Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection

Nanomedicine ◽  
2020 ◽  
Vol 15 (26) ◽  
pp. 2609-2624
Author(s):  
Bhawna Dahiya ◽  
Tulika Prasad ◽  
Vishwajeet Singh ◽  
Anish Khan ◽  
Ekta Kamra ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Mycobacterium Tuberculosis ◽  
Diagnostic Accuracy ◽  
Magnetic Beads ◽  
Pcr Assay ◽  
Pulmonary Tb ◽  
Functionalized Gold Nanoparticles ◽  
Bodily Fluids ◽  
Smear Negative ◽  
Improved Technology

Aim: To improve the diagnostic accuracy of immuno-PCR (I-PCR) in tuberculosis (TB) patients by using functionalized gold nanoparticles (AuNPs) coupled with detection antibodies and oligonucleotides, and magnetic beads (MBs) conjugated with capture antibodies in the liquid phase. Materials & methods: MB-coupled AuNP-based I-PCR (MB-AuNP-I-PCR) assay was designed to detect a cocktail of Mycobacterium tuberculosis MPT64 and CFP-10 proteins in bodily fluids of TB patients. Results: The sensitivities of 89.3 (n = 94) and 78.1% (n = 73) were observed in pulmonary TB and extrapulmonary TB patients, respectively, with specificities of 97.9–98.3%. Notably, the sensitivities attained by MB-AuNP-I-PCR in smear-negative pulmonary TB and extrapulmonary TB patients were significantly higher (p < 0.05–0.001) than Magneto-ELISA and GeneXpert assay. Conclusion: The improved technology, as well as enhanced diagnostic accuracy of MB-AuNP-I-PCR, may lead to development of an attractive diagnostic kit.

scholarly journals Evaluation of a IS6110-Taqman real-time PCR assay to detect Mycobacterium tuberculosis in sputum samples of patients with pulmonary TB

2013 ◽  
Vol 114 (4) ◽  
pp. 1103-1108 ◽  
Author(s):  
L.A.S. Lira ◽  
F.C.F. Santos ◽  
M.S.Z. Carvalho ◽  
R.A. Montenegro ◽  
J.F.C. Lima ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Pulmonary Tb

scholarly journals Diagnostic challenges and Gene-Xpert utility in detecting Mycobacterium tuberculosis among suspected cases of Pulmonary tuberculosis

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251858
Author(s):  
Shaila Kabir ◽  
M. Tanveer Hossain Parash ◽  
Nor Amalina Emran ◽  
A. B. M. Tofazzal Hossain ◽  
Sadia Choudhury Shimmi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pulmonary Tuberculosis ◽  
Predictive Value ◽  
Demographic Data ◽  
Nucleic Acid Amplification ◽  
Age Group ◽  
Cross Sectional ◽  
Care Clinic ◽  
Pulmonary Tb ◽  
Smear Negative

The incidence of pulmonary tuberculosis (PTB) can be reduced by preventing transmission with rapid and precise case detection and early treatment. The Gene-Xpert MTB/RIF assay is a useful tool for detecting Mycobacterium tuberculosis (MTB) with rifampicin resistance within approximately two hours by using a nucleic acid amplification technique. This study was designed to reduce the underdiagnosis of smear-negative pulmonary TB and to assess the clinical and radiological characteristics of PTB patients. This cross-sectional study included 235 participants who went to the Luyang primary health care clinic from September 2016 to June 2017. The demographic data were analyzed to investigate the association of patient gender, age group, and ethnicity by chi-square test. To assess the efficacy of the diagnostic test, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated. The area under the curve for sputum for both AFB and gene-Xpert was analyzed to compare their accuracy in diagnosing TB. In this study, TB was more common in males than in females. The majority (50.71%) of the cases belonged to the 25–44-year-old age group and the Bajau ethnicity (57.74%). Out of 50 pulmonary TB cases (smear-positive with AFB staining), 49 samples were positive according to the Gene-Xpert MTB/RIF assay and was confirmed by MTB culture. However, out of 185 smear-negative presumptive cases, 21 cases were positive by Gene-Xpert MTB/RIF assay in that a sample showed drug resistance, and these results were confirmed by MTB culture, showing resistance to isoniazid. In comparison to sputum for AFB, Gene-Xpert showed more sensitivity and specificity with almost complete accuracy. The additional 21 PTB cases detection from the presumptive cases by GeneXpert had significant impact compared to initial observation by the routine tests which overcame the diagnostic challenges and ambiguities.

scholarly journals 1346. Ruling out TB in New York City: Are Two NAATs (Nucleic Acid Amplification Testing) Enough?

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S487-S488
Author(s):  
William G Greendyke ◽  
Janett Pike ◽  
Lilibeth V Andrada ◽  
Krystal Balzer ◽  
Thelesha Gray ◽  
...  
Keyword(s):  
New York ◽  
New York City ◽  
Nucleic Acid ◽  
Diagnostic Accuracy ◽  
York City ◽  
Median Number ◽  
Nucleic Acid Amplification ◽  
Pulmonary Tb ◽  
Nucleic Acid Amplification Testing ◽  
Smear Negative

Abstract Background Prompt diagnosis of pulmonary Mycobacterium tuberculosis (TB) infection can prevent nosocomial exposure. However, sputum smears are insensitive, and turnaround time for cultures can take weeks. Rapid diagnostics, such as nucleic acid amplification testing (NAAT), on respiratory specimens of patients suspected to have TB can improve diagnostic accuracy. Current practice at our institution is to obtain ≥ 3 NAATs in high-risk patients prior to discontinuing airborne isolation, but some studies have suggested that 2 negative NAATs may be sufficient. We conducted a retrospective study of patients at our institution diagnosed with TB. Methods The study was conducted at an academic adult hospital, an academic pediatric hospital, and a community hospital in New York City. Line lists of positive cultures for TB and positive NAATs from 2014 to mid-2018 were obtained from microbiology. Chart review was performed. Patient demographics, comorbidities, and radiographic findings were collected. Concordance between culture results and NAATs was evaluated. Incidence of inpatient TB exposure was noted. Results 82 cases of TB were found in the study period (see Figure 1). 45 cases were new inpatient diagnoses of pulmonary TB. The most common presenting symptoms were cough (69%), weight loss (49%), and fever (42%, see Table 1). 38/45 (84%) of patients were originally from a country other than the United States. 43/45 (96%) of patients had abnormal lung imaging. Cavitary disease was seen in 29%; other upper lobe disease was seen in 42%. Among smear-negative pulmonary TB cases, NAAT was positive in 11/16 (69%) of patients. Within this subgroup, the sensitivity of one NAAT was 41% when compared with culture. In smear-negative, NAAT-positive patients, NAATs were fully concordant with cultures in 4/11 patients (36%, see Table 2). The median number of positive NAATs was 1; the median number of positive cultures was 2. Five patients with pulmonary TB had negative NAATs altogether (median = 3); 2/5 resulted in TB exposure investigations after airborne precautions were discontinued following NAAT results. Overall, 13/45 (28%) of new diagnoses resulted in an exposure investigation. Conclusion Obtaining ≥ 3 NAATs in patients suspected of pulmonary TB improved diagnostic accuracy compared with obtaining 2 or fewer. Disclosures All authors: No reported disclosures.

Detection of Mycobacterium tuberculosis lipoarabinomannan and CFP-10 (Rv3874) from urinary extracellular vesicles of tuberculosis patients by immuno-PCR

2019 ◽  
Vol 77 (5) ◽  
Author(s):  
Bhawna Dahiya ◽  
Anish Khan ◽  
Preeti Mor ◽  
Ekta Kamra ◽  
Netrapal Singh ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Mycobacterium Tuberculosis ◽  
Extracellular Vesicles ◽  
Rapid Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Tuberculosis Patients ◽  
Pulmonary Tb ◽  
Polymerase Chain

ABSTRACT Extracellular vesicles (EVs), the small circulating vesicles released from urine samples of tuberculosis (TB) patients, contain a pool of biomarkers. We recently detected Mycobacterium tuberculosis lipoarabinomannan (LAM) and CFP-10 (Rv3874) biomarkers from the urinary EVs of pulmonary TB (PTB) and extrapulmonary TB (EPTB) patients by immuno-polymerase chain reaction (I-PCR) assay and the results were compared with the analogous enzyme-linked immunosorbent assay (ELISA). The detection limits of both purified LAM and CFP-10 were determined to be 1 fg/mL with I-PCR, which was 106 times lower than ELISA. Detection of LAM and CFP-10 biomarkers in urinary EVs of TB patients by I-PCR showed superiority over ELISA. Notably, LAM I-PCR revealed sensitivities of 74.3 and 67.9% in PTB (n = 74) and EPTB (n = 53) patients, respectively, with specificities of 91.5–92.8% (n = 116). Moreover, the sensitivities attained with LAM I-PCR were significantly higher (P < 0.01) than with CFP-10 I-PCR. After further improving the sensitivity and specificity of the assay, our I-PCR based on LAM detection in urinary EVs may be used as an adjunct test for rapid diagnosis of TB.

TB-PCR and drug resistance pattern in BALF in smear-negative active pulmonary TB

2017 ◽  
Vol 21 (12) ◽  
pp. 1294-1299 ◽  
Author(s):  
K. Chavalertsakul ◽  
V. Boonsarngsuk ◽  
S. Saengsri ◽  
P. Santanirand
Keyword(s):  
Predictive Value ◽  
Resistance Pattern ◽  
Sputum Smear ◽  
Pcr Assay ◽  
Tertiary Referral Hospital ◽  
Standard Drug ◽  
Pulmonary Tb ◽  
Drug Resistance Pattern ◽  
Smear Negative ◽  
Tb Pcr

SETTING: A tertiary referral hospital in Bangkok, Thailand.OBJECTIVES: To evaluate the efficacy of a bronchoalveolar lavage fluid (BALF) tuberculosis (TB) polymerase chain reaction (PCR) assay for the diagnosis of sputum smear-negative pulmonary TB (PTB) and the usefulness of a drug-resistant (DR) TB-PCR assay compared with standard drug susceptibility testing (DST).DESIGN: BALF samples from 918 patients with acid-fast bacilli (AFB) negative sputum smears who underwent bronchoscopy for diagnostic evaluations of pulmonary diseases were prospectively determined for specific genetic elements of TB using the AnyplexTM MTB/NTM Real-Time Detection kit. Positive TB-PCR samples were subsequently evaluated for DR-TB using the Anyplex II MTB/MDR Detection kit.RESULTS: A total of 224 patients were finally diagnosed with PTB. The sensitivity, specificity, positive predictive value and negative predictive value of the TB-PCR assay were respectively 38.8%, 100%, 100%, and 83.5%. The TB-PCR assay was more sensitive than culture (30.4%) and smear (6.7%). Of the 68 TB-positive culture samples, three cases with either isoniazid (INH) or rifampicin (RMP) resistance were detected by DST. The Anyplex II MTB/MDR assay provided similar results.CONCLUSIONS: The BALF TB-PCR assay is a useful tool in the diagnosis of sputum smear-negative PTB. It can also provide INH and RMP susceptibility patterns similar to those of standard DST.

scholarly journals Nilai Diagnostik Metode “Real Time” PCR GeneXpert pada TB Paru BTA Negatif

2016 ◽  
Vol 5 (3) ◽  
Author(s):  
Eka Kurniawan ◽  
Raveinal Raveinal ◽  
Fauzar Fauzar ◽  
Zulkarnain Arsyad
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Diagnostic Value ◽  
Cross Sectional Study ◽  
Sectional Study ◽  
Cross Sectional ◽  
Pulmonary Tb ◽  
Smear Negative

 AbstrakTuberkulosis (TB) paru adalah penyakit menular yang disebabkan oleh kuman Mycobacterium tuberkulosis. TB masih tetap menjadi masalah kesehatan dunia karena lebih kurang 1/3 penduduk dunia terinfeksi oleh kuman ini dan sumber penularannya berasal dari Basil Tahan Asam (BTA) positif maupun negatif. TB paru BTA negatif didiagnosis berdasarkan gambaran klinis dan rontgen torak yang sesuai TB serta pertimbangan dokter sehingga hal ini dapat menimbulkan under atau over diagnosis TB. GeneXpert merupakan pemeriksaan molekuler dengan metode “real time“ PCR dan merupakan penemuan terobosan untuk mendiagnosis TB secara cepat. Tujuan penelitiian ini adalah melakukan penilaian validitas GeneXpert pada TB paru BTA negatif dibandingkan dengan kultur Loweinstein Jensen. Desain penelitian uji diagnostik ini adalah cross sectional study. Penelitian dilakukan terhadap 40 orang pasien TB paru BTA negatif di Puskesmas sekitar kota Padang dan pasien yang dirawat di Bagian Penyakit Dalam RS dr. M. Djamil Padang. Dilakukan pemeriksaan sputum dengan GeneXpert dan dibandingkan dengan kultur Loweinstein Jensen. Hasil uji diagnostik dengan GeneXpert untuk mendiagnosis TB paru BTA negatif didapatkan sensitivitas 83.33%, spesifisitas 95.46%, nilai prediksi positif 93.75%, nilai prediksi negatif 87.5% dan akurasi 90% serta hasil uji kappa didapatkan 0.796. Disimpulkan GeneXpert memiliki sensitivitas, spesifisitas, nilai prediksi positif, nilai prediksi negatif dan akurasi yang tinggi pada TB paru BTA negatif.Kata kunci: nilai diagnostik, TB paru BTA negatif, GeneXpert AbstractPulmonary tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. TB is still a global health problem. Approximately one third of the world population is infected by Mycobacterium tuberculosis and the source of infection came from smear positive and negative patient. Smear negative pulmonary TB can be considered based on clinical symptom and chest x-ray as well as description of TB and it’s depend on doctor's decision, so this can lead to under or over-diagnosis. Molecular examination with real time PCR method of GeneXpert is a breakthrough invention to diagnose TB quickly. The objective of this study was to assess the validity of GeneXpert in smear negative pulmonary TB and it’s compared with Loweinstein Jensen culture. Study design was diagnostic test with cross sectional study. Research conducted on 40 patients with smear negative pulmonary TB in public health centers around the city of Padang and the patients who were treated at the Internal Medicine department of dr. M. Djamil hospital Padang. Sputum examination conducted by GeneXpert compared with Loweinstein Jensen culture. Diagnostic value of GeneXpert for diagnosing smear negative pulmonary tuberculosis are sensitivity 83.33%, specificity 95.46%, positive predictive value 93.75%, negative predictive value 87.5% and accuracy 90%. Kappa value is 0.796. GeneXpert has a high sensitivity, specificity, positive predictive value, negative predictive value and accuracy on smear negative pulmonary tuberculosis.Keywords: diagnostic value, smear negative pulmonary tuberculosis, GeneXpert

scholarly journals Detection of Mycobacterium tuberculosis (MTB) in Fecal Specimens From Adults Diagnosed With Pulmonary Tuberculosis Using the Xpert MTB/Rifampicin Test

2015 ◽  
Vol 2 (2) ◽  
Author(s):  
Hiroyuki Kokuto ◽  
Yuka Sasaki ◽  
Shoji Yoshimatsu ◽  
Kazue Mizuno ◽  
Lina Yi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Sputum Smear ◽  
Proof Of Concept ◽  
Retrospective Case ◽  
Pulmonary Tb ◽  
Smear Negative ◽  
Positive Results ◽  
Control Study

Abstract Background.  The Xpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) is a fully automated diagnostic test that allows for the detection of MTB including its RIF resistance. Although the test is used for the diagnosis of tuberculosis (TB) in sputum samples worldwide, studies using fecal specimens are scarce. We therefore evaluated the efficacy of the Xpert MTB/RIF test for detection of MTB in fecal specimens obtained from adult pulmonary TB patients, confirmed by culture and/or molecular diagnostic methods. Methods.  We conducted a retrospective case-control study to provide proof-of-concept regarding the efficacy of the Xpert MTB/RIF test using fecal samples for diagnosing pulmonary TB via detection of MTB in adult patients (≥20 years) at the Fukujuji Hospital in Tokyo, Japan. Results.  Fecal specimens were obtained from 56 active pulmonary TB patients (including 48 sputum smear-positive and 8 sputum smear-negative patients), 10 non-TB patients (including 4 Myocobacterium avium complex infections), and 27 healthy individuals who were exposed to active pulmonary TB patients. The sensitivity of the fecal Xpert MTB/RIF was 100% (81.7%–100%) for detection of MTB in specimens from sputum smear-positive (1+ to 3+) patients, 81.0% (58.1%–94.6%) in specimens from sputum smear scanty positive patients, and 50.0% (15.7%–84.3%) in specimens from sputum smear-negative patients. Meanwhile, each of the fecal specimens from the non-TB group was negative for MTB (specificity 100%; 95% confidence interval, 86.2–100). Conclusions.  The fecal Xpert MTB/RIF test could detect MTB in a large proportion of smear-positive pulmonary TB patients, without frequent false-positive results at a TB referral hospital in Japan.

scholarly journals Molecular Detection of Mycobacterium tuberculosis: Impact on Patient Care

2001 ◽  
Vol 47 (8) ◽  
pp. 1553-1558 ◽  
Author(s):  
Karen L Kaul
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Patient Care ◽  
Molecular Detection ◽  
Clinical Care ◽  
Antibiotic Sensitivity ◽  
Cost Of Care ◽  
Pcr Assay ◽  
Smear Negative ◽  
Pcr Assays ◽  
The Impact

Abstract Background: Nucleic acid amplification technologies such as PCR are revolutionizing the detection of infectious pathogens such as tuberculosis (TB). Amplification technology offers the potential for the diagnosis of TB in a few hours with a high degree of sensitivity and specificity. However, molecular assays neither replace nor reduce the need for conventional smear and culture, speciation, and antibiotic sensitivity assays. Methods: We undertook prospective studies of sputum samples to assess the performance of two PCR-based assays for the detection of TB as well as the impact of more rapid availability of test results on patient care. Results: The sensitivity of both the in-house and Amplicor PCR assays was 100% for smear-positive sputa. For smear-negative sputa (two sputum samples collected during the first 24 h of hospitalization), the sensitivity was 85% for our in-house PCR assay and 74% for the Roche PCR assay. Approximately 10% of the smear- and culture-negative sputa yielded positive PCR results; however, more than one-half of these were positive with both the in-house and Amplicor assays, suggesting the presence of TB DNA or organisms. Several of these came from patients whose other samples grew Mycobacterium tuberculosis during the same admission, and others came from patients who had previously treated TB. Overall, the specificities of the in-house and Amplicor PCR assays in smear-negative patients were 86% and 93%, respectively. Conclusions: Molecular detection of slow-growing pathogens such as M. tuberculosis have the potential to improve clinical care through a dramatic reduction in the time required for detection and may provide substantial savings in the overall cost of care of a patient compared with conventional smear, culture, and speciation alone, despite the fact that conventional assays must still be performed for speciation of nontuberculous mycobacteria and for full assessment of antibiotic sensitivity.

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