scholarly journals Prevalence of antibodies againstEntamoeba histolyticain Mexico measured by ELISA

1995 ◽  
Vol 115 (3) ◽  
pp. 535-543 ◽  
Author(s):  
C. R. González ◽  
A. Isibasi ◽  
V. Ortiz-Navarrete ◽  
J. Paniagua ◽  
J. A. García ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Confidence Limit ◽  
Metropolitan Areas ◽  
Solid Phase ◽  
Axenic Culture ◽  
Amoebic Liver Abscess ◽  
Serum Samples ◽  
Indirect Haemagglutination ◽  
Younger Age ◽  
Triton X

SummaryThe prevalence of antibodies againstEntamoeba histolyticawas studied in the Mexican population using an immunoenzyme assay in solid phase (ELISA) and semiautomatic equipment. The antigen was a mixture of membrane proteins obtained by Triton X-100 extraction from an axenic culture ofEntamoeba histolyticaHM1-IMSS. The method was standardized by comparing serum samples from amoebic liver abscess patients with healthy volunteers. From the 60538 samples suppliedbythe National Seroepidemiology Survey, antibodies were found in 4·49 % (4·32–;4·65% at 95 % confidence limit). More significant titres occurred in the central region of the country. The ratio female to male was 1·25:1. The population living in metropolitan areas had probably been infected at a younger age than those living in the country.Important differences were found in the seroprevalence obtained by ELISA compared with a study which used indirect haemagglutination (IHA) in the same sample frame.

scholarly journals Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

2011 ◽  
Vol 18 (11) ◽  
pp. 1913-1917 ◽  
Author(s):  
Weng Kin Wong ◽  
Zi Ning Tan ◽  
Nurulhasanah Othman ◽  
Boon Huat Lim ◽  
Zeehaida Mohamed ◽  
...  
Keyword(s):  
Human Serum ◽  
Entamoeba Histolytica ◽  
Amoebic Liver Abscess ◽  
Serum Samples ◽  
Pyruvate Phosphate Dikinase ◽  
Content Type ◽  
Human Serum Samples ◽  
Antigenic Proteins ◽  
Secretory Antigen ◽  
Excretory Secretory Antigen

ABSTRACTSerodiagnosis of amoebiasis remains the preferred method for diagnosis of amoebic liver abscess (ALA). However, the commercially available kits are problematic in areas of endemicity due to the persistently high background antibody titers. Human serum samples (n= 38) from patients with ALA who live in areas of endemicity were collected from Hospital Universiti Sains Malaysia during the period of 2008 to 2010. Western blots using excretory-secretory antigen (ESA) collected from axenically grownEntamoeba histolyticawere probed with the above serum samples. Seven antigenic proteins of ESA with various reactivities were identified, i.e., 152 kDa, 131 kDa, 123 kDa, 110 kDa, 100 kDa, 82 kDa, and 76 kDa. However, only the 152-kDa and 110-kDa proteins showed sensitivities above 80% in the Western blot analysis. All the antigenic proteins showed undetectable cross-reactivity when probed with healthy human serum samples (n= 30) and serum samples from other infections (n= 33). From the matrix-assisted laser desorption ionization–two-stage time of flight (MALDI-TOF/TOF) analysis, the proteins were identified as heavy subunits ofE. histolyticalectin andE. histolyticapyruvate phosphate dikinase, respectively. Use of theE. histolyticalectin for diagnosis of ALA has been well reported by researchers and is being used in commercialized kits. However, this is the first report on the potential use of pyruvate phosphate dikinase for diagnosis of ALA; thus, this molecule merits further evaluation on its diagnostic value using a larger panel of serum samples.

Circulating antibodies to histolysain, the major cysteine proteinase ofEntamoeba histolytica, in amoebic liver abscess patients

Parasitology ◽  
1992 ◽  
Vol 105 (2) ◽  
pp. 207-210 ◽  
Author(s):  
L. M. Osorio ◽  
T. Picóf ◽  
A. Luaces
Keyword(s):  
Immune Response ◽  
Liver Abscess ◽  
Humoral Immune Response ◽  
Solid Phase ◽  
Cysteine Proteinase ◽  
Amoebic Liver Abscess ◽  
Healthy Controls ◽  
Serum Samples ◽  
Humoral Immune ◽  
Circulating Antibodies

SUMMARYA solid-phase enzyme immunoassay (EIA) was used to detect circulating antibodies to histolysain, the major cysteine proteinase ofEntamoeba histolytica. Serum samples from 40 healthy controls, 33 asymptomaticE. histolyticacyst passers and 22 patients with amoebic liver abscess were tested. Antibodies to histolysain were found in 72·7% of cases of amoebic liver abscess, 18·1% of the cyst passers and 2·5% of healthy controls, which suggests that a humoral immune response is induced by histolysain during amoebic liver abscess.

scholarly journals Overexpression of Specific Cysteine Peptidases Confers Pathogenicity to a Nonpathogenic Entamoeba histolytica Clone

mBio ◽  
2013 ◽  
Vol 4 (2) ◽  
Author(s):  
Jenny Matthiesen ◽  
Ann-Katrein Bär ◽  
Anne-Kathrin Bartels ◽  
Dennis Marien ◽  
Susann Ofori ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Liver Abscess ◽  
Clinical Symptoms ◽  
Axenic Culture ◽  
Protozoan Parasite ◽  
Culture Conditions ◽  
Amoebic Liver Abscess ◽  
Content Type ◽  
Pathogenicity Factors ◽  
Cysteine Peptidases

ABSTRACTCysteine peptidases (CPs) ofEntamoeba histolyticaare considered to be important pathogenicity factors. Previous studies have found that under standard axenic culture conditions, only four (ehcp-a1,ehcp-a2,ehcp-a5, andehcp-a7) out of 35 papain-likeehcpgenes present in theE. histolyticagenome are expressed at high levels. Little is known about the expression of CPs inE. histolyticaduring amoebic liver abscess (ALA) formation. In the current study, a quantitative real-time PCR assay was developed to determine the expression of the variousehcpgenes during ALA formation in animal models. Increased expression of fourehcpgenes (ehcp-a3,-a4,-a10, and-c13) was detected in the gerbil and mouse models. Increased expression of another threeehcpgenes (ehcp-a5,-a6, and-a7) was detected in the mouse model only, and two otherehcpgenes (ehcp-b8and-b9) showed increased expression in the gerbil model only. Trophozoites of the nonpathogenicE. histolyticaHM-1:IMSS clone A1, which was unable to induce ALAs, were transfected with vectors enabling overexpression of those CPs that are expressed at high levels under culture conditions or during ALA formation. Interestingly, overexpression ofehcp-b8,-b9, and-c13restored the pathogenic phenotype of the nonpathogenic clone A1 whereas overexpression of various other peptidase genes had no effect on the pathogenicity of this clone.IMPORTANCEEntamoeba histolyticais a widespread and clinically important protozoan parasite. It normally exists in the human intestine without causing clinical symptoms but can invade the intestinal mucosa, which causes serious intestinal (amoebic colitis) and extraintestinal (amoebic liver abscess [ALA]) diseases. The identification of factors responsible for the invasion of the parasite and disease formation is a major topic in the field. Here, we investigate the roles of different papain-like cysteine peptidases (CPs) as pathogenicity factors. We show that the expression of some of the peptidases that are normally expressed at low levels increases during ALA formation. Furthermore, nonpathogenic amoebae can be transformed to pathogenic amoebae, simply by specific overexpression of some of these CPs. Our findings reinforce the importance of CPs as pathogenicity factors ofE. histolytica.

scholarly journals UHPLC-ESI-MS/MS Quantification of Relevant Substrates and Metabolites of the Kynurenine Pathway Present in Serum and Peritoneal Fluid from Gastric Cancer Patients—Method Development and Validation

2021 ◽  
Vol 22 (13) ◽  
pp. 6972
Author(s):  
Ilona Sadok ◽  
Katarzyna Jędruchniewicz ◽  
Karol Rawicz-Pruszyński ◽  
Magdalena Staniszewska
Keyword(s):  
Gastric Cancer ◽  
Cancer Patients ◽  
Peritoneal Fluid ◽  
Method Development ◽  
Solid Phase ◽  
Kynurenine Pathway ◽  
Serum Samples ◽  
Esi Ms ◽  
Gastric Cancer Patients ◽  
Development And Validation

Metabolites and enzymes involved in the kynurenine pathway (KP) are highly promising targets for cancer treatment, including gastrointestinal tract diseases. Thus, accurate quantification of these compounds in body fluids becomes increasingly important. The aim of this study was the development and validation of the UHPLC-ESI-MS/MS methods for targeted quantification of biologically important KP substrates (tryptophan and nicotinamide) and metabolites(kynurenines) in samples of serum and peritoneal fluid from gastric cancer patients. The serum samples were simply pretreated with trichloroacetic acid to precipitate proteins. The peritoneal fluid was purified by solid-phase extraction before analysis. Validation was carried out for both matrices independently. Analysis of the samples from gastric cancer patients showed different accumulations of tryptophan and its metabolites in different biofluids of the same patient. The protocols will be used for the evaluation of tryptophan and kynurenines in blood and peritoneal fluid to determine correlation with the clinicopathological status of gastric cancer or the disease’s prognosis.

scholarly journals Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2

2004 ◽  
Vol 50 (9) ◽  
pp. 1607-1617 ◽  
Author(s):  
Ville Väisänen ◽  
Susann Eriksson ◽  
Kaisa K Ivaska ◽  
Hans Lilja ◽  
Martti Nurmi ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Solid Phase ◽  
Specific Antigen ◽  
Control Group ◽  
Serum Samples ◽  
Human Kallikrein 2 ◽  
Kallikrein 2 ◽  
Capture Antibody ◽  
Total Psa

Abstract Background: Free and total human kallikrein 2 (hK2) might improve the discrimination between prostate cancer and benign prostatic hyperplasia. Concentrations of hK2 are 100-fold lower than concentrations of prostate-specific antigen (PSA); therefore, an hK2 assay must have a low detection limit and good specificity. Methods: PSA- and hK2-specific monoclonal antibodies were used in solid-phase, two-site immunofluorometric assays to detect free and total hK2. The total hK2 assay used PSA-specific antibodies to block nonspecific signal. The capture antibody of the free hK2 assay did not cross-react with PSA. To determine the hK2 concentrations in the male bloodstream, total hK2 was measured in a control group consisting of 426 noncharacterized serum samples. Free and total hK2 were measured in plasma from 103 patients with confirmed prostate cancer. Results: All 426 males in the control group had a total hK2 concentration above the detection limit of 0.0008 μg/L. The median total hK2 concentration was 0.022 μg/L (range, 0.0015–0.37 μg/L). hK2 concentrations were 0.1–58% of total PSA (median, 3.6%). hK2 concentrations were similar in men 41–50 and 51–60 years of age. The ratio of hK2 to PSA steadily decreased from 5–30% at PSA <1 μg/L to 1–2% at higher PSA concentrations. In 103 patients with prostate cancer, the median hK2 concentration in plasma was 0.079 μg/L (range, 0.0015–16.2 μg/L). The median free hK2 concentration was 0.070 (range, 0.005–12.2) μg/L. The proportion of free to total hK2 varied from 17% to 131% (mean, 85%). Conclusions: The wide variation in the free-to-total hK2 ratio suggests that hK2 in blood plasma is not consistently in the free, noncomplexed form in patients with prostate cancer. The new assay is sufficiently sensitive to be used to study the diagnostic accuracies of free and total hK2 for prostate cancer.

scholarly journals Ribavirin Quantification in Combination Treatment of Chronic Hepatitis C

2003 ◽  
Vol 47 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Sylvie Larrat ◽  
Françoise Stanke-Labesque ◽  
Agnès Plages ◽  
Jean-Pierre Zarski ◽  
Germain Bessard ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Combination Treatment ◽  
High Performance ◽  
Solid Phase ◽  
Ammonium Phosphate ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Immunodeficiency Virus

ABSTRACT Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C18-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4°C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 ± 1.06 μg/ml (n = 24) at week 12 of treatment (W12) and 3.24 ± 1.35 μg/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

scholarly journals Entamoeba histolytica acetyl–CoA synthetase: biomarker of acute amoebic liver abscess

2014 ◽  
Vol 4 (6) ◽  
pp. 446-450
Author(s):  
Lim Boon Huat ◽  
Alfonso Olivos Garcia ◽  
Tan Zi Ning ◽  
Wong Weng Kin ◽  
Rahmah Noordin ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Liver Abscess ◽  
Amoebic Liver Abscess ◽  
Acetyl Coa

scholarly journals LC-MS/MS for Identifying Patients with CYP24A1 Mutations

2016 ◽  
Vol 62 (1) ◽  
pp. 236-242 ◽  
Author(s):  
Hemamalini Ketha ◽  
Rajiv Kumar ◽  
Ravinder J Singh
Keyword(s):  
Genetic Testing ◽  
Solid Phase ◽  
Internal Standard ◽  
Measurement Range ◽  
High Ratio ◽  
Unknown Etiology ◽  
Loss Of Function ◽  
Serum Samples ◽  
Dihydroxyvitamin D ◽  
Cytochrome P450 Family

Abstract BACKGROUND Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing. METHODS After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia. RESULTS The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%–15% across the analytical measurement range of 0.1–25 ng/mL (0.2–60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7–35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99–467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing. CONCLUSIONS Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.

scholarly journals Quantitative Determination of Azithromycin in Human Plasma by Liquid Chromatography–Mass Spectrometry and its Application in Pharmackokinetic Study

2012 ◽  
Vol 11 (1) ◽  
pp. 55-63 ◽  
Author(s):  
Maizbha Uddin Ahmed ◽  
Mohammad Safiqul Islam ◽  
Tasmin Ara Sultana ◽  
AGM Mostofa ◽  
Muhammad Shahdaat Bin Sayeed ◽  
...  
Keyword(s):  
Mobile Phase ◽  
Solid Phase ◽  
Internal Standard ◽  
Extraction Process ◽  
Serum Samples ◽  
Limit Of Quantification ◽  
Relative Standard ◽  
Linear Concentration Range ◽  
Linear Concentration

Azithromycin is an effective and well-known antimicrobial agent. In the present study, a simple, sensitive and specific LC/MS/MS method has been developed and validated for the quantification of Azithromycin in  human serum samples using Clarithromycin as internal standard. Azithromycin was extracted from biological matrix  by using solid phase extraction process. The chromatographic separation was performed on Luna C18 (3 ?, 2x150   mm) column with a mobile phase consisting of 35 mM ammonium acetate buffer (mobile phase-A) and acetonitrile  and methanol in ratio of 90:10 ( as mobile phase-B) at a flow rate of 0.25 mL/min. The method was validated over a  linear concentration range of 0.5?50.0 ng/mL and limit of quantification (LOQ) was 0.5 ng/mL with a coefficient of  correlation (r2) = 0.9998. The intra-day and inter-day precision expressed as relative standard deviation were 1.64% – 8.43% and 2.32% – 9.92%, respectively. The average recovery of azithromycin from serum was 98.11%. The method  was successfully applied to a pharmacokinetic study after oral administration of Azithromycin 200 mg/5 ml suspension in healthy Bangladeshi volunteers. DOI: http://dx.doi.org/10.3329/dujps.v11i1.12488 Dhaka Univ. J. Pharm. Sci. 11(1): 55-63, 2012 (June)

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