Other Automated Culture Systems
Other recent developments for the rapid detection of mycobacteria include manual methods like the MB-Redox (Heipha Diagnostika Biotest, Heidelberg, Germany) based on the reduction of a tetrazolium salt indicator in liquid medium, and automated equipment-based methods like the MB/ BacT jystem
(Organon Teknika, Boxtel, Holland) based on the colorimetric detection of carbon dioxide produced by myco-bacterial growth in a closed system, and the ESP culture system II (Trek Diagnostics, Inc., Cleveland, OH, USA) based on the detection of pressure changes in the culture medium of a sealed vial during mycobacterial growth. These systems have not gained widespread use outside laboratories in industrialised countries."
The FastPlaque TB Assay
The FastPlaqueTB Assay is a novel, patent protected, phage amplification technology that has been developed for rapid detection and enumeration of M. tuberculosis complex from respiratory specimens. This method uses specific mycobacteriophages (viruses that infect M. tuberculosis complex) to detect the presence of viable TB bacilli in the clinical specimen.
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Mycobacteria are mixed with phages, which are allowed to adsorb and infect the cells. All unadsorbed extracellular phages are then inactivated using a virucidal chemical; while the phages that have infected the viable TB bacilli
remain protected and continue to replicate. After replication the progeny
bacteriophages are released and detected by mixing with fast growing
non-pathogenic helper cells (M. smegmatis) on an agar plate. The
mycobacteriophages in turn infect, replicate and lyse these helper cells and
lysis is detected as plaques (clear zones). The number of plaques visualized
from a given sample is related to the number of viable tubercle bacilli in the
original sample.
A meta-analysis by Kalantri and colleagues found that phage-based assays were
highly specific, but had lower and variable sensitivity in different studies.
They concluded that improvement in the sensitivity of the tests was required
before phage-based assays could replace other diagnostic tests.
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Advances In Microscopy
In many countries diagnosis of TB is performed by microscopic examination of a stained sputum smear by the Ziehl-Neelsen (ZN) method. Although easy to perform and specific, it lacks sensitivity. Several studies have been performed to assess the usefulness of adding a chemical reagent, such as sodium hypochlorite, to liquefy and then concentrate the sputum by further centrifugation to increase sensitivity. In most of these studies, a statistically significant improvement in the proportion of positive smears or sensitivity was obtained; however, for several reasons, sodium hypochlorite, also known as the "bleach" method is not used routinely in many settings.
In high-income countries, fluorescence microscopy rather than conventional
microscopy has become the standard diagnostic method.17 The use of auramine as a
fluorescent method to detect mycobacteria in sputum was proposed many years ago
and re-evaluated later using a combination of auramine-O and rhodamine. This
fluorescent method is associated with a higher rate of detection, since slides
can be examined at lower magnifications.
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Examination of smear may take less time with fluorescence microscopy.
Fluorescence microscopy is credited with increased sensitivity and lower work
effort than conventional microscopy, but there is concern that specificity may
be lower. It is also more expensive than the conventional ZN staining requiring
a fluorescent microscope.