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Background Homozygous familial hypercholesterolemia (HoFH) is a rare, treatment-resistant disorder characterized by early-onset atherosclerotic and aortic valvular cardiovascular disease if left untreated. Contemporary information on HoFH in the United States is lacking, and the extent of underdiagnosis and undertreatment is uncertain. Methods and Results Data were analyzed from 67 children and adults with clinically diagnosed HoFH from the CASCADE (Cascade Screening for Awareness and Detection) FH Registry. Genetic diagnosis was confirmed in 43 patients. We used the clinical characteristics of genetically confirmed patients with HoFH to query the Family Heart Database, a US anonymized payer health database, to estimate the number of patients with similar lipid profiles in a "real-world" setting. Untreated low-density lipoprotein cholesterol levels were lower in adults than children (533 versus 776 mg/dL; P=0.001). At enrollment, atherosclerotic cardiovascular disease and supravalvular and aortic valve stenosis were present in 78.4% and 43.8% and 25.5% and 18.8% of adults and children, respectively. At most recent follow-up, despite multiple lipid-lowering treatment, low-density lipoprotein cholesterol goals were achieved in only a minority of adults and children. Query of the Family Heart Database identified 277 individuals with profiles similar to patients with genetically confirmed HoFH. Advanced lipid-lowering treatments were prescribed for 18%; 40% were on no lipid-lowering treatment; atherosclerotic cardiovascular disease was reported in 20%; familial hypercholesterolemia diagnosis was uncommon. Conclusions Only patients with the most severe HoFH phenotypes are diagnosed early. HoFH remains challenging to treat. Results from the Family Heart Database indicate HoFH is systemically underdiagnosed and undertreated. Earlier screening, aggressive lipid-lowering treatments, and guideline implementation are required to reduce disease burden in HoFH.
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Anticolesterolemiantes , Aterosclerosis , Enfermedades Cardiovasculares , Hipercolesterolemia Familiar Homocigótica , Hiperlipoproteinemia Tipo II , Estados Unidos/epidemiología , Humanos , Enfermedades Cardiovasculares/tratamiento farmacológico , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiología , Hiperlipoproteinemia Tipo II/genética , LDL-Colesterol , Aterosclerosis/diagnóstico , Aterosclerosis/epidemiología , Aterosclerosis/genética , Sistema de Registros , Anticolesterolemiantes/uso terapéutico , HomocigotoRESUMEN
PURPOSE: Polymerase chain reaction (PCR) is a molecular technique for the diagnosis of ocular infectious disease. In this large patient sample and multiyear study, the impact of PCR for detecting infectious agents from ocular samples was reviewed in comparison with nonmolecular diagnostic techniques. DESIGN: A retrospective laboratory review of PCR testing. PARTICIPANTS: Three thousand fifty-six patient samples with a differential of ocular infection. METHODS: The daily laboratory logs for diagnostic testing were reviewed for PCR, cell culture isolation, shell vial isolation, and Acanthamoeba isolation from January 1997 through May 2010 for herpes simplex virus (HSV), adenovirus, varicella zoster virus (VZV), Chlamydia trachomatis, Acanthamoeba, and infrequent pathogens of intraocular inflammation. MAIN OUTCOME MEASURES: Incidence of the positive presence of ocular infectious agents. RESULTS: Polymerase chain reaction results were positive more often than culture results for HSV (P = 0.0001), VZV (P = 0.00001), C. trachomatis (P = 0.00005), and Acanthamoeba (P = 0.04). For adenovirus, cell culture isolation results were positive more often than PCR results (P = 0.001). Polymerase chain reaction was the primary diagnostic test for detecting cytomegalovirus and Toxoplasma. CONCLUSIONS: The current study demonstrated the importance of PCR as a routine diagnostic test for detecting both common and infrequent ocular pathogens. Cell culture isolation is still a definitive test for adenovirus and a confirmatory test for HSV and Acanthamoeba.
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Infecciones del Ojo/microbiología , Infecciones del Ojo/parasitología , Reacción en Cadena de la Polimerasa , Acanthamoeba/aislamiento & purificación , Adenoviridae/aislamiento & purificación , Línea Celular Tumoral , Chlamydia trachomatis/aislamiento & purificación , Citomegalovirus/aislamiento & purificación , Herpesvirus Humano 3/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa/normas , Estudios Retrospectivos , Simplexvirus/aislamiento & purificación , Toxoplasma/aislamiento & purificaciónRESUMEN
Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivière and Qvarnstrom assays) were developed for the detection of genus-specific Acanthamoeba DNA but lacked clinical validation. We have adapted these assays for the Cepheid SmartCycler II system (i) by determining their real-time PCR limits of detection and amplification efficiencies, (ii) by determining their ability to detect trophozoites and cysts, and (iii) by testing a battery of positive and negative samples. We also examined the inhibitory effects of a number of commonly used topical ophthalmic drugs on real-time PCR. The results of the real-time PCR limit of detection and amplification efficiency of the Rivière and Qvarnstrom assays were 11.3 DNA copies/10 microl and 94% and 43.8 DNA copies/10 microl and 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoeba cysts/10 microl and 2.3 Acanthamoeba trophozoites/10 microl by both real-time PCR assays. The overall agreement between the assays was 97.0%. The clinical sensitivity and specificity of both real-time PCR assays based on culture were 100% (7 of 7) and 100% (37 of 37), respectively. Polyhexamethylene biguanide was the only topical drug that demonstrated PCR inhibition, with a minimal inhibitory dilution of 1/640 and an amplification efficiency of 72.7%. Four clinical samples were Acanthamoeba culture negative and real-time PCR positive. Our results indicate that both real-time PCR assays could be used to diagnose Acanthamoeba keratitis. Polyhexamethylene biguanide can inhibit PCR, and we suggest that specimen collection occur prior to topical treatment to avoid possible false-negative results.
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Queratitis por Acanthamoeba/diagnóstico , Acanthamoeba/genética , Parasitología/métodos , Reacción en Cadena de la Polimerasa/métodos , Acanthamoeba/aislamiento & purificación , Animales , Biguanidas/farmacología , Cartilla de ADN/genética , Inhibidores Enzimáticos/farmacología , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To compare the SmartCycler II system (Cepheid, Sunnyvale, Calif) results with those of standard cell culture, to compare the SmartCycler II system results with those of a dedicated polymerase chain reaction facility, and to establish the SmartCycler II system as a polymerase chain reaction method for detecting viral and chlamydial DNA from ocular specimens. METHODS: True-positive samples (test-positive specimens based on standard testing) and true-negative samples (test-negative specimens based on standard testing) were processed for polymerase chain reaction using the SmartCycler II system for adenovirus, herpes simplex virus type 1, varicella-zoster virus, and Chlamydia trachomatis. Sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were based on the testing of true-positive and true-negative specimens. RESULTS: The descriptive statistics for adenovirus, herpes simplex virus type 1, varicella-zoster virus, and C trachomatis were, respectively, as follows: sensitivity, 85%, 98%, 100%, and 94%; specificity, 98%, 100%, 100%, and 100%; positive predictive value, 98%, 100%, 100%, and 100%; negative predictive value, 85%, 91%, 100%, and 98%; and efficiency, 92%, 95%, 100%, and 99%. Test sensitivity for the SmartCycler II system was equivalent to that from a central molecular laboratory. CONCLUSION: The descriptive statistics of the SmartCycler II system obtained in a small laboratory were comparable to those of a central molecular laboratory for detecting viruses and Chlamydia species. Clinical Relevance Polymerase chain reaction has great potential in the routine diagnosis of ocular infections in any conventional laboratory.
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Chlamydia trachomatis/genética , ADN Bacteriano/análisis , ADN Viral/análisis , Infecciones Bacterianas del Ojo/diagnóstico , Infecciones Virales del Ojo/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Virus/genética , Adenovirus Humanos/genética , Infecciones Bacterianas del Ojo/microbiología , Infecciones Virales del Ojo/virología , Reacciones Falso Positivas , Herpesvirus Humano 1/genética , Herpesvirus Humano 3/genética , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Varied concentrations of moxifloxacin (MOX) and gatifloxacin (GAT) and the addition of 0.005% benzalkonium chloride (BAK) were evaluated for eliminating Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and coagulase-negative Staphylococcus (CNS). DESIGN: In vitro laboratory investigation. METHODS: The time-kill survival of SA, PA, and CNS were tested at one, two, three, six, eight, and 24 hours to: (1) Mueller-Hinton broth, (2) BAK, (3) 0.5% MOX, (4) 0.5% GAT, (5) 0.3% MOX, (6) 0.3% GAT, (7) 0.3% GAT plus BAK, (8) 0.5% MOX plus BAK, (9) 8 microg/ml GAT, and (10) 8 mug/ml MOX. Antibiotic interactions (GAT and BAK) were determined by checkerboard testing. The outcome measures were (1) time-to-kill, (2) killing-rates, and (3) fractional inhibitory concentration (FIC) indices. RESULTS: MOX and GAT at either 0.5% or 0.3% had equivalent antibacterial effects. BAK alone or the addition of BAK to either antibiotic eliminated SA and CNS within one hour, whereas 0.3% GAT plus BAK eliminated bacteria faster than 0.5% MOX (P = .016). For PA, BAK alone had no antibacterial effect. The kill rates of MOX and GAT were equivalent. FIC indices indicated that GAT and BAK were indifferent against SA and CNS, but antagonistic to PA. CONCLUSION: As a preservative, MOX and GAT have equivalent antibacterial activity with similar killing rates. BAK appears to independently complement GAT for eliminating SA and CNS, but has no effect on PA. The in vitro predictive clinical effect due to varied antibiotic concentration and the addition of BAK requires confirmatory clinical studies for validation.
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Antiinfecciosos/farmacología , Compuestos Aza/farmacología , Compuestos de Benzalconio/farmacología , Fluoroquinolonas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Quinolinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Farmacorresistencia Bacteriana , Gatifloxacina , Pruebas de Sensibilidad Microbiana , Moxifloxacino , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Factores de TiempoRESUMEN
We provide hematologic (n = 34) and biochemical (n = 30) blood values for wild-caught Australian bush rats (Rattus fuscipes). Hematology values have similar range limits compared with other rat species. Biochemistry values for glucose, alanine transaminase, aspartate aminotransferase, and creatine kinase have higher maximum ranges compared with other rats.
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Ratas/sangre , Animales , Animales Salvajes/sangre , Animales Salvajes/metabolismo , Glucemia/análisis , Femenino , Hemoglobinas/análisis , Recuento de Leucocitos/veterinaria , Masculino , Nueva Gales del Sur , Ratas/metabolismo , Valores de ReferenciaRESUMEN
AIM: Anaerobic bacteria can cause ocular infections. We tested the OxyPlate™ Anaerobic System (OXY) to isolate pertinent anaerobic bacteria that can cause ocular disease. METHODS: OXY, which does not require direct anaerobic conditions (i.e. bags, jars), was compared to conventional isolation of incubating culture media in anaerobic bags. Standard colonies counts were performed on anaerobic ocular bacterial isolates under aerobic and anaerobic conditions (anaerobic bags) using agar media: 1) OXY (aerobic only), 2) 5% sheep blood (SB), 3) Chocolate, and 4) Schaedler. The bacteria tested were de-identified ocular isolates cultured from endophthalmitis and dacryocystitis that include 10 Propionibacterium acnes and 3 Actinomyces species. The colony counts for each bacteria isolate, on each culturing condition, were ranked from largest to smallest, and non-parametrically compared to determine the best culturing condition. RESULTS: All anaerobic conditions were positive for all of the anaerobic isolates. SB and Schaedler's agar under aerobic conditions did not support the growth of anaerobic bacteria. Sparse growth was noted on chocolate agar with Propionibacterium acnes. As an anaerobic system, SB in an anaerobic bag isolated higher colony counts than OXY (P=0.0028) and chocolate agar (P=0.0028). CONCLUSION: Although OXY did not test to be more efficient than other anaerobic systems, it appears to be a reasonable alternative for isolating anaerobic bacteria from ocular sites. The use of an agar medium in a specially designed plate, without the requirement of an anaerobic bag, rendered OXY as an advantage over other anaerobic systems.
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AIM: To determine a species-specific real-time polymerase chain reaction (PCR) assay to detect Pseudomonas aeruginosa (PA), a secondary DNA target for PA that may provide a universal target for other bacterial pathogens, and validate both assays for diagnostic testing. METHODS: PCR detection was established against the ecfX PA gene and the 16S rRNA gene using known PA keratitis isolates. The outcome parameters for both assays were "limit of detection" (LOD), amplification efficiency (AE), and PAGE amplified product analysis. Both assays were validated against 20 true-positive clinical samples positive for PA DNA and 20 true-negative samples containing no PA DNA. Descriptive statistics and PAGE analysis were used as outcome parameters. RESULTS: AE of the ecfX assay was 96.6%, and LOD was 33.6 copies of target DNA per microliter. AE of the 16S rRNA assay was 103.4%, and LOD was 8.12 copies per microliter. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency for the ecfX and 16S rRNA assays were [75%, 95%, 94%, 79%, and 85%], and [70%, 100%, 100%, 77%, and 85%], respectively. Both PCR assays were validated, followed by confirmation of DNA patterns from PAGE analysis. CONCLUSION: The PCR methodology described here may be a useful adjunct to standard methods in the diagnosis of PA keratitis.
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AIM: We compared polymerase chain reaction (PCR) to cell culture isolation for the laboratory diagnosis of ocular herpes simplex virus (HSV) disease. METHODS: Laboratory and medical records of consecutive patients were reviewed for results of 1) HSV PCR testing, 2) HSV cell culture isolation, and 3) clinical diagnosis. PCR results were statistically compared to cell culture isolation and patients initially diagnosed for ocular HSV infection. RESULTS: Of 581 cases submitted for laboratory testing, 520 were PCR negative, cell culture negative (89.6%); 0 were PCR negative, cell culture positive (0%); 27 were PCR positive, cell culture negative (4.6%); and 34 were PCR positive, cell culture positive (5.8%). PCR tested more positive than cell culture isolation (McNemar's, P=0.0001). Of 47 HSV PCR positive cases with complete medical records, 19 were cell culture negative for HSV and 28 were cell culture positive for HSV. Fourteen of 19 cell culture negative cases (74%) (Without PCR, 5 cases of HSV would be missed) and 25 of the 28 cell culture positive cases (89%) (Laboratory testing was necessary for diagnosing 3 cases) were clinically diagnosed with HSV at the initial examination. CONCLUSION: PCR was a more definitive test for diagnosing HSV ocular infection than cell culture isolation. Cell culture isolation alone can miss an atypical presentation of HSV ocular infection.
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AIM: Povidone iodine (PI) and SteriLid (SL) are biocides used to decrease bacterial load on the eyelid margin. We compared the antibacterial activity of PI and SL to determine the better antiseptic. METHODS: Time-kill studies of PI and SL against a battery of bacteria that included Staphylococcus epidermidis, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, and Branhamella catarrhalis were conducted at time points 1, 2, 10, and 30 min. The comparative outcome measures were based on 90% and 99.9% decreases in bacterial colony counts. RESULTS: PI was more effective at a 90% kill than SL at time points, 1 min (8/9 vs. 7/9), 2 min (9/9 vs. 6/9), 10 min (9/9 vs. 8/9), and 30 min (9/9 vs. 8/9). PI was more effective at a 99.9% kill than SL at time points, 1 min (5/9 vs. 3/9), 2 min (7/9 vs. 4/9), 10 min (9/9 vs. 4/9), and 30 min (9/9 vs. 6/9). CONCLUSIONS: Our study supports PI as an effective antiseptic for decreasing the bacterial load that can exist on the eyelid margin. SL appears to be a less effective alternative.
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Bacterias/efectos de los fármacos , Desinfectantes/farmacología , Monoterpenos/farmacología , Povidona Yodada/farmacología , Monoterpenos Acíclicos , Antiinfecciosos Locales/farmacología , Recuento de Colonia Microbiana , Párpados/microbiología , Factores de TiempoRESUMEN
Many Serratia marcescens strains produce the red pigment prodigiosin, which has antimicrobial and anti-tumor properties. Previous reports suggest that cyclic AMP (cAMP) is a positive regulator of prodigiosin production. Supporting this model, the addition of glucose to growth medium inhibited pigment production in rich and minimal media. Unexpectedly, we observed highly elevated levels of prodigiosin production in isogenic strains with mutations in genes involved in cAMP production (cyaA and crr) and in cAMP-dependent transcriptional signaling (crp). Multicopy expression of the Escherichia coli cAMP-phosphodiesterase gene, cpdA, also conferred a striking increase in prodigiosin production. Exogenous cAMP decreased both pigment production and pigA-lacZ transcription in the wild-type (WT) strain, and pigA-lacZ transcription was significantly increased in a crp mutant relative to WT. Suppressor and epistasis analysis indicate that the hyperpigment phenotype was dependent upon pigment biosynthetic genes (pigA, pigB, pigC, pigD and pigM). These experiments establish cAMP as a negative regulator of prodigiosin production in S. marcescens.