Bacteriology
Accuracy of loop-mediated isothermal amplification for the diagnosis of Clostridium difficile infection: a systematic review

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Abstract

Loop-mediated isothermal DNA amplification (LAMP) is currently used as standalone diagnostic test for C. difficile infection (CDI). We assessed the diagnostic accuracy of LAMP for the diagnosis of CDI. We searched 5 databases to identify studies that compared LAMP with culture cytotoxicity neutralization assay or anaerobic toxigenic culture (TC) of C. difficile. We used the random-effects model to calculate pooled sensitivities, specificities, diagnostic odds ratios, and their 95% confidence intervals (CIs). The search of the databases yielded 16 studies (6979 samples) that met inclusion criteria. When TC was used as the gold standard (6572 samples), bivariate analysis yielded a mean sensitivity of 0.95 (95% CI, 0.93–0.97; I2 = 67.4) and a mean specificity of 0.99 (95% CI, 0.96–1.00; I2 = 97.0). LAMP is a useful diagnostic tool with high sensitivity and specificity for detecting CDI. The results should, however, be interpreted only in the presence of clinical suspicion and symptoms of CDI.

Introduction

Clostridium difficile is the most common bacterial cause of inhospital healthcare-associated diarrhea in North America and Europe with an estimated incidence of 3.85 cases per 1000 patient-days in US acute care hospitals (Zimlichman et al., 2013). Over the last several years, new, highly virulent strains, such as BI-NAP1-027, have caused several global outbreaks (Loo et al., 2005). These strains have been shown to cause more severe disease than the non-NAP1 strains and are associated with high level fluoroquinolone resistance (Deshpande et al., 2008, Loo et al., 2005, McDonald et al., 2005). In addition to the associated morbidity and mortality, the cost of hospital-acquired C. difficile infections (CDIs) has been estimated to exceed $1.5 billion a year in the United States (Zimlichman et al., 2013). Nearly all antimicrobials have been associated with CDI over the years, with longer exposure and exposure to multiple antimicrobial agents increasing the risk of CDI further (Pepin et al., 2005). The diagnosis of CDI is usually made based on the presence of symptoms (diarrhea, abdominal pain, and fever) and either a stool test result positive for C. difficile toxins or toxigenic C. difficile or colonoscopic findings demonstrating pseudomembranous colitis (Cohen et al., 2010). Laboratory test methods available for identification of CDI include anaerobic toxigenic culture (TC), culture cytotoxicity neutralization assay (CCNA), enzyme immunoassay (EIA) for toxin A and B, glutamate dehydrogenase (GDH) EIA, real-time polymerase chain reaction (RT-PCR), and loop-mediated isothermal amplification assay (LAMP) (O'Horo et al., 2012). There is currently no accepted gold standard test for diagnosis of CDI, but CCNA (sensitivity of 70–100%, specificity of 90–100%) and anaerobic TC (sensitivity of 90–100%, specificity of 98–100%) are often used as reference tests for evaluation of each other as well as evaluation of novel testing methods (Massey et al., 2003, Poutanen and Simor, 2004). CCNA and anaerobic TC are time-consuming and resource-intensive tests. In practice, many labs perform the rapid and easy-to-perform EIA for toxin A and B detection, though this test lacks sensitivity and is considered a suboptimal approach by current clinical practice guidelines (Cohen et al., 2010). More recently, nucleic acid amplification tests including RT-PCR and LAMP have been developed for diagnosis of CDI. Some hospitals have already begun to implement these tests in order to improve the rapidity of CDI testing and detection rates. Two previous meta-analyses have evaluated at the diagnostic characteristics of RT-PCR. They found that while it is highly sensitive and specific, it is also dependent on CDI prevalence (Deshpande et al., 2011, O'Horo et al., 2012). While most commercially available RT-PCR assays are designed to detect a conservative region of tcdB, it has been reported that in C. difficile variant strains, tcdA is more conserved (Rupnik, 2008). The Illumigene™ C. difficile Assay (Meridian Bioscience, Cincinnati, OH, USA) uses loop-mediated isothermal amplification technology to detect a 204-bp sequence in the conserved 5′ region of tcdA. While some variant C. difficile strains have deletions at the 3′ end of the tcdA gene, the 5′ portion remains intact for these strains (Rupnik et al., 1998). A recent study confirmed the ability of the Illumigene assay to detect these Toxin A−/B+ strains (Couturier et al., 2013). The Illumigene assay is currently the only commercial LAMP assay approved for US laboratory use. The Food and Drug Administration now requires all vendors to include toxigenic culture as a comparator for any new diagnostic test for C. difficile detection. One systematic review evaluated the diagnostic capabilities of LAMP for detection of CDI and concluded that LAMP was a promising test, but further investigation was necessary to evaluate LAMP as a diagnostic tool (O'Horo et al., 2012). The most recent Infectious Diseases Society of America (IDSA)/Society for Healthcare of America (SHEA) guidelines on diagnostic testing of C. difficile suggest that more data are needed on nucleic acid amplification tests before it can be implemented for routine use (Cohen et al., 2010). We performed a meta-analysis to assess the capabilities of LAMP in the diagnosis of CDI. The aim of this study was to investigate whether LAMP is sensitive and specific enough for the diagnosis of CDI when used as a standalone test.

Section snippets

Methods

This review was performed with a standardized written protocol that followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement guidelines (Moher et al., 2009).

Study characteristics

Our search yielded 60 articles; 30 potentially relevant citations were selected based on relevance to the study topic. After reviewing the titles and abstracts, 21 articles were selected for full-text evaluation (Fig. 1). Sixteen articles with 18 studies published between 2005 and 2014 reported the sensitivity and specificity of LAMP on stool samples for the diagnosis of CDI and were included in our meta-analysis (Bruins et al., 2012, Buchan et al., 2012, Calderaro et al., 2013, Dubberke et

Discussion

The LAMP assay evaluated here, which amplifies the C. difficile tcdA gene, demonstrated high sensitivity and specificity in comparison to anaerobic TC as the reference standard for diagnosing CDI. Despite statistical heterogeneity, the diagnostic accuracy was consistently similar in the direction of effect in the majority of the studies. The likelihood ratios for the LAMP assay indicate that the test is useful in determining posttest probability of CDI. Also, the PPV increases as the prevalence

Strengths and limitations of the review

We used a comprehensive search strategy to identify all relevant studies. Our search involved 5 unique and large databases with no language restrictions. Also, there was relatively low likelihood of publication bias, as assessed by Deek's method.

The present study has some limitations. First, the varied study designs and the substantial heterogeneity among the pooled estimates soften any conclusions drawn from our meta-analysis. To minimize this, only studies that used TC or CCNA as the

Conclusions

LAMP assay appears to be a highly sensitive and specific method for the rapid diagnosis of CDI. However, as a relatively newer assay, and considerable heterogeneity among studies, a cautious approach may be needed, with more large scale, well-designed studies to assess its true potential.

The following are the supplementary data related to this article.

Supplemental Appendix 1.

Reporting

The study was reported according to the PRISMA Guidelines.

Funding

None

Conflict of Interest

All authors report no conflict of interest.

Acknowledgments

This research was first presented at ID Week Annual Meeting, Philadelphia, USA, October 2014.

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    AL and VP contributed equally to this manuscript.

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