scholarly journals Urine lipoarabinomannan in HIV uninfected, smear negative, symptomatic TB patients: effective sample pretreatment for a sensitive immunoassay and mass spectrometry

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anita G. Amin ◽  
Prithwiraj De ◽  
Barbara Graham ◽  
Roger I. Calderon ◽  
Molly F. Franke ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Sample Pretreatment ◽  
Positive Association ◽  
Proteinase K ◽  
Clinical Samples ◽  
Sputum Smear ◽  
Smear Negative ◽  
Tuberculostearic Acid ◽  
Negative Sputum ◽  
Adult Culture

AbstractOur study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD450) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d-arabinose (d-ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

scholarly journals Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the First Rapid Multiplex PCR Commercial Assay for SARS-CoV-2 Detection

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Benoit Visseaux ◽  
Quentin Le Hingrat ◽  
Gilles Collin ◽  
Donia Bouzid ◽  
Samuel Lebourgeois ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Limit Of Detection ◽  
Clinical Sample ◽  
Point Of Care ◽  
Sample Pretreatment ◽  
Tracheal Aspirate ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Viral Neutralization ◽  
Efficient Detection

ABSTRACT In the race to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), efficient detection and triage of infected patients must rely on rapid and reliable testing. In this work, we performed the first evaluation of the QIAstat-Dx respiratory SARS-CoV-2 panel (QIAstat-SARS) for SARS-CoV-2 detection. This assay is the first rapid multiplex PCR (mPCR) assay, including SARS-CoV-2 detection, and is fully compatible with a non-PCR-trained laboratory or point-of-care (PoC) testing. This evaluation was performed using 69 primary clinical samples (66 nasopharyngeal swabs [NPS], 1 bronchoalveolar lavage fluid sample [BAL], 1 tracheal aspirate sample, and 1 bronchial aspirate sample) comparing SARS-CoV-2 detection with the currently WHO-recommended reverse transcription-PCR (RT-PCR) (WHO-RT-PCR) workflow. Additionally, a comparative limit of detection (LoD) assessment was performed for QIAstat-SARS and WHO-RT-PCR using a quantified clinical sample. Compatibility of sample pretreatment for viral neutralization or viscous samples with the QIAstat-SARS system were also tested. The QIAstat-Dx respiratory SARS-CoV-2 panel demonstrated a sensitivity comparable to that of the WHO-recommended assay with a limit of detection at 1,000 copies/ml. The overall percent agreement between QIAstat-Dx SARS and WHO-RT-PCR on 69 clinical samples was 97% with a sensitivity of 100% (40/40) and specificity at 93% (27/29). No cross-reaction was encountered for any other respiratory viruses or bacteria included in the panel. The QIAstat-SARS rapid multiplex PCR panel provides a highly sensitive, robust, and accurate assay for rapid detection of SARS-CoV-2. This assay allows rapid decisions even in non-PCR-trained laboratory or point-of-care testing, allowing innovative organization.

scholarly journals Immobilization of Proteinase K for urine pretreatment to improve diagnostic accuracy of active tuberculosis

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257615
Author(s):  
Yosita Panraksa ◽  
Anita G. Amin ◽  
Barbara Graham ◽  
Charles S. Henry ◽  
Delphi Chatterjee
Keyword(s):  
Point Of Care ◽  
Sample Pretreatment ◽  
Urine Samples ◽  
Proteinase K ◽  
World Health ◽  
Clinical Samples ◽  
Hiv Status ◽  
Capture Elisa ◽  
Resource Limited ◽  
Health Organization

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.

scholarly journals Detection of Mycobacterium tuberculosis in AFB smear-negative sputum specimens through MTB culture and GeneXpert® MTB/RIF assay

2019 ◽  
Vol 33 ◽  
pp. 205873841982717 ◽  
Author(s):  
Ghulam Rasool ◽  
Arif Muhammad Khan ◽  
Raza Mohy-Ud-Din ◽  
Muhammad Riaz
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Methods ◽  
Public Health Issue ◽  
Sputum Smear ◽  
Smear Microscopy ◽  
Drug Resistant ◽  
Resistant Tuberculosis ◽  
Study Population ◽  
Smear Negative ◽  
Negative Sputum

Tuberculosis (TB) is an important public health issue around the globe which is a chronic infectious disease and is still one of the major challenges for developing countries. The emergence of drug-resistant TB makes the condition worse and there is an urgent need of fast, highly sensitive diagnostic methods. This study was undertaken to evaluate the performance of GeneXpert® MTB/RIF assay and MTB culture for the detection of Mycobacterium tuberculosis (MTB) in sputum smear-negative pulmonary TB/drug-resistant tuberculosis (DR-TB) suspects. A total of 168 sputum smear-negative TB suspects were recruited for the study. Among the suspected TB cases, 52.98% were male and 47.02% were females with the mean age of 42 ± 17.6 years. All the sputum specimens collected from the study population were subjected to Ziehl–Neelsen (ZN) smear microscopy, GeneXpert MTB/RIF assay, and MTB culture. The results revealed that, out of 168 acid-fast bacilli (AFB)/ZN smear microscopy–negative sputum specimens, 48 (28.57%) and 58 (34.52%) were detected MTB positive by GeneXpert MTB/RIF assay and MTB culture, respectively, while 120 (71.43%) and 110 (65.48%) suspected TB cases were confirmed negative by GeneXpert MTB/RIF assay and MTB culture, respectively. The study concluded that GeneXpert assay was found to be a rapid and accurate tool for MTB detection in smear-negative sputum specimens. GeneXpert has advantage over ZN smear microscopy and MTB culture as it detects MTB and rifampicin resistance simultaneously within 2 h with minimal biohazards.

scholarly journals Comparison of LJ Medium and BACTEC MGIT 960 Culture System for the Diagnosis of Tuberculosis

2020 ◽  
Author(s):  
Pinki Kumari ◽  
Jiwesh Kumar Thakur ◽  
Prashant Kumar ◽  
Rakesh Kumar ◽  
Deval Parekh
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Public Health Problem ◽  
Categorical Variables ◽  
Clinical Samples ◽  
Sputum Smear ◽  
Major Public Health Problem ◽  
Continuous Variables ◽  
Cross Sectional ◽  
The Mean ◽  
Smear Negative

Introduction: Sputum negative pulmonary Tuberculosis (TB) is a major public health problem. So, the emergence of new techniques for a more precise and rapid microbiological identification of Mycobacterium tuberculosis in clinical samples is of great importance to improve the management of TB. Aim: To determine and compare the sensitivity and turnaround time for Mycobacterium tuberculosis detection by the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system, Lowenstein Jensen (LJ) medium and Ziehl-Neelsen (ZN) staining. Materials and Methods: An Institution based, observational, cross-sectional study was conducted at Rajendra Institute of Medical Sciences (RIMS), Ranchi, Jharkhand, India, from July 2013-March 2016. Sputum, pericardial fluid, pleural fluid, peritoneal fluid, pus and endometrial tissue samples were collected from 80 patients of suspected TB cases. All were Acid-Fast stained by ZN staining method and cultured on solid culture LJ medium and on liquid medium (MGIT). Data was analysed using Statistical Package for the Social Sciences (SPSS) software, Version 20.0 (SPSS Inc, Chicago, IL, USA). Fisher’s-Exact test was used to show association of categorical variables. Non-parametric Mann-Whitney U test was used to show median difference of non-normally distributed continuous variables of two groups. Results: Out of the 80 samples, 41 cases were positive by either of the all methods. The positive specimen for ZN staining, LJ media and MGIT were 21, 29 and 41 cases respectively. The mean Time To Detection (TTD) was shorter for MGIT system than LJ media. Both LJ medium and MGIT 960 detected all cases of sputum smear positive cases and in addition significantly higher number than ZN stain in sputum smear negative cases. MGIT 960 detected significantly higher number of cases of sputum negative cases than LJ Media. The mean TTD was also significantly shorter in case of smear positive cases than the smear negative cases by both the solid and liquid culture mediums. Conclusion: The use of liquid media (MGIT) is more accurate and rapid method for the diagnosis of TB. The combination of more than one method is also highly recommended for rapid detection and early treatment of TB.

scholarly journals Diagnostic Outcomes After Chest Radiograph Interpretation in Patients With Suspected Tuberculosis and Negative Sputum Smears in a High-Burden Human Immunodeficiency Virus and Tuberculosis Setting

2017 ◽  
Vol 4 (3) ◽  
Author(s):  
Patrick G. T. Cudahy ◽  
Rodney Dawson ◽  
Brian W. Allwood ◽  
Gary Maartens ◽  
Douglas Wilson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Pulmonary Tuberculosis ◽  
Chest Radiographs ◽  
World Health ◽  
Sputum Smear ◽  
Lower Sensitivity ◽  
Immunodeficiency Virus ◽  
Suspected Tuberculosis ◽  
Smear Negative ◽  
Negative Sputum

Abstract Background Evaluation of patients with suspected tuberculosis and negative sputum smears for acid-fast bacilli (AFB) is challenging, especially in high human immunodeficiency virus coinfection settings where sputum smears have lower sensitivity for detecting AFB. Methods We examined the utility of chest radiographs for detecting smear-negative pulmonary tuberculosis. Three hundred sixty sputum smear–negative patients who were referred from primary care clinics in the KwaZulu-Natal province of South Africa were evaluated. Chest radiographs were read by experienced pulmonologists using a previously validated Chest X-Ray Reading and Recording System (CRRS). Results Agreement between observers using CRRS was high at 91% with a Cohen’s kappa of 0.64 (95% confidence interval [CI] = 0.52–0.76). Against a reference standard of sputum culture, sensitivity was 93% (95% CI = 86%–97%), whereas specificity was 14% (95% CI = 10%–19%). Performance against clinical diagnosis (following World Health Organization guidelines) was similar with sensitivity of 92% (95% CI = 88%–95%) and specificity of 20% (95% CI = 13%–28%). Conclusion The low specificity of CRRS in this setting indicates poor diagnostic utility for detecting pulmonary tuberculosis.

Detection of Mycobacterium tuberculosis in smear negative sputum by PCR

2012 ◽  
Vol 6 (2) ◽  
pp. 2-6 ◽  
Author(s):  
Mohammad Jobayer ◽  
SM Shamsuzzaman ◽  
Kazi Zulfiquer Mamun
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pulmonary Tuberculosis ◽  
Accurate Method ◽  
Major Health Problem ◽  
Major Health ◽  
Tuberculosis Patients ◽  
Diagnostic Potential ◽  
Total Death ◽  
Smear Negative ◽  
Negative Sputum

Pulmonary tuberculosis is a major health problem in Bangladesh that is responsible for about 7% of total death in a year. This study was conducted to isolate and identify Mycobacterium tuberculosis from sputum and to evaluate the efficacy of PCR as a modern diagnostic tool, for diagnosis of pulmonary tuberculosis, especially in the smear negative cases. One hundred and fifty suspected pulmonary TB patients (male/ female: 97/53) were included in this study. Single morning sputum was collected from each patient and diagnostic potential of PCR was compared with staining and culture. Twenty five (16.7%) sputum were positive by ZN stained smear. Among 125 smear negative samples, 13 (10.4%) yielded growth in culture in LJ media and 21 (16.8%) samples were positive by PCR. The sensitivity and specificity of PCR in smear negative cases was 100% and 92.9% respectively. Mean detection time in PCR was 24 hours. PCR detected M. tuberculosis in 21 smear negative and 9 culture negative samples. For diagnosis of tuberculosis in smear negative cases, PCR directly from sputum was a very sensitive and accurate method. In conclusion, PCR may be done, especially in clinically suspected pulmonary tuberculosis patients who remain negative by conventional methods.DOI: http://dx.doi.org/10.3329/bjmm.v6i2.19368 Bangladesh J Med Microbiol 2012; 06(02): 2-6

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