Advantages and Limitations of Sequencing for Identification of Mycobacteria: The advantages of DNA sequencing for the identification of mycobacteria are that it allows for the objective identification of a wide variety of mycobacteria species including Mycobacterium tuberculosis. Also, a fairly rapid turnaround time is possible with same day or next day identification to the species level after the organism grows in culture. Limitations of sequencing include the fact that it’s fairly labor-intensive and requires highly skilled, trained technologists for performance. In addition, the equipment and reagents costs are not insignificant. Finally, one cannot stress strongly enough how dependent the use of a high- quality library database. Sequencing is generally used for the identification of nontuberculous mycobacteria, but Mycobacterium tuberculosis complex can also be identified by sequence analysis if the specific nucleic acid hybridization probe is not available. Limitations of LTD PCR for Mycobacteria: Limitations of a laboratory-developed PCR assay for Mycobacterium species include some of those limitations found for the commercially available tests, namely a lower sensitivity for smear-negative specimens, the fact that a negative result does not rule-out Mycobacterium infection, and a mycobacterial culture is still needed, and the fact that nucleic acid amplification tests detect the presence of amplifiable nucleic acid, but they don’t indicate if the organism is still viable. In addition, the cost of an LDT test can also be high. Limitataions of MALDI-TOF MS for Identification of Mycobacteria: Limitations of MALDI-TOF MS for the identification of mycobacteria include the need to grow the organism in culture first, which is often a rate limiting step. MALDI-TOF requires a pure isolate so mixtures of organisms often give a “no identification” result. In addition, the phase of growth and the media used can affect the spectra produced. For best performance, the spectral library needs to be composed of spectra produced under comparable conditions to your everyday working practices in the laboratory. The manufacturer’s databases need expansion for less common organisms and instrument downtime, whether for scheduled maintenance or due to a breakdown, can be a problem if you are using a single instrument and if it has been the main method of organism identification in your laboratory. Regulatory issues can be a concern for groups of organisms which have not yet received FDA-clearance and, specifically for mycobacteria, the technique may, at times, be a little slower than sequencing because of the need to have more growth for MALDI-TOF MS than for sequencing. Limitations of MTD Test: The limitations of the MTD test include the observation that this is a technically difficult test for laboratory technologists to perform. It is an open PCR system, which requires opening of tubes and transfer of amplified products so the risk for a false-positive res.

1. Advantages and Limitations of Sequencing for Identification of Mycobacteria:
The advantages of DNA sequencing for the identification of mycobacteria are that it allows for
the objective identification of a wide variety of mycobacteria species including Mycobacterium
tuberculosis. Also, a fairly rapid turnaround time is possible with same day or next day
identification to the species level after the organism grows in culture.
Limitations of sequencing include the fact that it’s fairly labor-intensive and requires highly
skilled, trained technologists for performance. In addition, the equipment and reagents costs are
not insignificant. Finally, one cannot stress strongly enough how dependent the use of a high-
quality library database.
Sequencing is generally used for the identification of nontuberculous mycobacteria, but
Mycobacterium tuberculosis complex can also be identified by sequence analysis if the specific
nucleic acid hybridization probe is not available.
Limitations of LTD PCR for Mycobacteria:
Limitations of a laboratory-developed PCR assay for Mycobacterium species include some of
those limitations found for the commercially available tests, namely a lower sensitivity for
smear-negative specimens, the fact that a negative result does not rule-out Mycobacterium
infection, and a mycobacterial culture is still needed, and the fact that nucleic acid amplification
tests detect the presence of amplifiable nucleic acid, but they don’t indicate if the organism is
still viable. In addition, the cost of an LDT test can also be high.
Limitataions of MALDI-TOF MS for Identification of Mycobacteria:
Limitations of MALDI-TOF MS for the identification of mycobacteria include the need to grow
the organism in culture first, which is often a rate limiting step. MALDI-TOF requires a pure
isolate so mixtures of organisms often give a “no identification” result. In addition, the phase of
growth and the media used can affect the spectra produced. For best performance, the spectral
library needs to be composed of spectra produced under comparable conditions to your everyday
working practices in the laboratory. The manufacturer’s databases need expansion for less
common organisms and instrument downtime, whether for scheduled maintenance or due to a
breakdown, can be a problem if you are using a single instrument and if it has been the main
method of organism identification in your laboratory. Regulatory issues can be a concern for
groups of organisms which have not yet received FDA-clearance and, specifically for
mycobacteria, the technique may, at times, be a little slower than sequencing because of the need
to have more growth for MALDI-TOF MS than for sequencing.
Limitations of MTD Test:
The limitations of the MTD test include the observation that this is a technically difficult test for
laboratory technologists to perform. It is an open PCR system, which requires opening of tubes

2. and transfer of amplified products so the risk for a false-positive result due to sample cross-
contamination or amplicon contamination is high. In addition, potential inhibitors from specimen
components is a concern.
A negative MTD test does not rule-out Mycobacterium infection and a mycobacterial culture still
needs to be performed. In addition, the MTD test detects the presence of nucleic acid, but it
doesn’t indicate if the organism is still viable. Cross reactions can occur with some rare
mycobacteria and the test can be costly.
Solution
Advantages and Limitations of Sequencing for Identification of Mycobacteria:
The advantages of DNA sequencing for the identification of mycobacteria are that it allows for
the objective identification of a wide variety of mycobacteria species including Mycobacterium
tuberculosis. Also, a fairly rapid turnaround time is possible with same day or next day
identification to the species level after the organism grows in culture.
Limitations of sequencing include the fact that it’s fairly labor-intensive and requires highly
skilled, trained technologists for performance. In addition, the equipment and reagents costs are
not insignificant. Finally, one cannot stress strongly enough how dependent the use of a high-
quality library database.
Sequencing is generally used for the identification of nontuberculous mycobacteria, but
Mycobacterium tuberculosis complex can also be identified by sequence analysis if the specific
nucleic acid hybridization probe is not available.
Limitations of LTD PCR for Mycobacteria:
Limitations of a laboratory-developed PCR assay for Mycobacterium species include some of
those limitations found for the commercially available tests, namely a lower sensitivity for
smear-negative specimens, the fact that a negative result does not rule-out Mycobacterium
infection, and a mycobacterial culture is still needed, and the fact that nucleic acid amplification
tests detect the presence of amplifiable nucleic acid, but they don’t indicate if the organism is
still viable. In addition, the cost of an LDT test can also be high.
Limitataions of MALDI-TOF MS for Identification of Mycobacteria:
Limitations of MALDI-TOF MS for the identification of mycobacteria include the need to grow
the organism in culture first, which is often a rate limiting step. MALDI-TOF requires a pure
isolate so mixtures of organisms often give a “no identification” result. In addition, the phase of
growth and the media used can affect the spectra produced. For best performance, the spectral
library needs to be composed of spectra produced under comparable conditions to your everyday
working practices in the laboratory. The manufacturer’s databases need expansion for less

3. common organisms and instrument downtime, whether for scheduled maintenance or due to a
breakdown, can be a problem if you are using a single instrument and if it has been the main
method of organism identification in your laboratory. Regulatory issues can be a concern for
groups of organisms which have not yet received FDA-clearance and, specifically for
mycobacteria, the technique may, at times, be a little slower than sequencing because of the need
to have more growth for MALDI-TOF MS than for sequencing.
Limitations of MTD Test:
The limitations of the MTD test include the observation that this is a technically difficult test for
laboratory technologists to perform. It is an open PCR system, which requires opening of tubes
and transfer of amplified products so the risk for a false-positive result due to sample cross-
contamination or amplicon contamination is high. In addition, potential inhibitors from specimen
components is a concern.
A negative MTD test does not rule-out Mycobacterium infection and a mycobacterial culture still
needs to be performed. In addition, the MTD test detects the presence of nucleic acid, but it
doesn’t indicate if the organism is still viable. Cross reactions can occur with some rare
mycobacteria and the test can be costly.