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1.
Braz. J. Pharm. Sci. (Online) ; 57: e19073, 2021. tab, graf
Article in English | LILACS | ID: biblio-1345463

ABSTRACT

A reversed-phase high performance liquid chromatography (RP-HPLC) method with ultraviolet detection was developed and validated for the simultaneous quantification of antiretroviral drugs lamivudine (3TC), stavudine (d4T), and zidovudine (AZT) in perfusate samples obtained from the Single-Pass Intestinal Perfusion studies. The chromatographic analysis was performed using a Gemini C18 column and didanosine as internal standard (IS). The following parameters were considered for the validation procedure: system suitability, accuracy, precision, linearity and selectivity. The limits of detection were 0.32 µg/mL for 3TC, 0.11 µg/mL for d4T and 0.45 µg/mL for AZT and the limits of quantification were 1.06 µg/mL for 3TC, 0.38 µg/mL for d4T and 1.51 µg/mL for AZT. Repeatability and intermediate precision ranged from 1.05 to 1.31 and 1.50 to 1.87, respectively, and are expressed as percent of relative standard deviation (RSD). Based on these results, the developed and validated RP-HPLC method can be used for simultaneous determination of 3TC, d4T, and AZT in perfusate samples. Furthermore, this method is simple and adequate for measurements of the antiretroviral drugs in the same sample, since those compounds are mostly co-administered. Besides, this work can be used as an initial base for the development of similar methods in the same conditions presented in our study.


Subject(s)
Zidovudine/pharmacology , Chromatography, High Pressure Liquid/methods , Lamivudine/pharmacology , Validation Study , Anti-Retroviral Agents/pharmacology , Perfusion/instrumentation , Permeability , Measurements, Methods and Theories , Pharmaceutical Preparations/administration & dosage , Limit of Detection
2.
Braz. J. Pharm. Sci. (Online) ; 57: e18899, 2021. tab, graf
Article in English | LILACS | ID: biblio-1339302

ABSTRACT

Microbiological quality of pharmaceuticals is fundamental in ensuring efficacy and safety of medicines. Conventional methods for microbial identification in non-sterile drugs are widely used; however they can be time-consuming and laborious. The aim of this paper was to develop a chemometric-based rapid microbiological method (RMM) for identifying contaminants in pharmaceutical products using Fourier transform infrared with attenuated total reflectance spectrometry (FTIR-ATR). Principal components analysis (PCA) and linear discriminant analysis (LDA) were used to obtain a predictive model capable of distinguishing Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), Enterococcus faecium (ATCC 8459), Escherichia coli (ATCC 8739), Micrococcus luteus (ATCC 10240), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538), and Staphylococcus epidermidis (ATCC 12228) microbial growth. FTIR-ATR spectra provide data on proteins, DNA/RNA, lipids, and carbohydrates constitution of microbial growth. Microbial identification provided by PCA/LDA based on FTIR-ATR method were compatible with those obtained using traditional microbiological methods. The chemometric-based FTIR-ATR method for rapid identification of microbial contaminants in pharmaceutical products was validated by assessing the sensitivity (93.5%), specificity (83.3%), and limit of detection (17-23 CFU/mL of sample). Therefore, we propose that FTIR-ATR spectroscopy may be used for rapid identification of microbial contaminants in pharmaceutical products and taking into account the samples studied


Subject(s)
Spectrum Analysis/instrumentation , Pharmaceutical Preparations/analysis , Discriminant Analysis , Spectroscopy, Fourier Transform Infrared/methods , Fourier Analysis , Pseudomonas aeruginosa/classification , Bacillus subtilis/classification , Candida albicans/classification , Quality Management , Limit of Detection
3.
Article in English | WPRIM | ID: wpr-762457

ABSTRACT

BACKGROUND: The interferon-gamma (IFN-γ) releasing assay (IGRA) is widely used for latent tuberculosis infection (LTBI) diagnosis. We evaluated the analytical performance of a new automated chemiluminescent immunoanalyzer-based IGRA (CLIA-IGRA), AdvanSure I3 (LG Life Sciences, Seoul, Korea) and compared it with that of the QuantiFERON-TB Gold In-Tube (QFT-GIT) assay. METHODS: Repeatability and reproducibility were evaluated at four levels. Detection capability, including limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), was evaluated using IFN-γ standard material (National Institute for Biological Standards and Control code: 87/586). Agreement between the results of two assays was evaluated using 341 blood samples from healthcare workers and patients at a tertiary care hospital. To determine the cut-off value of CLIA-IGRA for diagnosing LTBI, the ROC curve was analyzed. RESULTS: Repeatability and reproducibility were 4.86–7.00% and 6.36–7.88% CV, respectively. LoB, LoD, and LoQ were 0.022, 0.077, and 0.249 IU/mL, respectively. IFN-γ values between CLIA-IGRA and QFT-GIT showed a strong correlation within the analytical measurable range of both assays, especially when the value was low. Qualitative comparison of the two assays yielded a 99.1% overall agreement (kappa coefficient=0.98). A cut-off value of 0.35 IU/mL was appropriate for diagnosing LTBI. CONCLUSIONS: CLIA-IGRA is a reliable assay for LTBI diagnosis, with performance similar to that of QFT-GIT.


Subject(s)
Biological Science Disciplines , Delivery of Health Care , Diagnosis , Humans , Interferon-gamma , Latent Tuberculosis , Limit of Detection , ROC Curve , Seoul , Tertiary Healthcare
4.
Article in English | WPRIM | ID: wpr-762451

ABSTRACT

Accurate detection of BCR-ABL fusion transcripts at and below molecular response (MR) 4 (0.01% International Scale [IS]) is required for disease monitoring in patients with chronic myeloid leukemia (CML). We evaluated the analytical performance of the QXDx BCR-ABL %IS (Bio-Rad, Hercules, CA, USA) droplet digital PCR (ddPCR) assay, which is the first commercially available ddPCR-based in vitro diagnostics product. In precision analysis, the %CV was 9.3% and 3.0%, with mean values of 0.031% IS and 9.4% IS, respectively. The assay was linear in the first order, ranging from 0.032% IS to 20% IS. The manufacturer-claimed limit of blank, limit of detection, and limit of quantification were verified successfully. There was a very strong correlation between the results of the QXDx BCR-ABL %IS ddPCR assay and the ipsogen BCR-ABL1 Mbcr IS-MMR (Qiagen, Hilden, Germany) real-time quantitative PCR assay (r=0.996). In conclusion, the QXDx BCR-ABL %IS ddPCR assay can provide reliable results for CML patients.


Subject(s)
Humans , In Vitro Techniques , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Limit of Detection , Polymerase Chain Reaction
5.
Article in English | WPRIM | ID: wpr-816637

ABSTRACT

Sexually transmitted infections (STIs) are caused by the spread of pathogens via sexual activity and can cause serious complications if left untreated, regardless of their symptoms. Therefore, early diagnosis of STI is important, and molecular diagnostic methods for rapid detection and monitoring are needed. In this study, we evaluated a multiplex polymerase chain reaction (PCR) kit for simultaneously detecting 13 different bacterial, fungal, and viral microorganisms that cause STIs. The kit performance was evaluated for its sensitivity, lot-to-lot variation, and interference in detecting different pathogens. Additionally, its clinical usefulness was evaluated by estimating its sensitivity and specificity for clinical samples. The limit of detection (LOD) was 0.021–50.104 copies for each pathogen. In the tests of lot-to-lot, 100% of positive samples were detected at low concentrations and negative samples all showed negative results. This result confirms that there is no the variation of lot-to-lot. In the test for interference between pathogens, the efficiency of amplification for each pathogen was not significantly reduced and no nonspecific amplification product was formed. We tested 322 vaginal swab samples using the multiplex PCR kit and confirmed that its clinical sensitivity and specificity were 100% for all pathogens. This multiplex PCR kit can be used widely for rapid diagnosis and monitoring of STIs.


Subject(s)
Diagnosis , Early Diagnosis , Limit of Detection , Multiplex Polymerase Chain Reaction , Pathology, Molecular , Sensitivity and Specificity , Sexual Behavior , Sexually Transmitted Diseases
6.
Con-ciencia (La Paz) ; 7(1): 67-77, abr. 2019.
Article in Spanish | LILACS | ID: biblio-1178663

ABSTRACT

El presente estudio describe la validación del método para la cuantificación de Ciclosporina A en sangre total por Cromatografía Líquida de Alta Resolución (HPLC) en Fase Reversa con Detector de Arreglo de Diodos. La Ciclosporina A es un fármaco inmunosupresor con un estrecho margen terapéutico además de su amplia variabilidad en los procesos farmacocinéticos, justifican su monitorización de dosis a pacientes con trasplantes de órganos para evitar los efectos secundarios y el posible rechazo del órgano trasplantado. La separación de la Ciclosporina A de una matriz compleja como la sangre fue llevada a cabo de manera exitosa utilizando como fase estacionaria una columna C8 5um (250 mm x 4,6mm) o equivalente a L7 según la USP 37, la fase móvil fue una mezcla de acetonitrilo y agua en gradiente, flujo 1.4 ml/min, temperatura de la columna 75ºC y detección con Arreglo de Diodos a 210nm. El método fue validado con los siguientes parámetros: especificidad, linealidad, precisión, exactitud, límite de detección y límite de cuantificación. También se realizó la prueba de aptitud del sistema. El método fue específico para la Ciclosporina A, la respuesta fue lineal en el rango de 100 a 1000 ng/mL de concentración del analito. El valor del coeficiente de variación o desviación estándar relativa (C.V. o DSR) para la precisión fue óptimo. La recuperación media fue de 103,06%. Y el límite de detección y cuantificación resultaron óptimos para la cuantificación de Ciclosporina en sangre total en el rango definido. Finalmente el método cromatográfico nos permitió separar y cuantificar a la Ciclosporina A presente en las muestras de sangre de pacientes con transplante renal.


This study describe the validation method for the quantification of Cyclosporin A in Whole Blood by Liquid Chromatography High Efficiency (HPLC) in reverse phase. Cyclosporine A is immunosuppressant United Nations UN the narrow scam therapeutic margin: In addition to its extensive ¿variability in pharmacokinetic processes justify their monitoring dose Patients with organ transplants paragraph Avoid Side Effects and poaible organ rejection transplanted. Separation of Cyclosporine A of a matrix complex as the blood was carried out successfully using as the stationary phase C8 column, the mobile phase is a mixture of acetonitrile and water gradient, flow 1.4 ml / min, Temperature column was 75 ° C detection 210 nm in one. The method was validated with the following parameters: specificity, linearity, precision, accuracy, limit of detection and limit of quantification. Aptitude Test System was also performed. Was Method Specific for Cyclosporin A, the response was linear in the range 100 a 1000 ng/mL concentration of the analyte. The coefficient of variation or relative standard deviation (RSD or CV) for the precision was optimal. Recovery media WAS 103,06%. And the limit of detection and quantification were optimal for the quantification of total Cyclosporine in blood in the range defined. Finally Chromatographic Method allowed us to separate and quantify Cyclosporin A in samples of patients with renal transplantation.


Subject(s)
Cyclosporine , Dosage , Limit of Detection , Sensitivity and Specificity , Kidney Transplantation , Reference Parameters
7.
Natural Product Sciences ; : 238-243, 2019.
Article in English | WPRIM | ID: wpr-760566

ABSTRACT

In this study, the marker compounds of Curcumae Rhizoma (CR) were simultaneously quantified by high-performance liquid chromatography equipped with a photodiode array detector and the anti-inflammatory effects of CR extract and marker compounds in human benign prostatic hyperplasia epithelial-1 (BPH-1) cell lines were investigated. The marker components (4S,5S)-(+)-germacrone-4,5-epoxide, furanodienone, and germacrone, were separated on Gemini C₁₈ columns (250 mm × 4.6 mm, 5 µm) at 40 ℃ by using a gradient of two mobile phases eluting at 1.0 mL/min. Prostaglandin E₂ (PGE₂) levels in Human BPH-1 cells were determined with an ELISA kit. The coefficients of determination in a calibration curve of each analyte were all 0.9997. The limits of detection and quantification of the three compounds were 0.10 – 0.32 µg/mL and 0.30 – 0.98 µg/mL, respectively. The content of three compounds, (4S,5S)-(+)-germacrone-4,5-epoxide, furanodienone, and germacrone, in the CR sample were found to be 5.79 – 5.92 mg/g, 4.72 – 4.86 mg/g, and 1.06 – 1.09 mg/g, respectively. Regarding pharmacological activity against benign prostatic hyperplasia, CR and its components significantly suppressed PGE₂ levels of BPH-1 cells. The established analysis method will help to improve quality assessment of CR samples and related products. In addition, CR and its components exhibit anti-inflammatory activity in BPH-1 cells, suggesting the inhibitory efficacy of these compounds against the pathogenesis of BPH.


Subject(s)
Calibration , Cell Line , Chromatography, Liquid , Curcuma , Enzyme-Linked Immunosorbent Assay , Humans , Limit of Detection , Methods , Prostatic Hyperplasia , Rhizome
8.
Article in Korean | WPRIM | ID: wpr-759579

ABSTRACT

BACKGROUND: A nucleic acid amplification test was adopted to detect transfusion-transmitted infectious agents. In the case of HTLV, however, there was no internal control (IC) because the laboratory developed polymerase chain reaction (laboratory-developed PCR) was used. In this study, noncompetitive IC was constructed for the laboratory-developed PCR of HTLV and the effectiveness was compared with the competitive test that was constructed in a previous study. METHODS: As a competitive IC, plasmid DNA, including the primer recognition sequence for the amplification of the HTLV pX region, was constructed. As a noncompetitive IC, an additional primer was constructed for the amplification of the housekeeping gene, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The performance of the competitive and noncompetitive IC was verified and compared using 10 HTLV positive samples and 10 negative samples. In addition, the detection limits in the assay adopting competitive IC and noncompetitive IC were compared. RESULTS: In the case of competitive IC applications, all 10 positive samples were positive and all 10 negative samples were negative. In the case of noncompetitive IC applications, however, one positive sample was not detected. The detection limit of the assay using competitive IC was 100 pg and that of the assay using noncompetitive IC was 1 ng. CONCLUSION: Although the manufacturing processes is not required using noncompetitive IC, the adoption of competitive IC is more effective to ensure the assay results because the ability of detection of the assay adopting competitive IC was better than that using noncompetitive IC.


Subject(s)
DNA , Genes, Essential , Glyceraldehyde 3-Phosphate , Limit of Detection , Nucleic Acid Amplification Techniques , Oxidoreductases , Plasmids , Polymerase Chain Reaction
9.
Article in English | WPRIM | ID: wpr-758912

ABSTRACT

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.


Subject(s)
Anti-Bacterial Agents , Chickens , Ciprofloxacin , DNA , Drug Resistance, Microbial , Enterococcus , Limit of Detection , Linezolid , Methods , Multiplex Polymerase Chain Reaction , Prevalence , Tetracycline , Vancomycin , Virulence
10.
Chinese Journal of Biotechnology ; (12): 901-909, 2019.
Article in Chinese | WPRIM | ID: wpr-771320

ABSTRACT

To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.


Subject(s)
Animals , Chromatography, Affinity , Diagnostic Tests, Routine , Reference Standards , Gold Colloid , Chemistry , Haemophilus Infections , Diagnosis , Haemophilus influenzae , Humans , Limit of Detection , Rabbits , Sensitivity and Specificity
11.
Article in English | WPRIM | ID: wpr-762556

ABSTRACT

BACKGROUND: 5-chloro-2-(2,4-dichlorophenoxy)phenol (triclosan) is used as an antiseptic and is a potential endocrine-disrupting chemical that can affect thyroid hormone levels. This study evaluated the relationship between triclosan exposure and thyroid hormones. METHODS: Data from the second Korean National Environmental Health Survey (2012–2014) were analyzed. Triclosan exposure was evaluated using urinary triclosan concentrations and classified into 2 groups: ‘below detection (< limit of detection [LOD])’ vs. ‘detected (≥ LOD).’ Multiple linear regression analysis was conducted to determine the relationship between triclosan exposure and the serum thyroid hormone concentrations, adjusting for age, body mass index, urinary creatinine, and smoking status. RESULTS: When grouped by sex, triclosan exposure was positively associated with the serum thyroid-stimulating hormone (TSH) concentrations in females with marginal significance (β = 0.066, p = 0.058). However, no significant association was identified between triclosan exposure and serum total triiodothyronine and thyroxine in both males and females, and TSH in males. CONCLUSIONS: This study is the first human study to evaluate the relationship between triclosan exposure and serum thyroid hormone concentrations in the Korean population. There was suggestive positive association between triclosan exposure and the serum TSH in females. Further studies need to evaluate the relationship between long-term exposure to low-dose triclosan and thyroid hormones.


Subject(s)
Body Mass Index , Creatinine , Environmental Health , Female , Humans , Limit of Detection , Linear Models , Male , Smoke , Smoking , Thyroid Gland , Thyroid Hormones , Thyrotropin , Thyroxine , Triclosan , Triiodothyronine
12.
Article in Korean | WPRIM | ID: wpr-719666

ABSTRACT

Viral respiratory infections are one of the most common infections worldwide. It is important to detect the virus early and precisely. In this study, we evaluated the limit of detection (LoD) and usefulness of the Real-Q RV Detection kit (BioSewoom, Seoul, Korea). We measured the LoD of the Real-Q RV Detection kit using 10 strains of standard viruses. We then compared the detection results by the Allplex Respiratory Panel Assay kit (Seegene, Seoul, Korea) using 123 clinical specimens. The discrepant results were confirmed by sequencing. Among the 10 standard viruses, the LoD of human rhinovirus (HRV) was the lowest and that of parainfluenza virus 2 and 3 was relatively high as detected by Real-Q RV Detection kit. Agreements of the two kits ranged from 95.9% to 100%. Three specimens detected negative by the Allplex Respiratory Panel kit were detected as adenovirus (AdV) by the Real-Q RV Detection kit and were confirmed by sequencing. Similarly, a specimen detected negative by the Allplex Respiratory Panel kit was detected as HRV by the Real-Q RV Detection kit and was confirmed by sequencing. A specimen detected as human enterovirus by the Allplex Respiratory Panel kit was detected as HRV by the Real-Q RV Detection kit and was confirmed by sequencing. Real-Q RV Detection kit showed good diagnostic performance and can be useful for detecting major viruses that cause respiratory infections.


Subject(s)
Adenoviridae , Enterovirus , Humans , Limit of Detection , Paramyxoviridae Infections , Respiratory Tract Infections , Rhinovirus , Seoul
13.
Article in English | WPRIM | ID: wpr-719648

ABSTRACT

BACKGROUND: The Automated Fluorescent Immunoassay System (AFIAS) rotavirus assay (Boditech Med Inc., Chuncheon, Korea) is a new rapid antigen test for rotavirus detection. We evaluated the performance of this assay for detecting rotaviruses and their specific genotypes in clinical stool samples. METHODS: AFIAS rotavirus assay was performed in 103 rotavirus-positive and 103 rotavirus-negative stool samples (confirmed by both PCR and ELISA), and its results were compared with those of PCR, ELISA, and immunochromatographic assay (ICA). We evaluated diagnostic sensitivity/specificity, the detectability of rotavirus subtypes, lower limit of detection (LLOD), reproducibility, cross-reactivity, and interference of AFIAS rotavirus assay. RESULTS: Based on PCR and ELISA results, diagnostic sensitivity and specificity of the AFIAS rotavirus assay were both 99.0%. LLOD results showed that the AFIAS assay had sensitivity similar to or greater than ICA and ELISA. High reproducibility was confirmed, and no cross-reactivity or interference was detected. This assay could detect genotypes G1P[8], G2P[4], G3P[8], G4P[6], G4P[8], G8P[4], G8P[8], G9P[4], and G9P[8]. CONCLUSIONS: The AFIAS rotavirus assay showed high reproducibility, sensitivity, and specificity as well as excellent agreement with ELISA, PCR, and ICA. It detected the most common as well as unusual genotypes of rotavirus prevalent in Korea. It could be a useful on-site assay for rapid, convenient, and cost-effective detection of rotavirus infection.


Subject(s)
Enzyme-Linked Immunosorbent Assay , Genotype , Immunoassay , Chromatography, Affinity , Korea , Limit of Detection , Polymerase Chain Reaction , Rotavirus Infections , Rotavirus , Sensitivity and Specificity
14.
Gut and Liver ; : 471-478, 2019.
Article in English | WPRIM | ID: wpr-763852

ABSTRACT

BACKGROUND/AIMS: Metallic stents designed to relieve malignant biliary obstruction are susceptible to occlusive tumor ingrowth or overgrowth. In a previous report, we described metallic stents covered with paclitaxel-incorporated membrane (MSCPM-I, II) to prevent occlusion from tumor ingrowth via antitumor effect. This new generation paclitaxel-eluting biliary stent is further endowed with sodium caprate (MSCPM-III) for enhanced drug delivery. The purpose of this study is to examine the safety of its drug delivery system in the porcine biliary tract. METHODS: MSCPM-III (10% [wt/vol] paclitaxel) and covered metal stents (CMSs) were endoscopically inserted in porcine bile ducts in vivo. Histologic biliary changes, levels of paclitaxel released, and various serum analytes (albumin, alkaline phosphate, aspartate transaminase, alanine transaminase, total protein, total bilirubin, and direct bilirubin) were assessed. RESULTS: Based on the intensity of reactive inflammation and fibrosis, changes in porcine biliary epithelium secondary to implanted MSCPM-III were deemed acceptable (i.e., safe). Histologic features in the MSCPM-III and CMS groups did not differ significantly. In a related serum analysis, paclitaxel release from MSCPM-III stents was below the limit of detection for 28 days. Biochemical analyses were also similar for the two groups, and no evidence of hepatic or renal toxicity was found in animals receiving MSCPM-III stents. CONCLUSIONS: In a prototypic porcine trial, this newly devised metal biliary stent incorporating both paclitaxel and sodium caprate appears to be safe in the porcine bile duct.


Subject(s)
Alanine Transaminase , Animals , Aspartate Aminotransferases , Bile Ducts , Biliary Tract Neoplasms , Biliary Tract , Bilirubin , Drug Delivery Systems , Drug-Eluting Stents , Epithelium , Fibrosis , Inflammation , Limit of Detection , Membranes , Paclitaxel , Pancreatic Neoplasms , Self Expandable Metallic Stents , Sodium , Stents
15.
Asia Pacific Allergy ; (4): e5-2019.
Article in English | WPRIM | ID: wpr-750169

ABSTRACT

BACKGROUND: Previous studies have reported that exposure to paraben (Pb) and triclosan (TCS) is associated with allergies. However, Pb and TCS exposure in the Japanese population is not fully understood. OBJECTIVES: The present study was aimed to examine such exposure among Japanese individuals with allergic diseases. METHODS: This cross-sectional study included the International Study of Asthma and Allergies in Childhood questionnaire survey to evaluate allergic outcomes and the collection of urine samples to examine Pb and TCS exposure. RESULTS: Pb containing daily commodities was used in 84.8% children. Pb use was positively associated with current atopic dermatitis (adjusted odds ratio, 4.61; 95% confidence interval, 1.23–17.3). Urinary Pb concentrations were increased significantly in those with current atopic dermatitis (AD) (median, 4.58 vs. 0; p < 0.0001), and showed an increased tendency in those with current wheeze (median, 3.45 vs. 1.81; p = 0.0535) in participants ≤15 years old. Urinary TCS concentration was under the limit of detection in all children. CONCLUSION: Urinary levels of Pb were associated with current AD in children. We should pay more attention about Pb and TCS.


Subject(s)
Asian Continental Ancestry Group , Asthma , Child , Cross-Sectional Studies , Dermatitis, Atopic , Humans , Hypersensitivity , Limit of Detection , Odds Ratio , Respiratory Sounds , Triclosan
16.
Braz. j. med. biol. res ; 52(3): e8186, 2019. tab, graf
Article in English | LILACS | ID: biblio-989465

ABSTRACT

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.


Subject(s)
Nucleic Acid Amplification Techniques/methods , Genes, Bacterial , Klebsiella pneumoniae/genetics , Plasmids/isolation & purification , Plasmids/genetics , Temperature , Time Factors , Polymerase Chain Reaction/methods , Reproducibility of Results , Sequence Analysis, DNA , DNA Primers/isolation & purification , DNA Primers/genetics , Limit of Detection , Klebsiella pneumoniae/isolation & purification
17.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 16(3): 6-12, dic. 2018. tab, ilus
Article in Spanish | LILACS, BDNPAR | ID: biblio-998219

ABSTRACT

El cáncer de cuello uterino es el segundo cáncer femenino más común a nivel mundial. El agente causal es el virus de papiloma humano (VPH). Se han identificado 13 tipos de virus de papiloma humano de alto riesgo oncogénico (VPH-AR), entre los cuales el VPH 16 y VPH 18 son los más frecuentemente detectados en cáncer de cuello uterino, siendo en Paraguay detectados en el 70% de casos de cáncer invasor. Por ello, el objetivo fue estandarizar y determinar el límite de detección de una técnica de PCR convencional para la detección de VPH 16 y 18. Para la detección de ADN de VPH 16 y 18, se observaron mejores resultados con 2mM de MgCl2 y 60°C para la temperatura de alineamiento. El límite de detección para las PCR fue de 14,6x10-11ng/µL para VPH 16 y 21,7x10-12ng/µL para VPH 18. Este trabajo servirá de base a otros estudios de detección e identificación de estos tipos virales por PCR, con miras a identificar un grupo de mujeres positivas para VPH-AR que poseen mayor riesgo de desarrollo de lesión y cáncer de cuello uterino y precisan de un seguimiento más cercano(AU


Cervical cancer is the second most common female cancer worldwide. It is caused by the human papilloma virus (HPV). Thirteen genotypes of high oncogenic risk human papilloma viruses (HPV-HR) have been identified, among which types 16 and 18 are the most frequently detected in cervical cancer. In Paraguay, they are detected in 70% of the invasive cancer cases. Therefore, the objective was to standardize and determine the detection limit of a conventional PCR technique for the detection of HPV 16 and 18. Better results were observed with 2mM MgCl2 and 60°C for the alignment temperature in detection of HPV 16 and 18 DNA. The limit of detection was 14.6x10-11ng/µL for HPV 16 and 21.7x10-12ng/µL for HPV 18. This work will help other studies for the detection and identification of these viral types by PCR in order to identify a group of HPV-HR positive women who have higher risk for the development of lesions and cervical cancer and need a closer follow-up(AU)


Subject(s)
Humans , Female , Uterine Cervical Neoplasms/virology , Polymerase Chain Reaction/methods , Papillomavirus Infections/virology , Human papillomavirus 16/genetics , Human papillomavirus 18/genetics , Base Sequence , Genome, Viral , DNA Primers , Electrophoresis, Polyacrylamide Gel , Limit of Detection
18.
Con-ciencia (La Paz) ; 6(1): 15-25, jun. 2018. ilus., tab.
Article in Spanish | LILACS, LIBOCS | ID: biblio-1178720

ABSTRACT

Staphylococcus aureus puede contaminar una gran gama de alimentos, siendo los quesos frescos un medio diferencial y selectivo para el desarrollo de este microorganismo, llegando a la producción de enterotoxina termoestable ocasionando una intoxicación estafilocóccica trasmitida por alimentos. El empleo del método 3M Petrifilm Staph Express (STX) supone considerables ventajas frente al método convencional o de referencia, facilitando y favoreciendo muchos aspectos técnicos y económicos, dentro de ellos tenemos la optimización de tiempo en cuanto se refiere a la preparación de medios de cultivo, necesidad de equipamiento, requerimiento de infraestructura, menor requerimiento de personal y la disminuye los costos del uso de consumo eléctrico, disminuye la generación de residuos sólidos, entre otros. La comparación entre el método alternativo frente al método convencional fue hecha por medio de la contaminación artificial de una matriz libre de S. aureus y un recuento bajo de microbiota acompañante, donde se establecieron diferentes niveles de contaminación, evaluándose los indicadores de desempeño (exactitud relativa, precisión relativa, linealidad, curva de calibración y límite de cuantificación). En indicador de exactitud relativa se obtuvo un 95,6% de recuperación para el nivel más bajo (aproximadamente 8 UFC/g) y 98,7% de recuperación para el nivel de contaminación más alto (aproximadamente 5300 UFC/g), teniendo un rango de 70 a 120% de aceptación. Para la precisión relativa se calcularon la RSD de todos los niveles ensayados, obteniéndose datos que aceptan la precisión relativa del método de placa seca rehidratable porque los valores fueron menores al RSD teórico calculado. El límite de detección fue ensayado con <10UFC/g obteniéndose un CV de 1,16%. Por los resultados obtenidos podemos concluir que el método 3M Petrifilm Staph Express (STX) cumple con los indicadores de desempeño frente a la norma ISO 6888-1:2003.


Staphylococcus aureus can contaminate a wide range of foods, being fresh cheeses a differential and selective medium for the development of this microorganism, reaching the production of thermostable enterotoxin causing a staphylococcal intoxication transmitted by food. The use of the 3M Petrifilm Staph Express (STX) method has considerable advantages over the conventional or reference method, facilitating and favoring many technical and economic aspects, within them we have the optimization of time as regards the preparation of culture media, need for equipment, infrastructure requirements, lower personnel requirements and decreases the costs of the use of electricity consumption, decreases the generation of solid waste, among others. The comparison between the alternative method and the conventional method was made by means of the artificial contamination of a free matrix of S. aureus and a low accompanying microbiota count, where different levels of contamination were established, evaluating the performance indicators (relative accuracy, relative precision, linearity, calibration curve and limit of quantification). In the relative accuracy indicator, 95.6% recovery was obtained for the lowest level (approximately 8 CFU / g) and 98.7% recovery for the highest level of contamination (approximately 5300 CFU / g), with a range of 70 to 120% acceptance. For the relative precision, the RSD of all the tested levels was calculated, obtaining data that accept the relative accuracy of the dry rehydratable plate method because the values were lower than the calculated theoretical RSD. The limit of detection was tested with <10 CFU /g, obtaining a CV of 1.16%. Based on the results obtained, we can conclude that the 3M Petrifilm Staph Express (STX) method complies with the performance indicators against the ISO 6888-1: 2003 standard.


Subject(s)
Staphylococcus aureus , Cheese , Calibration , Limit of Detection , Food
19.
Article in English | WPRIM | ID: wpr-758796

ABSTRACT

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.


Subject(s)
Animals , Diagnosis , DNA , Enterobacteriaceae , Enzyme-Linked Immunosorbent Assay , Genome , Limit of Detection , Methods , Mycobacterium avium , Mycobacterium , Paratuberculosis , Point-of-Care Testing , Polymerase Chain Reaction , Recombinases , Ruminants , Sensitivity and Specificity
20.
Article in English | WPRIM | ID: wpr-716058

ABSTRACT

PURPOSE: Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, which is a pathogenic mycobacterial species grouped under Mycobacterium tuberculosis complex (MTBC) with four other pathogenic mycobacterial species. The mycobacteria not included in MTBC are known as nontuberculous mycobacteria (NTM), and cause several pulmonary diseases including pneumonia. Currently, NTM occurrences in TB-suspected respiratory specimens have increased, due to which, precise detection of MTBC and NTM is considered critical for the diagnosis and vaccination of TB. Among the various methods available, real-time PCR is frequently adopted for MTBC/NTM detection due to its rapidness, accuracy, and ease of handling. In this study, we evaluated a new real-time PCR kit for analytical and clinical performance on sputum, bronchial washing, and culture specimens. MATERIALS AND METHODS: For assessing its analytical performance, limit of detection (LOD), reactivity, and repeatability test were performed using DNA samples. To evaluate clinical performance, 612 samples were collected and clinically tested at a tertiary hospital. RESULTS: LOD was confirmed as 0.584 copies/µL for MTBC and 47.836 copies/µL for NTM by probit analysis (95% positive). For the reactivity test, all intended strains were detected and, in the repeatability test, stable and steady results were confirmed with coefficient of variation ranging from 0.36 to 1.59. For the clinical test, sensitivity and specificity were 98.6%–100% and 98.8%–100% for MTBC and NTM, respectively. CONCLUSION: The results proved the usefulness of the kit in TB diagnosis. Furthermore, it could be adopted for the assessment of vaccine efficacy.


Subject(s)
BCG Vaccine , Diagnosis , DNA , Limit of Detection , Lung Diseases , Mycobacterium tuberculosis , Nontuberculous Mycobacteria , Pneumonia , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Sputum , Tertiary Care Centers , Tuberculosis , Vaccination
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