Clinical management and humoral immune responses to rabies post-exposure prophylaxis among three patients who received solid organs from a donor with rabies.

BACKGROUND: The rabies virus causes a fatal encephalitis and can be transmitted through organ transplantation. In 2013, a man developed rabies 18 months after receiving a kidney from a donor with rabies, who was not known to have been infected when the organs were procured. Three additional persons who received organs from the same donor (liver, kidney, heart), all of whom were not vaccinated for rabies before transplantation, received rabies post-exposure prophylaxis (PEP) with rabies immune globulin and 5 doses of rabies vaccine as soon as the diagnosis of rabies was made in the donor (18 months after their transplant surgeries). We describe their clinical management. METHODS: As the 3 recipients were all on immunosuppressive medications, post-vaccination serologic testing was performed using the rapid fluorescent focus inhibition test to measure rabies virus neutralizing antibodies (RVNAs). An acceptable antibody response to administration of rabies vaccine was defined as detection of RVNAs at a concentration ≥0.1 IU/mL from a serum specimen collected ≥7 days after the fifth vaccine dose. RESULTS: All 3 recipients demonstrated an acceptable antibody response despite their immunosuppressed states. More than 36 months have passed since their transplant surgeries, and all 3 recipients have no evidence of rabies. CONCLUSIONS: The survival of 3 previously unvaccinated recipients of solid organs from a donor with rabies is unexpected. Although the precise factors that led to their survival remain unclear, our data suggest that PEP can possibly enhance transplant safety in settings in which donors are retrospectively diagnosed with rabies.

Amazon River enhances diazotrophy and carbon sequestration in the tropical North Atlantic Ocean.

The fresh water discharged by large rivers such as the Amazon is transported hundreds to thousands of kilometers away from the coast by surface plumes. The nutrients delivered by these river plumes contribute to enhanced primary production in the ocean, and the sinking flux of this new production results in carbon sequestration. Here, we report that the Amazon River plume supports N(2) fixation far from the mouth and provides important pathways for sequestration of atmospheric CO(2) in the western tropical North Atlantic (WTNA). We calculate that the sinking of carbon fixed by diazotrophs in the plume sequesters 1.7 Tmol of C annually, in addition to the sequestration of 0.6 Tmol of C yr(-1) of the new production supported by NO(3) delivered by the river. These processes revise our current understanding that the tropical North Atlantic is a source of 2.5 Tmol of C to the atmosphere [Mikaloff-Fletcher SE, et al. (2007) Inverse estimates of the oceanic sources and sinks of natural CO(2) and the implied oceanic carbon transport. Global Biogeochem Cycles 21, doi:10.1029/2006GB002751]. The enhancement of N(2) fixation and consequent C sequestration by tropical rivers appears to be a global phenomenon that is likely to be influenced by anthropogenic activity and climate change.

Modeling the Seasonal Cycle of Iron and Carbon Fluxes in the Amundsen Sea Polynya, Antarctica.

The Amundsen Sea Polynya (ASP) is distinguished by having the highest net primary production per unit area in the coastal Antarctic. Recent studies have related this high productivity to the presence of fast-melting ice shelves, but the mechanisms involved are not well understood. In this study we describe the first numerical model of the ASP to represent explicitly the ocean-ice interactions, nitrogen and iron cycles, and the coastal circulation at high resolution. The study focuses on the seasonal cycle of iron and carbon, and the results are broadly consistent with field observations collected during the summer of 2010-2011. The simulated biogeochemical cycle is strongly controlled by light availability(dictated by sea ice, phytoplankton self-shading, and variable sunlight). The micronutrient iron exhibits strong seasonality, where scavenging by biogenic particles and remineralization play large compensating roles. Lateral fluxes of iron are also important to the iron budget, and our results confirm the key role played by inputs of dissolved iron from the buoyancy-driven circulation of melting ice shelf cavities (the "meltwater pump"). The model suggests that westward flowing coastal circulation plays two important roles: it provides additional iron to the ASP and it collects particulate organic matter generated by the bloom and transports it to the west of the ASP. As a result, maps of vertical particulate organic matter fluxes show highest fluxes in shelf regions located west of the productive central ASP. Overall, these model results improve our mechanistic understanding of the ASP bloom, while suggesting testable hypotheses for future field efforts.

A rapid diffusion immunoassay in a T-sensor.

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 microl of reagents and sample, and is applicable to a wide range of analytes.

Ocean biogeochemistry modeled with emergent trait-based genomics.

Marine ecosystem models have advanced to incorporate metabolic pathways discovered with genomic sequencing, but direct comparisons between models and "omics" data are lacking. We developed a model that directly simulates metagenomes and metatranscriptomes for comparison with observations. Model microbes were randomly assigned genes for specialized functions, and communities of 68 species were simulated in the Atlantic Ocean. Unfit organisms were replaced, and the model self-organized to develop community genomes and transcriptomes. Emergent communities from simulations that were initialized with different cohorts of randomly generated microbes all produced realistic vertical and horizontal ocean nutrient, genome, and transcriptome gradients. Thus, the library of gene functions available to the community, rather than the distribution of functions among specific organisms, drove community assembly and biogeochemical gradients in the model ocean.

A disposable chemical heater and dry enzyme preparation for lysis and extraction of DNA and RNA from microorganisms.

Sample preparation, including bacterial lysis, remains a hurdle in the realization of complete point-of-care tests for many pathogens. Here, we developed a sample preparation methodology for enzymatic lysis and sample heating for low-resource, point-of-care applications. We show an instrument-free chemical heater system for rapid lysis of a gram-positive bacterium (Staphylococcus aureus) and an RNA virus (human respiratory syncytial virus) using a dried lysis enzyme mixture (achromopeptidase) for S. aureus. After a lysis step (<1 minute), lysis enzymes are heat deactivated (<5 minutes) using a simple disposable chemical heater. We demonstrated that both DNA and RNA in the heat-treated sample could be directly amplified without purification, even in the presence of a clinically-obtained human nasal sample. This simple approach to dry enzyme storage and sample heating is adaptable to many applications where samples need to be lysed, including use in low-resource laboratories and in single-use or cartridge-based point-of-care diagnostic devices.

Perturbation of the chain melting transition of DPPC by galactose, agarose and Laurdan as determined by differential scanning calorimetry.

Differential scanning calorimetry was used to determine the effects of agarose hydrogel, galactose and the fluorophore Laurdan on the thermal behavior of multilamellar liposomes of DPPC. Although the effect of agarose on the phase transition was found to be negligible at low concentrations (< or = 2.5%), higher concentrations result in an endotherm that broadens and splits into two calorimetric events, one of which is at a higher temperature than that of hydrated DPPC. Equal weight fractions of galactose produce similar effects, although both fractions have raised melting temperatures. The higher melting components may be produced by osmotically-driven dehydration of the inner liposomal monolayers, a physical interaction between the carbohydrates and lipid headgroups, or a combination of both. Laurdan has little effect on the phase transition of DPPC vesicles at the concentration used in the sensor (0.67 mol%); concentrations up to 5.4 mol% only slightly lowered the melting temperature.

The kinetics of the main phase transition of aqueous dispersions of phospholipids induced by pressure jump and monitored by Raman spectroscopy.

The sensitivity of the melting transition temperature of aqueous dispersions of dipalmitoyl- and distearoylphosphatidylcholine to hydrostatic pressure is used to allow measurement of the rates of isothermal freezing and melting of the lipids by rapidly changing the pressure. The degree of order of the lipids is measured by monitoring a ratio of two points in the Raman spectrum of the lipids which changes sharply at the melting temperature. Use of this Raman order ratio allows correlation between the order of the sample and the rates of transition in a manner which is impossible by monitoring only turbidity. Our longest relaxation times range upwards from a few seconds for both compounds. The freezing rates are slowest when the samples are initially fully melted, and the melting rates are slowest when the samples are initially frozen. These results imply that nucleation of the growing phase dominates the kinetics of both freezing and melting.

Changes in size and shape of liposomes undergoing chain melting transitions as studied by optical microscopy.

Changes in the shape and size of dipalmitoylphosphatidylcholine liposomes at the phase transition at 41.5 degrees C have been monitored by light microscopy. All liposomes change size or shape at the transition and those with simple topologies such as spheres and cylinders can be readily measured. The surface area of these is some 24% greater above the transition than below. This surface area change is virtually identical to that predicted by crystallographic measurements on this system. Also, the rate of transition from one state to another is seen to proceed more rapidly in the smaller liposomes. Optical microscopic observation provides a rapid simple method for monitoring the dependence of the lipid bilayer area on temperature.

Formation of high axial ratio microstructures from peptides modified with glutamic acid dialkyl amides.

A growing number of amphiphiles are known to form high axial ratio microstructures (HARMs) such as the hollow cylindrical microstructures called lipid tubules. As a prelude to exploring the potential of HARMs formed from lipopeptides in controlled release drug delivery, several microstructure formation conditions were investigated. We report the preparation of several glutamic acid dialkyl amides with varying alkyl chain lengths bearing a verity of peptides (1-4 amino acids) [peptide-Glu-(NHCnH2n+1)2, n=12, 14, 16]. These surfactants have been rapidly and efficiently converted into HARMs in aqueous buffer at physiological pH and ionic strength, or in buffer containing MeOH or EtOH. Helical ribbons and tubular HARMs were produced that were stable for as long as 6 months below the phase transition temperatures of the compounds. To estimate the stability of HARMs in vivo, HARMs formed from (Pro)3-Glu(NHC16H33)2 were incubated with DOPC liposomes or fetal calf serum at 40 degreesC. HARM size and shape did not change significantly, suggesting that such lipopeptide particles can retain their morphology long enough in vivo to be useful as drug delivery vehicles.

One-step purification and concentration of DNA in porous membranes for point-of-care applications.

The emergence of rapid, user-friendly, point-of-care (POC) diagnostic systems is paving the way for better disease diagnosis and control. Lately, there has been a strong emphasis on developing molecular-based diagnostics due to their potential for greatly increased sensitivity and specificity. One of the most critical steps in developing practical diagnostic systems is the ability to perform sample preparation, especially the purification of nucleic acids (NA), at the POC. As such, we have developed a simple-to-use, inexpensive, and disposable sample preparation system for in-membrane purification and concentration of NAs. This system couples lateral flow in a porous membrane with chitosan, a linear polysaccharide that captures NAs via anion exchange chromatography. The system can also substantially concentrate the NAs. The combination of these capabilities can be used on a wide range of sample types, which are prepared for use in downstream processes, such as qPCR, without further purification.

Precision chemical heating for diagnostic devices.

Decoupling nucleic acid amplification assays from infrastructure requirements such as grid electricity is critical for providing effective diagnosis and treatment at the point of care in low-resource settings. Here, we outline a complete strategy for the design of electricity-free precision heaters compatible with medical diagnostic applications requiring isothermal conditions, including nucleic acid amplification and lysis. Low-cost, highly energy dense components with better end-of-life disposal options than conventional batteries are proposed as an alternative to conventional heating methods to satisfy the unique needs of point of care use.

Electromechanical cell lysis using a portable audio device: enabling challenging sample preparation at the point-of-care.

Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.

Encapsulation of hemoglobin in phospholipid vesicles.

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 A as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4 degrees C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4 degrees C.

Generation of natural pH gradients in microfluidic channels for use in isoelectric focusing

As a part of an ongoing investigation of the use of isoelectric focusing (IEF) in microfluidic devices, pH gradients were electrochemically formed and optically quantified in microfluidic channels using acid-base indicators. The microchannels consisted of two parallel 40-mm-long electrodes with an interelectrode gap of 2.54 mm; top and bottom transparent windows were separated by 0.2 mm. Gradients in pH were formed as a result of the electrochemical decomposition of water at an applied potential not higher than 2.5 V to avoid generation of gas bubbles. Solutions contained low concentrations of a single buffer. The stability of the pH gradients and their sensitivity to changes in initial conditions were investigated under static (nonflow) conditions. Isoelectric focusing of sample biological analytes, bovine hemoglobin and bovine serum albumin, was performed to illustrate the potential of "microfluidic transverse IEF" for use in continuous concentration and separation systems.

Early antibody responses to rabies post-exposure vaccine regimens.

The aim of post-exposure rabies vaccine treatment is to induce immunity, measured as neutralizing antibody, as fast as possible. This is especially important in the tropical rabies-endemic areas where simultaneous passive prophylaxis with hyperimmune serum is not practicable in the majority of cases. We compared the rate of production of antibody during the first two weeks, by six vaccine regimens in 118 subjects using two tissue culture vaccines, human diploid cell strain vaccine (HDCSV) and purified Vero cell rabies vaccine (PVRV). No antibody was detected on day 5. On day 7, the highest seroconversion rate was seen in subjects given HDCSV intramuscularly at two sites on days 0 and 3 (7 of 15), but this was not significantly different from the group with the lowest rate: the conventional single-site intramuscular regimen. All subjects had antibody by day 14, at which time the highest geometric mean titer was in the group vaccinated with 0.25 ml doses of diploid cell vaccine given subcutaneously at eight sites. This regimen, together with the standard single-site diploid cell vaccine and an eight-site intradermal regimen of the same product gave significantly higher titers than the two-site intramuscular regimens of either product. No single immunization schedule emerges as best, so the speed of antibody response, economy, and the skill needed for intradermal injection should be considered when deciding on the optimum regimen for use in a particular geographic area.

Laboratory investigation of human deaths from vampire bat rabies in Peru.

In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.

Continuous and highly variable rate controlled release of model drugs from sphingolipid-based complex high axial ratio microstructures.

Sphingolipids have been synthesized that contain as polar headgroups, model drugs ester-linked to the primary hydroxyl group of the ceramide core. These lipids, when allowed to self assemble below their chain-melting temperatures, either as single molecular species or in combination with other sphingolipid-derived amphiphiles, are shown to form supramolecular assemblies of varying morphologies including complex high axial ratio microstructures (CHARMs). Within these microstructures, the lipid esters are highly resistant to hydrolysis as compared to the esters dispersed as solitary monomers in aqueous solution or in a matrix of fluid phosphatidylcholine vesicles. The rate of headgroup hydrolysis within CHARMs may be manipulated over a broad range (days to years) by varying the length of the amide-linked fatty acyl chain in the ceramide core or the distance between the ester and the C-1 ceramide of the core. These microstructures, which have exceptionally high surface area display of attached headgroups, may be useful for controlled release of pharmacological agents.

The relationship between the structure of the headgroup of sphingolipids and their ability to form complex high axial ratio microstructures.

Ceramides with chemically modified polar headgroups were prepared and examined for their ability to form complex high axial ratio microstructures (CHARMS), potential drug delivery vehicles. In general, if the modified ceramide had either a hydrogen bond donor or acceptor at C-1 and C-3, including hydrophobic or hydrophilic groups attached to C-1 microstructures formed. Tolerated groups include amides, esters, sulfonates, and ethers. If modification at C-3 added significant bulk (greater than four carbons regardless of hydrophilicity), then amorphous aggregates formed. Ceramides with C-1 and C-3 bridged through a cyclic structure also made microstructures. By using a sphingolipid with an amine headgroup, CHARMs may be modified covalently after formation.

Structure of polymerizable lipid bilayers. I--1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a tubule-forming phosphatidylcholine.

This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.