A Simple Microfluidic Assay for Diagnosing Tuberculous Meningitis in HIV-Uninfected Patients

Abstract

We evaluated the diagnostic performance of a simple and label-free pathogen enrichment method using homobifunctional imidoesters (HI) and a microfluidic system, called the SLIM assay, followed by real-time PCR from cerebrospinal fluid (CSF) in human immunodeficiency virus (HIV)-uninfected patients with suspected tuberculous meningitis (TBM). Patients with suspected TBM were prospectively enrolled in a tertiary hospital in an intermediate tuberculosis (TB)-burden country during a 30-month period. TBM was classified according to the uniform case definition. Definite and probable TBM were regarded as the reference standards for TBM, and possible TBM and not-TBM as the reference standards for not-TBM. Of 72 HIV-uninfected patients with suspected TBM, 10 were diagnosed with definite (n ϭ 2) and probable (n ϭ 8) TBM by the uniform case definition. The sensitivity of the SLIM assay was 100% (95% confidence interval [CI], 69 to 100%) compared with definite or probable TBM, and it was superior to those of mycobacterial culture (20% [95% CI, 3 to 56%]) and the Xpert MTB/RIF assay (0% [95% CI, 0 to 31%]). Of 21 possible TBM and 41 not-TBM patients by the uniform case definition, 5 possible TBM and no not-TBM patients gave positive results in the SLIM assay. The specificity of the SLIM assay was 92% (95% CI, 82 to 97%; 5/62). We demonstrated that the SLIM assay had a very high sensitivity and specificity with small samples of 10 cases of definite or probable TBM. Further studies are needed to confirm this finding and to compare the SLIM assay with mycobacterial culture, Xpert MTB/RIF, and Xpert MTB/ RIF Ultra assays in a larger prospective cohort of patients with suspected TBM, including both HIV-infected and HIV-uninfected cases. KEYWORDS SLIM assay, Xpert MTB/RIF, tuberculous meningitis T uberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis infection and accounts for about 1% of all tuberculosis cases (1). The clinical course of TBM can deteriorate rapidly, and the mortality rate is high in the absence of prompt diagnosis and treatment (2). The gold standard for diagnosing tuberculosis is mycobacterial culture, but it takes 2 to 8 weeks to get the results, and the sensitivity is relatively low because of the paucibacillary nature of TBM (2). In 2013, the World Health Organization (WHO) endorsed the Xpert MTB/RIF assay as the initial diagnostic test for suspected TBM patients in preference to other tests, such as mycobacterial culture and M. tuberculosis PCR (3). The 43 to 72% sensitivity of the Xpert MTB/RIF assay was still not high enough to rule out TBM (4-7). In a recent report, the sensitivity of the Xpert MTB/RIF Ultra assay (70 to 95%) was superior to those of the Xpert MTB/RIF assay (43 to 45%) and mycobacterial culture (43 to 45%) (7). However, only human immunodeficiency virus (HIV)-infected patients with suspected TBM were enrolled in that study.

Gene-based diagnosis of tuberculosis with a new-generation pathogen enrichment technique

“…In most conventional assays, only a small quantity of clinical sample is used for pathogen detection uses, then the remaining sample unused. However, the SLIM assay uses larger sample volumes (up to 50 mL) with simultaneous pathogen concentration and extraction in a single system [3,4], with comparable time to Xpert MTB/RIF (2.5-4.5 h for SLIM) but with significantly lower cost ($5-6 for SLIM) and instrument requirements for sample processing. In the current study, we showed superior sensitivity of the SLIM assay compared to the Xpert MTB/RIF, especially for detecting smear-negative PTB.…”

“…The specialists choosing TB treatment were blinded to SLIM assay results. SLIM assay principles and detailed methods have been described previously [3,4]. Briefly, a mixture of sputum sample and liquefaction solution with HIs reagent were added to the SLIM system to enrich M. tuberculosis and DNA extracted without the need for detergents or bulky instruments.…”

“…1,6 , Young Ae Kang2,6 , Choong Eun Jin3,6 , Ho Cheol Kim1 , Geun Su Noh3 , Hyo Joo Lee3 , Joung Ha Park4 , Yong Seo Koo 5 , Yong Shin3,7 and Sung-Han Kim4,7 1 Dept of Pulmonary and Critical Care Medicine, Asan Medic1al Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. 2 Division of Pulmonology, Dept of Internal Medicine, Yonsei University College of Medicine, Severance hospital, Seoul, Republic of Korea.…”

Gene-Based Diagnosis of Tuberculosis from Oral Swabs with a New Generation Pathogen Enrichment Technique

“…However, the sensitivity of the Xpert assay is not high enough for paucibacillary TB cases ( 4 , 12 ), and is thus limited for use in smear-negative TB cases that require more sensitive diagnostic methods. To meet this unmet clinical need, we applied the SLIM assay, a new generation pathogen enriching technique, and demonstrated its efficacy in PTB and other infectious diseases ( 9 , 10 , 13 ).…”

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India

“…The sensitivity of GeneXpert, a cartridge based rapid molecular test was still not high enough to rule out TBM (45-67% sensitivity to detect microbiologically proven TBM). GeneXpert Ultra has 70% sensitivity versus clinical case definition and misses up to 30% of cases [5]. However, a recent study from Donovan et al reported that GeneXpert Ultra was not superior to GeneXpert for TBM diagnosis in both HIV-uninfected and HIV-infected adults [6].…”