Assessment of blood-brain barrier penetration of miltefosine used to treat a fatal case of granulomatous amebic encephalitis possibly caused by an unusual Balamuthia mandrillaris strain.

Balamuthia mandrillaris, a free-living ameba, causes rare but frequently fatal granulomatous amebic encephalitis (GAE). Few patients have survived after receiving experimental drug combinations, with or without brain lesion excisions. Some GAE survivors have been treated with a multi-drug regimen including miltefosine, an investigational anti-leishmanial agent with in vitro amebacidal activity. Miltefosine dosing for GAE has been based on leishmaniasis dosing because no data exist in humans concerning its pharmacologic distribution in the central nervous system. We describe results of limited cerebrospinal fluid (CSF) and serum drug level testing performed during clinical management of a child with fatal GAE who was treated with a multiple drug regimen including miltefosine. Brain biopsy specimens, CSF, and sera were tested for B. mandrillaris using multiple techniques, including culture, real-time polymerase chain reaction, immunohistochemical techniques, and serology. CSF and serum miltefosine levels were determined using a liquid chromatography method coupled to tandem mass spectrometry. The CSF miltefosine concentration on hospital admission day 12 was 0.4 µg/mL. The serum miltefosine concentration on day 37, about 80 h post-miltefosine treatment, was 15.3 µg/mL. These are the first results confirming some blood-brain barrier penetration by miltefosine in a human, although with low-level CSF accumulation. Further evaluation of brain parenchyma penetration is required to determine optimal miltefosine dosing for Balamuthia GAE, balanced with the drug's toxicity profile. Additionally, the Balamuthia isolate was evaluated by real-time polymerase chain reaction (PCR), demonstrating genetic variability in 18S ribosomal RNA (18S rRNA) sequences and possibly signaling the first identification of multiple Balamuthia strains with varying pathogenicities.

Vaccine-associated paralytic poliomyelitis and BCG-osis in an immigrant child with severe combined immunodeficiency syndrome - Texas, 2013.

Poliovirus transmission has been eliminated in most of the world through the use of inactivated poliovirus vaccine (IPV) and live, attenuated oral poliovirus vaccine (OPV). In the United States, use of OPV was discontinued by the year 2000 because of the potential for vaccine-associated paralytic polio (VAPP); an average of eight cases were reported each year in the United States during 1980-2000. Polio eradication efforts in other parts of the world continue to rely on OPV to take advantage of transmission of poliovirus vaccine strains to unvaccinated persons in the population, lower cost, and ease of administration. In 2013, an infant aged 7 months who recently immigrated to the United States from India was referred to a hospital in San Antonio, Texas. The infant had fever, an enlarging skin lesion in the deltoid region with axillary lymphadenopathy, decreased activity, and inability to bear weight on the left leg, progressing to paralysis of the left leg over a 6-week period. Recognition of lymphopenia on complete blood count led to immune evaluation, which revealed the presence of severe combined immunodeficiency syndrome (SCIDS), an inherited disorder. A history of OPV and bacille Calmette-Guérin (BCG) vaccination in India led to the diagnoses of VAPP and BCG-osis, which were confirmed microbiologically. This report demonstrates the importance of obtaining a comprehensive clinical history in a child who has recently immigrated to the United States, with recognition that differing vaccine practices in other countries might require additional consideration of potential etiologies.

A national outbreak of Ralstonia mannitolilytica associated with use of a contaminated oxygen-delivery device among pediatric patients.

OBJECTIVES: In August 2005, the Centers for Disease Control and Prevention was notified of a Ralstonia species outbreak among pediatric patients receiving supplemental oxygen therapy with the Vapotherm 2000i (Vapotherm, Inc, Stevensville, MD). The Vapotherm 2000i is a reusable medical device that was used in >900 hospitals in the United States in 2005. Ralstonia are waterborne bacilli that have been implicated in hospital-acquired infections. We initiated an investigation to determine the source of the outbreak and implement infection control and prevention measures. PATIENTS AND METHODS: We performed a case-control study at 1 hospital and conducted national case findings to obtain clinical and environmental samples for laboratory analysis. Case-patients had health care-acquired Ralstonia colonization or infection. Isolates were compared by using pulsed-field gel electrophoresis. We tested manufacturer-recommended disinfection protocols for the Vapotherm 2000i under simulated-use conditions. RESULTS: Case-patients at the hospital (n = 5) were more likely to have received Vapotherm therapy than controls. Nationally, Ralstonia mannitolilytica was confirmed in 38 patients (aged 5 days to 7 years); 35 (92%) of the patients were exposed to the Vapotherm 2000i before recovery of the organism. Pulsed-field gel electrophoresis showed related R. mannitolilytica strains from isolates sent from 18 hospitals in 12 states. A Vapotherm machine reprocessed with a protocol proposed by the manufacturer grew Ralstonia spp after 7 days of simulated use. In December 2005, Vapotherm recalled the 2000i. CONCLUSIONS: Our findings suggest intrinsic contamination of Vapotherm devices with Ralstonia spp. New medical devices may provide therapy equivalent to current devices yet pose novel reprocessing challenges.

The beneficial effects of specialist thoracic surgery on the resection rate for non-small-cell lung cancer.

We aimed to evaluate the effect of the appointment of a dedicated specialist thoracic surgeon on surgical practice for lung cancer previously served by cardio-thoracic surgeons. Outcomes were compared for the 240 patients undergoing surgical resection for lung cancer in two distinct 3-year periods: Group A: 65 patients, 1994-1996 (pre-specialist); Group B: 175 patients, 1997-1999 (post-specialist). The changes implemented resulted in a significant increase in resection rate (from 12.2 to 23.4%, P < 0.001), operations in the elderly (over 75 years) and extended resections. There were no significant differences in stage distribution, in-hospital mortality or stage-specific survival after surgery. Lung cancer surgery provided by specialists within a multidisciplinary team resulted in increased surgical resection rates without compromising outcome. Our results strengthen the case for disease-specific specialists in the treatment of lung cancer.

Alpha-Synuclein is degraded by both autophagy and the proteasome.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of aggregates (Lewy bodies) in neurons. alpha-Synuclein is the major protein in Lewy bodies and rare mutations in alpha-synuclein cause early-onset PD. Consequently, alpha-synuclein is implicated in the pathogenesis of PD. Here, we have investigated the degradation pathways of alpha-synuclein, using a stable inducible PC12 cell model, where the expression of exogenous human wild-type, A30P, or A53T alpha-synuclein can be switched on and off. We have used a panel of inhibitors/stimulators of autophagy and proteasome function and followed alpha-synuclein degradation in these cells. We found that not only is alpha-synuclein degraded by the proteasome, but it is also degraded by autophagy. A role for autophagy was further supported by the presence of alpha-synuclein in organelles with the ultrastructural features of autophagic vesicles. Since rapamycin, a stimulator of autophagy, increased clearance of alpha-synuclein, it merits consideration as a potential therapeutic for Parkinsons disease, as it is designed for chronic use in humans.

Human neuropathy target esterase catalyzes hydrolysis of membrane lipids.

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues in Drosophila and yeast and is detected in vitro by assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727-1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A(2) (iPLA(2)) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA(2) inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V(max) approximately 0.01 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of (14)C-labeled products from two differentially labeled (14)C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V(max) approximately 20 micromol/min/mg and K(m) approximately 0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V(max) approximately 1 micromol/min/mg and K(m) approximately 0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.

Monomers of the catalytic domain of human neuropathy target esterase are active in the presence of phospholipid.

NEST is a hydrophobic recombinant polypeptide comprising the catalytic domain (residues 727-1216) of neuropathy target esterase. NEST in bacterial lysates has potent esterase activity, which is lost after its solubilization and purification in detergent-containing solutions. Activity in purified NEST preparations was restored by the addition of phospholipids before the removal of detergent by dialysis. The pattern of digestion by proteinase K of NEST-phospholipid complexes suggested that NEST might incorporate in a topologically random fashion into nascent liposomes and that the bulk of each NEST molecule might be exposed either to the liposome lumen or the external medium. Significant quantities of NEST were liberated from NEST-phospholipid complexes by treatment with dilute acid or alkali, suggesting that charge interactions might contribute to the association; however, NEST was irreversibly denatured at these pH values. Treatment of NEST-phospholipid complexes with glutaraldehyde afforded some protection against the inactivation of esterase activity by detergent but the pattern of cross-linked forms of NEST generated did not indicate pre-existing oligomers. Similarly, the inactivation of esterase activity in NEST-phospholipid complexes by radiation indicated that NEST monomers are catalytically active. The foregoing observations are not compatible with structural algorithms predicting that the catalytic serine residue lies at the centre of one of three transmembrane helices in NEST.