Acanthamoeba griffini. Molecular characterization of a new corneal pathogen

Purpose. Acanthamoeba was isolated from the cornea of a soft contact lense wearer who had keratitis. The protozoan was also isolated from the contact lens storage case and the domestic water supply used to clean the case. Using morphologic features, all three isolates were identified tentatively as A. griffini, a species not previously associated with keratitis. Complete small subunit ribosomal RNA gene (18S rDNA) sequence analysis was used to characterize further the three isolates. Methods. 18S rDNA was polymerase chain reaction-amplified from whole cell DNA derived from amoebal lysates. The genes were cloned and sequenced. Complete sequences of approximately 2800 base pairs were obtained from each culture and compared with those stored in a data base of homologous Acanthamoeba sequences. Results. The isolates were unequivocally identified as A. griffini both by comparison of the gene sequence available for the type strain of the species and the presence of a unique group I intron located within the small subunit rDNA. Sequences obtained for the three isolates were identical, indicating that they were the same strain. Conclusions. The first direct connection between human disease and A. griffini is reported from a case of Acanthamoeba keratitis. The type strain of this species was isolated from a marine environment, but the disease<ausing strain was isolated from a domestic water supply. The DNA sequences obtained confirmed unequivocally the epidemiologic association between a keratitis-causing strain of Acanthamoeba, the contact lens storage case, and the domestic water supply

Detection of Bacterial Endosymbionts in Clinical Acanthamoeba Isolates

PURPOSE: To determine the presence of four clinically relevant bacterial endosymbionts in Acanthamoeba isolates obtained from patients with Acanthamoeba keratitis (AK) and the possible contribution of endosymbionts to the pathogenesis of AK. DESIGN: Experimental study PARTICIPANTS: Acanthamoeba isolates (N=37) recovered from cornea and contact lens paraphernalia of 23 patients with culture proven AK and 1 environmental isolate. METHODS: Acanthamoeba isolates were evaluated for the presence of microbial endosymbionts belonging to the bacterial genera Legionella, Pseudomonas, Mycobacteria and Chlamydia using molecular techniques (Polymerase chain reaction and sequence analysis, fluorescent in situ hybridization) and transmission electron microscopy. Corneal toxicity and virulence of Acanthamoeba isolates with and without endosymbionts were compared using a cytopathic effect (CPE) assay of human corneal epithelial cells in vitro. Initial visual acuity (VA), location and characteristics of the infiltrate, time to detection of the infection and symptoms duration at presentation were evaluated in all patients. MAIN OUTCOME MEASURES: Prevalence and potential pathobiology of bacterial endosymbionts detected in Acanthamoeba isolates recovered from AK. RESULTS: Twenty-two of the 38 (59.4%) cultures examined contained at least one bacterial endosymbiont. One isolate contained two endosymbionts, Legionella and Chlamydia, confirmed by fluorescence in situ hybridization. Corneal toxicity (CPE) was significantly higher for Acanthamoebae hosting endosymbionts compared to isolates without endosymbionts (p<0.05). Corneal pathogenic endosymbionts such as Pseudomonas and Mycobacterium enhanced Acanthamoeba CPE significantly more than Legionella (p<0.05). In the presence of bacterial endosymbionts, there was a trend toward worse initial VA (p>0.05), central location (p<0.05), absence of radial perineuritis (p<0.05), delayed time to detection (p>0.05) and longer symptoms duration at presentation (p>0.05). CONCLUSION: The majority of Acanthamoeba isolates responsible for AK harbors one or more bacterial endosymbionts. The presence of endosymbionts enhances the corneal pathogenicity of Acanthamoeba isolates and might impact detection time and clinical features of AK

Cysticidal Activity of Antifungals against Different Genotypes of Acanthamoeba

Antifungal drugs have been proposed as a novel treatment for Acanthamoeba keratitis. The cysticidal activity of several antifungal compounds was tested against different genotypes of culture collection and clinical isolates of Acanthamoeba. Only voriconazole and posaconazole were found to be cysticidal, with no differences in activity observed between clinical and culture collection isolates

Thyroid hormone reverses aging-induced myocardial fatty acid oxidation defects and improves the response to acutely increased afterload.

Subclinical hypothyroidism occurs during aging in humans and mice and may contribute to the development of heart failure. Aging also impairs myocardial fatty acid oxidation, causing increased reliance on flux through pyruvate dehydrogenase (PDH) to maintain function. We hypothesize that the metabolic changes in aged hearts make them less tolerant to acutely increased work and that thyroid hormone supplementation reverses these defects.Studies were performed on young (Young, 4-6 months) and aged (Old, 22-24 months) C57/BL6 mice at standard (50 mmHg) and high afterload (80 mmHg). Another aged group received thyroid hormone for 3 weeks (Old-TH, high afterload only). Function was measured in isolated working hearts along with substrate fractional contributions (Fc) to the citric acid cycle (CAC) using perfusate with (13)C labeled lactate, pyruvate, glucose and unlabeled palmitate and insulin.Old mice maintained cardiac function under standard workload conditions, despite a marked decrease in unlabeled (presumably palmitate) Fc and relatively similar individual carbohydrate contributions. However, old mice exhibited reduced palmitate oxidation with diastolic dysfunction exemplified by lower -dP/dT. Thyroid hormone abrogated the functional and substrate flux abnormalities in aged mice.The aged heart shows diminished ability to increase cardiac work due to substrate limitations, primarily impaired fatty acid oxidation. The heart accommodates slightly by increasing efficiency through oxidation of carbohydrate substrates. Thyroid hormone supplementation in aged mice significantly improves cardiac function potentially through restoration of fatty acid oxidation

Drug-resistant severe Acanthamoeba keratitis caused by rare T5 Acanthamoeba genotype

To describe a case of severe and drug-resistant Acanthamoeba keratitis in a contact lens wearer caused by atypical T5 Acanthamoeba genotype (Acanthamoeba lenticulata). Report of a case, Acanthamoeba DNA amplification and sequencing. A 61-year-old patient was referred to our clinic with a 2-week history of keratitis. Acanthamoeba keratitis (AK) was diagnosed using confocal microscopy and corneal scraping culture. Using polymerase chain reaction (PCR) and DNA sequencing, the organism was classified as a T5 genotype (A. lenticulata). The keratitis continued to progress despite topical antiamoebic therapy and ultimately led to enucleation of the affected eye. T5 genotype Acanthamoeba can cause severe AK. Atypical Acanthamoeba genotypes could be associated with worse prognosis and resistance to therapy

Evaluation of a Novel Dry Powder Surfactant Aerosol Delivery System for Use in Premature Infants Supported with Bubble CPAP.

Aerosolized lung surfactant therapy during nasal continuous positive airway pressure (CPAP) support avoids intubation but is highly complex, with reported poor nebulizer efficiency and low pulmonary deposition. The study objective was to evaluate particle size, operational compatibility, and drug delivery efficiency with various nasal CPAP interfaces and gas humidity levels of a synthetic dry powder (DP) surfactant aerosol delivered by a low-flow aerosol chamber (LFAC) inhaler combined with bubble nasal CPAP (bCPAP). A particle impactor characterized DP surfactant aerosol particle size. Lung pressures and volumes were measured in a preterm infant nasal airway and lung model using LFAC flow injection into the bCPAP system with different nasal prongs. The LFAC was combined with bCPAP and a non-heated passover humidifier. DP surfactant mass deposition within the nasal airway and lung was quantified for different interfaces. Finally, surfactant aerosol therapy was investigated using select interfaces and bCPAP gas humidification by active heating. Surfactant aerosol particle size was 3.68 µm. Lung pressures and volumes were within an acceptable range for lung protection with LFAC actuation and bCPAP. Aerosol delivery of DP surfactant resulted in variable nasal airway (0-20%) and lung (0-40%) deposition. DP lung surfactant aerosols agglomerated in the prongs and nasal airways with significant reductions in lung delivery during active humidification of bCPAP gas. Our findings show high-efficiency delivery of small, synthetic DP surfactant particles without increasing the potential risk for lung injury during concurrent aerosol delivery and bCPAP with passive humidification. Specialized prongs adapted to minimize extrapulmonary aerosol losses and nasal deposition showed the greatest lung deposition. The use of heated, humidified bCPAP gases compromised drug delivery and safety. Safety and efficacy of DP aerosol delivery in preterm infants supported with bCPAP requires more research

Echocardiographic analysis of the left ventricle at baseline and after thyroid hormone treatment.

<p>HR, heart rate; BPM, beats per minute; MV, mitral valve; IVST, interventricular septal thickness; EDD, end diastolic dimension; ESD, end systolic dimension; PWT, posterior wall thickness; LV%FS, left ventricular per cent fractional shortening. Values are means±SEM. n = 4 paired mice.</p