Fast detection of Mycobacterium tuberculosis in culture-positive sputum samples by nitrate reductase activity

Abstract

Microscopy and bacterial culture are the main tools in the diagnosis of tuberculosis. Since the slow growth of Mycobacterium tuberculosis impairs rapid diagnosis strategies, especially in countries where the latter are the only available resources, the ongoing development of new and inexpensive tools based on mycobacterial metabolism optimizing growth detection with preliminary identification is greatly welcome. When compared to the other species from the M. tuberculosis complex, M. tuberculosis is a strong nitrate reducer. Current assay compares the nitrate reductase activity of M. tuberculosis from pulmonary specimens cultivated in nitrate-supplemented media. Fifty-five sputum samples were decontaminated and inoculated in conventional (Middlebrook 7H9, Ogawa Kudoh-OK) and in nitratesupplemented media (Middlebrook 7H9-N, Ogawa Kudoh-N). An aliquot from the media directly reacted with Griess reagent (7H9-N and OK-N) every five days, or transferred to a nitrate substrate solution (7H9, OK). Nitrate to nitrite reduction was considered positive, revealed by the pink color, indicating bacterial growth. As reference method, the Mycobacteria Growth Indicator Tube (MGIT) was used for sensitivity calculations and statistical analysis. 7H9-N and OK-N assays proved to perform better in detecting M. tuberculosis than conventional assays (7H9 and OK). Indeed, broth nitrate-supplemented medium (7H9-N) was comparable to MGIT to detect M. tuberculosis, except in growth detection time. Results show that 7H9-N may be used as an alternative tool particularly in low-income countries since it is a simple and cheap technique, and does not restrict diagnosis to single-source products.

The Contribution of Nitrate Dissimilation to Nitrate Consumption in narG - and napA -Containing Nitrate Reducers with Various Oxygen and Nitrate Supplies

“…First, the Griess reagent was used to primarily screen nitrate-reducing strains by determining nitrite production of the isolated strains ( 42 ). Subsequently, these primarily selected strains were cultured overnight in EM, and then the genomic DNA of each strain was extracted using a TIANamp bacterial DNA kit (TianGen Biotech, China).…”

Mycobacterium avium ssp. Paratuberculosis and Crohn’s Disease—Time to Consider New Therapeutic Approaches?

“…Other researchers have shown little difference in the performance of supplemented 7H9 medium and MGIT tubes, when used in nonautomated manual procedures. [37,38] In-house fluid media has advantages as an experimental vehicle in the development of CWDM culture media, including the ability to supplement the media with specific nutrients, and monitoring for variations in replication times of isolates in response to additives. The authors believe that because of the numerous variables, (nutrients, osmolarity, atmosphere etc) that in-house bespoke fluid media is cost-effective and sufficiently flexible for further development of media for CWDM, until such time that consensus is reached and commercially manufactured CWDM is available.…”

Mycobacterium Avium ssp Paratuberculosis, and the Detection of Cell Wall-Deficient Forms in Human Subjects

“…Bacterial culture is necessary for confirmation of the ZN stain result and for the subsequent antimycobacterial susceptibility tests and molecular studies. Other researchers have shown little difference in the performance of supplemented 7H9 medium and MGIT tubes, when used in nonautomated manual procedures [37,38]. In-house fluid media has advantages as an experimental vehicle in the development of CWDM culture media, including the ability to supplement the media with specific nutrients, and monitoring for variations in replication times of isolates in response to additives.…”