scholarly journals Recent Development in the Molecular Diagnosis of Tuberculosis

2018 ◽  
Vol 5 (2) ◽  
pp. 10-15
Author(s):  
Supriya Sarkar
Keyword(s):  
Drug Resistance ◽  
Nucleic Acid ◽  
Diagnostic Tests ◽  
Control Program ◽  
Molecular Techniques ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Second Line ◽  
Childhood Tb ◽  
Smear Negative

ABSTRACT Molecular diagnostic tests are the molecular techniques used to detect a nucleic acid of organisms. They have revolutionized the diagnosis of tuberculosis (TB) and drug resistance. Cartridge-based nucleic acid amplification tests (CBNAAT) and line probe assay (LPA) have been accepted in our national tuberculosis control program. CBNAAT has the advantage that it can be done from crude samples and smear negative samples and results can be obtained by 2 hours. But CBNAAT can detect rifampicin resistance only. On the contrary, LPA can detect rifampicin and isoniazid resistance, but the test can only be done at regional or national levels. LPA cannot be done from smear negative or crude specimens. The recent version of LPA (version 2.0) can overcome that problem. Second line LPA can detect the drug resistance against fluoroquinolones, second line injectable drugs and ethambutol (in version 1.0 only). GeneXpert Omni is a portable technique having 4-hour lasting battery using the same cartridge. GeneXpert Ultra has more sensitivity due to larger chamber size and due to two additional probes. Ultra may replace Xpert soon as it will be more effective against smear-negative TB, TB in HIV positive persons, childhood TB, and extrapulmonary TB. With the introduction of molecular diagnostic tests for TB, we will be able to reduce the time of diagnosis of TB and drug resistance. Thereby, we will be able to start first line or second line anti-tubercular chemotherapy earlier, and that will help us to achieve our goal in controlling TB.

scholarly journals A Retrospective Cross-Sectional Study of the Utility of Cartridge-Based Nucleic Acid Amplification Test in Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in People Living with HIV in Second Highest HIV Prevalent State in India

2021 ◽  
Author(s):  
Tade Bagbi ◽  
Ningthoukhongjam Reema ◽  
S. Bhagyabati Devi ◽  
Thangjam Gautam Singh ◽  
Mohammad Jaleel ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cross Sectional Study ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Rifampicin Resistance ◽  
Sputum Smear ◽  
Smear Microscopy ◽  
Sectional Study ◽  
Cross Sectional ◽  
Smear Negative

Abstract Introduction Tuberculosis (TB) in people living with human immunodeficiency virus (PLHIV) is difficult to diagnose due to fewer organisms in sputum and extrapulmonary samples. Sputum culture takes 4 to 8 weeks for growth of the mycobacteria. Delayed treatment for TB in PLHIV leads to increased mortality. This study evaluated cartridge-based nucleic acid amplification test (CBNAAT) as a diagnostic tool for diagnosis of pulmonary TB (PTB) and extrapulmonary TB (EPTB) in PLHIV in the second most HIV prevalent state in India and for comparing its efficacy between Ziehl–Neelsen (ZN) staining sputum smear–positive and sputum smear–negative TB. Methods This cross-sectional study was conducted in RIMS, Imphal, with 167 PLHIV patients, age 15 years or older, having signs and symptoms of TB. Appropriate samples for sputum microscopy and CBNAAT were sent. Conclusion The overall sensitivity of sputum smear for acid-fast bacillus (AFB) was found to be 30.71% and that of CBNAAT was 38.57%. Sensitivity of CBNAAT for sputum smear–positive and sputum smear–negative TB was 100 and 11.3%, respectively. Sensitivity of ZN smear for AFB of EPTB sample was 48.1% and that of CBNAAT was 59.25%. In both PTB and EPTB, CBNAAT showed an increase in diagnosis of microbiologically confirmed PTB cases by 7.8 and 11.1%, respectively, over and above the cases diagnosed by ZN smear microscopy. Rifampicin resistance was detected in five patients. We conclude that CBNAAT is a rapid test with better sensitivity in diagnosis of PTB and EPTB in PLHIV, compared with ZN smear microscopy. It detects rifampicin resistance for multidrug-resistant TB and helps in early treatment intervention.

scholarly journals Overinterpretation of Clinical Applicability in Molecular Diagnostic Research

2009 ◽  
Vol 55 (4) ◽  
pp. 786-794 ◽  
Author(s):  
Blanca Lumbreras ◽  
Lucy A Parker ◽  
Miquel Porta ◽  
Marina Pollán ◽  
John P A Ioannidis ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Odds Ratio ◽  
Adjusted Odds Ratio ◽  
Diagnostic Value ◽  
Molecular Techniques ◽  
Impact Factors ◽  
Molecular Diagnostic ◽  
Original Research ◽  
Clinical Applicability ◽  
Diagnostic Research

Abstract Background: We evaluated whether articles on molecular diagnostic tests interpret appropriately the clinical applicability of their results. Methods: We selected original-research articles published in 2006 that addressed the diagnostic value of a molecular test. We defined overinterpretation of clinical applicability by means of prespecified rules that evaluated study design, conclusions regarding applicability, presence of statements suggesting the need for further clinical evaluation of the test, and diagnostic accuracy. Two reviewers independently evaluated the articles; consensus was reached after discussion and arbitration by a third reviewer. Results: Of 108 articles included in the study, 82 (76%) used a design that used healthy controls or alternative-diagnosis controls, only 15 (11%) addressed a clinically relevant population similar to that in which the test might be applied in practice, 104 articles (96%) made definitely favorable or promising statements regarding clinical applicability, and 61 (56%) of the articles apparently overinterpreted the clinical applicability of their findings. Articles published in journals with higher impact factors were more likely to overinterpret their results than those with lower impact factors (adjusted odds ratio, 1.71 per impact factor quartile; 95% CI, 1.09–2.69; P = 0.020). Overinterpretation was more common when authors were based in laboratories than in clinical settings (adjusted odds ratio, 18.7; 95% CI, 1.41–249; P = 0.036). Conclusions: Although expectations are high for new diagnostic tests based on molecular techniques, the majority of published research has involved preclinical phases of research. Overinterpretation of the clinical applicability of findings for new molecular diagnostic tests is common.

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals Evaluation of AMPLILINK Software for the COBAS AMPLICOR System

1999 ◽  
Vol 37 (2) ◽  
pp. 436-437 ◽  
Author(s):  
Harald H. Kessler ◽  
Donald Jungkind ◽  
Evelyn Stelzl ◽  
Sue Direnzo ◽  
Srinivas K. Vellimedu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Internal Control ◽  
Diagnostic Procedures ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Single Specimen ◽  
Clinical Laboratories ◽  
And Control

The use of AMPLILINK version 1.0 software was evaluated for the operation and control of one COBAS AMPLICOR instrument and for two COBAS AMPLICOR instruments run simultaneously to perform and detect nucleic acid amplification reactions. A total of 3,384 results were analyzed. The initial accuracy of the results was 99.91%. Three errors of omission of transfer of data from the COBAS AMPLICOR to the AMPLILINK system were observed. Two of these errors were from a single specimen, where both the analyte and internal control results were not transmitted. These errors did not interfere with the correctness of any other data. There were no interruptions of runs, and no data were mixed. AMPLILINK increased convenience, saved labor, and was found to be a very useful addition for clinical laboratories performing molecular-diagnostic procedures with the COBAS AMPLICOR system.

Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.

1997 ◽  
Vol 10 (2) ◽  
pp. 242-256 ◽  
Author(s):  
M Ieven ◽  
H Goossens
Keyword(s):  
Nucleic Acid ◽  
Respiratory Tract ◽  
Respiratory Tract Infections ◽  
Clinical Laboratory ◽  
Pneumocystis Carinii ◽  
Diagnostic Techniques ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Amplification Techniques ◽  
Tract Infections

Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for excluding false-positive and false-negative results.

Diagnosing Emerging and Reemerging Infectious Diseases: The Pivotal Role of the Pathologist

2011 ◽  
Vol 135 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Juan P. Olano ◽  
David H. Walker
Keyword(s):  
Nucleic Acid ◽  
Infectious Diseases ◽  
Molecular Diagnostics ◽  
Clinical Setting ◽  
Driving Forces ◽  
Molecular Techniques ◽  
Nucleic Acid Amplification ◽  
Pivotal Role ◽  
Laboratory Diagnostics ◽  
Molecular Tools

Abstract Context—Molecular diagnostics continues to evolve very rapidly, and its impact in the diagnosis of infectious diseases is undeniable. Molecular tools have played a pivotal role in discovering and characterizing several emerging infectious agents and have now become the gold standard for the diagnosis of infectious diseases caused by fastidious or uncultivable agents. Multiple challenges still remain for the widespread use of cost-effective, validated, and commercially available molecular tools. Automated instruments capable of sample processing and multiplex nucleic acid amplification and postamplification analysis have already been approved by the US Food and Drug Administration (FDA) for use in the clinical setting. Nanobiotechnology is beginning to impact laboratory diagnostics in the clinical setting. Objective—To address current nucleic acid techniques used in the clinical laboratory for diagnosis of infectious diseases. FDA-approved tests are listed, as well as molecular techniques (amplification and postamplification analysis). A comprehensive list of emerging pathogens during the last 4 decades is also presented. Biosurveillance systems are discussed in the context of molecular tools. The rapidly evolving field of nanobiotechnology is briefly addressed. Data Sources—Original publications, major reviews, and book chapters were used to present a comprehensive, yet short, review of molecular diagnostics in infectious diseases. Conclusions—We will continue to witness an exponential growth of molecular techniques used for the initial diagnosis of infectious diseases. Molecular tools will also continue to have an impact on disease prognosis and response to therapeutic interventions. Automation, multiplexing, and miniaturization will continue to be driving forces in the development of new instruments.

scholarly journals Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples

2004 ◽  
Vol 53 (12) ◽  
pp. 1215-1219 ◽  
Author(s):  
T D McHugh ◽  
C F Pope ◽  
C L Ling ◽  
S Patel ◽  
O J Billington ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clinical Diagnosis ◽  
Nucleic Acid Amplification ◽  
Prospective Evaluation ◽  
Strand Displacement ◽  
Strand Displacement Amplification ◽  
Fine Needle ◽  
Fine Needle Aspirates ◽  
Time To Diagnosis ◽  
Smear Negative

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis.

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.

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