Clinical value of IS6110-based loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis complex in respiratory specimens
Introduction
Tuberculosis (TB) continues to be a major public health challenge today. In 2011, an estimated 8.7 million new cases of TB and 1.4 million TB-associated deaths, mostly in developing countries, were reported.1
Clinical laboratories play a crucial role in prompt and accurate diagnosis of TB required for ultimate control of the disease. Traditionally, acid-fast staining of smears and mycobacterial culture are used as the routine TB diagnostic methods particularly in developing countries. However, smear microscopy and culture are respectively insensitive and time-consuming methods, although the latter remains the reference standard test for diagnosis of TB.2 Additionally, molecular approaches such as amplification of specific gene targets could shorten the turnaround time needed for laboratory detection of the members of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. A wide variety of in-house and commercial PCR assays targeting different chromosomal elements have been developed for this purpose, which in turn respectively suffer from heterogeneity of results and high costs.3
To address the pressing need for a rapid and dependable assay to detect M. tuberculosis (MTB); several studies have employed the innovative nucleic acid amplification technique, namely loop-mediated isothermal amplification (LAMP).4, 5, 6, 7, 8, 9, 10, 11, 12 This method, which was introduced in the last decade by Notomi et al.,13 has the advantages of being: (i) specific, as six primers recognizing eight different regions on the target sequence are used; (ii) efficient, because amplification is performed with an enzyme with strand displacement activity under fixed temperature; and (iii) simple, rapid and inexpensive since obviating the need for a post amplification detection step, and therefore applicable in resource-limited settings.
We recently reported the promising preliminary results of a newly developed IS6110-based LAMP assay.12 To demonstrate the diagnostic value of this novel assay for the detection of MTBC in respiratory specimens, it was evaluated in the present study in comparison with Amplicor MTB test (Roche Diagnostics) and home-made conventional IS6110-based PCR and nested PCR (nPCR) assays. Mycobacterial culture or clinical recovery of the patients following anti-TB therapy were used as the reference points to determine the diagnostic sensitivities and specificities of these different nucleic acid amplification-based methods. To the best of our knowledge, this is the first report on the clinical use of IS6110-LAMP assay compared with an FDA-approved commercial test (Amplicor) for detection of MTBC in respiratory specimens.
Section snippets
Clinical samples
In this cross-sectional study, a total of 101 sputum specimens were collected from 93 patients who were highly suspected of having pulmonary TB. All the patients were admitted to the Center for Public Health and Care (Western Division), Ahvaz Jundishapour University of Medical Sciences, Ahvaz, Iran, between January 2008 and April 2009. Patients were included in the study based on their clinical features including: a suggestive chest radiograph, a productive cough for more than 3 weeks (with or
Results
After obtaining the results of the molecular assays, the results of smear and culture and available clinical data were unblinded, in order to be compared with each other. Among 101 sputum specimens collected from 93 patients, 67 (66.3%) and 74 (73.3%) specimens resulted positive respectively by smear analysis for acid-fast bacilli (AFB) and by MTB culture (Table 1). Twenty-four out of 27 culture-negative specimens were clinically ruled out to be from TB patients. All the four molecular assays
Discussion
In addition to improved sensitivity and specificity, an innovative technology for diagnosis of TB must also address simplicity, rapidity and capability of becoming a point-of care test with one-day result.
The nucleic acid amplification-based LAMP technique has the potential to become such a diagnostic test for the detection of MTBC but also of other infectious agents.17 To date, several studies have evaluated the LAMP method targeting various genomic regions for the detection of MTBC in
Acknowledgments
The authors would like to thank Samad Aryan for taking high-quality photographs from LAMP reaction tubes. This study was initiated at and financially supported by Ahvaz Jundishapoor University of Medical Sciences, Ahvaz, Iran, and completed at Mashhad University of Medical Sciences, Mashhad, Iran, which kindly supported facilities needed for repeating some experiments.
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A novel, rapid (within hours) culture-free diagnostic method for detecting live Mycobacterium tuberculosis with high sensitivity
2020, EBioMedicineCitation Excerpt :The major drawback of culture techniques, however, is that it takes weeks to more than a month before a positive culture for M. tuberculosis can be identified, and the methods require at least a moderately well-equipped laboratory [5]. On the other hand, nucleic acid amplification tests (NAATs) including the Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA), Amplicor Mycobacterium Tuberculosis Test (Roche, Basel, Switzerland), and Loop-Mediated Isothermal Amplification (LAMP; Eiken Chemical, Tokyo, Japan), are now widely used, even in low- and middle-income countries due to financial assistance from various global foundations [6-8]. It is estimated that NAATs can detect M. tuberculosis in suspensions containing as few as 10–1000 CFU/mL, making this technology highly sensitive [9,10].
Evaluation of improved IS6110 LAMP assay for diagnosis of pulmonary and extra pulmonary tuberculosis
2017, Journal of Microbiological MethodsCitation Excerpt :It may be due to the reason that other methods used in CRS were nucleic acid amplification methods which can detect DNA extracted from dead/non-viable bacteria in culture negative specimens. LAMP was able to detect one additional specimen as positive in comparison to PCR targeting same sequence which is in accordance with reported literature (Aryan et al., 2013). The performance of modified IS6110 LAMP assay was also compared with in-house sdaA LAMP assay which showed significant concordance even in culture negative specimens.
Loop-mediated isothermal amplification assay for detection of Mycobacterium tuberculosis complex in infertile women
2016, Indian Journal of Medical MicrobiologyThe frequency of Helicobacter pylori in dental plaque is possibly underestimated
2015, Archives of Oral BiologyCitation Excerpt :The loci and sequences of the primers are shown in Table 1. The LAMP reactions were performed as described earlier with some modifications.20,21 The Optimal LAMP reaction was prepared in a total volume of 30 μl containing 10 mM KCl, 10 mM (NH4)2SO4, 10 mM MgSO4, 0.1% Triton X-100 and 0.8 M betaine (Sigma–Aldrich), 1.6 mM each of HP-ureC FIP and HP-ureC BIP, 0.2 mM each of HP-ureC FOP and HP-ureC BOP, 0.8 mM each of HP-ureC LF and HP-ureC LB, 2 mM dNTPs, and 6 μl of template DNA.
Real-time fluorescence Loop-Mediated Isothermal Amplification (LAMP) for rapid and reliable diagnosis of pulmonary tuberculosis
2015, Journal of Microbiological MethodsCitation Excerpt :In this study we integrated the amplification and detection stages of LAMP into one portable and simple to use platform in an attempt to not only make this method easily usable but also acquire real time data. Primers were designed for targeting IS6110 region, a high-copy-number gene of MTB (Aryan et al., 2013). All of 14 standard MTB strains were successfully detected by real-time fluorescence LAMP, except for standard non-MTB strains, and there were no false-positive and false-negative results.