: 50% chosen at random, 50% in the centre of the array and 20% in the South of the array. Additionally, we simulated and analysed data under a suite of trap designs and home range sizes. In the black bear study, we found that results were similar across trap arrays, except when only 20% of the array was used. Black bear density was approximately 10 individuals per 100 km(2). Our simulation study showed that SCR models performed well as long as the extent of the trap array was similar to or larger than the extent of individual movement during the study period, and movement was at least half the distance between traps. SCR models performed well across a range of spatial trap setups and animal movements. Contrary to non-spatial capture-recapture models, they do not require the trapping grid to cover an area several times the average home range of the studied species. This renders SCR models more appropriate for the study of wide-ranging mammals and more flexible to design studies targeting multiple species.     

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging. Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.     

Probing Mixed-Genotype Infections I: Extraction and Cloning of Infections from Hosts of the Trypanosomatid Crithidia bombi

We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single Crithidia bombi cells from bumblebee faeces using flow cytometry. We report a precision of >99% in obtaining single trypanosomatid cells for further culture and analysis (“cloning”). In the study, we have investigated the use of different liquid media to cultivate C. bombi and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve C. bombi cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.