Background: Tuberculosis (TB) remains a burden to Sri Lanka, where the incidence of the disease has been increasing over the past decade. The lack of early and accurate detection of the disease has been the main obsta...Background: Tuberculosis (TB) remains a burden to Sri Lanka, where the incidence of the disease has been increasing over the past decade. The lack of early and accurate detection of the disease has been the main obstacle to its control. Microscopy or the culturing of mycobacteria from clinical samples is the most commonly used TB diagnostic tools in Sri Lanka. All these methods have their own limitations. Alternative diagnostic methods are therefore of high importance. Objectives: In this study, an attempt was made to validate loop mediated isothermal amplification (LAMP), which specifically amplifies a DNA sequence very rapidly at a low cost with limited resources. Methods: Crude DNA extractions of fifty culture isolates prepared from sputum samples, which were collected from patients with suspected TB extracts, were subjected to three separate LAMP assays. One assay was specific for 16S ribosomal RNA (16S rRNA) gene in genus Mycobacterium, and could detect the bacteria up to the genus level. The other two contained MTB specific primers targeting rimM or gyrB gene sequences in Mycobacterium tuberculosis (MTB), which enabled detection up to the species level. The sensitivity and specificity of the LAMP assays in the identification of mycobacteria or MTB were compared to microscopy and culture techniques. Results: Forty three out of the 47 Mycobacterium cultures were Mycobacterium-positive for LAMP assays with universal primers indicating a sensitivity of 92% in identifying Mycobacterium genus. However, thirteen out of 14 culture negatives were also positive with LAMP assays, which showed a specificity of only 7% in identifying MTB. The results suggested a high percentage of false positives by LAMP assays as compared to culture. Based on the colour changing of ZYBR Green dye and gel electrophoresis of the LAMP-amplified product, the detection of a non-specific amplification, even in the absence of target DNA, was recurrently observed. The result was the same even after following strict safety operations and labora展开更多
Fusarium wilt of melon, Fusarium oxysporum f.sp. melonis is one of the most important diseases, causing tremendous losses in melon growing areas in Iran. There is a real need for a rapid and inexpensive assay to facil...Fusarium wilt of melon, Fusarium oxysporum f.sp. melonis is one of the most important diseases, causing tremendous losses in melon growing areas in Iran. There is a real need for a rapid and inexpensive assay to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. One of the procedures designed for detection of this disease is loop-mediated isothermal amplification(LAMP)assay; its efficiency has been contrasted with polymerase chain reaction(PCR). The translation elongation factor 1-alpha gene is basically used for designing the LAMP(i.e. F3, B3, FIP, and BIP) together with PCR(F and B). Using hydroxynaphthol blue(HNB) dye, LAMP was placed in a water bath after the optimization was done. The results show LAMP is an advantageous method because it is highly sensitive(100-fold), quite cheap,user-friendly, and safe; in addition, it is performed quickly by visual detection and does not require DNA extraction(in direct-LAMP). The LAMP is believed to be a simple and reliable tool for laboratory purposes because it needs only very basic instruments and the results can be observed and contrasted visually.展开更多
Tuberculosis represents a main concern for public health worldwide. In poor countries, the most prevalent method for bacteriological confirmation re- mains Smear Sputum Microscopy (SSM). This study objective was to as...Tuberculosis represents a main concern for public health worldwide. In poor countries, the most prevalent method for bacteriological confirmation re- mains Smear Sputum Microscopy (SSM). This study objective was to assess clinical performances of Loop Mediated Isothermal Amplification for TB detection (Lamp-TB). Sputum of patients presenting symptoms consistent with tuberculosis were collected according to the National Tuberculosis Control Programme guidelines in Centre Antituberculeux de Yopougon. SSM after Ziehl-Neelsen staining and TB-Lamp were blindly performed with spot sputum specimen. Samples, transported at Institut Pasteur de Cote d’Ivoire were decontaminated according to N-acetyl-L-cystein (NALC) method. In Mycobacteria Growth Indicator Tube (MGIT), 500 μl of pellet were inoculated and incubated in MGIT 960 instrument. MPT64 antigen was detected on positive culture. Of 500 patients enrolled, 469 were included. Clinical isolates of M. tuberculosis Complex were detected for 157 (33.5%). Comparatively to culture, Sensitivity and Specificity of SSM were 86% (95% Confidence interval (CI): 81% - 91%) 96% (95%IC: 94% - 98%) respectively. TB-Lamp Sensitivity was 92% (95%CI: 88% - 96%), and Specificity 94% (95%CI: 91% - 97%). Positive Predictive Value of SSM and TB-Lamp was 91.8% and 88.8% respectively. Negative Predictive Value of TB-Lamp assay was 95.7% whereas this of SSM was 93.3%. Positive Likelihood Ratio was 15.3 for TB-Lamp and 21.5 for SSM 21.5 whereas negative Likelihood of TB-Lamp was lower than SSM. Active tuberculosis was detected in162/469 (34.5%) with TB-Lamp and 147 (31.3%) with SSM. TB-Lamp assay performances estimated from sputum samples may improve detection of active TB cases in routine.展开更多
<span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Background: </span></b></span><span><span><span style="font-family:&...<span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Background: </span></b></span><span><span><span style="font-family:""><span style="font-family:Verdana;">Due to the limitations of diagnosis by Xpert MTB/RIF assay, WHO suggests Loop</span><b><span style="font-family:Verdana;">—</span></b><span style="font-family:Verdana;">Mediated Isothermal Amplification (TB-LAMP) instead of sputum-smear microscopy for pulmonary TB diagnosis in patients. Dr. Thongchai Kaewphinit <i></span><i><span style="font-family:Verdana;">et al</span></i><span style="font-family:Verdana;"></i></span><i><span style="font-family:Verdana;">.</span></i><span style="font-family:Verdana;"> invented a fast TB detection kit called TB d-tect (LAMP-LFD assay). There was no clinical trial to estimate the performance of TB d-tect. </span><b><span style="font-family:Verdana;">Objective:</span></b><span style="font-family:Verdana;"> This study was aimed to find the performance of LFD assay and Xpert MTB/RIF ultra for pulmonary TB. </span><b><span style="font-family:Verdana;">Material</span></b> <b><span style="font-family:Verdana;">and</span></b> <b><span style="font-family:Verdana;">methods:</span></b><span style="font-family:Verdana;"> A cross-sectional study was conducted. Suggestive pulmonary TB patients were enrolled from June 2020-28 February 2021.</span></span></span></span><span><span><span style="font-family:""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">Respiratory specimen</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">s</span></span></span><span><span><span style="font-family:""><span style="font-family:Verdana;"> were collected from each patient and sent for AFB smear, LAMP-LFD assay, Xpert MTB/RIF ultra and culture TB. </span><b><span style="font-family:Verdana;">Result:</span></b><span style="font-family:Verdana;"> 139 patients with suspected pulmonary TB we展开更多
文摘Background: Tuberculosis (TB) remains a burden to Sri Lanka, where the incidence of the disease has been increasing over the past decade. The lack of early and accurate detection of the disease has been the main obstacle to its control. Microscopy or the culturing of mycobacteria from clinical samples is the most commonly used TB diagnostic tools in Sri Lanka. All these methods have their own limitations. Alternative diagnostic methods are therefore of high importance. Objectives: In this study, an attempt was made to validate loop mediated isothermal amplification (LAMP), which specifically amplifies a DNA sequence very rapidly at a low cost with limited resources. Methods: Crude DNA extractions of fifty culture isolates prepared from sputum samples, which were collected from patients with suspected TB extracts, were subjected to three separate LAMP assays. One assay was specific for 16S ribosomal RNA (16S rRNA) gene in genus Mycobacterium, and could detect the bacteria up to the genus level. The other two contained MTB specific primers targeting rimM or gyrB gene sequences in Mycobacterium tuberculosis (MTB), which enabled detection up to the species level. The sensitivity and specificity of the LAMP assays in the identification of mycobacteria or MTB were compared to microscopy and culture techniques. Results: Forty three out of the 47 Mycobacterium cultures were Mycobacterium-positive for LAMP assays with universal primers indicating a sensitivity of 92% in identifying Mycobacterium genus. However, thirteen out of 14 culture negatives were also positive with LAMP assays, which showed a specificity of only 7% in identifying MTB. The results suggested a high percentage of false positives by LAMP assays as compared to culture. Based on the colour changing of ZYBR Green dye and gel electrophoresis of the LAMP-amplified product, the detection of a non-specific amplification, even in the absence of target DNA, was recurrently observed. The result was the same even after following strict safety operations and labora
基金the Young Researchers and Elites Club,North Tehran Branch,Islamic Azad University, Tehran, Iran for financial support
文摘Fusarium wilt of melon, Fusarium oxysporum f.sp. melonis is one of the most important diseases, causing tremendous losses in melon growing areas in Iran. There is a real need for a rapid and inexpensive assay to facilitate accurate disease diagnosis and surveillance for better management of Fusarium wilt outbreaks. One of the procedures designed for detection of this disease is loop-mediated isothermal amplification(LAMP)assay; its efficiency has been contrasted with polymerase chain reaction(PCR). The translation elongation factor 1-alpha gene is basically used for designing the LAMP(i.e. F3, B3, FIP, and BIP) together with PCR(F and B). Using hydroxynaphthol blue(HNB) dye, LAMP was placed in a water bath after the optimization was done. The results show LAMP is an advantageous method because it is highly sensitive(100-fold), quite cheap,user-friendly, and safe; in addition, it is performed quickly by visual detection and does not require DNA extraction(in direct-LAMP). The LAMP is believed to be a simple and reliable tool for laboratory purposes because it needs only very basic instruments and the results can be observed and contrasted visually.
文摘Tuberculosis represents a main concern for public health worldwide. In poor countries, the most prevalent method for bacteriological confirmation re- mains Smear Sputum Microscopy (SSM). This study objective was to assess clinical performances of Loop Mediated Isothermal Amplification for TB detection (Lamp-TB). Sputum of patients presenting symptoms consistent with tuberculosis were collected according to the National Tuberculosis Control Programme guidelines in Centre Antituberculeux de Yopougon. SSM after Ziehl-Neelsen staining and TB-Lamp were blindly performed with spot sputum specimen. Samples, transported at Institut Pasteur de Cote d’Ivoire were decontaminated according to N-acetyl-L-cystein (NALC) method. In Mycobacteria Growth Indicator Tube (MGIT), 500 μl of pellet were inoculated and incubated in MGIT 960 instrument. MPT64 antigen was detected on positive culture. Of 500 patients enrolled, 469 were included. Clinical isolates of M. tuberculosis Complex were detected for 157 (33.5%). Comparatively to culture, Sensitivity and Specificity of SSM were 86% (95% Confidence interval (CI): 81% - 91%) 96% (95%IC: 94% - 98%) respectively. TB-Lamp Sensitivity was 92% (95%CI: 88% - 96%), and Specificity 94% (95%CI: 91% - 97%). Positive Predictive Value of SSM and TB-Lamp was 91.8% and 88.8% respectively. Negative Predictive Value of TB-Lamp assay was 95.7% whereas this of SSM was 93.3%. Positive Likelihood Ratio was 15.3 for TB-Lamp and 21.5 for SSM 21.5 whereas negative Likelihood of TB-Lamp was lower than SSM. Active tuberculosis was detected in162/469 (34.5%) with TB-Lamp and 147 (31.3%) with SSM. TB-Lamp assay performances estimated from sputum samples may improve detection of active TB cases in routine.
文摘<span style="font-family:Verdana;"><b><span style="font-family:Verdana;">Background: </span></b></span><span><span><span style="font-family:""><span style="font-family:Verdana;">Due to the limitations of diagnosis by Xpert MTB/RIF assay, WHO suggests Loop</span><b><span style="font-family:Verdana;">—</span></b><span style="font-family:Verdana;">Mediated Isothermal Amplification (TB-LAMP) instead of sputum-smear microscopy for pulmonary TB diagnosis in patients. Dr. Thongchai Kaewphinit <i></span><i><span style="font-family:Verdana;">et al</span></i><span style="font-family:Verdana;"></i></span><i><span style="font-family:Verdana;">.</span></i><span style="font-family:Verdana;"> invented a fast TB detection kit called TB d-tect (LAMP-LFD assay). There was no clinical trial to estimate the performance of TB d-tect. </span><b><span style="font-family:Verdana;">Objective:</span></b><span style="font-family:Verdana;"> This study was aimed to find the performance of LFD assay and Xpert MTB/RIF ultra for pulmonary TB. </span><b><span style="font-family:Verdana;">Material</span></b> <b><span style="font-family:Verdana;">and</span></b> <b><span style="font-family:Verdana;">methods:</span></b><span style="font-family:Verdana;"> A cross-sectional study was conducted. Suggestive pulmonary TB patients were enrolled from June 2020-28 February 2021.</span></span></span></span><span><span><span style="font-family:""> </span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">Respiratory specimen</span></span></span><span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">s</span></span></span><span><span><span style="font-family:""><span style="font-family:Verdana;"> were collected from each patient and sent for AFB smear, LAMP-LFD assay, Xpert MTB/RIF ultra and culture TB. </span><b><span style="font-family:Verdana;">Result:</span></b><span style="font-family:Verdana;"> 139 patients with suspected pulmonary TB we
