Elsevier

Journal of Infection

Volume 66, Issue 6, June 2013, Pages 487-493
Journal of Infection

Clinical value of IS6110-based loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis complex in respiratory specimens

https://doi.org/10.1016/j.jinf.2013.02.005Get rights and content

Summary

Objectives

A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens.

Methods

In this cross-sectional study (2008–2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases.

Results

The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69/77 specimens; 95% confidence interval [CI], 80.5–95.4%), 76.6% (59/77 specimens; CI, 65.6–85.5%), 79.2% (61/77 specimens; CI, 68.5–87.6%) and 59.7% (46/77 specimens; CI, 47.9–70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.828), and between LAMP and Amplicor (k 0.746), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens.

Conclusion

The better diagnostic performance of IS6110-LAMP compared to Amplicor (p = 0.009), nPCR (p = 0.013) and PCR (p < 0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings.

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.

References (21)

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