scholarly journals Integrated qPCR and Staining Methods for Detection and Quantification of Enterocytozoon hepatopenaei in Shrimp Litopenaeus vannamei

2020 ◽  
Vol 8 (9) ◽  
pp. 1366
Author(s):  
Lijun Wang ◽  
Qing Lv ◽  
Yantong He ◽  
Ruocheng Gu ◽  
Bingqian Zhou ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Shrimp Farming ◽  
Economic Losses ◽  
Amplification Efficiency ◽  
Qpcr Method ◽  
Staining Methods ◽  
Microscopy Method ◽  
Optimum Annealing Temperature ◽  
Enterocytozoon Hepatopenaei ◽  
Sensitivity Specificity

Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

scholarly journals miR-34a-5p, miR-148a-3p and miR-181a-5p: potential biomarkers for the diagnosis and prognosis of esophageal cancer detected by a novel absolute quantitative RT-qPCR method

2020 ◽  
Author(s):  
Zhiyuan Lin ◽  
Yongquan Chen ◽  
Yanling Lin ◽  
Huayue Lin ◽  
Hongwei Li ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
High Sensitivity ◽  
Diagnosis And Prognosis ◽  
Qpcr Method ◽  
Sensitivity Specificity ◽  
Combined Detection ◽  
Normal Controls ◽  
Progressive Dysphagia ◽  
Potential Biomarkers ◽  
Main Clinical Manifestation

Abstract Background Esophageal cancer (EC) is a malignant tumor of esophagus with progressive dysphagia as the main clinical manifestation. miRNAs are expected to become potential biomarkers in diagnosis and prognosis of EC. Methods Based on AllGlo probes, a novel absolute quantitative RT-qPCR method was established to detect miRNAs. And the clinical diagnosis significance of the screened miRNAs was explored with 213 patients (166 cases with EC and 47 cases with benign diseases) and 170 normal controls. Results Through a series of screening, miR-34a-5p, miR-148a-3p and miR-181a-5p were selected as EC-associated candidate miRNAs. Based on AllGlo probes, a novel absolute quantitative RT-qPCR method for detecting miRNAs was established with high sensitivity, specificity, good accuracy and no carryover contamination. Compared with normal controls, the level of miR-34a-5p increased while miR-148a-3p and miR-181a-5p decreased in EC and benign patients ( P <0.001), and the level of miR-181-5p in early EC patients was significantly lower ( P <0.001). According to logistic regression analysis, the combined detection of miR-34a-5p, miR-148a-3p and Cyfra21-1 provided the highest diagnosis efficiency of 85.07% with sensitivity and specificity reaching 85.45% and 84.71%. Compared with preoperative samples, the level of miR-34a-5p significantly decreased while the levels of miR-148a-3p and miR-181a-5p significantly increased in the postoperative samples ( P <0.001). Conclusions Based on AllGlo probes, this first developed, novel absolute quantitative RT-qPCR method exhibits high application value in detecting miRNAs. miR-34a-5p, miR-148a-3p and miR-181a-5p may serve as potential biomarkers in the diagnosis and prognosis of EC, especially, miR-181-5p probably could be used as a new biomarker for early EC.

scholarly journals COMPARISON AMONG THREE TECHNIQUES IN THE PARASITOLOGICAL DIAGNOSIS OF Fasciola hepatica IN CATTLE

2014 ◽  
Vol 19 (1) ◽  
Author(s):  
Anderson Silva Dias ◽  
Jackson Victor de Araújo ◽  
Fábio Ribeiro Braga ◽  
Odilon Azevêdo Calian ◽  
Vinícius Longo Ribeiro Vilela ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Linear Regression ◽  
Analysis Of Variance ◽  
Fasciola Hepatica ◽  
High Sensitivity ◽  
Economic Losses ◽  
Kappa Test ◽  
Parasitological Diagnosis ◽  
Correlation Tests ◽  
Sensitivity Specificity

Helminthosis are relevant because they are responsible by substantial economic losses.   Many techniques have been developed to facilitate parasitological diagnosis and comparisons among these techniques are essential. The present work aimed a comparison among three techniques of quantitative sedimentation for parasitological diagnosis of Fasciola hepatica in cattle. The modified sedimentation techniques of Dennis, Stone & Swanson (DSS), Girão and Ueno (quatro tamises - QT; four sieves) and Foreyt were compared using analysis of variance, linear regression and correlation tests. Evaluations of sensitivity, specificity and agreement were performed using the kappa test. It was found that the modified DSS technique had a high mean egg count and high sensitivity (p<0.01) (48.60%). The three techniques showed a specificity of 100%. There was no correlation among the values of egg count obtained by the three techniques, and significant coefficients were not observed by regression analysis. The egg count results of F. hepatica obtained by the techniques of quatro tamises and Foreyt showed excellent concordance by the kappa test. The modified DSS technique appeared to be the most effective for F. hepatica diagnosis in cattle (p<0.01).

scholarly journals Diagnosis of bacterial vaginosis: Comparison of Nugent and novel microscopic method

2020 ◽  
pp. 105-105
Author(s):  
Dane Nenadic ◽  
Snezana Ribis ◽  
Milos Pavlovic ◽  
Dejan Baskic
Keyword(s):  
Bacterial Vaginosis ◽  
Scoring System ◽  
Relative Number ◽  
High Sensitivity ◽  
Kappa Index ◽  
Predictive Values ◽  
Microscopy Method ◽  
Index Sensitivity ◽  
Sensitivity Specificity ◽  
Good Agreement

Background/Aim: Bacterial vaginosis (BV) is common cause of vaginal discomfort in women. The aim of this study was comparison of Nugent's scoring system and novel microscopy method, introduced in our laboratory and used in BV diagnosis. Methods: 705 pregnant and asymptomatic women between 24 and 28 weeks of pregnancy participated in this prospective study. Degree of agreement between methods was determined by kappa index. Sensitivity, specificity, positive and negative predictive value of novel microscopy method was compared to Nugent's score as standard. Results: Based on scoring system of both methods, Nugent and novel microscopy method, BV was diagnosed in 21%, and 25% of women, respectively. Despite the disparities among diagnostic criteria, which mainly concerned classification of intermediary samples, the degree of agreement between categories, determined by kappa index, was satisfactory: Nugent vs novel microscopy method (?=0,68; good agreement), and Nugent vs novel microscopy method without intermediary results (?=0,83; very good agreement). We also demonstrated that compared to Nugent, as golden standard, novel microscopy method had high sensitivity and specificity (ranging from 75%-99.3%), and positive and negative predictive values (ranging from 88.8%-99.5%). Conclusion: novel microscopy method in diagnosis of BV, corresponded well with Nugent's scoring system which allows it to be an alternative method in diagnosing of BV. Our method is based on relative number of bacterial morphotypes, either rod forms (? 1.5?m, lactobacilli) or non-rod forms (< 1.5?m, bacterial vaginosis associated bacteria) under 200x magnification, which extends the surface of examined preparation, but without prolongation of observer's working time. Furthermore, novel microscopy method appeared to be flexible and can be reorganized in the way to categorize findings into only two groups: normal and BV, which makes it comparable to dichotomous Amsel's clinical criterion.

scholarly journals Potential miRNA biomarkers for the diagnosis and prognosis of esophageal cancer detected by a novel absolute quantitative RT-qPCR method

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhiyuan Lin ◽  
Yongquan Chen ◽  
Yanling Lin ◽  
Huayue Lin ◽  
Hongwei Li ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
High Sensitivity ◽  
Benign Diseases ◽  
Diagnosis And Prognosis ◽  
Qpcr Method ◽  
Sensitivity Specificity ◽  
Combined Detection ◽  
Normal Controls ◽  
Potential Biomarkers ◽  
High Application

AbstractmiRNAs are expected to become potential biomarkers in the diagnosis and prognosis of Esophageal cancer (EC). Through a series of screening, miR-34a-5p, miR-148a-3p and miR-181a-5p were selected as EC-associated miRNAs. Based on AllGlo probe, a novel absolute quantitative RT-qPCR method with high sensitivity, specificity and accuracy was established for detecting miRNAs. Then the clinical significance of these 3 miRNAs was explored with 213 patients (166 cases with EC and 47 cases with benign diseases) and 170 normal controls. Compared with normal controls, the level of miR-34a-5p increased while miR-148a-3p and miR-181a-5p decreased in EC and benign patients (P < 0.001), and the level of miR-181a-5p in early EC patients was significantly lower (P < 0.001). According to logistic regression analysis, combined detection of miR-34a-5p, miR-148a-3p and Cyfra21-1 provided the highest diagnosis efficiency of 85.07% with sensitivity and specificity reaching 85.45% and 84.71%. Compared with preoperative samples, the level of miR-34a-5p decreased while miR-148a-3p and miR-181a-5p increased in postoperative samples (P < 0.001). Collectively, this first developed, novel absolute quantitative RT-qPCR method exhibits high application value in detecting miRNAs, miR-34a-5p, miR-148a-3p and miR-181a-5p may serve as potential biomarkers in the diagnosis and prognosis of EC, and miR-181a-5p probably could serve as a new biomarker for early EC.

scholarly journals DETEKSI DINI Enterocytozoon hepatopenaei (EHP) PADA UDANG VANAME (Litopenaeus vannamei) MENGGUNAKAN METODE PCR (POLYMERASE CHAIN REACTION)

2018 ◽  
Vol 13 (3) ◽  
pp. 267
Author(s):  
Annisa Fitriah Faisal ◽  
Adi Pancoro
Keyword(s):  
Spore Wall ◽  
Shrimp Farming ◽  
Test Results ◽  
Pcr Products ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Whiteleg Shrimp ◽  
Optimum Annealing Temperature ◽  
Enterocytozoon Hepatopenaei

Sejak akhir tahun 2014, wabah kotoran putih atau yang sering disebut juga WFD (White Feces Disease), merupakan salah satu masalah yang sering terjadi pada petambak udang di Indonesia. Wabah ini diketahui disebabkan oleh Enterocytozoon hepatopenaei (EHP) dan telah mengakibatkan retardasi pertumbuhan hingga kematian pada udang. Hingga saat ini, penyakit WFD dapat dideteksi dengan cara uji histologi, hibridisasi in situ, dan PCR. Penelitian ini bertujuan untuk mendapatkan metode deteksi dini penyakit EHP pada udang vaname dengan metode PCR melalui perancangan primer yang spesifik dan sensitif. Pada penelitian ini dilakukan isolasi EHP pada udang vaname yang terinfeksi, kemudian dideteksi dengan metode PCR yang mentarget SWP (spore wall protein) dari EHP serta pengujian spesifitas dan sensitivitasnya. Hasil yang diperoleh menunjukkan bahwa EHP dapat diisolasi dari udang yang terinfeksi dan dapat didesain dua pasang primer yaitu SWP-EHP1 dan SWP-EHP3 yang mentarget spore wall protein EHP. Kedua primer ini dapat digunakan untuk deteksi EHP menggunakan PCR, dengan produk PCR pada primer SWP-EHP1 yaitu 398 bp dan primer SWP-EHP3 sebesar 415 bp, serta nilai suhu annealing optimal pada 48oC.Hasil pengujian sensitivitas primer, diketahui bahwa primer SWP-EHP1 dapat mendeteksi EHP hingga jumlah DNA target sebanyak 7,74 x 102 kopi sedangkan primer SWP-EHP3 dapat mendeteksi hingga 16,2 x 102 kopi.Since 2014, white feces disease (WFD) is one of the emerging problems for whiteleg shrimp farming industries in Indonesia. This outbreak is known to be caused by Enterocytozoon hepatopenaei (EHP) infection to shrimp. EHP infection resulted in growth retardation to a mass mortality in shrimp. To date, WFD can be detected by histology, in situ hybridization and PCR. This study aimed to obtain an early detection method of EHP on whiteleg shrimp by PCR method through specific and sensitive primers design. In this study, we isolated the DNA of EHP from infected whiteleg shrimp, then detected by PCR method which targeted spore wall protein (SWP) from EHP as well as sensitivity and specificity testing. As a result, EHP can be isolated from infected shrimp and can be designed 2 pairs of primers (SWP-EHP1 and SWP-EHP3) targeting spore wall protein of EHP. These primers could be used for EHP detection using PCR, with PCR products from primers SWP-EHP1 was 398 bp and from SWP-EHP3 primers was 415 bp, with an optimum annealing temperature of 48oC. Primers sensitivity test results revealed that primers SWP-EHP1 could detect EHP to 7.74 x 102 copies while the primers SWP-EHP3 could detect up to 16.2 x 102 copies.

scholarly journals Detection and quantification of Bacillus cereus and its spores in raw milk by qPCR, and distinguish Bacillus cereus from other bacteria of the genus Bacillus

2022 ◽  
Author(s):  
Lijiao Liang ◽  
Ping Wang ◽  
Tianming Qu ◽  
Xiaomei Zhao ◽  
Yiqiang Ge ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Pathogenic Bacteria ◽  
High Sensitivity ◽  
Raw Milk ◽  
Raw Material ◽  
Amplification Efficiency ◽  
Genus Bacillus ◽  
Application Range ◽  
Spoilage Bacteria ◽  
Qpcr Method

Abstract Introduction The raw milk is the basic raw material of dairy products, Bacillus cereus is a typical conditional pathogenic bacteria and cold-phagocytic spoilage bacteria in raw milk. This study established a qPCR method for detecting B. cereus in raw milk Materials and Methods In this study, a qPCR method for detecting B. cereus in raw milk was established. The specificity of the method was verified by using other Bacillus bacteria and pathogenic bacteria, the sensitivity of the method was evaluated by preparing recombinant plasmids and simulated contaminated samples, and the applicability of the method was verified by using pure spore DNA. The actual sample detection was completed by using the established qPCR method Results The qPCR established in this study can specifically detect B. cereus in raw milk. The LOD of the method was as low as 200 CFU/mL, and the LOQ ranged from 2 × 10 2 to 2 × 10 8 CFU/ml, the amplification efficiency of qPCR was 96.6% Conclusins The method established in this study can distinguish B. cereus from other Bacillus bacteria, and spore DNA can be used as the detection object. This method has the advantages of strong specificity, high sensitivity, wide application range and short detection time, which is expected to be applied in the dairy industry.

TO SEPARATELY ASSESS THE DIAGNOSTIC POTENTIAL OF TVS & MRI IN THE TERMS OF SENSITIVITY AND SPECIFICITY BY CORRELATING THE IMAGING FINDINGS WITH HISTOPATHOLOGY

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Suraj Mathur
Keyword(s):  
Transvaginal Ultrasound ◽  
High Sensitivity ◽  
Imaging Evaluation ◽  
Medical College ◽  
Conventional Mri ◽  
Diagnostic Potential ◽  
Adc Mapping ◽  
Malignant Lesions ◽  
Sensitivity Specificity

This prospective study was done in the Department of Radio diagnosis Govt. Medical College, Kozhikode. A total of 65 patients who were referred to our department with clinical suspicion of endometrial lesions and incidentally detected endometrial lesions on ultrasonography underwent transvaginal ultrasound and subsequent Imaging evaluation of pelvis MRI has very high sensitivity (95%) and specificity (98%) and is almost as accurate (97%) as histopathology in differentiating benign from malignant lesions. Addition of DWI with ADC mapping to conventional MRI increases its accuracy even more. However there is inherent limitation to MRI in detecting carcinoma in situ and micrometastasis. Keywords: TVS, MRI, Sensitivity, Specificity, Histopathology.

scholarly journals Use of β-D-glucan in diagnosis of suspected Pneumocystis jirovecii pneumonia in adults with HIV infection

2021 ◽  
pp. 095646242110222
Author(s):  
Thomas Juniper ◽  
Chris P Eades ◽  
Eliza Gil ◽  
Harriet Fodder ◽  
Killian Quinn ◽  
...  
Keyword(s):  
Predictive Value ◽  
Diagnostic Tests ◽  
Elevated Serum ◽  
Clinical Information ◽  
High Sensitivity ◽  
Pneumocystis Pneumonia ◽  
Pneumocystis Jirovecii Pneumonia ◽  
Alternative Diagnosis ◽  
Positive Threshold ◽  
Sensitivity Specificity

Objectives: An elevated serum (1-3)-β-D-glucan (BDG) concentration has high sensitivity for a diagnosis of Pneumocystis pneumonia (PCP) in people with HIV (PWH). At the current manufacturer-recommended positive threshold of 80 pg/mL (Fungitell), specificity for PCP is variable and other diagnostic tests are required. We evaluated the utility of serum BDG for diagnosis of suspected PCP in PWH at three inner-London hospitals to determine BDG concentrations for diagnosis and exclusion of PCP. Methods: From clinical case records, we abstracted demographic and clinical information and categorised patients as having confirmed or probable PCP, or an alternative diagnosis. We calculated sensitivity, specificity and positive predictive value (PPV) of serum BDG concentrations >400 pg/mL and negative predictive value (NPV) of BDG <80 pg/mL. Results: 76 patients were included; 29 had laboratory-confirmed PCP, 17 had probable PCP and 30 had an alternative diagnosis. Serum BDG >400 pg/mL had a sensitivity of 83%, specificity of 97% and PPV 97% for diagnosis of PCP; BDG <80 pg/mL had 100% NPV for exclusion of PCP. Conclusions: In PWH with suspected PCP, BDG <80 pg/mL excludes a diagnosis of PCP, whereas BDG concentrations >400 pg/mL effectively confirm the diagnosis. Values 80–400 pg/mL should prompt additional diagnostic tests.

scholarly journals The Child Behavior Checklist as a Screening Instrument for PTSD in Refugee Children

Children ◽  
2021 ◽  
Vol 8 (6) ◽  
pp. 521
Author(s):  
Ina Nehring ◽  
Heribert Sattel ◽  
Maesa Al-Hallak ◽  
Martin Sack ◽  
Peter Henningsen ◽  
...  
Keyword(s):  
Child Behavior ◽  
Child Behavior Checklist ◽  
High Sensitivity ◽  
Roc Curves ◽  
Structured Interview ◽  
Screening Instrument ◽  
Screening Tools ◽  
Refugee Children ◽  
Post Traumatic Stress ◽  
Sensitivity Specificity

Thousands of refugees who have entered Europe experienced threatening conditions, potentially leading to post traumatic stress disorder (PTSD), which has to be detected and treated early to avoid chronic manifestation, especially in children. We aimed to evaluate and test suitable screening tools to detect PTSD in children. Syrian refugee children aged 4–14 years were examined using the PTSD-semi-structured interview, the Kinder-DIPS, and the Child Behavior Checklist (CBCL). The latter was evaluated as a potential screening tool for PTSD using (i) the CBCL-PTSD subscale and (ii) an alternative subscale consisting of a psychometrically guided selection of items with an appropriate correlation to PTSD and a sufficient prevalence (presence in more than 20% of the cases with PTSD). For both tools we calculated sensitivity, specificity, and a receiver operating characteristic (ROC) curve. Depending on the sum score of the items, the 20-item CBCL-PTSD subscale as used in previous studies yielded a maximal sensitivity of 85% and specificity of 76%. The psychometrically guided item selection resulted in a sensitivity of 85% and a specificity of 83%. The areas under the ROC curves were the same for both tools (0.9). Both subscales may be suitable as screening instrument for PTSD in refugee children, as they reveal a high sensitivity and specificity.

scholarly journals Evaluation of HiCrome KPC Agar for the Screening of Carbapenem-Resistant Enterobacterales Colonization in the ICU Setting of a Tertiary Care Hospital

2021 ◽  
Author(s):  
Ashoka Mahapatra ◽  
K Nikitha ◽  
Sutapa Rath ◽  
Bijayini Behera ◽  
Kavita Gupta
Keyword(s):  
Tertiary Care ◽  
High Sensitivity ◽  
Care Hospital ◽  
Predictive Values ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Carbapenem Resistant ◽  
Rectal Swabs ◽  
Control And Prevention ◽  
Indian Setting ◽  
Sensitivity Specificity

Abstract Background Spread of carbapenem-resistant Enterobacterales (CRE) is a significant concern in intensive care unit (ICU) settings. Approaches to routine screening for CRE colonization in all ICU patients vary depending on institutional epidemiology and resources. The present study was aimed to evaluate the performance of HiCrome Klebsiella pneumoniae carbapenemase (KPC) agar for the detection of CRE colonization in ICU settings taking the Centers for Disease Control and Prevention (CDC) recommended method as reference. Methods Two-hundred and eighty rectal swabs (duplicate) from 140 patients were subjected to CRE detection in HiCrome KPC agar and MacConkey agar (CDC criteria). Results Using CDC method, total 41 CRE isolates were recovered comprising of 29 E scherichia coli, 11 Klebsiella, and 1 Enterobacter spp. On the other hand, 49 isolates of CRE recovered from 140 rectal swabs using HiCrome KPC agar, out of which 33 were E. coli, 15 Klebsiella, and 1 Enterobacter sp. Statistical Analysis Sensitivity, specificity, negative, and positive predictive values of CRE screening by HiCrome KPC agar were found to be 100% (91.4–100), 91.9% (84.8–95.8), 83.6% (70.9–91.4), and 100% (95.9–100), respectively, taking the CDC recommended method as reference. Conclusion HiCrome KPC agar has high sensitivity in screening CRE colonization. Further studies are needed to establish its applicability for detecting the predominant circulating carbapenemases in the Indian setting.

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