Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations

Vanessa Deggim-Messmer,Guido V Bloemberg,Claudia Ritter,Antje Voit,Rico Hömke,Peter M Keller,Erik C Böttger

doi:10.1016/j.ebiom.2016.06.016

Vanessa Deggim-Messmer, Guido V Bloemberg + Show 5 more

Open Access

https://doi.org/10.1016/j.ebiom.2016.06.016

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Journal: EBioMedicine	Publication Date: Jun 14, 2016
Citations: 34	License type: cc-by

Affiliation: University of Zurich

Abstract

Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology.Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1–2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB.MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy.Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

Highlights

Conventional methods for detection of mycobacteria include smear microscopy (AFB) and isolation by culture followed by phenotypic drug susceptibility testing (DST)
In the first part of our study we evaluated the performance of polymerase chain reaction (PCR) based detection of M. tuberculosis in patients' specimens in comparison to culture (Fig. 1)
Discrepant culture/PCR results were resolved by evaluating clinical data, the patients' clinical history and the availability of additional patient specimens that were analyzed by culture and PCR

Summary

Introduction

The genus Mycobacterium is separated into the pathogenic species Mycobacterium tuberculosis complex, Mycobacterium leprae, and Mycobacterium ulcerans, and various nontuberculous mycobacteria (NTM) (Manual of Clinical Microbiology, 2015). Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis. Global priorities for tuberculosis (TB) care and control are to improve case-detection and to enhance rapid diagnosis of tuberculosis disease (WHO, 2014). Observed standardized short-course chemotherapy (DOTS) is an effective treatment for drug-susceptible tuberculosis disease, with cure rates N95.0% (Hopewell et al, 2006). The current situation is characterized by increasing numbers of drug-resistant tuberculosis disease. The treatment of multidrug-resistant (MDR) or extensively drug-resistant (XDR) TB requires the use of second-line drugs, which are less effective, more expensive, and more toxic than first-line drugs (WHO, 2014; Hopewell et al, 2006)

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