Low mycobacterial recovery, especially in MGIT, observed in the present study suggests that OM-S reagent might not be compatible with the MGIT system. More extensive field evaluations of the OM-S reagent are warranted to demonstrate a significant benefit over currently used methods.

f Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks. In recent years, studies employing whole-genome sequencing (WGS) of Mycobacterium tuberculosis isolates have demonstrated its value in understanding transmission patterns, recurrent tuberculosis (TB), development of drug resistance, and bacterial evolution (1-9). In parallel, improvements of next-generation sequencing platforms and library preparation workflows make it possible to determine the genome sequence from bacterial DNA samples in the time span of 1 week. Reculturing isolates for DNA isolation has been reported as a necessary step in published WGS studies (2,4,7,10). As culturing of slow-growing bacteria, such as M. tuberculosis, takes from 1 week to several weeks, it constitutes the main time-consuming process in WGS projects (11)(12)(13)(14). While initially large amounts of DNA were required for reliable WGS library preparation, newly developed library preparation protocols for bacterial samples typically require about 1 to 10 ng of DNA (e.g., Illumina Nextera XT [1 ng], New England BioLabs NEBNext Ultra [5 to 1,000 ng], and Bioo Scientific NEXTflex ChIP-Seq [1 to 10 ng]). Therefore, faster and reliable methods for DNA isolation would enable a considerable decrease in the time needed to perform WGS analyses. In this regard, a recent study proposed a WGS workflow starting from early positive liquid cultures of the MGIT system (15), potentially enhancing the speed of WGS procedures as part of routine diagnostics.Furthermore, reculturing of isolates from even well-maintained frozen stocks can fail entirely, usually excluding the respective isolate from any further analysis. A recent publication reported failure rates of up to 50% for M. tuberculosis glycerol stocks (16). New methods enabling WGS analysis directly from frozen stocks without reculturing can rescue genotype information, especially from historic collections of isolates.In this study, we investigated and tested a protocol for performing WGS of DNA extracted directly from frozen glycerol stocks, which were all historic isolates from patients diagnosed with fully susceptible TB between 1992 and 2012, circumventing the step of reculturing altogether.For validation, we sequenced DNA from cultured aliquots of the same frozen stocks in parallel. In total, we included 40 frozen glycerol stocks (1992 to 2012) stored at Ϫ80°C at the International Reference Laboratory of Mycobacteriology at the Statens Serum Institut (Copenhagen). All were processed according to a standard lysis protocol with heat inactivation and sonication as is usually used for PCR (17,18). Lysates were concentrated with Microcon filters (Merck KGaA, Darmstadt, Germany), followed by a purification with ethanol (EtOH) precipitation and be...

Quality improvement measures implemented in TB laboratories showed a positive impact on various outcomes. Due to the high heterogeneity of outcome reporting and interventions and the low quality of the studies, the effect size was not clear. Identification of standardized quality indicators and their link to the quality of patient care would improve knowledge in this field.

BackgroundHigh quality diagnostic services are crucial for tuberculosis (TB) diagnosis, treatment and control. A strong laboratory quality management system (QMS) is critical to ensuring the quality of testing and results. Recent initiatives to improve TB laboratory quality have focused on low and middle-income countries, but similar issues also apply to high-income countries.Methods and findingsUsing a multipronged approach reviews of facilities, equipment, processes (purchasing, pre-analytic, analytic and post-analytic), staff, health and safety, documentation, information management and organization based on the ISO 15189 and the twelve quality system essentials were conducted between October 2015 and January 2016 at the National TB Reference Laboratory in Germany. Outcome assessment included proportion of smear positive slides, proportion of contaminated liquid cultures and DNA contamination rates before and after implementation of QMS. The odds ratio for these outcomes was calculated using a before/after comparison. Reviews highlighted deficiencies across all twelve quality system essentials and were addressed in order of priority and urgency. Actions aimed at improving analytical quality, health and safety and information management were prioritised for initial implementation in parallel with each other. The odds ratio for a sample to be tested as microscopically positive increased by 2.08 (95%CI 1.41–3.06) comparing the time before with the time after implementation of quality managed fluorescence microscopy. Liquid culture contamination rates decreased from 23.6- 7.6% in April-July 2016 to <10% in November 2017-March 2018. The proportion of negative controls showing evidence of DNA contamination decreased from 38.2% in 2013 to 8.1% in 2017, the corresponding odds ratio was 0.14 (95%CI 0.07–0.29).ConclusionThis study showed marked improvement on quality indicators after implementation of a QMS in a National TB Reference Laboratory. The challenges and lessons learned in this study are valuable not just for high-income settings, but are equally generalizable to other laboratories.

Background Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test. Methods Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types. Results We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts. Conclusions Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.